TRENDS IN BIOSCIENCES JOURNAL

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Trends
in
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Volume 2 Number 2 December, 2009
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Trends in Biosciences
(A Biannual Scientific Journal)
International Advisory Board
Dr. A. Coomans, Ex-Professor State University of Ghent, Belgium
Dr. Randy Gaugler, Director Centre for Vector Biology, Rutgers University, USA
Dr. S.B. Sharma, Director Plant Security, South Perth, Australia
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Dr. Masood Ali, Ex-Director, Indian Institute of Pulses Research (IIPR), Kanpur
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Editor in Chief: Dr. S.S. Ali, Emeritus Scientist, Indian Institute of Pulses Research (IIPR), Kanpur
Associate Editors
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Dr. S.K. Jain, Coordianator, AICRP Nematode, Indian Agriculture Research Institute, New Delhi
Dr. R. Ahmad, Ex- Principal Scientist, IIPR, Kanpur
Mr. Naimuddin, Scientist, IIPR, Kanpur
Dr. Mohd. Ovais, Ex-Professor, Barkatullah University, Bhopal, M.P.
Dr. Rashid Pervez, Indian Institute of Spices Research, Khozicod, Kerala
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ISSN 0974-8431
Trends
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Volume 2 Number 2 December, 2009
Online version available at
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Trends in Biosciences
Volume 2 No. 2 December, 2009
MINI REVIEW
1. Breeding for Cold Tolerance in Chickpea 1-6
S. K. Chaturvedi, D.K. Mishra, P. Vyas and Neelu Mishra
RESEARCH PAPERS
2. Antifungal Activity of Some Medicinal Plants on Clinically Isolated Candida tropicalis 7-10
Ch. Tanushree Das, Ritarani Das and R. C. Mohanty
3. A Dye Based Assay for Lignin and Manganese Peroxidases of Phanerochaete chrysosporium RP-78 11-13
K. Haritha and K.R.S., Sambasiva Rao
4. Conventional Indoor Rearing Leads to Abnormalities in the Ultrastructure of Muga Silkworm (Antheraea 14-17
assamensis), Mid-Gut Cell Microvilli
Sudip Dey, Begonia Dkhar, Rahul Chakraborty, Sudipto Chaudhury and Dhirendra K. Sharma
5. Toxic Effect of Synthetic Pyrethroid Deltamethrin and Neem Based Formulation Achook on Zebrafish, 18-20
Danio rerio
Badre Alam Ansari and Dilip K. Sharma
6. Studies on Genotype × Environment Interaction and Stability for Seed Yield in Common Bean (Phaseolus 21-22
vulgaris L.)
S. A. Dar, F. A. Pir, Abu Manzar and Ajaz A. Lone
7. Seasonal Population Fluctuation of Hoplolaimus indicus on Pigeon Pea 23-24
S.S. Ali
8. Integrated Phosphorus Management in Mungbean in Kashmir Valley 25-26
F.A. Pir, F.A. Nehvi, Abu Manzar, S.A. Dar and B.A. Allai
9. Anti-mutagenic Effects of Flavonoids in Human Lymphocytes Culture 27-30
Md. Sultan Ahmad, Mrs. Sheeba, Afsar Ali, Rakesh Yadav and B.R.Gautam
10. Effect of Irrigation Intervals on the Incidence of Shoot Borer, Chilo infuscatellus Snellen in Sugarcane 31-32
Crop in Punjab
H. S. Randhawa, K. S. Thind. and J. S. Chhina
11. Efficacy of Three Antibiotics on Reduction of Mortality Rate in Mulberry Silkworm (Bombyx mori L.) 33-35
in the Monsoon Season of Lucknow
Amit Srivastava and R. Venkatesh Kumar
12. A New Species of Hoplolaimus (Basirolaimus) (Hoplolaiminae : Tylenchida) from Pigeon Pea Ecosystem 36-38
of Bumdelkhand Region
S. Sharafat Ali, Azra Shaheen and Rashid Pervez
13. Characterization of Aromatic Hydrocarbon Degrading Bacteria Isolated from Effluents of Thermal Power 39-41
Archana Mishra, Rais Ahmad and Shilpa Kaistha
14. Effect of Plant Extracts on Activity and Mortality of Second-Stage Juveniles of Root-knot Nematode, 42-44
Meloidogyne javanica
S.S. Ali and Rashid Pervez
CONTENTS

15. Heterosis Estimates of Garden Pea Crosses (Pisum sativum L. hortense) 45-47
Saumya Awasthi, G. Roopa Lavanya and Rashmi Jain
16. Estimation of Unavoidable Yield Losses in Certain Rabi Pulse Crops Due to the Root-Knot Nematode, 48-49
Meloidogyne javanica
S.S. Ali
17. Efect of Intercrops on Quantitative and Qualitative Leaf Yield of Mulberry (Morus Alba L.) 50-52
K.A. Murugesh
18. Description of Some Aphid Species (Homoptera : Aphididae : Aphidinae) Belonging to 53-58
Tribe Macrosiphini Recorded from Aligarh Region
M. K. Usmani and Uzma Rafi
19. Nitrogenase Activity in Cyanobacterial Biofilm 59-60
P. Parameswaran, S. Palani and D.Venkatesan
20. Morphological Traits as Descriptors for Characterization of Important Composites of Maize in 61-63
Kashmir Valley
F.A. Nehvi, Ajaz, A. Lone, Vaseem Yousaf, M. I. Makhdoomi and S. A. Dar
21. Avoidable Yield Loss Due to Meloidogyne incognita and Other Parasitic Nematode in Fieldpea 64-65
(Pisum sativum) under Field Condition
M.G. Haidar, T.H. Askary and S.S. Ali
22. Management of Shoot and Capsule Borer, Conogathes punctifaralis L in Castor by Intercropping 66-67
B.S.Patel and I.S.Patel
23. Seed Vigour Studies in Relation to Position of Pod in Pea 68-69
Prabha Shankar Shukla and Rajendra Prasad
24. Infectivity of Spodoptera litura (F.) (Lepidoptera: Noctuidae) by Certain Native Entomopathogenic 70-73
Nematodes and Their Penetration in Test Insect and in vivo Production
Rashid Pervez and S. S. Ali
25. Seasonal Prevalence of Oesophagostomum sp. in Goats and Sheep from Kanpur 74-75
Siddiqua Bano, N. Sultana and Ved Prakash
26. Evaluation of Relatively Eco-friendly Insecticides Against Pigeon Pea Podfly Melanagromyza obtusa 76-78
(Malloch) in Late Pigeon Pea
Manzoor Hussain Dar, P.Q. Rizvi and Hem Saxena
27. Studies on the Anamorph Characters and Management of Powdery Mildew of Mustard 79-80
Shabbir Ashraf and Bhawana Yadav
SHORT COMMUNICATIONS
28. Resupination and Flower Structure: the Adaptation for Pollination in Impatiens L.? 81-82
R. Ramasubbu, A.K. Sreekala and A.G. Pandurangan
29. Comparative Efficacy of Ranide and Fasinex Against Fascioliasis in Sheep. 83-84
Siddiqua Bano, N. Sultana and Ved Prakash
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Trends in Biosciences 2 (2): 1-6, 2009
MINI REVEIEW
Breeding for Cold Tolerance in Chickpea
S.K. CHATURVEDI, D. K. MISHRA, P. VYAS AND NEELU MISHRA
Indian Institute of Pulses Research, Kanpur 208 024
e-mail: chaturvedi5463@yahoo.co.in
ABSTRACT
Chickpea (Cicer arietinum L.), a winter season crop, is the 4 th
largest grain-legume crop in the world. In India, among various
grain legumes grown chickpea ranks 1 st covering 8.25 m ha
area during 2008-09. Being a cool-season crop, chickpea faces
low temperature to the tune of 0-5 o C for about 15-20 days in
the northern states. The sensitivity of varieties at flowering to
chilling temperature below 10°C has adverse effect on chickpea
production (15-20% yield losses). The development of varieties
possessing cold/low temperature tolerance is viable option for
enhancing chickpea production and productivity in low
temperature environments of countries like India, Canada and
Australia. In present paper, attempts have been made to define
cold/low temperature stress in relation to chickpea for different
growing environments/countries. Various screening techniques
and scoring index in vogue, availability of donors, genetics of
cold tolerance, different aspects of varieties development
including problems and prospects have been discussed.
Key words
Chickpea, cold tolerance, production, environment,
review
Chickpea (Cicer arietinum L.), a winter season legume,
is the 4 th largest grain-legume crop in the world covering more
than 11 m ha. Chickpea productivity records in the last four
decades revealed interesting trend like, productivity
consistently increased in India and Mexico while it declined
in Turkey, Pakistan, and Iran. In India, 7.05 million tonnes of
chickpea was produced from 8.25 m ha area during 2008-09
with 855 kg/ha average yield indicating all time high production
and productivity. This is mainly due to more availability of
seeds of high yielding varieties and mild temperature during
crop season in most part of the country, especially in northern
India leading optimum crop growth. Being a cool-season food
legume, chickpea faces low temperature to the tune of 0-5 o C
for about 15-20 days in the northern states as the crop is
highly sensitive to mean temperatures below 15°C at flowering
leading to flower drop or pod abortion. The sensitivity of
varieties at flowering to chilling temperature below 10°C has
adverse effect on chickpea yield (Ali and Kumar, 2005; Sharma,
et al., 2005). Any advantage derived from early flowering is
negated by flower drop and pod abortion/loss due to low
temperature. Similarly, crop sown after harvest of rice faces
low temperature i.e. cold stress at emergence/vegetative stage
leading to less biomass production. The breeding varieties
possessing cold/low temperature tolerance will certainly help
Dr. S. K. Chaturvedi, Principal
Scientist and Head, Division of Crop
Improvement, Indian Institute of
Pulses Research and Nodal Officer
(Seed) is well known chickpea
breeder. He has developed four
chickpea varieties namely DCP 92-3,
SCS 3 (IPC 97-67), Shubhra (IPCK
2002-29) and IPCK 2004-29. For the first time, he has
developed extra large seeded (>50 g/100 seed wt.) kabuli
chickpea genotype IPCK02. He has contributed in
identification of extra large seeded kabuli chickpea variety
Phule G 0517. Dr. Chaturvedi has developed and identified
many donors prominent among these are wilt resistant IPC
2004-52 and IPC 97-29, green seeded kabuli IPCK2002-112
and triple poded IPC 99-18 chickpea genotypes. He has
handled ad-hoc projects funded by ACIAR, Australia,
Ministry of Agriculture, Govt. of India and ICAR, New Delhi.
Recognizing the chickpea improvement work carried out
by Dr. Chaturvedi and his team, ICRISAT honoured
Chickpea Improvement Team of Indian Institute of Pulses
Research by presenting King Boudein and Dooreen
Mashler Award 2002. He is a fellow of Indian Society of
Genetics & Plant Breeding and Indian Society of Pulses
Research and Development (ISPRD). As secretary of ISPRD
he has organized National Symposium on “Pulses for
Sustainable Agriculture and Nuitritional Security” in 2001
at New Delhi. Dr. Chaturvedi initiated and organized
Chickpea Scientist meet to avoid duplication of research
efforts in generating breeding material and organized three
such meets. He has visited Australia, Syria and Spain
worked with ACIAR as visiting scientist at CLIMA/Ag
west, Australia. Dr. Chaturvedi has published more than
105 research papers including two edited books and six
technical bulletins and training manual.

2 Trends in Biosciences 2 (2), 2009
in enhancing chickpea production and productivity in
northern India. Efforts have been to assess the worth of
genetic options for alleviating cold tolerance to regain
traditional crop area of chickpea in north India. Chickpea
varieties released for cultivation in Indo-gangetic plains in
the country, often suffer in yield due to low temperature at
flowering causing 15-20% yield losses ICAR, 2006). Clarke
and Siddique, 2003 have also suggested use of chilling tolerant
varieties with early pod sets to harvest benefit from chickpea
cultivation.
Low temperature/cold stress Low temperature stress
like other abiotic stress is highly heterogeneous in time and
space, and is extremely unpredictable. This makes it very
difficult to collect and collate the observations recorded at
different sites during yield loss assessment and drawing
logical conclusions from field based studies. Even low
temperature has different meanings for chickpea cultivation
in Indian sub-continent, Australia and in Middle East. In India
and Australia usually chickpea crop suffers from low
temperature at flowering or pod development resulting either
flower drop or pod abortion whereas in middle east low
temperature restricts the crop growth and its effect is mainly
at vegetative stage. In northern India, late sown chickpea
crop also suffer to low temperature in terms of poor crop growth
(less biomass production) resulting in low productivity.
Therefore, there is need to develop low temperature tolerant
varieties of chickpea for Indian conditions possessing ability
to set pods at lower temperature (

CHATURVEDI et al., Breeding for Cold Tolerance in Chickpea 3
labelled and individual flowers are tagged on the day of
anthesis. These individual flowers are observed for pod set
and data is correlated to the daily minimum and maximum
temperatures. The genotypes exhibiting pod set below 8ºC
temperature are considered as cold tolerant. Development of
chickpea genotypes which set seed when minimum
temperature ranged from -1 to 7ºC at ICRISAT demonstrated
the worth of this screening procedure (ICRISAT, 1988).
Systematic screening for cold tolerance has only been started
relatively recently (Clarke, et al., 2004; Bhardwaj and Sandhu,
2009). Based on experiments conducted under controlled
environments, it was found that i) pod set can occur at night
temperatures of 0-5ºC if day time temperatures exceeds 20ºC,
ii) seed development requires a higher threshold temperature
than pod set and iii) 15/5 and 15/0ºC (day/night) thermal regimes
are useful in preliminary screening of germplasm lines for cold
tolerance (Srinivasan, et al., 1998).
Evidences for presence of genetic variability
Srinivasan, et al., 1998 examined genetic variation in
pod and seed set at low temperatures and observed
substantial variation both in field and controlled environments.
Two early maturing advance breeding lines, ICCV 88502 and
ICCV 88503, showed good pod set in cold spells, but seed
development in them was limited. Evaluation of the parents
involved in these breeding lines indicated possible
contribution of cold tolerance from genotype K 1189 which
originated from former Soviet Union. Srinivasan, et al., 1999
further observed that cold tolerance of ICCV88502 and
ICCV88503 was associated with a high pollen vigour
(germination and tube growth) and ovule viability at low
temperature. At the same time the presence of total number of
ovules was not affected by cold stress in any cultivars/lines.
Clarke, et al., 2004 suggested two novel methods for chickpea
improvement; firstly the selection of desirable alleles during
the haploid phase of growth by pollen selection and secondly
screening for molecular markers closely associated with
chilling tolerance. The precise location and timings at location
and timings of sensitivity to low temperature (0-15ºC) during
floret development were identified in controlled environments.
Comparisons between and sensitive genotypes were used to
examine the effect of chilling stress on reproduction. A pollen
selection technique was then used for breeding, whereby the
ovule of a susceptible variety is successfully fertilised by a
pollen grain from a tolerant parent under chilling stress
conditions. Molecular markers were associated with the
chilling tolerance trait in a segregating population from a cross
between a chilling tolerant breeding line and a sensitive
cultivar was studied. Bulk segregant analysis was used to
rapidly screen a large number of bands based on Amplified
Fragment Length Polymorphisms and low temperature
genotypes were pin pointed.
Screening index for cold tolerance
Based on survival and killing per cent various scales
including 1-3, 1-5 or 1-9 have been developed and used by
various workers. Attempts were made to develop a more
reliable field screening technique for evaluation of cold
tolerance in chickpea at ICARDA, Tel Heldya, Syria, and the
main research site of ICARDA at Aleppo, Syria (Singh, et al.,
1989) and screening procedure was developed. They also
developed a more precise 1-9 scale (Table 1), using a
combination of per cent plants killed and visual damage on
leaflets and branches on individual plants which can be used
to evaluate even individual plants.
Later, Saccardo and Calcagno, 1990 used a 0-5 scale
(0=all plants killed; 5= all plants survived) to screen chickpea
material for cold tolerance and to develop lines for winter
sowing in Italy. They identified 27 lines as cold tolerant ones
at site where the minimum temperature was -12ºC and the plant
survival rate was 50-70%. Wery, 1990 reported variation among
the chickpea cultivars, which were evaluated for frost
resistance (minimum temperature -10ºC to -18.5ºC) and
suggested that phenological stage as most important in
Table1. Scoring of cold tolerance in field conditions in chickpea (Singh, et al., 1989)
Scale Category
Reaction
1 - No visible symptoms of damage
2 Highly tolerant Upto 100% leaflets show withering and drying , no killing
3 Tolerant 11 to 20% leaflets show withering and upto20% branches show withering and drying,
no killing
4 Moderately tolerant 21 to 40% leaflets and upto 20%branches show withering and dryings, no killing
5 Intermediate 41 to 60% leaflets and 21 to 40%branches show withering and drying, upto 5% plant
killing
6 Moderately susceptible 61 to 80% leaflets and from 41 to 0%branches show withering and drying, to 25% plant
killing
7 Susceptible 81 to 99% leaflets and 61 to 80% branches show withering and drying,26 to 50% plant
killing
8 Highly susceptible 100% leaflets and 81 to 99% branches show withering and drying, 51 to 99% plant
killing
9 - 100% plant killing

4 Trends in Biosciences 2 (2), 2009
determining the response of the crop to cold; cold resistance
decreased with progress in growth from germination to the
flowering stage. He used a “frost resistance ratio” (the number
of plants at harvest/ the number of plants emerged) as a
parameter for cold tolerance and grouped the genotypes in
following categories: ‘ fall type’ (frost resistance); ‘winter type’
(frost tolerant); and ‘spring type’ (susceptible to frost) and
also confirmed that early sowing dates are more suitable for
screening for cold tolerance under mediterranean
environments.
Genetics of cold/low temperature tolerance
Both additive and non-additive gene effects governing
cold tolerance in chickpea, with preponderance of former has
been reported. The cold tolerance was found under control of
at least five sets of genes and cold tolerance was dominant
over susceptibility to cold (Malhotra and Singh, 1990). The
results from generation mean analysis showed the presence
of genic interactions in addition to additive and dominance
gene effects (Malhotra and Singh, 1991). Genetic studies of
cold tolerance based on pollen viability and pod formation at
low temperature indicated that cold tolerance in ICCV 93929 is
under control of single recessive gene (Bhardwaj and Sandhu,
2009).
Sources of resistance to cold tolerance
In order to search for the germplasms possessing a higher
level of tolerance to cold than the cultivated species the large
number of accessions of eight wild Cicer species were
evaluated (Table 2). Eight annual wild Cicer species were
evaluated for cold tolerance and only C. reticulatum and C.
echinospermum were adjudged as cold tolerant (Malhotra,
1998). Later, several progenies selected from crosses involving
wild Cicer spp. have shown tolerance to cold temperature
(Malhotra, et al., 2003). Search for donors, which can retain
flowers and set pods under low temperature has led to
identification of M 450, ILC 8262, ICCV 88501, ICCV 88502,
ICCV 88503, ICCV 88506, FLIP 84-70C, FLIP 84-71C and FLIP
84-79 C (Singh and Saxena, 1993). Several introductions from
Russia and wild species have also shown fairly good level of
cold tolerance (Singh, et al., 1990). Based on per se tolerance
and avoidance mechanism, ICCV 88503 was rated as the cold
tolerant genotype when this genotype set pod at optimum
temperature (10-15°C at 08.30 h). The mechanisms for
Table 2.
Sources of tolerance to cold in cultivated and annual wild species in chickpea (updated after Malhotra and Saxena,
1993)
Species Accession(s) Source
C. arietinum ILC-794,-1071,-1251,-1256,-1444,-1455,-1464,-1875,-3465,-
3498,-3746,-3747,-3791,-3857,-3861, FLIP 82- 85C, -82-
131C, -84-112C,-85-4C,-85-49C,-85-81C, K 1189, ICCV
88501, ICCV 88502, ICCV 88503
Singh, et al., 1989,
Srinivasan, et al.,1998
M450, ICCV88501, ICCV88502, ICCV88503, ICCV 88506
and ILC8262
FLIP 84-70C, FLIP 84-71C, FLIP 84-79 C
ICCV 88503
ICCV88502, ICCV88503, ICCV88506, ICCV 88510 and
ICCV 88516
ICCV 93929
C. bijugum ILWC-7-1, -7-2,-7-4-1,-7/S-1,-7/S-3,-7/S-4,-7/S-5,-7/S-11,-
7/S-12, 7/S-13, 7/S-14, 7/S-15, 7/S-17, 7/S-18,-8-33,-8-4,-8S-
1,-8S-3, -32-2, -34/S-1, -34/S-2, -42/1,-42/2
ILWC 32, 62, 73, 79
C. pinnatifidum ILWC29/S-10
ILWC 236
C. echinospermum ILWC35/S-3
Singh, et al., 1990
Singh and Saxena, 1993
Dua, 2001
Gaur, et al., 2007
Bhardwaj and Sandhu, 2009
Singh, et al., 1990
Sandhu, 2004 Pers. Comm.
Singh, et al., 1990
Sandhu, 2004 Pers. Comm.
Singh et al. , 1990
C reticulatum
ILWC 39, 181
ILWC88/2,-21-1-2/2,-21-2/1,-21-2/3,-21-2/5,-21-11,-21-15,-
21-17,-21-21,-21-30,-21-31,-21-32,-36/3
ILWC 81, 112, 142
Sandhu, 2004 Pers. Comm.
Singh, et al., 1990
Sandhu, 2004 Pers. Comm.

CHATURVEDI et al., Breeding for Cold Tolerance in Chickpea 5
withstanding cold temperature include per se tolerance,
avoidance and escape was also suggested (Dua, 2001). Genetic
variation in pod set at low temperatures was also noticed
earlier under field conditions (ICRISAT, 1988) and confirmed
both in field and controlled environments by others (Singh, et
al., 1993; Srinivasan, et al., 1998). Variation in pollen viability
among the genotypes was also noticed by Srinivasan, et al.,
1999 when identified ICCV88501 and ICCV88502 as genotypes
that had the ability to develop good number of productive
pods at low temperature. Srinivasan, et al., 1998 further
reported cold tolerance in these two genotypes at low
temperature under field conditions during December and
January, and identified the third genotype (ICCV88503) which
had better tolerance to low temperature.
Breeding efforts and genetic options
Chickpea improvement programs targeting the
insulation of varieties against low temperature/cold stress has
been initiated by many centres globally and particularly in
India under ambit of All India Coordinated Research Project
on Chickpea since its inception in 1993. Screening against
low temperature has been taken up vigorously in recent years.
At the Centre for Legumes in Mediterranean Agriculture
(CLIMA), in Australia, chilling tolerance have transferred
from ICCV 88516 and two desi chickpea varieties WACPE
2075 (Sonali) and WACPE 2095 (Rupali) have been developed
(Clarke, et al., 2004). Breeding efforts made at ICARDA, Syria
has demonstrated the release of more genetic variability for
flowering at low temperatures using cultivated x wild Cicer
crosses. This shows that genes responsible for flowering at
low temperatures should be transferred from wild to cultivated
species, C. arietinum. Cold tolerance at flowering can also be
achieved through an accelerated breeding programme based
on haploid selection. Development and identification of
molecular markers and QTLs offer promise for mitigating low
temperature stress at genetic level. Molecular markers assisted
breeding can be a viable option in targeting the desired gene(s)
or QTLs. Good scope exists for exploitation of transgenic
technology in development of low temperature/cold tolerant
genotypes.
Per se tolerance to abiotic stresses appears to be a
difficult research aim to be tackled by conventional breeding
due to several technical limitations. In changing climatic
conditions where crop has to face abrupt low temperature
during reproductive phase, concerted efforts for the
development of low temperature/ cold tolerant chickpea
varieties are needed. An integrated approach involving
molecular biologists, conventional breeders, physiologists
and agronomists should be adopted to mitigate the low
temperature/cold stress for better crop productivity. This may
include defining target environment, development of reliable
screening techniques, identification of desirable traits and
donors, transferring the targeted gene(s) in desirable
agronomic backgrounds. Critical assessment of cold
temperature genotypes under target areas (proper
phenotyping) will certainly help in identification of high
yielding chickpea varieties for cultivation in low temperature/
cold prone areas.
LITERATURE CITED
Ali, M. and Kumar, S. 2005. Chickpea (Cicer arietinum L.) research in
India: Accomplishments and future strategies. Indian Journal of
Agricultural Sciences, 75 (3): 125-33.
Bhardwaj, R. And Sandhu, J.S. 2009. Pollen viability and pod formation
in chickpea (Cicer arietinum L.) as criteria for screening and genetic
studies of cold tolerance. Indian Journal of Agricultural Sciences,
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Recieved on 10-7-2009 Accepted on 15-10-2009

Trends in Biosciences 2 (2): 7-10, 2009
Antifungal Activity of Some Medicinal Plants on Clinically Isolated Candida tropicalis
CH. TANUSHREE DAS, RITARANI DAS AND R.C. MOHANTY
Post Graduate Department of Applied and Industrial Microbiology, Department of Botany, Utkal University,
Bhubaneswar 751 004, Orissa
e-mail: tanushree_das@live.com, das.ritarani@yahoo.com
ABSTRACT
Speciation of Candida tropicalis isolates from clinical specimen
is described and its necessity is emphasized. Aqueous and
methanolic extracts of Allium cepa, Allium sativum,
Cinnamomum zeylanicum , Eugenia caryophyllus, Piper nigrum
and Zingiber officinale were screened for antifungal study by
disc diffusion method against C.tropicalis . The crude extract in
five different amounts (2g/5ml, 4g/5ml, 6g/5ml, 8g/5ml, 10g/
5ml) were tested on the C.tropicalis. Both the aqueous and
methanolic extracts of A.sativum and E.caryophyllus showed
maximum inhibition zones compared to those of A.cepa,
C. zeylanicum , P. nigrum and Z. officinale. The inhibitory effects
of these medicinal plants varied greatly with increase in their
crude extract concentrations.
Key words
Speciation, Candida tropicalis., medicinal plants,
antifungal activity, selected concentrations.
Recent years have shown a global increase in the incident
of mycotic infections. Although rare in healthy people,
candiduria is common in hospitalized patients (Rivett, et al.,
1986).In tertiary care facilities, as many as 10% of positive
urine cultures yield Candida isolates (Schaberg, et al.,1991),
which reflects the cumulative pressure of contributing factors
such as urinary instrumentation and prolonged use of broadspectrum
antibiotics (Hamory and Wenzel, 1978, Wise and
Silver, 1993). Candida tropicalis is the second most common
Candida spp. colonization humans (Zhang, et al., 1997) and
the third most frequently isolated Candida spp. from urine
cultures (Kaufmann, et al., 2000). In addition, the nosocomial
transmission of Candida strains through medical waste and
equipment has rarely been reported. C. tropicalis is a major
cause of septicemia and disseminated candidiasis, especially
in patients with lymphoma, leukemia and diabetes. It is also
found as part of the normal human muco-cutaneous flora.
Sucrose negative variants of C. tropicalis have also been
increasingly found in case of disseminated candidiasis .
Moreover the so called non-albicans Candida are also
implicated with greater frequency as opportunistic pathogens
in the compromised hosts (Pfaller,1996).Rapid and accurate
identification of yeasts have thus become important not only
for effective management as various species respond
differently to different antifungals but also to prevent drug
resistance. Because of the side effects and the resistance that
pathogenic micro-organisms build against the antibiotics, much
attention has recent been paid to extract biological active
compounds from plant species that used in herbal medicine.
In many parts of the world, medicinal plants are used for their
antimicrobial, antifungal and antiviral activities. These plant
extracts were used as a source of medicinal agents to cure
urinary tract infections, cervicities ,vaginitis ,gastro-intestinal
disorders and skin infections such as herpes simplex virus
type. Scientific experiments since the late 19 th century have
documented the antimicrobial properties of some spices, herbs
and their components (Shobana and Naidu,2000). Many herbs
and spices are known to exert antioxidant activity and are
useful for preventing lipid oxidation in living organism as well
as in foods.
The present study explores the antifungal activity of 7
aqueous and methanolic extracts from spices against clinically
isolated C. tropicalis .Some spice plants previously screened
for biological activity by other investigators were included in
this study, which provided a comparison base.
MATERIALS AND METHODS
C. tropicalis from urine, sputum, pus ,blood, throatswab,
stool and high vaginal swab of patients attending to
S.C.B.Medical College, Cuttack was isolated by the
conventional methods using bacteriological media
(MacConkey agar and blood agar ) as Candida is able to
grow in such media while carrying out the bacteriological
analysis. Then identification up to species level was made
using chromagar Candida tube test. All the species identified
by this method were reconfirmed by using other conventional
methods.
Smears were made from the collected urine, sputum, pus,
blood , throat-swab, stool and high vaginal swab of patients.
Gram stained smears were used to look for presence of gram
positive budding yeast cells with pseudohyphae. The suitable
samples were cultured on plain Sabouraud’s Dextrose Agar
(SDA) and also in chromagar. Both plates and slants were
incubated for 48hr at room temperature ( 25±1°C - 35±1°C ) to
look for growth to cream coloured pasty colonies on SDA
plates and light blue coloured colonies on chromagar slants
suggestive of C. tropicalis.
A single colony was inoculated in human serum and
incubated at 37±1º C. After 2-4 hours, wet mount was prepared
and examined under the microscope to look for the presence
of germ tube. The absence of microscopically visible germtubes
within this time is indicative of non-albicans Candida .All

8 Trends in Biosciences 2 (2), 2009
Candida isolates were tested for the production of
chlamydospores in corn meal agar with Tween 80. The Candida
spp. was inoculated in corn meal agar (CMA) and then
incubated at 25±1ºC. After 72 hours, the plates were examined
under microscope for the presence of chlamydospores.
Chlamydospore formation is generally associated with C.
albicans.
All Candida isolates were subjected to carbohydrate
fermentation test. Carbohydrate solutions used were 6%
solution of glucose, maltose, lactose and sucrose with basal
media. Sugar tests were performed in beef extract broth with
bromothynol blue indicator and 1% final concentration of the
respective sugars means for species identification (Gray and
Glenn, 1970).
Random samples of the respective plant parts of each
test plant were collected and washed first in tap water and
then in distilled water . These were macerated in mortar with a
pestle . Selected concentrations such as 2g/5ml, 4g/5ml, 6g/
5ml, 8g/5ml and 10g/5ml were prepared with solvents (distilled
water and methanol ). They were filtered off with sterile
Whattman No.1 filter paper in to clean sterile glass plates.
Antifungal activity was performed by disc diffusion
method (Bauer, et al., 1966). Savored dextrose agar (SDA)
plates were prepared under sterile conditions. These plates
were kept for one hour in the oven at 37°C for drying before
inoculation of culture.
The discs of alcoholic and aqueous extracts of the six
plants viz., Allium cepa, Allium sativum, Cinnamomum
zeylanicum , Eugenia caryophyllus, Piper nigrum and
Zingiber officinale were prepared by soaking Whattman’s
filter paper No.1 , so that each disc ( diameter 6mm ) contained
same concentration of the extracts. SDA plates were inoculated
with 0.1 ml of 48hr broth culture of C. tropicalis containing
1.0×10 7 cells and spread with sterile cotton swabs. Sterile filter
paper discs containing various concentrations of aqueous
and alcoholic extracts were put in SDA plates. Standard
antibiotic discs of Nystatin (100 units/disc) were used for
comparison. The plates were incubated for 48hr at room
temperature (25±1°C - 35±1°C). Zones of inhibition were
measured at the end of the incubation period including the
disc diameter.
Statistical analysis
The experiment for the antifungal activity was conducted
and analyzed as a factorial experiment with three replications
in a completely randomized design. The results were presented
as the means with SEM (Standard error of the mean).
Positively index was calculated by comparing the zone
of inhibition of the extract with standard antibiotic action in
plates.
Activity index = Inhibition area of test sample / Inhibition
area of standard antibiotic.
RESULTS AND DISCUSSION
The expected results of speciation tests for Candida,
including the sugars used for fermentation and for general
tests, are listed in Table 1 and 2. Based on these criteria, the
Candida spp. was identified as Candida tropicalis .It has
been the impression of clinicians that patients infected by
Candida other than C. albicans were not in life-threatening
situations and therefore less concern was given to these
infections. Louria, et al., 1967 indicated that C. tropicalis and
perhaps other Candida species cause severe and fatal
infections. From a clinical point of view, this means that all
Candida isolates from patients should be speciated with the
tests which are readily available and easy to perform.
Table 1.
Speciation tests used in the identification of
Candida tropicalis.
Types of tests
HiCrome Candida agar
Results
Light blue colour of the
colony
Chlamydospore production --- (± )
Germ tube production ---
Glucose
AG
Maltose
AG
Sucrose
A / AG
Lactose ---
A-Acid, G- Gas, AG- Acid with Gas, (±) – Variable result
Table 2.
Sugar tests of medically important Candida
tropicalis.
Sugars
Glucose, Maltose, Galactose, D-Xylose, Soluble starch,
D-Mannitol, L-Arabinose(weak),
Sucrose , Cellobiose , D- Ribose, Ribitol , L-Sorbose ,
DL- Lactic acid.
L-Rhamnose , Lactose, Raffinose , Inositol , D-
Arabinose.
Results
Positive
Variable
Negative
The antifungal test results of the six plant species (A.
cepa, A. sativum, C. selenium, E. caryophyllus, P. nigrum and
Z. officinale) are presented in Table 3 and 4 against C.
tropicalis. The crude extracts from these plant species
showed different antifungal activity in the C.tropicalis. It was
observed that aqueous and methanolic extracts (with different
concentrations) of A. sativum and E. caryophyllus had better
antifungal effects with maximum inhibition zones.
With these two treatments C. tropicalis initially formed
23±1.03mm and 10±0.87mm zones of inhibition in aqueous
extraction at 10g/ml while in methanolic extraction 19±1.25mm
and 12.6±0.27mm zones of inhibition, respectively. Arora and
Kaur, 1999 assayed the sensitivity of yeasts to spices aqueous
extracts and found that garlic and clove extract were able to
inhibit C. tropicalis and some cases strong cidal effect was
observed.
On the other hand A.cepa, C. zeylanicum, P. nigrum
and Z.officinale showed comparatively less antifungal activity

DAS et al., Antifungal Activity of Some Medicinal Plants on Clinically Isolated Candida tropicalis 9
Table 3. (a)
Antifungal activity of aqueous extract of six
plants against Candida tropicalis. tested based
on disc diffusion method( including the disc
diameter in mm).
Plants 2g/5ml 4g/5ml 6g/5ml 8g/5ml 10g/5ml
Allium cepa 7±0.87 7.3±0.72 8±0.87 8.3±0.54 8.6±0.54
Allium sativum 20.16±1.06 21±1.08 21.5±1.18 21.66±1.19 23±1.03
Cinnamomum 7±0.47 8.3±0.27 9±0.47 9.3±0.54 9.6±0.72
zeylanicum
Eugenia 8±0.87 9±0.87 9.3±0.72 9.6±0.54 10±0.87
caryophyllus
Piper nigrum 7.6±0.54 8±0.47 8.3±0.27 8.5±0.24 8.6±0.27
Zingibar
officinale
----- 8.16±0.14 8.6±0.24 ------ -----
Table 3. (b)
Antifungal activity of methanol extract of six
plants against Candida tropicalis. tested based
on disc diffusion method( including the disc
diameter in mm).
Plants 2g/5ml 4g/5ml 6g/5ml 8g/5ml 10g/5ml
Allium cepa ----- ----- 7.5±0.62 8±0.82 8.3±0.54
Allium sativum 16±1.25 18±1.25 18.33±1.19 18.5±1.84 19±1.25
Cinnamomum 7±0.47 7.3±0.27 7.6±0.27 8±0.47 8.6±0.54
zeylanicum
Eugenia 10±0.82 10.3±0.72 10.6±0.27 12±0.47 12.6±0.27
caryophyllus
Piper nigrum ----- ----- ------ ----- -----
Zingibar
officinale
------ 7.3±0.36 ----- 8.8±0.36 -----
than standard antibiotic (Nystatin). The inhibition zone
diameters against C. tropicalis by the aqueous plant extract
of C. zeylanicum and A.cepa at 10g/ml concentration were
found to be 9.6±0.72mm and 8.6±0.54mm and on methanolic
extraction were 8.6±0.54mm and 8.3±0.54mm , respectively. The
aqueous extracts of P. nigrum exhibited inhibitory activity
against C. tropicalis , but methanolic extract did not.
Extractions of Z. officinale did not elicit remarkable inhibitory
effect properly in each concentration tested (Table- 3a and
3b). Barbosa-Canovas, et al., 1998 reported that cinnamon
and clove had a strong inhibitory activity against
microorganisms while cumin a moderate one and red pepper
had a weak inhibitory activity.
The data obtained on activity indices, through the
determination of MIC, from the association of standard
antibiotic (Nystatin) with the selected plant extracts are
presented in Tables 4(a) and 4(b). The results revealed
variability in the inhibitory concentration at 10gm/ml of each
extract in C. tropicalis, isolated from hospital samples. Of the
seven plant species, A.sativum showed highest activity index
i.e. 1.53 in aqueous extraction and 1.27 in methanolic extraction.
A recent report by Athar and Winner,1971 indicates that
clinical isolates of C.tropicalis are more resistant in vitro to
polyene antibiotics than is C.albicans. Simoes, et al.,1999
reported systematic screening for biological interactions
Table 4. (a)
Positive index: comparision of the zone of
inhibition of aqueous plant extract with
standard antibiotic (Nystatin) action in petri
plates.
Plant species 2g/5ml 4g/5ml 6g/5ml 8g/5ml 10g/5ml
Allium cepa 0.47 0.49 0.53 0.55 0.57
Allium sativum 1.34 1.40 1.43 1.44 1.53
Cinnamomum 0.47 0.53 0.60 0.62 0.64
zeylanicum
Eugenia
0.53 0.60 0.62 0.64 0.67
caryophyllus
Piper nigrum 0.51 0.53 0.55 0.57 0.57
Zingibar officinale ----- 0.54 0.57 ----- -----
*Inhibition zone diameter for Nystatin standard is 15mm.
Table 4. (b)
Positive index: Comparison of the zone of
inhibition of methanolic plant extract with
standard antibiotic(Nystatin) action in petri
plates.
Plant species 2g/5ml 4g/5ml 6g/5ml 8g/5ml 10g/5ml
Allium cepa ---- ----- 0.50 0.53 0.55
Allium sativum 1.07 1.20 1.22 1.23 1.27
Cinnamomum 0.47 0.49 0.51 0.53 0.57
zeylanicum
Eugenia
0.67 0.69 0.71 0.80 0.84
caryophyllus
Piper nigrum ---- ----- ---- ---- -----
Zingibar officinale ---- 0.49 ---- 0.59 ----
*Inhibition zone diameter for Nystatin standard is 15mm.
between microorganisms and showed that plant products had
been valuable source of new and effective antimicrobial
substances.
The broad antimicrobial action of the aqueous extract of
all the tested plants could be ascribed due to the components
such as allicin, cynnamic aldehyde , thiopropanol-S-oxide,
eugenol, piperine, zingiberene and allyl-isothiocyanate. These
are water soluble components naturally occurring in most
plant materials (Chalfoun, et al., 2004). However, some plant
extracts like P.nigrum , C.zeylanicum and Z.officinale showed
lower action as antifungal agents. This may be due to little
diffusion properties of these extracts in the solvent or because
fresh plant contains active substances which may be affected
or disappeared during extraction method. El Astal, et al., 2005
observed that the methanolic extract showed slightly better
killing action than the ethanolic extract suggesting that the
methanolic extract could be used more. Elloff, 1998 reported
that methanol was a better solvent for the consistent extraction
of antimicrobial substances from medicinal plants. Therefore,
methanol was used for plant extraction in this study and
antifungal activities were quantitatively assessed by the
inhibition zone diameters.
Little information is available concerning medicinal
plants and derivatives action on/in the fungal cell in order to
promote fungistatic or fungicide effect. In general, inhibitory

1 0 Trends in Biosciences 2 (2), 2009
action of natural products on mould involves cytoplasm
granulation ,cytoplasmic membrane rupture and inactivation
and / or inhibition of intercellular and extracellular enzymes
.These biological events could take place separately or
concomitantly culminating with mycelium germination
inhibition. Also, it is reported that plant lytic enzymes act in
the fungal cell wall causing breakage of â-1,3 glycan , â-1,6
glycan and chitin polymers ( Brull and Coote,1999) .
Present study suggest that the crude extract of A. cepa,
A. sativum, C. zeylanicum , E. caryophyllus, P. nigrum and Z.
officinale can be used as antimicrobial agents directly and
these can be the raw material in the development of new drugs
for the treatment of infectious diseases.
ACKNOWLEDGEMENT
The authors like to express thanks to the Lab personnels
of S.C.B.Medical College and Hospital, Cuttack and Head
Department of Applied & Industrial Microbiology , Utkal
University for co-opration in the study.
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Recieved on 9-11-2009 Acceted on 1-12-2009

Trends in Biosciences 2 (2): 11-13, 2009
A Dye Based Assay for Lignin and Manganese Peroxidases of Phanerochaete
chrysosporium RP-78
K. HARITHA AND K.R.S., SAMBASIVA RAO
Centre for Biotechnology, Acharya Nagarjuna University, Guntur 522 510, Andhra Pradesh
email: harithabio@gmail.com, krssrao@yahoo.com
ABSTRACT
Azodyes are important chemical pollutants of industrial origin.
The decolourisation and degradation efficiency of
Phanerochaete chrysosporium RP-78 with the reactive azodye
Procion Yellow H-E4 (Reactive yellow 84), was studied. A new
assay of lignin and manganese peroxidases that are involved in
dye degradation based on the oxidation of micromolar
concentrations of the dye Reactive yellow 84 and phenol red is
presented. Although it is as simple and rapid as the veratryl
alcohol assay, it appears to overcome some of the shortcomings
of that assay. In particular, interference from UV- and shortwavelength
visible-light-absorbing materials is greatly reduced
and assay specificity is improved. Finally it was identified that
the inducer (Veratryl alcohol) and inhibitor (Thiourea) of lignin
degradation also affect the dye degradation effectively.
Key words
Reactive azodyes, bioremediation, Phanerochaete
chrysosporium RP-78, lignin peroxidase, manganese
peroxidase.
Azodyes are aromatic structures having azo linkage (-
N=N-) Chromphore. As the electron withdrawing nature of
the azo linkage(s) obstructs the susceptibility of the dye
molecules to oxidative reactions. In contrast reductive
cleavage of azo linkages under anaerobic conditions results
in the formation of colorless but potentially harmful aromatic
amines.(Brown and Devito, 1993; Levin, et al., 2004).The earlier
attempts on biodegradation studies indicates that the
Phanerochaete chrysosporium a basidiomyces white rot fungi
is the best choice of organism having the ability of
biodegradation of dyes and recalcitrant compounds(Glenn
and Gold, 1983; Levin et al., 2004, Paszczynski and Crawford,
1991; Sureshbabu Naidu, et al., 2003).The biodegradation
ability of Phanerochaete chrysosporium is due to its lignin
degrading exoenzymes such as lignin and manganese
peroxidases (Chivukula and Renganathan, 1995; Schliephake,
et al., 2000). The initial detection and quantization of LP
activity were made by oxidation of a 1-0-4 model compound
(Tien, and Kirk, 1983.). Later, a gas chromatographic assay of
the ethylene released by the one-electron oxidation of 2-keto-
4-methiolbutyric acid (KMB) by Phanerochaete
chrysosporium LiP was used. However, p-0-4 model
compounds must be synthesized, and the KMB assay is
considerably more laborious than most cuvettespectrophotometer
assays. As a result, when a simple assay
based on the oxidation of veratryl (3,5- dimethoxybenzyl)
alcohol (VA) to veratraldehyde was introduced (Tien and Kirk,
1983). Assays employing VA are, in contrast, simple and rapid
and enable one to easily monitor reaction rates and the effect
of additions on rates. The purpose of the present study was
to investigate the degradation ability of Lignolytic cultures of
Phanerochaete chrysosporium RP-78 on procion yellow HE4R
(Reactive yellow 84) and also develop a new rapid cuvette
assay for peroxidase proteins that lacks many problems of VA
based assays.
MATERIALS AND METHODS
Maintenance of organisms
The Phanerochaete chrysosporium RP-78 was obtained
from the Forest Products Laboratory, Madison, WI. The pure
cultures of P. chrysosporium RP-78 was maintained in YMPG
medium in laboratory by serial sub culturing for every 15 days
and stored at 4 0 C.
Dyes
The dyes selected for decolourisation studies are
procion dyes, which are procured from Atul Dyes
Manufacturing Limited Gujarat, India.
Methodology
The experimental procedure for dye decolourisation was
followed as per the method described by Cellin Cripps, et al.,
1990. The modified and optimized YMPG media with 1%
Glucose, 0.6% Aspargine, 0.2% MgSO 4
7H 2
O and 0.5%
KH 2
PO 4
, PH-4.5 was used for the studies. The degradation
medium was prepared, sterilized and inoculated with 2%
overnight developed seed culture of P. chrysosporium RP-78.
The culture was allowed to grow for 6 days at 200 rpm. On the
sixth day the dye was added to the medium, The rate of
degradation was studied by decrease in the absorbance (at
lmax 408nm for procion yellow and 510 nm for phenol red) of
the samples collected by centrifuging broth at 10000rpm for
15 min at different time intervals during the course of
experiment.
Effect of Thiourea on Dye degradation
The effect of thiourea an inhibitor on degradation was
studied by adding 5mM thiourea to the degradation media.
Urea (5 mM), which served as a control for thiourea, was not
an inhibitor. Then the degradation rate was studied following

1 2 Trends in Biosciences 2 (2), 2009
the procedure detailed earlier. The biodegradation of the
azodyes was monitored by UV-Vis analysis.
Effect of Veratryl alcohol (VA) on degradation
The effect of inducer veratryl alcohol 3, 4-di methoxy
benzyl alcohol) on fungal decolourization was studied by
adding 0.125 g to 100 ml of the degradation medium. The
medium was inoculated and allowed to grow for 6 days. On
sixth day dye was added. Then the rate of degradation of the
dyes in that medium was determined by using the procedure
discussed earlier.
Enzyme assays
The dye based lignin peroxidase assay was run with
50mM sodium tartarate (P H 4.5), 0.1mM H 2
O 2
and 0.1 % reactive
Yellow 84.The dye and H 2
O 2
were made up as 100X stock
solutions and 20 µl of each was added to the 4ml reaction
volume contains 3.8 ml of supernatant of lignolytic cultures
of P. chrysosporium RP-78.The optical density decrease was
read at exactly 408nm and the amount of dye utilized by the
enzyme was determined from standard graph.
The manganese peroxidase (MnP) assay (total volume,
1.0 ml) contained 0.2 mM MnSO 4
, 0.1 mM H 2
0 2
, and 0.0025%
phenol red, all in 50 mm sodium tartrate buffer (p H 4.5).The
reaction was monitored at 431 nm (Kuwahara, et al.,1984)The
amount of substrate converted in to product was calculated
from standard graph. For the determination of the specific
activity of the enzymes the protein content of the supernatant
was estimated by using Lowry’s method.
RESULTS AND DISCUSSION
In the dye degradation studies, most of the colour loss
occurred within 24 hrs. (Fig.1-2), indicates the decolourisation
with the disruption of the chromophore group. The decrease
in the absorbance of the two peaks in the UV region (242and
307nm) suggest that there was changes in the aromatic group.
The dye based assays measure the disappearance of
the substrate i.e. the absorbance decrease caused by hyper
chromic shift of the visible absorbance peak of the substrate.
The extent of decolourisation was measured by using standard
curves of the dyes. As controls for the possibility that the
decolorization was due to a non biological oxidation, the dyes
were also incubated only with hydrogen peroxide. No change
in absorbance was seen with any of the dyes when incubated
only with H 2
O 2
. As controls for the possibility that the
decolorization was due to enzymes other than peroxidases,
the dyes were also incubated with both crude enzyme extracts,
in the absence of hydrogen peroxide. In this case also no dye
decolorization was observed. Thus, the remainder of the text
will refer to the activity of LiP or of Mn (II)-peroxidase (MnP).
The absorbance of the procion yellow at zero hour samples is
0.231 which was decreased to 0.127 with in 15 min. The
concentration of the dye corresponds to initial and final
absorbance were calculated as 315 µM and 175.43 µM
respectively (from standard graph of absorbance vs
concentration of the procion yellow) indicates that the amount
of substrate utilized by the 1ml of lignin peroxidase enzyme
source in 1 min was 2.339 µM i.e.2.339IU/ml or 23339IU/l.
Procion yellow also did not react with Mn3+ pyrophosphate
or MnPs. These findings are in coincidence with the results
of lignin peroxidase activity from P. chrysosporium 2100 IU/l
(Ganesh Kumar, et al., 2006).
In the same way phenol red is used in case of manganese
peroxidase even though it is oxidized by the lignin peroxidase
but the oxidation is slow, so that it can be good substrate for
the short term assay of manganese peroxidase .The initial
absorbance was found to be 0.389 which was decreased to
0.30 in 15 min after the addition of supernatant, that
corresponds to the 531.914 µM and 398.936 µM respectively
(from standard graph of absorbance vs. concentration of the
phenol red). From these results the activity of manganese
peroxidase was found to be 1.773IU/ml or 1773IU/l of
supernatant. The activity was comparable with the findings
of Ganesh Kumar, et al., 2006 P. chrysosporium (1200IU/L).
From the activities and protein concentration the specific
activities of crude lignin and manganese peroxidases are
calculated as 1.016 unit/mg and 0.770unit/mg respectively
Fig. 1. Degradation of procion yelow
Fig. 2. Degradation of phenol red

HARITHA et al., A Dye Based Assay for Lignin and Manganese Peroxidases of Phanerochaete chrysosporium RP-78 1 3
Brown, M. A. and De Vito, S.C. 1993. Predicting Azo Dye Toxicity.
Critical Reviews in Environmental Science and Technology, 23(3):
249-324.
Cellin Cripps, Bumpus, J.A. and Aust, S.D. 1990. Biodegradation of azo
and heterocyclic dyes by Phanerochaete chrysosponum. J. Appl.
Environ. Microbiol., 56: 1114-1118
Chivukula, M. and Renganathan, V. 1995. Phenolic azodyes oxidation
by laccase from Pyricularia oryzae. Appl environ microbial., 61:
4374-4377.
Cripps, C., Bumpus, J. A. and Aust, S D. 1990.Biodegradation of azo
and heterocyclic dyes by Phanerochaete chrysosporium. Appl
Environ Microbiol., 56(4): 1114-1118.
Fig. 3. Evaluation of thiourea and veratryl alcohol on
degradation.
Table 1.
Sample
Activity of lignin, manganese peroxidases.
Thiourea, a potent -OH scavenger and inhibitor of lignin
degradation, was very effective at inhibiting all the dyes
degradation (Glen and Gold, 1983), the extent of inhibition
procion yellow is decreased by16% (Fig. 3).Veratryl alcohol
(3,4 dimethoxy benzyl alcohol) is an inducer of the lignin
peroxidase (Arora, and Gill, 1996) .So in the presence of
inducer the production of the enzyme is more, that’s why as
shown in the Fig. 3 in the presence of the veratryl alcohol the
percentage of degradation is more than in the control but it
shows minor effect only. The presence of veratryl alcohol
increases the degradation of azodyes.
LITERATURE CITED
Activity
(unit/ml)
Protein
(mg/ml)
Specific
activity
(unit/mg)
Crude ligninase 2.339 IU/ml 2.30 1.016
Crude Mn (II) peroxidase 1.773 IU/ml 2.30 0.770
Arora, D.S. and Gill, P.K.1996. Comparison of two assay procedures
for lignin peroxidase. J.Biotechnology Techniques, 10(4): 273-
276
Ganesh Kumar, A., Sekaran, G. and Sarayu Krishnamoorthy.2006.Solid
state fermentation of Achras zapota lignocellulose by
Phanerochaete chrysosporium. Bioresourse technology, 97(13):
1521-1528.
Glenn, J. K. and Gold, M. H. 1983. Decolorization of several polymeric
dyes by the lignin-degrading basidiomycete Phanerochaete
chrysosporium. Appl. Environ. Microbiol., 45: 1741- 1747.
Kuwahara, M., Glenn, J. K., Morgan, M. A. and Gold, M. H. 1984.
Separation and characterization of two extracellular H 2
0 2
-dependent
oxidases from ligninolytic cultures of Phanerochaete chrysosponum.
FEBS Lett., 169: 247-250.
Levin, L., Papinutti, L. and Forchiassin, F.2004. Evaluation of
Argentinean white rot fungi for their ability to produce lignin
modifying enzymes and decolourize industrial dyes. J.Bioresource
technology, 94(2): 169-176.
Paszczynski, A. and Crawford, R.L. 1991. Degradation of azo
compounds by ligninase from Phanerochaete chrysosporium:
involvement of veratryl alcohol. Biochem. Biophys. Res. Commun.,
178: 1056-1063
Schliephake, K., Mainwaring, D.E., Loneragan, G.T, Jones, I.k, Baker,
W.L.2000.Transformation and degradation of the disazo dye
Chicago sky blue by a purified laccase from Pycnoporus
cinnabarinus. Enzyme. Mirob Technol., 27: 100-110.
Sureshbabu, Naidu, K., Reddy, N.S., Rao, G.V. and Sambasivarao, K.R.S.
2003. Biodegradation of Textile dyes using Phanerochaete
chrysosponum, Indian J. Ecol., 30(1): 268-370
Tien, M. and Kirk, T. K. 1983. Lignin-degrading enzyme from the
hymenomycete Phanerochaete chrysosponum. Burds. Science,
221: 661-663.
Received on 13-11-2009 Accepted on 30-11-2009

1Trends 4 in Biosciences 2 (2): 14-17, 2009
Trends in Biosciences 2 (2), 2009
Conventional Indoor Rearing Leads to Abnormalities in the Ultrastructure of Muga
Silkworm (Antheraea assamensis) Mid-Gut Cell Microvilli.
SUDIP DEY 1 , BEGONIA DKHAR 1 , RAHUL CHAKRABORTY 1 , SUDIPTO CHAUDHURY 2 AND
DHIRENDRA K. SHARMA 3
1
Electron Microscope Division, Sophisticated Analytical Instrument Facility, North Eastern Hill University,
Bijni Complex, Laitumkhrah, Shillong 793 003.
2
Department of Zoology, North Eastern Hill University, Umshning, Shillong.
3
Department of Zoology, Gauhati University, Guwahati 781 014, Assam,
e-mail: sudipdeyrsic@yahoo.com
ABSTRACT
Conventional indoor rearing of muga silkworm (Antheraea
assamensis) by tray-feeding (horizontal feeding posture),
associated with low food consumption was found to cause a
number of abnormalities in the mid-gut cell microvilli. In
indoor-reared horizontally fed worms the density of microvilli
was found to be less than that of the control. In the longitudinal
sections, microvilli showed abnormalities in the form of
irregular arrangements, discontinuity and less thickness than
that of normal worms. Some of the microvilli showed fusion at
places and swelling at the tip. In transverse sections, the
microvilli was found to be remarkably disturbed. The continuity
of the membrane was lost in some of the microvilli. The worms
fed at vertical posture, however, did not exhibit any of these
abnormalities and were found to be normal.
Key words
Ultrastructure, muga silkworm, microvilli, mid-gut,
TEM
Antheraea assamensis, popularly known as muga
silkworm is a multivoltine, sericogenic insect, endemic to
Assam and adjacent states of northeast India. The silk moth
has six broods in a year (Seitz, 1933) but, only two are popular
for commercial rearing (Thangavelu and Sahu, 1986) because
the climatic conditions during the period are favourable for
growth and development of muga silkworm.
Commercial rearing under the outdoor conditions causes
a substantial amount of loss to the worm at various stages of
development. Consequently this has been causing tremendous
adverse impact on the muga silk industry. Further, Antheraea
assamensis, one of the most important bioresources of the
northeast India is under tremendous stress mainly due to the
problems associated with outdoor rearing.
The worms reared under indoor rearing condition
through conventional tray feeding was found to have very
low food consumption , low larval weight, prolonged larval
period, delayed molting and spinning etc. as compared to
worms grown in natural environment. The head cuticle of muga
silkworm contain some gravity receptors (dorsal campaniform
sensilla), which governs a vertical feeding posture of the worm
(Dey, et. al., 2002). This could explain the reason for reluctance
in food consumption in tray feeding with compelled horizontal
feeding posture. Proving the worms with vertical feeding
posture under indoor rearing condition could overcome most
of the abnormalities in rearing performance. It was thus
confirmed that the conventional tree-feeding of the worm at
horizontal feeding posture is to a great extent responsible for
its poor rearing performance under indoor rearing condition.
Since the low food consumption at conventional tray
feeding is likely to affect the metabolism significantly, it was
felt that a study on the cellular and ultrastructural features of
mid-gut cells of the worm will be relevant to the understanding
of the possible adverse effects on digestion and assimilation.
MATERIALS AND METHODS
Insect Materials
The fifth instar muga silkworms (Antheraea assamensis)
were used in the present study. The insect material was
collected from the field station of Central Silk Board, Muga
Silk Development Project (MSDP), P–3 Unit, Nongpoh,
Meghalaya, India.
Experimental Set-up
Vertical Feeding Posture
To provide vertical feeding posture to the worms under
indoor conditions, they were kept in twigs along with leaves
of Machilus bombycina, placed in water-filled bottles.
Horizontal Feeding Posture
For providing horizontal feeding posture, the worms
were kept in bamboo trays with twigs along with leaves of
Machilus bombycina.
The leaves and twigs in the bottles as well as the trays
were changed frequently to maintain freshness of the leaves.
The outdoor reared worms were used as control. The
experiment was carried out in the indoor rearing room of Muga
Silk Development Project (MSDP), Cental Silk Board, Nongpoh,
Meghalaya, India.
Transmission Electron Microscopy
The mid-gut samples excised from the fifth instar larval

DEY et al., Conventional Indoor Rearing Leads to Abnormalities in the Ultrastructure of Muga Silkworm 1 5
stages of indoor reared vertically fed and horizontally fed
worms and outdoor reared worms (control) were prepared for
transmission electron microscopy as follows.
The samples were cut into small pieces of approximately
1mm × 1mm in size and were fixed in modified Karnovsky’s
fixative having the composition of 250ml of 0.2M sodium
cacodylate buffer, 20g of para-formaldehyde dissolved in it at
60°C, bringing the volume to 480ml by double distilled water.
To this 20ml of 25% glutaraldehyde and 12.5g of anhydrous
calcium chloride was added.
After 4 hours in the above primary fixative, the samples
were washed thoroughly in 0.1M sodium cacodylate buffer.
Post fixation was carried out in 1% osmium tetroxide in the
same buffer for 1 hour at 4°C.
Specimens were dehydrated in ascending grades of
acetone (30%, 50%, 70%, 80%, 90%, 95% and dry acetone)
with two changes 15 minutes each. The samples were then
cleared off acetone by propylene oxide for 30 minutes.
Infiltration was carried out gradually in different
proportions of propylene oxide with liquid resin, hardener,
plasticizer etc. [Araldite CY212 – 10ml, DDSA (dodecenyl
succinic anhydride – 10ml, DMP-30 {Tri-(dimethyllaminomethyl)
phenol} – 0.4ml, and dibutyle phthalate
– 1ml].
Embedding of tissue was carried out in the araldite
embedding medium using beem–capsules.
The embedding blocks were kept at 50°C in an embedding
oven for 24 hours. The temperature was then raised to 60°C
and the embedded tissues were kept for 48 hours to complete
polymerization.
Ultra-thin sections (600–800?) were cut in an RMC Ultra-
Microtome, MT-X, with a diamond knife. The sections were
collected on copper grids and stained with alcoholic solution
for 10 minutes at room temperature in the dark, followed by
lead citrate for 5 minutes (Reynolds, 1963).
The stained sections were examined in a Jeol JEM 100
CX II Transmission Electron Microscope at an accelerating
voltage of 80Kv. 500 sections of each of the control,
horizontally postured and vertically postured worms were
examined.
RESULTS AND DISCUSSION
The microvilli of the mid-gut cells in control worms
collected from the field revealed round contour in the crosssectional
view. The membrane exhibited continuity without
any disruption at any place. The size of the microvilli in their
cross-section was also found to be uniform [Fig. 1. (a, b)]. In
the longitudinal section, the microvilli showed more or less
erect and continuous arrangement [Fig. 2.(a)].
In the indoor reared horizontally postured worms, the
contour in the cross-sectional view was found to be remarkably
disturbed. The round shape was not maintained in many of
the microvilli, and they were found to assume irregular shape.
Further, the continuity of the membrane was also disturbed to
a great extent. Disruption of membrane at places were also
evident [Fig. 1. (c, d)]. In the longitudinal section, the microvilli
showed some abnormalities mainly in the form of irregular
arrangements, discontinuities and reduction of thickness of
individual microvilli [Fig. 2. (b, c)]. Some of the microvilli showed
fusion at places and swelling at the tip [Fig. 2. (d)].
In the mid-gut cells of indoor-reared vertically-postured
worms, the disturbances noted in case of horizontally postured
worms were not present. The contour of the microvilli was
round as in the case of control and the continuity of the
membrane was maintained without any disorganization and
disruption. No irregularity in the shape of microvilli was
observed unlike those in horizontally postured indoor reared
worms [Fig. 1. (e, f)]. The microvilli in longitudinal section
showed similarity with those of control. There was no
disturbance in continuity, and, thickness was like that of the
control [Fig. 2. (e)].
The observations in the present study on the reduction
in thickness, irregular arrangements and discontinuities etc.
of the gut-cell microvilli in longitudinal section and distortion
of their contour in cross-sectional view in horizontally fed
indoor reared muga silkworms appear to be related to the poor
feeding in the worm under the said feeding conditions. That
these abnormalities result from poor feeding is supported by
the fact that no such abnormalities were observed in indoorreared
vertically postured worms which have their proper
feeding like the control worms in natural habitat. In this context,
it is worth mentioning that although the overall function of a
cell determines the abundance or scarcity of microvillus
process, the size, shape and perhaps the number of microvilli
change with cell function (Aldewachi, et. al., 1975).
The microvilli obviously increase the area of the apical
cell surface and are therefore interpreted as being specialized
for absorption and resorption (Smith, 1968). On that
consideration, the observed abnormalities in gut-cell microvilli
in poorly fed horizontally postured muga silkworm under
indoor rearing conditions merits detailed elucidation. It is
known that reduced demand for a functional gut (i.e., during
food deprivation) often results in the diminution of those
processes responsible for the energetic cost of the tissues,
thereby leading to marked changes in both its functional and
morphological properties (McBride and Milligan, 1985).
The majority of information describing the effects of
food deprivation on the gastrointestinal tract primarily
concerns short duration fasts by laboratory vertebrate
models. In contrast little is known about the responses of the
gut to naturally extended episodes of food deprivation, which
occur in a wide range of species as a result of environmental

1 6 Trends in Biosciences 2 (2), 2009
Fig. 1. a, b. Normal shape of microvilli (circular contour) T/S in gut cells of outdoor reared fifth instar muga
silkworm
a × 27,000 b (magnified) × 54,000
c × 27,000 d (magnified) × 54,000
Fig. 1. e, f. Normal shape of microvilli (circular contour) T/S in gut cells of indoor, vertically reared fifth instar
e × 27,000 f × 54,00
Fig. 2. a. Normal shape of microvilli L/S in gut cells of of indoor vertically reared fifth instar, b, c. Microvilli
L/S showing reduction in width in indoor horizontally reared fifth instar
a × 7,560 b × 7,560 c × 7,560
d × 7,560 e × 7,5600

DEY et al., Conventional Indoor Rearing Leads to Abnormalities in the Ultrastructure of Muga Silkworm 1 7
stress. In this regard, the present observation on the
abnormalities in gut microvilli in response to food deprivation,
in horizontally postured indoor reared muga silkworm appears
to be relevant. Numerous studies on the response of microvilli
to food deprivation exist, but the reports are restricted mainly
to vertebrates and are often contradictory (Mayhew, 1990,
Waheed and Gupta, 1997, Secor, et. al., 2000). The reduction
in width of gut-cell microvilli in horizontally postured indoor
reared muga silkworm in contrast to the larger width of gut
microvilli in control as well as in vertically postured indoor
reared muga silkworms suggests that the horizontally postured
worms were starved due to their incorrect feeding posture
resulting in microvillar abnormalities. Since the microvilli in
the gut play important role in absorption, the abnormally thin
microvilli, irregularity in their contour and membrane breakage
at places in horizontally postured indoor reared muga silkworm
suggests the disturbances in function of this structure in the
muga silkworm, experiencing stress in food consumption.
Similar types of observations on ultrastructural abnormalities
in gut microvilli was reported in some frogs (Cramp, et. al.,
2005) and in some insects (Jarial, 2005) in response to food
deprivation.
ACKNOWLEDGEMENT
The authors are thankful to the Head, SAIF, North
Eastern Hill University, Shillong for encouragement and
support. First author is grateful to NEC, Govt. of India for
financial grant.
LITERATURE CITED
Aldewachi, H.A., Wright, N.A., Appleton, D.R. and Watson, J.A. 1975.
The effect of starvation and re-feeding on cell population kinetics
in the rat small bowel mucosa. Journal of Anatomy., 119: 105–121.
Cramp, R.L., Franklin, C,E. and Meyer, E.A. 2005. The impact of
prolonged fasting during aestivation on the structure of the small
intestine in the green- striped burrowing frog, Cyclorana
alboguttata. Acta Zoologica (Stockholm)., 86: 13–24.
Dey Sudip, Singh, S. Dhar, N. J., Biswas, N., Chakraborty, R., Das, P.K.,
Sharma, D.K. and Raghuvarman, A. 2002. Possible relationship
between Dorsal Campaniform sensilla in head surface and preferred
feeding posture of muga silkworm Antheraea assamaensis during
indoor rearing. Sericologia., 42(2): 171–180.
Jarial, M.S. 2005. Electron microscopic studies on the anterior midgut
in Cenocorixa bifida Hung. (Hemiptera : Corixidae) with reference
to its secretory function. Zoolog. Sci., 22(7): 783–790.
Mayhew, T. 1990. Striated brush border of intestinal absorptive epithelial
cells: stereological studies on micrivillous morphology in different
adaptive states. Journal of Electron Microscopy, Technology, 16:
45–55.
McBride, B.W. and Milligan, L.P. 1985. Influence of feed intake and
starvation on the magnitude of Na+, K+ –ATPase (EC 3.6.1.3)–
dependent respiration in duodenal mucosa of sheep. British Journal
of Nutrition., 53: 605–614.
Reynolds, E.S. 1963. The use of lead nitrate at high pH as an electron
opaque stain in electron microscopy. J. Cell. Biol., 17: 208.
Secor, S., Whang, E., Lane, J.S. and Diamond. J. 2000. Luminal and
systematic signals trigger intestinal adaptation in the juvenile python.
American Journal of Physiol., 279: G1177–G1187.
Seitz, A. 1933. The Macrolepidoptera of the World, Alfred Keren
Publishers, Stuttgart., 10: 909.
Smith, D.S. 1968. Insect cells: their structure and function. Oliver and
Boyd. Edinburgh.
Thangavelu, K. and Sahu, A.K. 1986. Further studies on the indoor
rearing of muga silkworm (Antheraea assama Ww.) (Saturniidae:
Lepidoptera). Sericologia., 26 (2): 215–224.
Waheed, A. A. and Gupta, P. D. 1997. Changes in the structural and
functional properties of rat intestinal brush border membrane during
starvation. Life Sciences, 61: 2425–2433.
Recieved on 19-11-2009 Accepted on 30-11-2009

1Trends 8 in Biosciences 2 (2): 18-20, 2009
Trends in Biosciences 2 (2), 2009
Toxic Effect of Synthetic Pyrethroid Deltamethrin and Neem Based Formulation
Achook on Zebrafish, Danio rerio
BADRE ALAM ANSARI AND DILIP K. SHARMA
Zebrafish Laboratory,Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273 009 (U.P.)
e-mail: ba.ansari@rediffmail.com
ABSTRACT
In the present study, the toxic effect of synthetic pyrethroid,
Deltamethrin and a neem based formulation, Achook on
zebrafish, Danio rerio was evaluated. Adult male and female
zebrafish were randomly selected and exposed to these two
pesticides. A 24h to 96h LC 50
values were calculated. It was
found that toxicity was time as well as concentration dependent.
The result shows that zebrafish is very sensitive to
Deltamethrin than that of Achook. It was also observed that
Achook which is a natural product and reported to be safe for
non-target animals, is also toxic to zebrafish at low
concentrations. zebrafish can be used as bioindicator to assess
the pesticidal pollution in aquatic environment.
Key words
Zebrafish, pesticides, deltamethrin, achook, toxicity
Presence of toxic chemicals in both abiotic and biotic
environment all over the world has escalated the concern about
their adverse effect on flora and fauna including human
beings. Synthetic pyrethroids are a diverse class of more
powerful, broad-spectrum insecticides used to control insect
pests in agriculture, households and stored products to
increase the crop production. Although, they are based on
the chemical structures and biological activity of pyrethrum,
an extract from plants in the genus Chrysanthemum. The
development of synthetic pyrethroids had involved extensive
chemical modifications to make compound that are more toxic
and has rapid degradability. Improper handling and abuse of
these potentially hazardous chemicals has not only made them
ubiquitous, but also induced serious health hazard among
workers during manufacture, formulation and field use (Dikshit
and Dutta, 1973; Anon., 1975; Kashyap, 1984; Ansari and
Kumar, 1988).
To overcome the problem of synthetic hazards, one of
the best control measures is the use of plant origin chemicals
because of their rapid biodegradability, least persistence and
least toxic to non-target organisms, economic and easy
availability. Today, about 200 plants containing insecticidal
activity are known (Tripathi, et al., 1999; Tripathi, et al., 2000;
Singh, et al., 2001).
Literature review reveals that most of the studies with
reference to neem based formulations as well as raw plant
extracts are limited up to the testing of its toxicity on insect
pests. Intensive research projects on the biological evaluation
of neem products are being conducted in several countries
(Jilani and Su, 1983; Islam, 1984; Saxena, et al., 1984; Sharma,
et al., 1984).
However, little work has been done on the toxic effect of
neem based pesticides on fish. Hence, a need was felt to
investigate the comparative effect of synthetic pyrethroid,
Deltamethrin and a neem based formulation, Achook on the
zebrafish, Danio rerio. This fish was selected as the test
species according to the recommendations of the International
Organization for Standardization, 1976.
MATERIALS AND METHODS
For toxicity tests, zebrafish, Danio rerio of similar age
were procured from the laboratory breed general culture.
Toxicity test was performed in laboratory to determine the 24,
48, 72, and 96h LC 50
values using four concentrations of
Deltamethrin (0.10, 0.30, 0.50 and 0.70 µg/L) and five
concentrations of Achook (0.30, 0.60, 0.90, 1.20 and 1.50 µg/L)
previously diluted in acetone. Two replicates of 10 fishes for
each concentration of pesticides were performed. Each
experiment was accompanied by a control having the same
volume of acetone but without the pesticide. The
Randomization of the fish in test aquaria was done according
to the method prescribed by the U.S. Federal Water Pollution
Control Administration, 1968.
The water was changed every 24h. A fish was
considered dead when its gill movements ceased and it did
not respond to gentle prodding. Dead fish was removed from
aquaria to avoid deterioration. Deltamethrin (2.8 % E. C.,
Solvents and others: 97.2% w/w) and Achook (Azadirachtin
0.15 %, Glycerol Fatty acid ester 25 %, adjuvants: Emulsifier
Ca-salt of alpha benzoic sulphonic acid and polyoxyethyl ester
of hydroxyetdeanoic acid ester 5.0 %. Stabilizer:
Epichlorohydrin 0.50 %, Solvent: Cyclohexanone + aromax
(1:1) q s) was purchased from local market.
The results were computed by Stat Plus ® 2008 computer
programme. Mortalities of Zebrafish were recorded for different
exposure periods viz., 24, 48, 72 and 96 h at different
concentrations. The LC 50
values, Upper and Lower confidence
limits (UCL and LCL), Slope, Chi-square values were
calculated.
RESULTS AND DISCUSSION
After the exposure of both the pesticides, the fish
showed behavioural changes. Initially the fish aggregated at

ANSARI AND SHARMA, Toxic Effect of Synthetic Pyrethroid Deltamethrin and Neem Based Formulation Achook 1 9
one corner of the aquarium. After sometime they swam
erratically, their gill movements increased accompanied by
loss of equilibrium. Their body color darkened and the pectoral
and pelvic fins got expanded, the fishes rolled vertically prior
to death.
The results of the toxic effects are illustrated in Table 1.
It is evident from the table that the LC 50
values decreases with
the increase in treatment period. It means that the toxicity of
these pesticides increases with the advancement of time. In
other words, the mortality of fishes increases with increase of
time.
Table 1.
Treated
Period
(Hrs.)
24
48
72
96
Toxic effect of Deltamethrin and Achook against
zebrafish, Danio rerio
LC 50Values
(µg/L)
0.947*
1.773**
0.493
1.157
0.200
0.816
0.121
0.595
Confidence limits
LCL(µg/L) UCL(µg/L)
0.538
1.251
0.260
0.869
0.080
0,595
0.043
0.407
24.50
5.810
6.357
2.016
0.310
1.124
0.188
0.766
Slope
5.20
2.83
9.10
2.31
4.72
3.828
3.70
3.704
* Data in upper case belongs to Deltamethrin treatment
** Data in lower case belongs to Achook treatment
Chisquare
values
0.220
0.035
0.254
0.107
0.246
0.435
0.047
2.006
From Table 1 it is evident that Deltamethrin is more toxic
than Achook. The concentration of Deltamethrin required for
killing the fish is lower than that of the concentration of
Achook. It was observed that during the exposure of
Deltamethrin the LC 50
value after 24 h was 0.947µg/L which
decreased to 0.121 µg/L after 96 h of exposure. On the other
hand, the 24 h LC 50
value of Achook was 1.733 µg/L which
decreased to 0.595 µg/L after 96 h of exposure. It is also evident
that after 24 h of exposure the Deltamethrin is approximately
two times more toxic than Achook while with the increase of
time the toxicity of Achook was approximately five times less
as compared to Deltamethrin. This proves that the zebrafish
is more sensitive to Deltamethrin.
The slope values shown in the table are steep. The LC 50
values of the pesticide showed a significant (P

2 0 Trends in Biosciences 2 (2), 2009
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Report to the subcommittee on administrative practices and
procedure on the committee on the judiciary of the US senate.
pp.32-34.
Anonymous. 1998. U S Enviromental Protection Agency. Fact Sheet
Number 171: Karate Washington, D C, pp. 321.
Ansari, B.A. 2004. Effect of essential oils of Callistemon lanceolatus
and Eupatorium capillifolium against the cotton pest. Earias vittella
Fab. (Lepidoptera: Noctuidae). Farm. Sci. J. 13 (1): 68-69.
Ansari, B.A. and Kumar, K. 1982. A record of Zebra danio (Brachydanio
rerio) (Cyprinidae) from Uttar Pradesh with notes on sexual
dimorphism. J. Adv. Zool., 3: 88.
Ansari, B.A. and Kumar, K. 1988. Cypermethrin toxicity Effect on
the carbohydrate metabolism of the Indian catfish, Hetropneustes
fossilis. The Sci. Total Environm., 72:161-166.
Bao, G.G., Wang, M.H., William, L.C., Dao, J.C., Zheng, J.S. 2007. Risk
assessment of cyhalothrin on aquatic organisms in paddy field in
China. Regulatory Toxicology and Pharmacology, 48:69-74.
Cakmak, M.N., Gorgon, A. 2003. Toxic effect of a synthetic pyrethroid
insecticide (Cypermethrin) on blood cells of rainobow trout
(Oncorhynchus mykiss, Walbaum). On Line Journal of Biological
Sciences, 3: 694-698.
Dikshit, T.S.S. and Dutta, K.K. 1973. Endrin induced cytological changes
in albino rat. Bull. Environm. Cantam. Toxicol., 9:65-69.
Final (Revised) Proposal for Screening Chemical and Other Products
for Acute Toxicity to Freshwater Fish. 1976. International
Organisation for Standardisation, document ISO/TC 147/SC.5/
WG.3(Secrariat-6),18 November
Islam, B.N. 1984. Pesticidal action of Neem and certain indigenous
plants and weeds of Bangladesh, Proc. 2 nd Int. Neem Conf.
(Rauischolzhausen, 1983). pp. 263-290.
Jilani, G. and Su, H.C.F. 1983. Laboratory studies on several plant
materials as insect repellents for protection of cereal grains. J.
Econ. Ent., 76:154-157.
Kashyap, S.K. 1984. Report of PTI In: International workshop on
pesticide formulation, held at New Delhi.
Koprucu, K. and Aydin. 2004. The toxic effects of pyrethroid
deltamethrin on the common carp (Cyprinus carpio L.) embryos
and larvae. Pestic. Biochem. Physiol., 80:47-53.
Miyamoto, J.1976. Degradation, metabolism and toxicity of synthetic
pyrethroids. Environmental health Perspectives, 14:15-28.
Narahashi, T. 1983. Neurophysiological study of pyrethroids In:
Molecular and Membrane Mechanism of Action, (eds. Miyamoto,
J., Kearney, P.C.), Pesticide Chemistry, Human Welfare and
Environment. Oxford, Pergamon Press. pp. 365.
Paul, E.A., Simonin, H.A. 2006. Toxicology of Three Mosquito
Insecticides to Crayfish. Bull. Evironm. Contam. Toxicol., 76:614-
621.
Polat, H., Erkoc, F. U., Viran, R. and Knock, O. 2002. Investigation of
acute toxicity of betacypermethrin on guppies Poecilia reticulate.
Chemosphere, 49(1):39-44.
Richard, J. and Brodie, M.E. 1985. Correlation of blood & brain levels
of the neurotoxic pyrethroid deltamethrin with the onset of
symptoms in rats. Pestic. Biochem.Physiol., 23:143-156.
Ruzo, L.O., Eugel, J.L. and Casida, J.E. 1979. Cecamethrin metabolites
from oxidative, hydrolytic and conjugative reactions in mice.
Journal of Agriculture and Food Chemistry, 27:725-731.
Saxena, R.C., Epino, P.B., Cheng-Wen, T.U. and Puma, B.C. 1984.
Neem chinaberry and custard apple Antifeedant and insecticidal
effects of seed oils on leaf hopper and plant hopper pests of rice.
Proc. 2 nd Int. Neem Conf. (Rauischlolzhausen, 1983), pp. 403-
412.
Schmitz, A., Schafers, C., Trob, R., Ruch, B. and Kleeberg, H. 2001.
Effects of Neemazal on fish: Full life cycle test with the Zebrafish,
Danio rerio, In: Proceeding of the 2. Workshop “Neem and
Pheromones.’’ University of Uberaba, Brazil, May 15.-16.pp.36-
49.
Sharma, R. N., Nagasampagi, B. A., Bhosale, A. S., Kulkarni, M. M. and
Tungikar, V. B. 1984. “Neem rich” The concept of enriched fraction
from Neem for behavioural and physiological control of insects.
Proc. 2 nd Int. Neem Conf. (Rauischlolzhausen, 1983) pp. 115-118.
Singh, R., Singh, B. and Verma, R. A. 2001. Efficiency of different
indigenous plant products as grain protectent against Callosobruchus
chinensis Linn. on pea. Indian J. Ent., 63:179-181.
Tripathi, A.K., Prajapati, V., Agarwal, K.K., Khanuja, S.P.S. and Kumar,
S. 2000. Repellency and toxicity of oil from Artemisia annua to
certain stored product beetles. J. Econ. Ent., 93: 43-47.
Tripathi, A.K., Prajapati, B., Jain, D.C. and Saxena, S. 1999. Antifeedant
oviposition deterrent and growth inhibitory activity of
Andrographis paniculata against Spilaretia obligue. Insect Sci.
Applic., 19:211-216.
U.S. National Technical Advisory Committee. 1968. Report of the
National Technical Advisory Committee on Water Quality Criteria
to the Secretary of the Interior U.S. Federal Water Pollution Control
Administration, Washington, pp. 234.
Received on 17-11-2009 Accepted on 30-11-2009

Trends in Biosciences 2 (2): 21-22, 2009
Studies on Genotype × Environment Interaction and Stability for Seed Yield in
Common Bean (Phaseolus vulgaris L.)
S. A. DAR 1 , F. A. PIR 1 , ABU MANZAR AND AJAZ A. LONE 2 .
1
Pulse Research Sub- Station, SKUAST-K, Srinagar, 2 K. D. Research Station, SKUAST-K, Srinagar;
e-mail: ubaid_dar@rediffmail.com
ABSTRACT
Twelve promising genotypes of common bean (Phaseolus
vulgaris L.) and a standard check were evaluated at three
locations for seed yield. Significant differences were observed
among the genotypes, G x E interaction as well as individual
environmental effects revealed the presence of variability for
the trait under investigation. Significant variance due to G x E
(linear) against pooled deviation indicating that the yield
performance of the genotypes could be predicated with greater
precision across environments. Genotype SKUA-R-91 possessed
high mean values for seed yield across the environments and
its regression co-efficient (bi) approaching unity with least
deviation from regression co-efficient (s 2 di). This particular
genotype can be utilized in hybridization for isolating high
yielding and stable segregants. The environmental conditions
at E 1
were the best for the expression of seed yield.
Key words
Stability, regression co-efficient, G x E, Phaseolus
vulgaris.
Common bean (Phaseolus vulgaris L.),popularly known
as rajmash is one of the important pulse crop. In India it is
grown in the hilly areas of north-western Himalayan region
during kharif season and rabi in plains. The low and unstable
yield are the most pertinent problems in common beans
production when grown under varied agro-climatic conditions
because the crop is highly sensitive to temperature and other
climatic variations. It is important that plant breeders develop
varieties which are stable in yield levels with better adaptability
across the varying environmental conditions.. However, in
the process of evolution of widely adapted cultivars the G x E
interaction are of major consequence to the breeders
(Witcombe,1988).Significant advances have been made in the
measurement of contribution to the genotype–environment
interaction and in making predictions of performance of
genotypes using regression technique. Therefore, the
objective of the study to assess the amount and nature of
genotype x environment interaction and evaluate to identify
promising genotype giving consistent performance in the
different agro- climatic conditions or which can be utilized in
the development of elite gene pool through combination
breeding.
MATERIALS AND METHODS
Twelve high yielding genotypes of common bean
selected from advanced trials at the source stations over two
seasons and a standard check were evaluated for their yield
performance in trials replicated thrice across the three diverse
agro-climatic conditions during Kharif 2008.These genotypes
grown in randomized block design with three replications at
each location. Each replicate has 13 treatments which are
SKUA-R-91, SKUA-R-121, SKUA-R-23, SKUA-R-153, SKUA-
R-132, SKUA-R-161, SKUA-R-171, SKUA-R-165, SKUA-R-
180, SKUA-R-170, SKUA-R-106, SKUA-R-21 and Shalimar
Rajmash-1 planted in 4 rows each of 4m long with inter and
intra row distance of 30 and 10 cm respectively. Recommended
agronomic practices were adapted during the entire crop
season at all the locations to raise a good crop. Mean seed
yield per plant was recorded on 10 randomly selected plants
and subsequently plot yield was obtained from each
replication. The stability test was done in respect of seed
yield using the linear model proposed by Eberhart and Russel,
1996. The parameters include general mean (x), regression
coefficient (bi) that measured the response of a genotype to
varying environments and deviation from regression of the
genotype (S 2 di) in a particular environment.
RESULTS AND DISCUSSION
The analysis of variance for individual environments
showed significant differences among the genotypes for seed
yield indicating the sufficient variability was present among
the genotypes (Table 1). A wide range of variability in rajmash
for seed yield has been reported by Kapila and Pawar, 1997
and Tripathi et al.,1997.
Table 1.
Source of variation
Analysis of variance in individual environments
for seed yield in Rajmash
d.f
Mean squares
E 1 E 2 E 3
Replication 2 0.005 0.001 0.003
Genotype 14 0.022** 0.018** 0.0029*
Error 24 0.0013 0.001 0.0012
*, ** Significant at 5% and 1% levels respectively.
Joint regression analysis of variance for stability
revealed that the mean square due to genotypes was
significant for seed yield (Table 2) indicating the presence of
substantial genetic variations among the genotypes. The
significant mean square due to environments revealed that

2 2 Trends in Biosciences 2 (2), 2009
Table 2.
Analysis of variance for stability of seed yield in
Rajmash
Source of variation d.f Mean sum of squares
Genotype 12 3120.46**
Environments 2 22829.98**
Genotype x Environment 24 1107.81*
Env + (G x E) 26 2778.75**
Environment (lin) 1 45659.96**
G x E (Lin) 12 1768.48*
Pooled deviation 13 412.76
Pooled Error 78 1910.28
*, ** Significant at 5% and 1% levels respectively.
the environment plays a major role in developing genetic
variation among the genotypes for the trait under
investigation. Significant genotype x environment (G x E)
interaction indicating differential expressions of genotypes
for the seed yield over environments. The results are similar
with the findings of Chandra et al.,1973 and Noor and Singh,
1989. The combined environment and Genotype x Environment
[E + (G x E)] interaction was significant revealing that the
genotypes interacted considerably with environmental
conditions that existed over three locations. The G x E
interaction effects were further partitioned into linear
components of G x E interaction and non- linear pooled
deviation. The significant linear component of variation
observed for the seed yield indicating that the differential
regression co-efficient pertaining to various genotypes on
the environments mean were real, whereas non- significant
pooled deviation suggested their predictable nature of the
Table 3.
Genotype
Stability parameters for seed yield in Rajmash
Mean
_
x
(Kg/ha)
b i
S 2 d i
SKUA-R-91 960 1.01 0.04
SKUA-R-121 838 1.09 -0.18
SKUA-R-23 751 1.44 -0.16
SKUA-R-153 876 2.35 -4.19
SKUA-R-132 938 1.73 -0.17
SKUA-R-161 833 0.60 0.25
SKUA-R-171 642 0.53 1.94
SKUA-R-165 759 -0.29 -0.03
SKUA-R-180 915 1.24 1.12
SKUA-R-170 854 0.86 -2.30
SKUA-R-106 760 1.47 -5.31
SKUA-R-21 751 -0.08 -4.81
Shalimar rajmash-1 928 0.94 -1.08
Mean 831
SEM± 0.30
variability for the trait. Similar results were also reported by
Vaid et al.,1985 and Durate and Zimmernam, 1995. The
magnitude of G x E linear interaction was greater than the
pooled deviation revealing prepondence of linear over nonlinear
component for the yield trait. Similar results were also
reported by Manivannan et al.,1996.
The values of environmental indices were positive at E 1
were as poor at E 2
and E 3
for the manifestation of seed yield in
these genotypes.
According to Eberhart and Russel,1996 a genotype is
considered to be stable in performance if it has high mean
yield (x), regression co-efficient (bi) of unity and least mean
square deviation ( s 2 di). A non-significant linear (bi) and nonlinear
(s 2 di) component estimates the genotypes indicate
average stability with high precision across environmental
change. In the present investigation, the yield of all the
genotypes ranged from 642 to 960 Kg/ha across the locations
(Table 3).The magnitude of regression and deviation
regression varied among the genotypes indicating that
genotypes exhibited different degrees of environmental
response possibly due to presence of different set of alleles
for stability. On the basis of stability parameters it was
observed that genotype SKUA-R-91 revealed high mean
performance for seed yield, regression co-efficient
approaching near to unity and low magnitude of deviation
from regression could be considered as most stable and
promising genotype. Further, it was observed in the present
study that SKUA-R-132, SKUA-R-180 would be stable and
ideal for all environments with respect to seed yield.
LITERATURE CITED
Chandra,S., Sahoo,M.S. and Singh, K.P. 1973. Genotype x environment
interaction for yield in grain. J. Res. Punjab Agri. Univ., 8 (2):
165-68.
Durate, J.B. and Zimmernam, M. J. Do. 1995. Correlation among yield
stability parameters in common bean. Crop Sci., 35:905-912.
Eberhart, S.A and Russel, W.A. 1966. Stability parameters for comparing
varieties. Crop Sci.,6:36-40.
Kapila, R. K. and Pawar, K. S. 1997. Genetic parameters and association
analysis in rajmash. Indian J. Pulses Res., 10 (1): 38-41.
Noor, M.F. and Singh, N.B. 1989. Phenotypic stability for yield and its
components in chickpea. In: National Symposium on New Frontiers
in Pulses Research and Development.10-12, Nov.DPR, Kanpur.
Manivannan, N., Murugesan, S., Ramamoorthi, N. and Nadarajan, N.
1996. Stability analysis for seed yield in Mungbean. Indian J.
Pulses Res., 9 (2):149-152
Tripathi, D. P., Asthana, A. N. and Pandey, P. S. 1997. Variability and
correlation studies in exotic germplasm lines of rajmash. Indian J.
Pulses Res., 10 (1): 93
Vaid, K., Gupta, V. P. and Singh, R. M. 1985. Stability analysis in dry
beans. Crop improvement., 12: 28-31.
Witcombe, J. R. 1988. Estimates of stability for comparing varieities.
Euphytica., 39: 11-18.
Recieved on 5-10-2009 Accepted on 4-12-2009

Trends in Biosciences 2 (2): 23-24, 2009
Seasonal Population Fluctuation of Hoplolaimus indicus on Pigeon Pea
S.S. ALI
Indian Institute of Pulses Research, Kanpur 208 024
e-mail: ss_ali@rediffmail.com
ABSTRACT
A field trial on seasonal fluctuation of Hoplolaimus
indicus on three genotypes on pigeonpea representing three
maturity groups i.e. early maturing (EM), UPAS 120, medium
maturing (MM), PDA 86-1 and late maturing (LM), T-7 was
carried out at Indian Institute of Pulses Research. At monthly
intervals, soil samples were drawn from each treatment for a
period of one cropping season. Nematode population growth
over a growing season of three genotypes was not found
identical but it varies with maturity groups. The maximum
population was encountered at the flowering stage at the
respective maturity group genotypes which suggested that
H. indicus was specific to pigeon pea crop. This study indicated
that management of nematodes can be under taken by
checking high build up of population prior to flowering stage
of pigeon pea crop
Key words
Hoplolaimus indicus, pigeon pea, populationfluctuation,
maturity groups
Among pulses, pigeonpea (Cajanus cajan) L. Millsp.)
occupies an important place. Globally it is cultivated on 4.9 m
ha and 3.58 m ha and 72.7% of it is confined to India alone.
The new pigeon pea cultivars are attracting rainfed farmers
and due to this, the cropped area has witnessed a significant
increase from 2.3 m ha in 1950 to 3.6 m ha in 2006 (Saxena,
2009). The low productivity of pigeon pea in the country
attributed to various biotic factors among these the nematodes
is one of the important factor in crop production and causes
appreciable monetary losses. An avoidable yield loss of 19 %
in pigeon pea due to Rotylechulus reniformis has been
reported in sandy loam soil. (Ali, 1996). The wide spread
occurrence of the lance nematode, Hoplolaimus spp. and its
relative abundance along with root-knot, reniform and cyst
nematode have been reported on the roots of the pigeon pea
plants from U.P., Gujarat (Sharma, et al., 1993 and 1996). The
lance nematode is an economically important ectoparasitic
nematode available in the rhizosphere of pigeon pea crop in
varying densities throughout the cropping season, therefore
an attempt was made to study the seasonal fluctuation of
lance nematode population, the most prevalent and persistent
nematode on three different maturity groups genotypes of
pigeon pea.
Institute of Pulses Research, Kanpur farm to study the
seasonal fluctuation of the lance nematode, Hoplolaimus
indicus population on three different genotypes of pigeon
pea representing three maturity groups, i.e. early maturing
(EM), UPAS 120, medium maturing (MM), PDA 86-1 and late
maturing (LM), T-7. There were four replications and three
treatments arranged in randomized block design (R.B.D). The
seeds of these three genotypes were sown in June in plot size
of 2 x 3 m with spacing of 75 x 10 cm. Fertilizer application,
weed management and other cultural practices were carried
out as per recommendation. Composite soil samples were
collected 15 to 20 cm deep with a 20 cm long soil sampler at
monthly intervals from each treatment for a period of one
cropping season (July to May). An aliquot of 100 cm 3 soils
samples from composite bulk sample was processed by using
Cobb’s sieving and decanting techniques for determining the
nematode population. The nematode population was identified
and lance nematode population was assessed using a
binocular microscope.
RESEULTS AND DISCUSSION
Data indicated that in pigeon pea early maturing (EM)
genotype, UPAS 120 maximum lance nematode population
was recoded during the month of October then it declined
gradually at harvest in December (Fig.1). In pigeon pea
genotype PDA 86-1 (MM) two peak population of lance
nematodes were observed during the month of October and
January when its flowering started then there was a sharp
decline in population at harvest in the month of March. In
pigeon pea genotype T-7 (LM) maximum lance nematode
population was encountered during February when its
flowering started while minimum population was recorded at
MATERIALS AND METHODS
During Kharif a field trial was laid down at Indian
Fig. 1.
Seasonal fluctuation of Hoplolaimus indicus population
in three different maturity group genotypes of pigeon
pea

2 4 Trends in Biosciences 2 (2), 2009
harvest in the month of November - December. In late maturity
group genotype there is a peak population of lance nematode
in the month of October and it declined afterwards. After
attaining the highest lance nematode population in February
it declined again sharply at the harvest during the month of
April- May. Good multiplication of Hoplolaimus indicus
population was commonly associated with irrespective of
maturity groups genotypes when temperature ranges from 30
to 35°C during October. While its population declined during
whole winter season. In the medium and late maturity
genotypes when the flowering is initiated in January and
February respectively, again the lance nematode population
attained highest population in both the medium and late
maturity groups genotype of pigeon pea. Nematode population
growth over a growing season of the three different maturity
genotypes of pigeon pea was not found identical however it
varied with maturity groups. The maximum population was
encountered at the flowering stage of the respective maturity
group genotypes which suggested that H. indicus is specific
to pigeon pea crop and its population density is genotype
specific. This study indicated that the management of lance
nematode can be effectively carried out by checking the high
build up population of lance nematode prior to the flowering
and during the month of October as well.
LITERATURE CITED
Ali, S.S. 1996. Estimation of yield losses in pigeon pea due to reniform
nematode. Indian Journal of Pulses Research., 9 (2) : 209-210.
Sharma, S.B., Ali, S.S., Patel, H.V., Patel, S.K. and Patel, D.J. 1993.
Prevalence of plant parasitic nematodes associated with pigeon
pea in Gujarat State in India. Afro-Asian J. Nematol., 3 (1): 55-59.
Sharma, S.B., Ali, S.S., Upadhyay, K.D. and Ahmad, F. 1996. Potential
nematode constraints of pigeon pea in Uttar Pradesh in northern
India. Afro-Asian J. Nematol., 6 (2) : 151-155.
Saxena, K.B. 2009. Evaluation of Hybrid Breeding Technology in pigeon
pea. In: Milestones in Food Legume Research (eds. Masood Ali,
Shiv Kumar). Indian Institute of Pulses Research Kanpur 208 024.
pp. 82-114
Recieved on 20-7-2009 Acceted on 30-11-2009

Trends in Biosciences 2 (2): 25-26, 2009
Integrated Phosphorus Management in Mungbean in Kashmir Valley
F.A.PIR, F.A.NEHVI, ABU MANZAR, S.A.DAR AND B.A.ALLAI
Pulses Research Sub-Station-Habak, Sher-e-Kashmir University of Agricultural Sciences & Technology of
Kashmir 190 006, Srinagar; e-mail: abumanzar@rediffmail.com
ABSTRACT
An experiment was conducted for three years at Pulses Research
Sub-Station, Habak, SKUAST-K during kharif seasons from
2006 to 2008 to study integrated phosphorus management in
mungbean. Ten treatments viz.,control; 30:50:30kg NPK/
ha;30:50:30kg NPK/ha+Rhizobium;5t FYM/ha+30:50:30kg
NPK/ha +Rhizobium +PSB; 10t FYM/ha +15:25:15kgNPK/
ha+Rhizobium+PSB; 10t FYM/ha +20kg P 2
O 5
as Phosphate
Rock + PSB; 5t FYM/ha +40kg P 2
O 5
as Phosphate Rock + PSB;
10t FYM/ha +20kg P 2
O 5
as DAP+PSB and 5t FYM/ha + 40kg
P 2
O 5
as DAP + PSB were replicated thrice in RBD. Growth,
yield contributing characters and grain yield were significantly
enhanced under different treatments. Highest grain yield (918
kg/ha) was recorded under 5t FYM/ha +30:50:30kg NPK/ha
+Rhizobium +PSB which was 72.22% higher over control that
gave the lowest yield (518 kg/ha). It also recorded highest values
of plant height, pods per plant, nodule count and test weight. 5t
FYM/ha conjugated with 40Kg P 2
O 5
through rock phosphate +
seed inoculation with PSB gave mungbean yield (801kg/ha)
that was at par with 30:50:30kg NPK/ha +Rhizobium (803kg/
ha) & 5t FYM/ha +40kg P 2
O 5
as DAP + PSB (829kg/ha). 5t
FYM/ha + 30:50:30kg NPK/ha + Rhizobium+PSB procured
highest net return (Rs. 18320) and B.C. ratio (1:34).
Key words
Mungbean, phosphorus, phosphate rock
Mungbean [Phaseolus radiatus (L.) Wilczek] is a major
pulse crop of Kashmir. It is cultivated in summer as kharif
crop mostly under rainfed conditions. Phosphorus is a key
input and neglect in its use or low level in soil is responsible
for low yield of this crop. For proper utilization of the available
and applied source of P in the soil, an integrated approach is
required so as to fully utilize the yield potential of this crop.
Phosphate rock which is available in different grades is a
source of phosphorus application suitable for use in soils
having high organic matter content. Direct application of
phosphate rock has received special attention in Asian
countries (Mathur and Sarkar, 1998) where phosphate fertilizer
costs are high and there are large areas under acid soils.
Cumulative effect of MPR (Mussoorie phosphate rock) over
5 years was 39 per cent more than SSP in soybean (Prasad,
2007). Inoculation of seed at sowing with PSB provides
substantial amount of P 2
O 5
by solubulising the insoluble soil
and added phosphorus through production of aliphatic
aromatic acids, phytase, phospholipase etc. (Gaur, 1990).
Chhonkar and Subba Rao, 1967 and Kucey, et al., 1983 have
reported increased efficiency of phosphate rock by mixing it
with phosphate solubulising organisms. With this in view, a
study was undertaken to investigate the influence of
integrated phosphorus management on growth, yield
attributes and yield of mungbean.
MATERIALS AND METHODS
The experiment was conducted for three years during
kharif seasons of 2006, 2007 and 2008 at Pulses Research Sub
Station, Habak, SKUAST-K, Srinagar. The soil was clay loam
with a neutral pH(6.89), medium in available nitrogen(370kg/
ha), phosphorus(31kg/ha) and low in available
potassium(78kg/ha). Nine treatments viz., control (T 1
); 30:50:30
kgNPK/ha (T 2
); 30:50:30kgNPK/ha + Rhizobium (T 3
); 5t FYM/
ha + 30:50:30kgNPK/ha + Rhizobium + PSB (T 4
); 10tFYM/ha
+ 15:25: 15kgNPK/ha + Rhizobium + PSB (T 5
); 10 FYM/ha +
20 kg P 2
O 5
as Phosphate Rock + PSB (T 6
); 5t FYM/ha + 40 kg
P 2
O 5
as Phosphate Rock + PSB (T 7
); 10t FYM/ha + 20 kg P 2
O 5
as DAP + PSB (T 8
) and 5t FYM/ha + 40 kg P 2
O 5
as DAP + PSB
(T 9
) were tested in a randomized block design using three
replications.Mungbean variety Shalimar Moong-1 was sown
in the month of June in the years of experimentation.
Observations on growth, yield attributing characters and yield
were recorded and data subjected to analysis of variance.
RESULTS AND DISCUSSION
There was a significant influence of different treatments
on growth, yield attributes and yield of mungbean. Plant height
was highest (34.0cm) in 5t FYM/ha + 30:50:30 kgNPK/ha +
Rhizobium + PSB (T 4
) that was significantly higher over other
treatments baring 30:50:30kgNPK/ha + Rhizobium (T 3
) and
10t FYM/ha + 15:25:15kgNPK/ha + Rhizobium + PSB (T 5
)
being at par with them.
T 3
and T 4
recorded comparable values of nodule count
being statistically higher over rest of the treatments giving
141 and 101 per cent more nodules/plant than control.
Maximum number of pods per plant (23.70) and highest test
weight (3.68 g) was recorded in 30:50:30kgNPK/ha in presence
of 5tFYM/ha fortified with Rhizobium and PSB. This might be
due to enhanced availability of phosphorus and nitrogen in
rhizosphere which in turn influenced the crop growth and
yield contributing characters.
Lowest values of pods per plant and test weight (13.36
and 2.26 g respectively) were registered in control. Kumar et
al., 2003 and Pramanik and Singh, 2003 also reported
improvement in yield attributes in mungbean due to
phosphorus application in conjugation with biofertilizers. Full

2 6 Trends in Biosciences 2 (2), 2009
Table 1.
Growth and yield attributes of mungbean as influenced by integrated phosphorus management (pooled over three
seasons)
S.No Treatment Plant height
(cm)
Nodules/
plant
Pods/
plant
100-seed
weight (g)
1. Control (T 1) 23.6 17 13.36 2.26
2. 30:50:30 kg NPK/ha (T 2) 30.7 31 19.70 3.13
3. 30:50:30 kg NPK/ha + Rhizobium (T 3) 32.3 41 21.40 3.56
4. 5t FYM/ha + 30:50:30 kg NPK/ha + Rhizobium + PSB(T4) 34.0 38 23.70 3.68
5. 10t FYM/ha + 15:25:15 kg NPK/ha + Rhizobium + PSB (T 5) 31.8 29 18.20 2.81
6. 10t FYM/ha + 20 kg P 2O 5 as Phosphate Rock + PSB (T 6) 31.5 26 17.90 2.87
7. 5t FYM/ha + 40 kg P 2O 5 as Phosphate Rock + PSB (T 7) 31.5 30 15.60 3.20
8. 10t FYM/ha + 20 kg P 2O 5 as DAP + PSB (T 8) 30.4 27 17.10 2.63
9. 5t FYM/ha + 40 kg P 2 O 5 as DAP + PSB (T 9 ) 29.5 33 18.30 2.74
CD (P=0.05) 2.31 3.40 1.72 0.33
Table 2.
Yield and economics of mungbean as influenced by integrated nutrient management
S.No.
Seed yield (kg/ha)
Straw yield Net profit B.C.
Treatment
Mean
2006 2007 2008
(kg/ha) (Rs/ha) ratio
1. Control (T 1) 547 539 468 518 720 7710 0.63
2. 30:50:30 kg NPK/ha (T 2) 849 703 665 739 1096 13945 1.16
3. 30:50:30 kg NPK/ha + Rhizobium (T3) 862 723 824 803 1187 15875 1.29
4. 5t FYM/ha + 30:50:30 kg NPK/ha + Rhizobium + PSB (T 4) 939 828 987 918 1235 18320 1.34
5. 10t FYM/ha + 15:25:15 kgNPK/ha + Rhizobium + PSB (T 5) 900 751 944 855 1027 15355 1.05
6. 10t FYM/ha + 20 kg P 2O 5 as Phosphate Rock + PSB (T 6) 827 701 779 759 966 12695 0.91
7. 5t FYM/ha + 40 kg P 2O 5 as Phosphate Rock + PSB (T 7) 841 690 872 801 1006 15415 1.22
8. 10t FYM/ha + 20 kg P 2O 5 DAP + PSB (T 8) 689 725 929 781 1081 13189 0.93
9. 5t FYM /ha + 40 kg P 2O 5 as DAP + PSB (T 9) 857 737 893 829 989 15843 1.20
CD (p=0.05) 36 51 64 53 71 - -
recommended dose of chemical fertilizer in combination with
5t FYM/ha with seed inoculation with Rhizobium and PSB
(T 4
) gave the highest grain and straw yield (918kg/ha and
1235 kg/ha respectively) that was significantly higher over
other treatments and was 72.22 and 76.15 per cent more than
control respectively that recorded the lowest values (518kg/
ha and 720 kg/ha respectively). This was followed by 10tFYM/
ha + 15:25:15kgNPK/ha with seed treatment with Rhizobium
and PSB (T 5
) that was comparable with 30:50:30kgNPK/ha +
Rhizobium (T 3
) and 5TFYM/ha + 40 kg P 2
O 5
as DAP + PSB
(T 9
). The treatment of 30:50:30kg NPK/ha + Rhizobium (T 3
);
5t FYM/ha + 40 kg P 2
O 5
as phosphate rock + PSB (T 7
) and 5t
FYM/ha + 40 kg P 2
O 5
as DAP + PSB (T 9
) were also at par
among themselves with regard to seed and straw yield of
mungbean suggesting substitution of phosphate fertiliser with
direct application of phosphate rock. The enhancement in the
yield of mungbean due to integrated phosphorus application
may be ascribed to increased phosphorus availability that
lead to better translocation of photosynthates towards sink
with consequent improvement in yield attributes (Table 2).
Similar findings have also been reported by Naik and Rajput,
2003.
Highest net profit (Rs. 18,320) and B.C. ratio (1.34) was
realised in the treatment of 5tFYM/ha + 30:50:30kg NPK/ha +
Rhizobium + PSB.
LITERATURE CITED
Chhonkar, P.K.and Subba Rao, N.S. 1967. Phosphate solubulization by
fungi associated with legume root nodules. Canadian Journal of
Microbiol., 13 : 749-753.
Gaur, A.C. 1990. Phosphate solubulizing micro-organisms as biofertilizer.
Omega Scientific Publishers, New Delhi.pp.240.
Kucey, R.M.N., Janzen, H.H. and Legget, M.E. 1983. Micro-bially
mediated increases in plant available phosphorus. Adv. Agron., 42
: 192-228.
Kumar, S., Singh, R.C. and Kadian, V.S. 2003. Performance of mungbean
as influenced by seed inoculation with Rhizobium and levels of
organic and inorganic source of nutrients. Indian Journal of Pulses
Research, 16(1) : 67-68.
Mathur, B.S. and Sarkar, A.K. 1998. The use of phosphate rock material
on red and leteritic soils of eastern Indian. In : Nutrient management
for sustainable crop production in Asia (Eds., A.E. Johnstan and
J.K. Syres ), CAB International, Wallingford, U.K. pp. 345.
Naik, K.R. and Rajput, A.S. 2003. Effect of biofertilisers on seed
production of summer green gram (Phaseolus radiatus). Agronomy
Digest, 3 : 38-39.
Pramanik, K. and Singh, R.K. 2003. Effect of phosphorus and biofertilizers
on growth, yield attributes, yield and nutrient uptake of
green gram (P. radiatus). Agronomy Digest. ,3 : 35-36.
Prasad, R. 2007. Phosphorus. In : Crop Nutrition-Principles and
Practices. New Vishal Publications, New Delhi. pp.272.
Recieved on 10-6-2009 Accepted on 10-8-2009

Trends in Biosciences 2 (2): 27-30, 2009
Anti-mutagenic Effects of Flavonoids in Human Lymphocytes Culture.
MD. SULTAN AHMAD, MRS.SHEEBA**, AFSAR ALI, RAKESH YADAV AND B.R.GAUTAM.
Department of Zoology, S.N. (P.G). College, Azamgarh, U.P., 276 001
**Department of Zoology, D.S. College, Aligarh, U.P., 202 002
e-mail: sultansnz@yahoo.com
ABSTRACT
The antigenotoxic potential of flavonoids was demonstrated on
the genotoxicity induced by hydrocortisone (steroid). Studies
were on human lymphocyte culture. Chromosomal aberration
and sister chromatid exchange and cell cycle kinetics, with and
without S 9
mix were taken as end point in in vitro experiments.
Four doses viz., 250, 300, 350, and 400 µg/ml were selected, and
found that flavonoids were significantly reduced the frequencies
of chromosomal aberration, sister chromatid exchanges and
enhances RI in vitro. It was also noticed that the antigenotoxic
potential of flavonoids showing dose-response relationship.
Key words
Flavonoids, chromosomal aberration, sister
chromatid exchange, anticarcinogen
It is estimated that humans consume about 1g/day of
flavonoids from the intake of foods, containing plant tissues
such as vegetables, fruits, cereals, tea, cocoa, coffee, wine,
bear, cola and peanuts (Wollenwever, et al., 1981). Flavonoids
are abundant in legumes; within plant tissues, they exist as
sugar derivatives called glycosides. These compounds
undergo hydrolysis in the human gut, yielding aglycones.
Like the lignans, these aglycones meet one of the three fates,
they may be excreted or absorbed from the gut or undergo
further metabolism. Products of flavonoids metabolism may
be either excreted or absorbed. If absorbed, the
phytoestrogens undergo conjugation in liver with glucuronic
acid or to a lesser extent, sulfate and are excreted in urine or in
the bile. Recently it has been suggested that flavonoids might
be useful as new chemopreventive agents in human
carcinogenicity (Gabor, 1991).
Flavonoids inhibit the activities of hyaluronidase
enzymes, as they are involved in a number of processes
including allergic reaction, inflammation, migration of cancer
cells and malignant cell proliferation (Kuppusamy, et al., 1990).
The ability of rutin and quercetin to react with superoxide
anion and lipid peroxyradicals as well as to form iron complexes
that are unable to catalyze the formation of active oxygen
radicals makes them useful for possible therapy in combating
cellular damage caused by radicals, “free radical pathologies”
(Afanas’ev, et al., 1989). Quercetin and rutin can reduce the
level of serum triglycerides and are antithrombotic (Kato and
Tosa, 1983).
In the present study, the antigenotoxic effects of
flavonoids in terms of reduction in CAs, SCE frequencies and
enhancement of RI, in different doses and durations of
treatment, have been studied both singly with and without
the presence of S 9
mix against hydrocortisone and induced
damage of lymphocytes in culture.
MATERIALS AND METHODS
Using the technique of Moorehead, et al., 1960, analysis
of metaphase chromosome for the detection of CAs was
performed. Human lymphocyte cultures were set by adding
0.5 ml of whole blood (from two adult and healthy donors,
occupationally not exposed to mutagens) to 4.5 ml of RPMI
1640 (Gibco USA), antibiotics (Penicillin and streptomycin
100 IU/ml each; Hoechst) and L-Glutamine (1 mM; Gibco,
USA). Lymphocytes were stimulated to divide by adding 0.1
ml of phytohaemagglutinin-M (PHA-M, Gibco). The cultures
were incubated at 37 o C with 5% CO 2
for 72 hrs. in dark.
Hydrocortisone (Sigma, St. Louis) at a final concentration of
50 mg was added at 0 hour and kept for 24, 48 and 72 hours of
duration, which served as positive control. Subsequently,
flavonoids (Sigma, St. Louis) in four different concentrations
(250, 300, 350, 400 mM/ml) were added along with
hydrocortisone and the cultures were kept for 24, 48 and 72
hours. Solutions of flavonoids, and hydrocortisone were
prepared in DMSO. In the metabolic activation experiments
cultures were treated along with S 9
mix (0.8 ml). The S 9
mix was
freshly prepared as per the standard procedures of Maron
and Ames, 1983. The S 9
fraction was complemented by the
addition of 5 mM NADP and 10 mM glucose –6- phosphate
just before use. After 6 h of incubation the cells were collected
by centrifugation and the pellets were washed twice in prewarmed
(37 o C) medium to remove the drug and S 9
mix and
reincubated for 24 h in fresh medium supplemented with
antibiotic and fetal calf serum. Parallel cultures receiving same
concentrations of different phytoproducts for similar treatment
duration without S 9
mix were simultaneously set for
comparison. Colchicines (0.20 mg/ml, Micro lab) were added
to the cultures, 2.5 hours prior to harvesting. The cells were
collected by centrifugation (10 min, 1200 rpm), hypo tonic
treatment (0.075 KCl) was given for 10-12 min at 37 0 C and the
recollected cells after centrifugation were fixed in methanol:
acetic acid (3:1). A total of 300 well - spread metaphases were
analyzed per treatment per duration for all types of chromatid
and chromosome type of aberrations. Aberrations were scored

2 8 Trends in Biosciences 2 (2), 2009
as per Hundal, et al., 1997. Analysis of SCE was carried out
following the fluorescent plus Giemsa technique (Perry and
Wolff, 1974). The cells in the cultures were exposed to 5-bromo-
2-deoxyuridine (BrdU 2 mg/ml; Sigma) after 24 hours of
initiation of culture. The test compounds with same
concentrations as in the case of CA analysis were added
together with the BrdU. To minimize photolysis of BrdU
another 48 hours cultures were maintained in the dark.
One day old slides were stained in Hoechst 33258 stain
(Sigma 0.5 mg/ml), exposed to UV lamp (254nm) for 30 min.
and incubated in 2X SSC (0.3 M NaCl, 0.03M Sodium citrate;
pH 7.0) at 60 o C for 90 min and stained for sister chromatids.
The slides were coded prior to scoring and 50 well- spread
metaphase cells were scanned per concentration and the
number of exchange scored (Hundal, et al., 1997). Cells
undergoing 1 st (M 1
), 2 nd (M 2
) and 3 rd (M 3
) metaphase divisions
were detected with BrdU-Harlequin technique for differential
staining of metaphase chromosome (Latt and Wohllel, 1975),
by studying 200 metaphases for each combination and
duration. The replication index (RI), an indirect measure of
studying cell cycle progression, was calculated by applying
the following formula (Tice, et al., 1976).
RI =
M
1x1
M2x2
M
100
3x3
RESULTS AND DISCUSSION
The flavonoids have long been used as antioxidant for
combating various degenerative actions of free oxygen radicals
and reactive oxygen species. All the controls show aberrations
around 3%, while hydrocortisone treatment has values ranging
from 21.5 to 55.0% with increasing durations when flavonoids
are used, the reduction varies from 19.50 to 49.0% at lower
concentration and 19.0 to 46.5% at the highest concentration
(Table1.). When augmented with S 9
mix, the per cent aberrations
are higher, and however these get reduced when flavonoids
are used concurrently with hydrocortisone. The least values
go with control and F2. The range and mean values of SCE get
reduced with flavonoids in the absence as well in the presence
of metabolic activation (Table 2.). The replication indices,
which are reduced due to hydrocortisone treatment level (1.46),
is elevated to 1.61, thus bringing the cell proliferation nearly
back to normal of 1.73. The S 9
activation also has been noticed
(Table 3.).
As a result, there is considerable interest and effort in
research on the therapeutic potential of flavonoids as drugs
for the prevention and treatment of certain human diseases
(Farkas,et al., 1986). Some enzymatic effects of flavonoids on
cell physiology are being explored as novel therapeutic targets
of anticancer agents. Similarly, dietary quercetin significantly
reduced the number of palpable rat mammary tumors and also
Table 1.
Treatments
Analysis of C.A. after treatment of hydrocortisone and flavonoids in presence of S 9
– mix in vitro.
Metabolic
activation
Metaphase scanned
Per cent aberration
metaphase
Including
gap
Excluding gap
Types of aberration (%)
Chromatid
Chromosome
Total
Aberration/ Cell SE
Hydrocortisone
(50g/ml)
-S 9
+S 9
300
200
19.00
25.25
17.00
24.00
17.50
22.50
9.50
19.50
27.00
42.00
0.27 0.04
0.42 0.05
Hydrocortisone
+ Flavonoids
Hyd + F 0
-S 9
+S 9
300
200
17.00
23.50
15.50
22.50
16.00
21.00
9.00
18.00
25.00
39.00
0.270.04
0.390.05
Hyd + F 1
-S 9
+S 9
300
200
15.35
21.00
14.25
20.00
14.75
19.50
8.50
17.00
23.25
36.50
0.230.04
0.370.05
Hyd + F 2
-S 9
+S 9
300
200
14.00
18.25
12.75
17.00
14.00
17.50
8.00
15.50
22.00
33.00
0.220.04
0.330.05
Hyd + F 3
Control
-S 9
+S 9
300
200
12.50
16.50
12.00
15.00
12.00
15.00
7.50
13.00
19.50
28.00
0.200.04
0.280.05
Normal + F 2
Normal
DMSO (5g/ml)
-S 9
+S 9
-S 9
+S 9
-S 9
+S 9
300
200
300
200
300
200
2.50
3.00
3.50
4.20
4.00
4.50
2.00
2.50
2.00
3.70
1.50
2.00
2.50
2.00
3.00
3.50
3.30
2.70
0.50
1.00
0.33
0.50
0.70
1.00
3.00
3.00
3.33
4.00
4.00
3.70
0.03 0.02
0.03 0.02
0.03 0.02
0.04 0.02
0.04 0.02
0.04 0.02

AHMAD et al., Anti-mutagenic Effects of Flavonoids in Human Lymphocytes Culture 2 9
Table 2.
Analysis of SCE, after treatment of hydrocortisone and flavonoids in vitro with and without metabolic activation.
Treatment
Duration (h)
Metabolic
activation
Metaphase scanned
Total
SCE
Range
SCE / Cell SE
Hydrocortisone
50g/ml 48
Hydrocortisone
+ Flavonoids
Hyd + F 0
48
Hyd + F 1
Hyd + F 2
Hyd + F 3
Control
48
48
48
-S9
+S9
-S9
+S9
-S9
+S9
-S9
+S9
-S9
+S9
50
50
50
50
50
50
50
50
50
50
235
315
210
252
195
220
175
195
180
200
2 – 11
1 - 11
1 – 10
2 – 12
1 – 10
1 – 9
1 – 10
2 – 12
1 – 10
1 – 9
4.70 1.20
6.30 1.25
4.20 0.80
5.04 1.20
3.90 0.50
4.40 1.00
3.50 0.50
3.90 0.75
3.60 0.50
4.00 1.00
Normal + F 2
Normal
DMSO(5g/ml)
Table 3.
Treatment
48
48
48
-S9
+S9
-S9
+S9
-S9
+S9
50
50
50
50
50
50
77
90
85
105
90
115
0 – 6
0 – 6
0 – 5
0 – 7
0 – 5
0 – 6
1.54 0.14
1.80 0.14
1.70 0.14
2.14 0.20
1.80 0.14
2.30 0.20
Analysis of cell cycle kinetics after treatment of hydrocortisone and flavonoids in vitro with and without metabolic
activation.
Cell scored
Metabolic
activation
Per cent cell in
M1 M2 M3
Replication
index
(RI)
2*3 Chi square
test
Hydrocortisone
50g/ml
Hydrocortisone
+ Flavonoids
200
200
-S9
+S9
57
59
40
36
03
05
1.46
1.46
Significant
Significant
Hyd + F 0
Hyd + F 1
Hyd + F 2
Hyd + F 3
Control
200
200
200
200
200
200
200
200
-S9
+S9
-S9
+S9
-S9
+S9
-S9
+S9
53
56
51
54
48
51
50
50
38
37
37
38
37
40
39
42
09
07
12
08
15
09
11
08
1.56
1.51
1.61
1.54
1.67
1.58
1.61
1.58
Significant Significant
Significant Significant
NotSignificant
Significant Significant
Significant
Normal + F 2
Normal
DMSO (5g/ml)
200
200
200
42
43
44
39
41
40
19
16
16
1.77
1.73
1.72
Not.significant
Normal
the number of rats with tumors, induced by intragastric
instillation of 7, 12 dimethylbenz(á)anthracene (DMBA) and
by I.V. injection of N–nitrodomethyleurea, both of which are
confirmed chemical carcinogens (Verma, et al.,1988). A fresh
or boiled juice from vegetables significantly suppresses the
incidence of aberrations (Ito, et al., 1986). Present finding
also confirmed such affects of flavonoids in human
lymphocytes culture, where it reduces chromosomal aberration
along with sister chromatid exchanges. In spite of the progress
made by many investigations in the last several years to
elucidate the mechanism by which food flavonoids exert
their protective effect in reducing carcinogenicity, the precise

3 0 Trends in Biosciences 2 (2), 2009
mechanism of action is still unclear.
It is expected that many of the above mentioned
observations, mainly from in vitro experiments, might reflect
the ability of flavonoids to counteract the adverse biological
effects of mutagens and carcinogens (Bhattacharya, 1990).
However not all modes of action are applicable to all
flavonoids which may act in more than one way, or may not
act at all. Recently, De Flora and Ramel, 1988 provided an
excellent discussion of the overall mechanism of
antimutagenesis and anticarcinogenesis. In their scheme, they
classified the inhibitors by the possible mechanism of action
into three broad categories: (a) those that act extracellularly
either by hindering uptake of carcinogens, or by favoring
removal or deactivation of mutagens/carcinogens, (b) those
that act intracellular, modulating metabolic process in several
different ways and by blocking DNA-mutagen/carcinogen
interaction, and (c) those that act on already initiated
neoplastic cells by modulating tumor promotion or by
preventing malignant cell invasion. It appears that the
mechanisms by which flavonoids, as a group, perform mode
of action on biological systems.
ACKNOWLEDGEMENT
I would like to acknowledge the Department of Science
and Technology for granting SERC Young Scientist Project.
LITERATURE CITED
Afanas’ev, I.B. Dorozhko,A.I.; Brodskii,A.V.; Kostyuk, V.A.; and
Potapovitch, A.I. 1989. Chelating and free radical scavenging
mechanism of inhibitory activity of rutin and quercetin in lipid
peroxidation. Biochem. Pharmacol., 38: 1763–1769
Bhattacharya, R.K. 1990. In; Flavonoids in Biology and Medicine III.
Current Issues in Flavonoid Research (ed. N.P. Das) National
University of Singapore,pp. 340–347.
De Flora, S. and Ramel, C. 1988. Mechanisms of inhitors of mutagenesis
and carcinogenesis classification and overview. Mutat. Res., 202:
285–306.
Farkas, L. Gabor, M. and Kallay, F. 1986. Flavonoids and Bioflavonoids,
(eds.) Elsevier, Amsterdam.
Gabor, M. 1991. In: Plant Flavonoids in Biology and Medicine, Cody.V,
(eds.); Middleton. Jr. E; Harborne J.B and. Beretz ,A Alan R. Liss,
Inc. New York, pp.1–15
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studies of flavonoids as inhibitors of hyaluronidase. Biochem.
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Hungerfold, D.A. 1960. Chromosome preparations of leucocytes
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Inhibition of 7, 12-dimethylbenz (a) anthracene- and N-
nitrosomethylurea-induced rat mammary cancer by dietary flavonol
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869.
Recieved on 16-11-2009 Acceted on 30-11-2009

Trends in Biosciences 2 (2): 31-32, 2009
Effect of Irrigation Intervals on the Incidence of Shoot Borer, Chilo infuscatellus
Snellen in Sugarcane Crop in Punjab
H.S. RANDHAWA, K.S. THIND AND J.S. CHHINA
Regional Research Station, Punjab Agricultural University, Gurdaspur 143 521
email: harpals_randhawa@yahoo.co.in
ABSTRACT
In the newly planted sugarcane crop, irrigation intervals of 08
days was very effective for decreasing the incidence of early
shoot borer, followed by 16, 24 and 32 days irrigation interval.
Higher cane yield per acre was also obtained under 08 days’
irrigation intervals as compared to longer period of irrigations
intervals.
Key words
Shoot borer, sugarcane, irrigation
In Punjab sugarcane occupies an area 94 thousands
hectares and the estimated production may be up to the 514
lac tones (Dass, 2009). The average cane yield is very low. It
is not due to the climatic barriers but also involves improper
cultural practices i.e. lack of irrigation, adaptation of uncertified
seed, late sowing and harvesting, imbalance nutrition, bad
ratooning, insect-pests and diseases (Riaz, 2003).
In sugarcane crop different insect pests like borers,
termites, pyrilla, whitefly, thrip, bugs and mites attack on
sugarcane crop and cause heavy losses in Punjab conditions.
Out of these insect-pests, the early shoot borer (Chilo
infuscatellus Snellen) is a serious pest and it causes
considerable losses to the sugarcane crop in Punjab. The
symptom of early shoot borer on sugarcane crop incidence
seen in the form of ‘dead heart’ at the apex of cane which
when pulled out shows blackish coloration and emits foul
smell. When the mother shoot gets infested and die, generally
clumps get destroyed and gaps occur in the field. When mother
shoot is affected tillering stimulated, but in case of early attack
of the pest about 50 per cent of the shoot get eliminated
(Anonymous, 2009b).
A numbers of insecticides are recommended for the
control of this insect. These insecticides are not only costly
but also have side effects such as residue, resistance,
resurgence, destruction of parasitoids and predators and
environmental problems. (Bull, et al., 1979)
For combating this insect-pest, there is dire need to
develop more economical, efficient and safe pest control
method, which could be used singly or in combination with
hazardous insecticides. The present study was, therefore
undertaken to find out the utility of timing of irrigation
application in the newly planted sugarcane crop for the control
of shoot borer.
MATERIALS AND METHODS
The experiment was conducted at Regional Research
Station, PAU, Gurdaspur, Punjab, on newly planted crop of
sugarcane crop variety CoJ 85 in plots size 8.0×4.5 m, with
four replications Recommended agronomic practices were
followed to raise sugarcane crop. Four irrigation intervals i.e.
08, 16, 24 and 32 days were planned during pre-monsoon
period. The incidence of early shoot borer was recorded at 20
days intervals from April to June from the two middle rows of
each plot by counting the healthy plants and characteristics
dead hearts formed by the early shoot borer.
At harvest, the data of cane yield was also recorded to
study the effect of irrigation intervals on cane yield.
RESULTS AND DISCUSSION
The data regarding per cent incidence of early shoot
borer (Chilo infuscatellus Snellen) with respect to different
irrigation intervals (Table 1). It revealed that during the year
2006 the incidence of shoot borer in 08, 16, 24 and 32 days
irrigation interval treatments was 5.23, 9.67, 13.78 and 19.28
per cent respectively and the differences between any two
irrigation intervals were significant. During the year 2007 similar
results were obtained as the incidence of shoot borer in 08,
16, 24 and 32 days irrigation interval treatments was 5.25, 8.50,
13.25 and 17.00 per cent, respectively and the differences
between any two irrigation intervals were also significant.
When the mean per cent incidence of early shoot borer
of the two years was taken into, considerations and it was
observed that per cent incidence in these four irrigation
intervals was 5.24, 9.01, 13.13 and 18.14 per cent in 08, 16, 24
and 32 days irrigation interval treatments, respectively and
variations in each of the two respective irrigations intervals
were also significant. It showed that the frequent irrigation
caused adverse effect on the borer multiplication, the lowest
early shoot borer incidence being recorded at 08 days intervals
as compared to 16, 24 and 32 days interval. Similar, results
were also achieved by different workers such as
Krishnamurthy and Sheshagiri Rao, 1971, Patil, et al., 1982
and Mrig, et al., 1995 were also observed the lesser number of
irrigations were responsible for increase in its attack. Similarly,
Sardana, 2000 suggested that population of early shoot and
root borer could be managed by flooding of sugarcane’s fields.

3 2 Trends in Biosciences 2 (2), 2009
Table 1.
Irrigation
interval (Days)
Effect of irrigation intervals on the incidence of early shoot borer Chilo infuscatellus Snellen of sugarcane during
2006 and 2007
Figures in the parenthesis are square root transformations
Per cent incidence during different years Cane yield during different years (quintal/ac )
2006 2007 Mean 2006 2007 Mean
08 5.23 (2.49) 5.25(2.48) 5.24(2.33) 348.00 350.00 349.00
16 9.67(3.22) 8.50(3.07) 9.01(3.09) 341.00 340.75 341.82
24 13.78(3.73) 13.25(3.77) 13.13(3.67) 332.75 331.08 332.75
32 19.28(4.61) 17.00(4.24) 18.14(4.25) 330.90 323.65 322.07
CD (0.05) (0.46) (0.39) (0.41) 5.77 6.51 5.89
Effect of irrigation intervals on cane yield
The pooled data regarding cane yield for both the years’
i.e. 2006 and 2007 with respect to different irrigations intervals
is presented in the Table 1. It was observed that mean cane
yield in case of 08, 16, 24 and 32 days irrigation intervals were
349.03, 341.82, 332.75 and 322.07 quintals per acre respectively,
the differences in cane yield in each respective two level of
irrigation intervals being significant. This showed that each
shorter irrigation intervals had significantly higher cane yield
as compared to longer irrigation interval. The data in present
studies also in conformity with the earlier observations of
Malik and Chaudhary, 1990 and Mrig, et al.,1995 were obtained
the highest cane yield in the form the sugarcane planted plots
with frequent irrigation. The growth of cane is maximum during
February to May. During this critical period if the crop is
subjected to drought, heavy losses in terms of yield and
recovery occur in the field (Anonymous, 2009a). Therefore, it
is desirous to provide adequate irrigations during these critical
months.
LITERATURE CITED
Anonymous, 2009a. All India Area. Production and yield of sugarcane
www. ficciagroindia.com/general/agriculture-ststistics/4.20/a-http:/
/icargoa.res.in/sugarcane1.htm., found on Google.ask.co
Anonymous, 2009b. Package of Practices for Kharif crops in Punjab.
pp. 90-108.
Bull, D. C., House, V. S., Ables, J. R. and Morrison, R. K. 1979. Selective
methods for managing insect pests of crops. J econ Entomol., 78:
841-46.
Dass, B. 2009. Punjab Government going to increase the area under
sugarcane crop. Daily Ajit 06-02-2007 : 09
Krishnamurthy, M. M. and Sheshagiri Rao, C. 1971. Control of early
shoot borer through trash mulching and closer irrigations. Co-op.
Sug., 2: 489
Malik, V. S. and Chaudhary, J. P. 1990. Integration of heptachlor with
light earthing for reducing shoot borer (Chilo infuscatellus Snellen)
incidence in Sugarcane. Indian Sug., 38: 171-182
Mrig, K. K., Chaudhary, J. P. and Mehla, J. C. 1995. Effect of irrigations
intervals on the incidence of shoot and root borers in sugarcane
ratoon. J. Insect Sci., 8 (2):188-191
Patil, A. S., Gajare, B.G. and Hapase, D. G. 1982. Effect of irrigation on
incidence of Chilo infuscatellus Snellen. Maharashtra Sug., 7: 13-
1 7
Riaz, S. 2003. Scenario of sugar production. “The Nation”, July P: 17
Sardana, H. R. 2000. Integrated management of sugarcane borer., Coop.
Sugar, 46(11): 879-881
Received on 17-11-2009 Accepted on 30-11-2009

Trends in Biosciences 2 (2): 33-35, 2009
Efficacy of Three Antibiotics on Reduction of Mortality Rate in Mulberry Silkworm
(Bombyx mori L.) in the Monsoon Season of Lucknow
AMIT SRIVASTAVA* AND R.VENKATESH KUMAR
Department of Applied Animal Sciences, Babasaheb Bhimrao Ambedkar University,Vidya Vihar, Raebareli
Road, Lucknow(U.P.)-226025; e-mail: Sri_amit77@rediffmail.com
ABSTRACT
Mulberry silkworm, Bombyx mori is prone to several infectious
diseases caused by various pathogenic micro-organisms like,
BmNPV, CPV, IFV etc. The diseases prevail throughout the
year, and in tropics it is significantly high. Bacterial flacherie
and cytoplasmic polyhedrosis accounts for major losses (48.9
and 35.4 per cent) in sericulture where as pebrine and
muscardine are known to account for minor losses of 3.4 and
0.3 per cent respectively. The present investigation aims to
check efficacy of three antibiotics viz., Oflaxacin, Acyclovir
and Griesovin fp. with three different concentrations on
bacterial, viral and fungal diseases in CSR 2
X CSR 4
race of
silkworm, Bombyx mori L.
Key words
Oflaxacin, acyclovir, BmNPV, CPV, IFV. antibiotics.,
efficacy
The silkworm Bombyx mori L. is a monophagous insect
and it solely depends on mulberry leaves for its growth and
development. It is known to suffer from several diseases,
causing extensive crop losses. There are many survey reports
from different sericultural areas in India, which have revealed
that the cocoon crop losses mainly due to diseases. Among
silkworm diseases, viral diseases cause considerable damage
to cocoon production. Nuclear Polyhedrosis caused by
Bombyx mori Nuclear polyhedrosis virus (BmNPV) is also
known as Grasserie indicates the milky fluid condition of the
haemolymph of the diseased larvae. Grasserie accounts for
more than 15% loss in yield and 25-58% in total disease
incidence. The infected silkworm appears completely normal
and feeds as usual till it is close to death. About five to six
days after infection the inter-segmental membranes swell and
the worm appears to be under stress and exhibit restless
behaviour. At this stage, various tissues like epidermis, tracheal
matrix, fat bodies and haemolymph become turbid and milky
due to multiplication of polyhedral bodies in large numbers,
the skin losses its elasticity, become fragile and gets ruptured
easily by releasing turbid white haemolymph. The onset of
death from time of swelling of intersegments is relatively rapid
usually ranging from a few hours to less than a day.
The symptoms of infectious flacherie are similar to
bacterial flacherie such as loss of appetite, transparent
cephalothorax, and shrinkage of the body, retarded growth
and empty foregut. This follows vomiting of gastric juice and
gut is filled with yellowish brown fluid. Although, viral diseases
causes major damage in monsoon seasons but bacterial and
fungal diseases causes minor losses too.
Attempts have been made to use various chemicals
(antibiotics) to prevent the diseases and to increase the
productivity of silk. Antibiotics play a significant role in
conferring immunity to silkworms thereby increasing the
effective rate of rearing (Anandakumar, et al., 2005). Oral
feeding of antibiotics along with mulberry leaves is known to
reduce the incidence of flacherie and grasserie significantly
(Radha, et al., 1980). Antibiotics used for clinical purposes
have therapeutic effects on silkworms infected with the
pathogens (S. aureus, Candida albicans) (Hamamoto,
Hiroshi, et al., 2003). Rifampicin proved to be significantly
effective in reducing mortality of NPV infected worms,
followed by Chloramphenicol (Bhattacharya, et al., 1998).
Ganciclovir, foscarnet, vidarabine and ribavirin (antiviral
agents) inhibit the proliferation of baculovirus in silkworm
body fluid and had therapeutic effects (Yukata Orihara, et al.,
2008).
MATERIALS AND METHODS
The present study was conducted at Department of
Applied Animal Sciences, Babasaheb Bhimrao Ambedkar
University Lucknow. The experiment was conducted to check
the efficacy of three antibiotics viz., Oflaxacin, Acyclovir and
Griesovin fp. with different concentrations on CSR2XCSR4
race of silkworm (Bombyx mori).
The antibiotics were procured from the drug stores. 0.04,
0.08 and 0.12 % concentrated solutions of these antibiotics
were prepared in distilled water. The antibiotics solution was
uniformly smeared on the mulberry leaves @ 0.5 ml per leaf
(approximately leaf area being 140cm 2 ). For control only
distilled water was smeared on the leaves. Every time freshly
prepared solutions of antibiotics were used for the purpose
of smearing. The smeared leaves were shade dried and fed to
the silkworm larvae. The treatment was started after the worms
came out of 3 rd moult. The schedules of treatments were as
follows:
1. Daily one feed of antibiotic treated leaves from the
beginning of 4 th instar
2. Antibiotic treated leaves in every instar as a first feed
after 3 rd and 4 th moult.

3 4 Trends in Biosciences 2 (2), 2009
3. Control- Distilled water smeared leaves in every instar
as the first feed after 3 rd and 4 th moult.
While giving treatments, care was taken to give same
quantity of treated leaves for each replication of all the
treatments. Fresh untreated mulberry leaves were provided
for the feeds other than feeds with treatment.
Data were collected for grasserie flacherie and
muscardine every day from hatching to onset of spinning. In
monsoon season average temperature and humidity were
recorded in natural condition. The data were analyzed following
completely randomized design.
Four rearing were conducted across four seasons in
each year i.e. 2006-2007 and the experiment was in 2007-2008
with the same treatment combinations.
Three replicas with 400 larvae of selected breeds were
maintained for control and field condition separately. Data
were subjected to statistical method for deriving the result.
Experiment was conducted in monsoon rearing seasons of
year during 2006-08 respectively under Lucknow climatic
conditions.
The standard rearing method was adopted as
recommended by Krishnaswami, 1994 Gabriel and Rapusas,
1976 were followed. During rearing, other precautionary
measures such as use of disinfectant, removal of diseased
silkworm larvae, general cleanliness and sanitation,
maintenance of required humidity and temperature were
maintained.
RESULTS AND DISCUSSION
Concentration is an important factor in antibiotics. At
low levels they have no effect and as the concentration is
increased to cytotoxic levels, a direct relationship exists
between the concentration of the antibiotic in the medium
and the anti- microbial effect, until a level of maximum
Fig. 1. Grasserie Infected Silkworm
Fig. 2. Flacherie Infected Silkworm
Table1.
Treatment
± indicates S.D.
Effect of antibiotic treatment on incidence of diseases in the silkworm Bombyx mori L. during 2006-07 and
2007- 2008
2006-2007 2007-2008
Grasserie Flacherie Muscardine Grasserie Flacherie Muscardine
Oflaxacin (0.04%) 14.50±0.01 9.00±0.02 2.95±0.01 13.50±0.02 8.10±0.01 2.95±0.02
Oflaxacin (0.08%) 14.00±0.02 8.85±0.02 2.65±0.01 13.10±0.01 8.10±0.02 2.60±0.01
Oflaxacin (0.12%) 14.10±0.02 7.55±0.01 3.00±0.01 13.15±0.01 7.90±0.01 3.00±0.01
Acyclovir (0.04%) 12.45±0.01 9.10±0.01 3.00±0.02 12.45±±0.03 8.90±0.02 2.52±0.01
Acyclovir (0.08%) 12.10±0.01 9.25±0.02 2.80±0.03 12.50±0.01 9.00±0.02 2.85±0.02
Acyclovir (0.12%) 11.25±0.02 9.10±0.01 2.85±0.02 11.80±±0.03 9.10±0.01 2.85±0.01
Grisovin fp (0.04%) 13.35±0.02 10.10±0.01 2.50±0.01 13.35±0.02 8.90±±0.03 2.40±0.01
Grisovin fp (0.08%). 14.10±0.02 9.50±0.02 2.45±0.02 14.00±0.01 9.55±0.01 2.45±0.02
Grisovin fp. (0.12%) 14.10±0.01 9.32±0.01 2.00±0.02 14.40±0.02 9.90±0.01 2.00±0.02
Control 15.84±0.02 10.52±0.01 3.50±0.03 15.90±0.01 9.90±0.02 3.00±0.02

SRIVASTAVA AND VENKATESH, Efficacy of Three Antibiotics on Reduction of Mortality Rate in Mulberry Silkworm 3 5
effectiveness is reached. Radha et al., 1980, reported decrease
in grasserie incidence due to antibiotics enterocycline,
penicillin, erythromycin and streptomycin. It is evident from
the above study that the mulberry leaves supplemented with
0.12 per cent concentrations of antibiotics oflaxacin (7.55%,
7.90%), acyclovir (11.25%, 11.80%) and griesovin (2.00%,
2.00%) along with feed were found to be most effective in not
only increasing the survival rate but also preventing the
occurrence of grasserie, flacherie and muscardine diseases
followed by 0.08 per cent, 0.04 per cent treated batches which
are effective in comparision to control batch (Table 1-2) during
2006-2008. These results are in accordance with Baig, et al.,1990
who observed that 0.1 per cent treated batch of gentamycin
resulted in reducing significantly both grasserie and flacherie
compared to 0.05 per cent fed batches of gentamycin.
Watanabe, 1984 observed the presence of chloramphenicol in
the diet was very effective to prolong the lethal time of fed
larvae infected with cytoplasmic Polyhedrosis (CPV). Taking
all treatments into consideration it was found that feeding
antibiotics at 0.12% concentration through mulberry leaves
in CSR 2
XCSR 4
variety has recorded minimum mortality rate
due to grasserie, flacherie and muscardine diseases, which
are the prime threat to sericulture industry.
LITERATURE CITED
Anandakumar, M. D., Anathanarayana, S.R., Narayanaswamy, M. and
Mukunda, N. 2005. Effect of application of Tetracycline and
Sulphamethaxazole on the Growth and Development of Silkworm
Bombyx mori L. Proceedings of Advances in Tropical Sericulture,
pp. 341-345.
Baig, M., Nataraju, B. and Samson, M. V. 1990. Studies on the Effect of
Antibiotics on Rearing Perfomance and Loss Due to Diseases in
Silkworm Bombyx mori L. Indian J. Seric., 29(1):54-58.
Bhattacharya, J., Krishnan, N., Chandra, A.K., Das, N.K. and Sen, S.K.
1998. Reduction of Mortality by Some Antibiotics in Nuclear
Polyhedrosis of Silkworm, Bombyx mori L. Sericologia, 38(1):161-
164.
Gabriel, B.R. and Rapusas, H.R. 1976.The growth and development of
Bombyx mori L. at different leaf maturity and variety of mulberry.
The Philippine Agriculturist, 60: 139-146.
Hamamoto, Hiroshi, Kurakawa, Kenji, Chikara, Kaito, Kamura,
Koushirou, Razanajatovo, Manintra, Iony, Kusuhara, Hiroyuki,
Santa, Tomofumi and Sekimizu, Kazuhisa, 2005. Quantitative
Evaluation of the Therapeutic Effects of Antibiotics Using Silkworm
Infected with Human Pathogenic Microorganisms. Antimicrobial
Agents and Chemotherapy, 48(3): 774-779.
Krishnaswami, S. 1994. A practical guide to mulberry silk cocoon
production in tropics. Sriyamula Sericulture Consultants, Bangalore,
India, pp. 49-50.
Radha, N. V., Natarajan, T., Muthukrishnan, T. S. and Oblisami, G.,
1980. Effect of antibiotics on the growth and productivity of
mulberry silkworm. Proceedings of Sericulture Synposium and
Seminar, Tamil Nadu Agricultural University, Coimbatore, pp. 173-
176.
Orihara Yutaka, Hamamoto, Hiroshi, Kasuga, Hiroshi, Shimada, Toru,
Kawaguchi, Yasushi and Sekimizu, Kazuhisa, 2008. A silkwormbaculovirus
model for assessing the therapeutic effects of antiviral
compounds: characterization and application to the isolation of
antivirals from traditional medicines. J. Gen Virol., 89:188-194.
Watanabe, H., 1984. Effect of an antibiotic on polyhedrosis virus
infection in the silkworm, Bombyx mori L. reared on artificial diet.
J. Seric. Sci. Japan, 52: 160-164.
Recieved on 12-9-2009 Accepted on 25-10-2009

3Trends 6 in Biosciences 2 (2): 36-38, 2009
Trends in Biosciences 2 (2), 2009
A New Species of Hoplolaimus (Basirolaimus) (Hoplolaiminae : Tylenchida) from
Pigeon Pea Ecosystem of Bumdelkhand Region
S. SHARAFAT ALI, AZRA SHAHEEN AND RASHID PERVEZ
Indian Institute of Pulses Research, Kanpur 208 024; email: ss_ali@rediffmail.com
ABSTRACT
A new species of plant parasitic nematodes, Hoplolaimus
(Basirolaimus) puriensis sp. n. was found from pigeon pea
ecosystem in Hamirpur district, Bundelkhand region of
Uttar Pradesh, India is described and illustrated. H.(B.)
puriensis is characterised by having complete aerolation
on lateral fields through out body, lateral lines terminate
before anal region and not reaching till tail end. One
phasmid located far anterior to vulva, another post vulval.
H.(B.) puriensis sp. n. resembles with Hoplolaimus
(Basirolaimus) seinhorsti but differs in having more lateral lines
(i.e. four lateral lines vs. one to three in Hoplolaimus (B.)
seinhorsti) different position of excretory pore, i.e. excretory
pore below nerve ring in H.(B.) puriensis vs. excretory pore
adjacent to nerve ring in Hoplolaimus (B.) seinhorsti. Most
important character of this new species is the presence of lesser
number of nuclei in dorsal oesophageal gland instead of six
nuclei, there are only four nuclei located on dorsal oesophageal
gland. Hoplolaimus (B.) seinhorsti has longer stylet i.e. 40-49
µm (vs. stylet 32-35 µm in H.(B.) puriensis sp.n.). H.(B.) puriensis
also resembles with Hoplolaimus (Basirolaimus) indicus but
differs in having smaller spicules 32 µm (vs. 34-42 µm in H. (B.)
indicus); H.(B.) puriensis sp. n. has smaller stylet 32-35 µm vs.
H. (B.) indicus stylet, 33-47 µm.
Key words
New species, Hoplolaimus (Basirolaimus) puriensis,
morphology, taxonomy, plant parasitic nematodes.
Pigeon pea Cajanus cajan (L.) Millspaugh is infested
by a range of nematode pests in pulse growing regions of
India (Ali and Askary, 2001). Association of Hoplolaimus
(Basirolaimus) indicus was reported in pigeon pea ecosystem
from Gwalior (Ali, 1993), its prevalence was reported greater
in sandy loam soils in Gujarat (Sharma, et al.,1993). Its 12%
infestation in various districts in U.P. while 92% frequency of
occurrence from Hamirpur district of U.P. (Sharma, et al.,1996).
In view of its heavy incidence in Hamirpur district of
Bundelkhand region a random survey was conducted in 2004
and a new species of Hoplolaimus (Basirolaimus) puriensis
was encountered in good numbers from pigeon pea
ecosystem. This new species differs in a number of
morphological characters known species of the genus
Hoplolaimus, subgenus Basirolaimus.
Shamsi, 1979 erected a new genus Basirolaimus, later
on Luc, 1981 synonimized Basirolaimus with Hoplolaimus
Daday, 1905. Siddiqui, 2000 proposed subgenus Basirolaimus,
Shamsi, 1979 (Grad. n.) under the genus Hoplolaimus Daday,
1905 (Hoplolaimina: Hoplolaimidae), with type species
Basirolaimus seinhorsti (Luc, 1958) Shamsi, 1979 and shifted
17 species of the genus Hoplolaimus to subgenus
Basirolaimus. The occurrence of nuclei in dorsal gland of
Basirolaimus sp. is a unique character of the Tylenchida, this
supplemented with characters of head and labial disc, lateral
fields, excretory position to hemizonid and geographical
distribution, differentiated Basirolaimus from Hoplolaimus
(Siddiqui, 2000). In the present study, classification of
Hoplolaimidae was follwed as proposed by Siddiqui, 2000.
MATERIALS AND METHODS
Soil samples were collected from the rhizosphere of
pigeon pea standing crop during the month of April 2004 when
temperature ranges from 40-45 o C in Modha village from field
of Mr. Siraj , tehsil Madhupura, Hamirpur district, Uttar
Pradesh. Nematodes were extracted from soil samples by
decanting and sieving followed by modified funnel technique.
Nematodes were heat relaxed and fixed in 4% formaldehyde
and infiltrated with glycerin. The glycerin mounted specimen
were observed under high resolution DMLB Leica research
microscope equipped with differential interference contrast
optics and illustrations were prepared from armed type camera
Lucida. Measurements were made using a micrometer. Glass
supports were used to avoid flattening of specimen while
preparing the slides of Hoplolaimus (Basirolaimus) puriensis
sp. n.
RESULTS AND DISCUSSION
Hoplolaimus (Basirolaimus) puriensis sp.n. (Figs.1-2)
Measurements (see Table 1)
Female
Body almost straight or slightly curved ventrally after
killing by gentle heat. Head offset by deep constriction, dome
shaped hemispherical, strongly sclerotized and bears 4-5 fine
labial annules. Labial disc present. Stylet strong, knobs
massive, slightly pointed anteriorly, sloping backward,
rounded. Conus bigger than shaft, dorsal oesophageal gland
opening 3.5 µm below the base of spear knobs. Median
oesophageal bulb well developed with strong valve plate.
Oesophageal glands in tandem forming a long dorsally
overlapping lobe on intestine, nucleus near anterior end with
4 nuclei. Excretory pore located at oesophageal gland.
Hemizonid one to two body annules long and 3-6 annules
posterior to excretory pore. Lateral fields with four equidistant

Table 1.
ALI et al., A New Species of Hoplolaimus (Basirolaimus) (Hoplolaiminae : Tylenchida) from Pigeon Pea Ecosystem 3 7
Morphometrics of Hoplolaimus (Basirolaimus) puriensis sp. n. Measurements are in micron and in the form:
mean ±sd (range).
Characters Holotype female Paratype females Paratype males
L 1217.3 1201.79±101.0 (1105.8-1293.9) 1055.83±3.44 (1053.4-1058.27)
a 33.91 31.86± 3.08 (29.25-36.05) 33.66±1.93 (31.17-35.03)
b 7.38 7.49± 0.51 (6.97-8.08) 7.11±0.16 (6.99-7.23)
c 44.81 46.42± 7.91 (38.0-53.46) 34.75±2.61 (32.9-36.36)
c 1 0.95 0.95±0.12 (0.83-1.11) 1.52±0.17 (1.4-1.65)
V 54.81 55.51±2.52 (51.89-57.7)
Maximum body diameter. 35.89 37.76±1.58 (35.89-39.77) 32.01±2.74 (30.07-33.95)
Height of lip region 6.79 7.03±0.48 (6.79-7.76) 5.8±0.01(5.8-5.9)
Diameter of lip region 11.64 13.33±0.48 (12.61-13.58) 11.64±0.01 (11.64-11.67)
Length of stylet 35.89 35.88±1.37 (33.95-36.86) 32.98±0.01 (32.98-32.99)
Conus 18.43 18.97±0.54 (18.25-19.4) 16.98±0.01 (16.98-16.99)
Shaft 15.52 16.66±0.75 (15.70-17.0) 16.00±0.01 (16.00-16.02)
Width of stylet base 6.79 6.54±0.48 (5.82-6.79) 5.4±0.56 (5.00-5.8)
DGO from anterior end 41.95 41.28±1.70 (38.8-42.68) 43.15±2.04 (41.71- 44.6)
Anterior end to center of median bulb 81.48 82.93±2.31 (80.5-85.36) 77.6±4.11 (74.69-80.51)
Length of median bulb 17.46 17.70±1.83 (16.49-20.37) 16.49±0.01 (16.49-16.51)
Diameter of median bulb 17.46 15.76±0.48 (15.52-16.49) 13.58±3.00 (11.64-15.52)
Length of oesophagus 164.9 160.29±4.77 (154.23-165.87) 14.84±4.10 (145.5-151.3)
Length of gland lobe 63.05 47.28±7.57 (37.83-56.26) 44.62±1.37 (43.65-45.59)
Anterior end to excretory pore 106.7 125.37±12.43 (107.67-136.77) 113.0±4.80 (109.6-116.4)
Annule width at mid body 1.0 1.0± 0.01 (1.0-1.1) 1.1±0.01 (1.0-1.2)
Vulva from anterior end 667.36 666.87±56.48 (625.65-746.9)
Tail length 27.16 26.17±2.38 (24.2-29.1) 30.55±2.05 (29.1-32.01)
Body diameter at anus 28.13 28.13±0.79 (27.16-29.1) 19.88±0.68 (19.4-20.37)
Length of spicules 31.00±0.04 (31.01-31.04)
Length of gubernaculum 17.46±1.37 (16.49-18.43)
longitudinal incisures, completely aerolated over whole body.
Four incisures terminate before anus level and not reaching
till tail end. Three body annules terminating at vulva, vulval
lips not protuberant, epiptygma absent. Both genital branches
almost similar in size, outstretched, spermathecae round, filled
with sperms. Oocytes arranged in multiple rows. Post rectal
intestinal sac absent. Scuttella distinct, rounded, enlarged,
not opposite to each other but one anterior to vulva another
post vulval, 2.5-3 µm in diameter. Tail hemispherical, some
times a notch present at terminus usually 12-13 annules long,
hyaline portion clearly visible.
Male
Body slightly slender than females, acquires C-shape to
ventrally curved upon fixation, cephalic region strongly
sclerotized, offset by deep constriction with 3-4 annules. Labial
disc indistinct, stylet strong, size and shape same as in females.
Median oesophageal bulb and dorsal oesophageal glands
well developed. Excretory pore located at oesophagealintestinal
junction. Lateral field same as that of females. Both
scutella shield like, 2.5 - 3 µm in diameter; present in anterior
region of body. Post rectal intestinal sac absent. Bursa,
enveloping tail with annulated margin. Spicules tylenchoid
with a symmetrical rounded manubrium, titillae present.
Gubernaculum slender, trough shaped, about one-third length
of spicule.
Diagnosis and relationship
Hoplolaimus (Basirolaimus) puriensis is characterised
by having complete aerolation on lateral fields through
out body, lateral lines terminate before anal region and
not reaching till tail end. One phasmid located far anterior
to vulva another post vulval. H.(B.) puriensis sp. n. resembles
with Hoplolaimus (Basirolaimus) seinhorsti Luc, 1958, but
differs in having more lateral lines (i.e. four lateral lines vs.
one to three in Hoplolaimus (B.) seinhorsti); different position
of excretory pore i.e. excretory pore below nerve ring in H.(B.)
puriensis vs. excretory pore adjacent to nerve ring in
Hoplolaimus (B.) seinhorsti Luc, 1958, hemizonid 3-6 annules
posterier to excretory pore. Most important character of this
new species of lesser number of nuclei in dorsal oesophageal
gland in case of six nuclei , there are only four nuclei in dorsal
oesophageal gland. Hoplolaimus (B.) seinhorsti has longer
stylet i.e. 40-49 um (vs. stylet 32-35 µm in H.(B.) puriensis

3 8 Trends in Biosciences 2 (2), 2009
A B C
100 µm A, G
20 µm
B, D, E, F
50 µm
C
E
G
D
F
Fig. 1.
Hoplolaimus (Basirolaimus) puriensis sp.n.
Female : A. entire body, C. anterior region, D. posterior
region, E. vulval region (showing scutella), Male: B.
anterior region, F. posterior end, G. entire body.
Fig. 2.
Hoplolaimus (Basirolaimus) puriensis sp.n.
Female: A. anterior region, B-C. head end, D. vulval region,
E-F. posterior ends. Male: G. anterior region, H. posterior
region. (B, D-F at 100 x, A, C, E, G, H at 63 x)
sp.n.). H.(B.) puriensis also resembles with Hoplolaimus
(Basirolaimus) indicus Sher, 1963 but differs in having smaller
spicules 32 µm (vs. 34-42 µm in H. (B.) indicus); H.(B.)
puriensis sp. n. has smaller stylet (33-36 µm) vs. H. (B.)
indicus) stylet, 33-47 µm.
Etymology
Named after Dr. S.N. Puri, Vice Chancellor, Central
Agricultural University, Imphal, Manipur, India.
Type habitat and locality
Soil around roots of pigeon pea (Cajanus cajan (L)
Millsp.), sandy loam soil, cultivated field of Mr. Siraj farmer,
Modha village ,Madhupura tehsil, district Hamirpur, Uttar
Pradesh, India.
Type specimens
Holotype female and 4 paratype females and one male
deposited at Nematology unit, Division of Crop Protection,
Indian Institute of Pulses Research, Kanpur, India. Two
paratype females and one male deposited at CAB Bioscience
(UK) center, Egham, UK.
Acknowledgement
The authors are highly grateful to Dr. Masood Ali,
Director, Indian Institute of Pulses Research, Kanpur for
providing necessary facilities and help.
LITERATURE CITED
Ali, S. S. 1993. Prevalence of plant parasitic nematodes associated with
pigeon pea in Gwalior. International Pigeonpea Newsletter, 17: 31-
32.
Ali, S. S. and Askary, T.H. 2001. Taxonomic status of phytonematodes
associated with pulse crops. Current Nematology, 12:75-84.
Shamsi, M.A. 1979. Basirolaimus gen.n. (Nematoda:Hoplolaimidae)
with the description of Basirolaimus sacchari n. sp. from India.
Nematol. medit., 7: 15-19
Sharma, S.B.; Ali, S.S; Patel, H.V.; Patel, S.K. and Patel, D.J. 1993.
Prevalence of plant parasitic nematodes associated with pigeonpea
in Gujarat State in India. Afro-Asian J. Nematol., 3 (1) : 55-59.
Sharma, S.B.; Ali, S.S. Upadhyay, K.D. and Ahmad, F. 1996. Potential
nematode constraints of pigeonpea in Uttar Pradesh in northern
India. Afro-Asian J. Nematol., 6 (2) : 151-155.
Sher, S.A. 1963. Revision of the Hoplolaiminae (Nematoda). II
Hoplolaimus Daday 1905 and Aorolaimus n.gen. Nematologica,
9: 267-295
Siddiqi, M.R. 2000. Tylenchida parasites of plants and insects 2 nd edition
U.K., CABI Publishing. pp.1-833
Received on 12.7.2009 Accepted on 1-12-2009

Trends in Biosciences 2 (2): 39-41, 2009
Characterization of Aromatic Hydrocarbon Degrading Bacteria Isolated from
Effluents of Thermal Power
ARCHANA MISHRA, RAIS AHMAD AND SHILPA KAISTHA
Department of Microbiology, C. S. J. M. University, Kanpur 208 024
email: ahmad.rais2006@gmail.com
ABSTRACT
A study was conducted to characterize the bacteria present in
the effluent of Panki Thermal Power and their bio-remedial
properties. The selected five bacterial isolates collected during
2006 were subjected to morphological and biochemical test.
On the basis of colony characteristics, microscopic and
biochemical observations, the bacterial isolates belong to group
IV, VIII, IX and group XI according to Bergey’s manual of
determinative bacteriology. Following this system, the bacterial
isolates were tentatively identified as Pseudomonas, Escherichia
coli, Corynebacterium, Neisseria and Proteus. Isolate P1 grew
well on napthalene and kerosene containing media followed
by P3 and P2 and P4 whereas P5 failed to grow. On phenol
containing media isolate P2 grew well followed by P4 and P1
whereas P3 and P5 failed to grow. Antibiotic sensitivity assay
revealed that isolate P2 was sensitive to all the four antibiotic
used in the study, whereas isolate P1 was sensitive to all except
norfloxacin. It is concluded that the isolate P1 has the potential
to be exploited for cleaning the thermal effluent.
Key words
Micro organism, hydrocarbons, effluents, thermal
power.
Industrialization and technological development
processes have led to the introduction of hazardous chemicals
into the environment - water, air and land. These have
increased the number and level of hazardous chemicals such
as heavy metals, herbicides, pesticides, halogenated
polycyclic hydrocarbons, sewage wastes and other allied
contaminants, thereby, exposing man and animals health wise
(Awobajo, 1981). Introduction of industrial effluent directly
into water bodies causes hazards to human health, harm to
living resources and ecological systems. The impact of these
anthropogenic activities has been so extensive that rivers
have lost their self purification capacity to a larger extent.
This has resulted in scarcity of potable water supply and loss
of biodiversity in aquatic ecosystems (Barnsley, 1983). Kanpur
is an industrial city and hosts industries like thermal power
plant, coke oven, petroleum refining, leather industries, plastic
industries, tanneries that along with and automobile exhaust
are making the environment of the city polluted day by day
and have resulted in giving Kanpur a tag of one of the most
polluted cities.
These industries are the main source of hazardous
aromatic hydrocarbons and other toxic materials like phenols,
cyanide, ammonia, heavy metals, crude oil and aliphatic
compounds. These toxic materials are released into water
bodies’ directly causing, death of fish, algae and hazards for
human health (Cerniglia, 1984). Fuel and lubricating oil spills
have become major environmental hazards. In addition
aromatic hydrocarbons are commonly present in aqueous
wastes of many industries such as thermal power plant,
petrochemicals, oil refineries, pharmaceuticals, coke-oven,
steel plants and plastic industries and imparts taste and odor
to water and are toxic to fish and other aquatic life even at
very low concentration. These wastes display a pronounced
undesirable effect in the form of skin irritation, damage to the
lungs, kidney, liver, gastro urinary tract and known as
suspected carcinogens.
The dangers and health hazards caused by the above
pollutants can be reduced by the use of microbes.
Technologies using micro organisms with extensive
biodegradation capacities have been developed for use in
both pollution prevention as well as site remediation. Many
micro organisms can use these compounds for their growth
or degrade those compounds by several mechanisms such as
co-metabolism and degradation under aerobic or anaerobic
conditions, depending on the target compound, micro
organism and environmental conditions (Sastry, 1986). Taking
this into consideration, the present study was conducted to
isolate and characterize the bacteria present in the effluent of
Panki Thermal Power and to study their bio-remedial
properties.
MATERIALS AND METHODS
Isolation of Bacteria
The bacteria were isolated from the samples collected in
sterile bottles from effluent of Panki Thermal Power Plant.
Serial dilutions were prepared in aseptic conditions and 0.1ml
of different dilutions were spread on nutrient agar medium
(NAM) with the help of sterile glass spreader. Plates were
incubated at 29±1°C for 24 hrs. Different types of single
colonies were streaked on fresh NAM plates for purification.
From these plates again single colony of each isolates was
sub-cultured on NAM slants and five bacterial isolates
(referred as P1, P2, P3, P4 and P5) were taken for further studies.
Microscopic characterization of isolated bacteria
Isolated purified cultures were stained using technique
of gram staining to find out their reaction.

4 0 Trends in Biosciences 2 (2), 2009
Biochemical characterization of isolated bacteria
Catalase, gelatinase activities, salt tolerance and
antibiotic sensitivity tests were performed with all the five
bacterial isolates. For performing the catalase test, a colony
of the tested isolates of bacteria was transferred from plate
and onto a microscopic glass slide in a drop of water. Few
drops of 3% H 2
O 2
were placed over the culture and observed
for the liberation of effervescence of oxygen around the
bacterial colony. To examine the gelatinase activity, nutrient
gelatin agar medium slants were prepared and stabbed them
with each bacterial isolates and then incubated at 29°C for 24
hrs. After 24 hrs slants were kept at 4°C for 1 hr and observed
for liquification. To check the salt tolerance of the bacterial
isolates under study, the bacterial isolates were streaked on
the surface of mannitol salt agar media and incubated at 29±1
°C for 24 hrs and observed for colour production.
For antibiotic sensitivity test, 24 hrs. old broth cultures
of each bacterial isolates were spread on the surface of nutrient
agar media plates with the help of a glass spreader. Disc of the
streptomycin, kanamycin, ampicillin and norfloxacin antibiotics
were placed on the nutrient agar medium plates and incubated
at 29±1°C for 24 hrs. and observed for zone of inhibition and
sensitivity to them.
Growth characteristics in the presence of Naphthalene,
Kerosene and Phenol
All the isolates were checked for their growth in
presence of naphthalene, kerosene and phenol @ 1% of each
in 100 ml minimal medium separately. The isolates were
inoculated in the minimal media tubes containing naphthalene,
kerosene and phenol separately and incubated at 29±1°C for
one week.
RESULTS AND DISCUSSION
Sampling and isolation of bacteria
Samples collected from effluent of Panki Thermal Power
were characterized. Out of several bacterial cultures only five
pure cultures were selected for further studies and designated
as P1, P2, P3, P4 and P5.
Colony characteristics of the isolates
Colony characters viz., size, shape, colour, margin,
surface texture, elevation, consistency, optical features, and
pigmentation were determined and are presented in Table 1.
Isolate P2 had largest colony with diameter of 1.2 mm, whereas
isolates P1 and P3 had colonies with 1mm diam. Isolates P4
and P5 had smallest colony with

Table 3.
Bacterial
isolates
MISHRA et al., Characterization of Aromatic Hydrocarbon Degrading Bacteria Isolated from Effluents of Thermal Power 4 1
Utilization of naphthalene, kerosene and phenol by the bacterial isolates and their antibiotic sensitivity assay in vitro
1 Naphthalene
Growth
2 Kerosene
Growth pattern= 1 Pellicle formation, 2 Turbid growth, 3 Submerged growth
+++ = Very good; ++ = Good; + = Moderate and (-) = No growth, S =Sensitive, R = Resistant
Sensitivity assay
3 Phenol Streptomycin Kanamycin Ampicillin Norfloxacin
P1 +++ +++ + S S S R
P2 + ++ +++ S S S S
P3 ++ ++ - S S R R
P4 + ++ ++ S R S S
P5 - + - S S R S
Identification of isolates
On the basis of colony characteristics and microscopic
and biochemical observations, the bacterial isolates studied
in this study appear to belong to group IV, VIII, IX and group
XI according to Bergey’s manual of determinative bacteriology.
Following this system, the bacteria isolated from effluent of
Panki Thermal Power Station, Kanpur were provisionally
identified as Pseudomonas, Escherichia coli,
Corynebacterium, Neisseria and Proteus.
Growth on hydrocarbons
Isolate P1 grew well on napthalene and kerosene
containing media followed by P3 and P2 and P4 whereas P5
failed to grow. On phenol containing media isolate P2 grew
well followed by P4 and P1 whereas P3 and P5 failed to grow
(Table 3).
Antibiotic sensitivity test of isolates
Results of antibiotic sensitivity assay (Table 3) revealed
that isolate P2 was sensitive to all the four antibiotic used in
the study, whereas isolate P1 was sensitive to all except
norfloxacin. Isolate P3 was sensitive to streptomycin and
kanamycin and resistant to ampicillin and norfloxacin. Isolate
P4 was resistant to kanamycin and sensitive to rest of the
antibiotics, whereas isolate P5 was resistant to ampicillin and
sensitive to rest of them (Table 3).
Utilization of hydrocarbons by bacterial isolates was
observed by several earlier workers (Cerniglia, 1984; Heitkamp
et al., 1988). Barnsley in 1983 exhibited the metabolism of
naphthalene by pseudomonad. It is concluded that the isolate
P1 has the potential to be exploited for cleaning the thermal
effluent, but it needs to be further studied.
LITERATURE CITED
Awobajo, A.O. 1981. An analysis of oil spill incidents in Nigeria.
Proceedings of National Seminar on Petroleum Industries and
Nigerian Environment, Warri, pp. 57- 63.
Barnsley, E.A. 1975. The induction of the enzymes of naphthalene
metabolism in pseudomonad by salicylate and 2-aminobenzoate. J
Gen Microbiol, 88(1):193–196.
Barnsley, E. A. 1983. Bacterial oxidation of naphthalene. Journal of
Bacteriology, 153: 1069- 1071.
Cerniglia, C. E. 1984. Microbial metabolism of poly cyclic aromatic
hydrocarbons. Advances in Applied Microbiology, 30: 31- 71.
Cerniglia, C. E. 1992. Biodegradation of poly cyclic aromatic
hydrocarbons. Biodegradation, 3: 351 - 368.
Heitkamp, M. A., Freeman, J. P. and Cerniglia C.E. 1987. Naphthalene
biodegradation in environmental microcosms: estimates of
degradation rates characterization of metabolites Applied and
Environmental Microbiology, 53: 129-136.
Sastry C.A. 1986. Industrial waste biodegradation part I. Oncology,
1:29-33.
Recieved on 30-10-2009 Accepted on 30-11-2009

4Trends 2 in Biosciences 2 (2): 42-44, 2009
Trends in Biosciences 2 (2), 2009
Effect of Plant Extracts on Activity and Mortality of Second-Stage Juveniles of Rootknot
Nematode, Meloidogyne javanica
S.S. ALI AND RASHID PERVEZ*
Indian Institute of Pulses Research, Kanpur, 208 024,
* Indian Institute of Spices Research, Calicut 673 012
email:ss_ali@rediffmail.com
ABSTRACT
The effects of aqueous plant extracts viz., Sorghum
concentrations (2.5, 5.0, 7.5, 10.0, 15.0, 20.0, and 25.0 ppm) and
latex of Calotropis procera (0.1, 0.2, 0.3, 0.4 and 0.5%) on the
activity and mortality of second-stage juveniles of M. javanica
were tested in vitro. Both plant extracts reduced the activity
and induced the mortality of the nematode. With Sorghum
extracts, the mortality increased with the increase of exposure
time and concentration. Highest mortality (100%) occurred at
20 ppm with in 48 h and lowest (13%) at 2.5 ppm with in 24 h.
No mortality was observed at 2.5 ppm with in 12 h and in control
treatment. C. procera resulted in more than 50% immobility in
all tested concentrations at 36 and 48 h, respectively and
maximum mortality (47%) was found at 4 and 5% within 48 h.
There is great potential for plant extracts application system
to be used as an effective and sustainable management option
to control M. javanica.
Key words
Sorghum, Calotropis procera, Meloidogyne javanica,
plant extract, efficacy
The management of the root-knot nematodes has been
accomplished primarily through the use of chemical
nematicides, crop rotation and resistant cultivars on a few
selected crops (Sasser and Carter, 1985). Nematicides are highly
toxic chemicals and have raised concern regarding
environmental contamination and human health. As a result,
the use of a number of nematicides has been banned,
voluntarily withdrawn from market, scheduled to be phasedout
in the near future, or is currently under review. Thus,
alternative measures to control nematodes are urgently
needed. Various plant extracts, such as Azadirachtin, Nimbin
and Salanin exhibited nematicidal activity against the rootknot
nematode, Meloidogyne incognita (Upadhyay, et. al.,
2003; Mojumdar, et al., 2002; Akhtar and Mehmood, 1993;
Alam, 1990).
Few studies have shown that Sorghum sudanense
(Sudan grass) and Sorghum bicolor are effective in
suppressing infection of and damage caused by Meloidogyne
spp. to vegetables when incorporated into the soil as green
manures (Mojiahedi, et al., 1993 and Widmer and Abawi, 1998).
Viaene and Abawi, 1998, tested various cover crops and their
green manures for suppressing the northern root-knot
nematodes, M. hapla. Although many of the cover crops
tested, including Sudan grass hybrid cv. Truden 8, were non
hosts for this nematode, only green manure of Sudan grass
was highly effective in reducing the egg production and rootgall
severity on the subsequent lettuce crops. Moreover,
Maqbool, et al., 1987 tested the effect of latex of Calotropis
procera L. on the mortality of juveniles of M. incognita. Latex
of C. procera at the rate of 1% as seed treatment enhanced
the yield and plant growth characters in chickpea (Ali, 2003).
The present study was conducted to find out the effect
of Sorghum sp. and latex of C. procera on the activity and
mortality of second-stage juveniles of root-knot nematode M.
javanica.
MATERIALS AND METHODS
Nematode cultures
Second-stage juveniles of root-knot nematode, M.
javanica were obtained from egg masses harvested from the
roots of chickpea, which collected from Nematology green
house, Indian Institute of Pulses Research, Kanpur.
Plant Extracts
Fresh plants of Sorghum sp. were collected from field
washed with mercuric chloride 1% solution, chopped,
macerated in grinder and soaked in 100 ml distilled water. All
the materials were filtered in 4 ply muslin cloth and than in
Whatman filter paper no. 1. The filtered solution was
considered as the standard stock solution from which several
tested dilution were prepared (2.5, 5.0, 7.5, 10.0, 15.0, 20.0, and
25.0 ppm) by adding required amount of distilled water.
Latex was collected from the leaves of C. procera and 1
ml latex put in a conical flask and added with 99 ml of sterilized
distilled water and then shaken for 15 minutes on a shekar.
This suspension was used as stock solution for preparing
further five tested dilutions viz., 0.1, 0.2, 0.3, 0.4 and 0.5%.
Bioassay
Effect of plant extract on the activity and mortality of M.
javanica
Fifty juveniles were inoculated into cavity block
containing 1 ml of each tested concentrations of Sorghum sp.
or latex of C. procera solution separately. The cavity blocks

ALI AND PERVEZ, Effect of Plant Extracts on Activity and Mortality of Second-Stage Juveniles of Root-knot Nematode 4 3
covered with the lid, leaving a gap for aeration and kept in
BOD incubator at 28±1ºC. Juveniles in distilled water served
as control. All treatments were replicated fifteen times. The
immobility and mortality of juveniles were observed after
exposure to different plant extract concentrations after 12, 24,
36 and 48 h under a stereoscopic microscope. Inactive juveniles
mobile responded after the pricking the tail considered being
immobile and that did not move after the pricking the tail were
considered to be dead. Per cent of immobility and mortality
was calculated.
RESULTS AND DISCUSSION
Effect of plant extract of Sorghum sp. on the activity and
mortility of M. javanica
Immobility indicated that 5 and 10 ppm of Sorghum
extract was the most effective rendering 93 and 87 % activity
of second-stage juveniles of M. javanica at 12 h. The minimum
immobility (7 per cent) of juveniles were recorded at 25 ppm
after 48 hrs. No immobility was found when tested at 20 ppm
of Sorghum concentration within 48 hrs (Fig. 1A).
The highest mortality (100 %) was found at 20 ppm with
in 48 hrs. Lowest (13 %) mortality was recorded at 10 and 15
ppm within 12 h and at 2.5 ppm within 24 and 36 h, respectively.
No mortality was found at 2.5 and 5 ppm within 12 h and also
in control treatment after required time of observation (Fig.1B).
Effect of latex of C. procera on the activity and mortility
of M. javanica
Among the latex of Calotropis concentrations resulted
in more than 50% immobility at 36 and 48 hrs, respectively
recorded in all tested concentrations. Maximum immobility
was found at 0.1 and 0.2% within 48 h. Lowest (13%)
immobility was recorded at 0.1% within 12 h (Fig. 2A).
Maximum mortality was found to be 47 % at 0.4 and
0.5% within 48 h. The minimum (7%t) mortality was recorded
at 0.3% within 24 h. Mortality of juveniles not to be found at
0.1% as well as control treatment at any observation time (Fig.
2B).
In this and previous studies (Mojiahedi, et al., 1993;
Viaene and Abawi, 1998 and Widmer and Abawi, 1998) on
Sudan grass was demonstrated to suppress infections by
Meloidogyne sp. to crops when incorporated as a green
manures. Suppression of plant pathogen by green manures
has been primarily attributed to two mechanism of action :
Fig. 1(A).
Effect of plant extract of Sorghum sp. on the
immobility of M. javanica
Fig. 1(B).
Effect of plant extract of Sorghum sp. on the
mortality of Meloidogyne javanica
Fig. 2(A).
Effect of latex of C. procera on the immobility of
M. javanica
Fig. 2(B).
Effect of latex of C. procera on the mortality of
Meloidogyne javanica

4 4 Trends in Biosciences 2 (2), 2009
chemicals or microbial ( Baker and Cook, 1974). Latex of
Calotropis procera effectively reduced root penetration by
M. incognita juveniles (Latif, et al., 1999). Maqbool, et al.,
1987 in vivo showed that extracts of C. procera was highly
toxic to juveniles of M. incognita and M. javanica . Haque, et
al., 1997 reported that nematicidal effect of Calotropis leaf
powder on mungbean and soil amendment with C. procera
leaves 0.5 and 1.0 % W/W significantly reduced the infection
of M. incognita on mungbean, while C. procera used @ 2%
(W/W) was phytotoxic. The present investigations indicated
that Sorghum and C. procera extracts inhibited activity and
rate of mortality of second-stage juveniles of M. javanica.
Sorghum is relatively more effective against juveniles of M.
javanica than the Calotropis procera L. There is great
potential for plant extracts application system to be used as
an effective and sustainable management option for the
management of M. javanica.
ACKNOWLEDGEMENT
Authors are grateful to Dr. Masood Ali, Director, Indian
Institute of Pulses Research, Kanpur for providing facilities
and constant encouragement and Dr. Lalit Kumar Scientis for
providing sorghum concentrates.
LITERATURE CITED
Akhtar, M. and Mahmood, I. 1993. Control of plant parasitic nematodes
with nimbin and some plant oil by bare-root dip treatment. Nematol.
medit., 21: 89–92.
Alam, M. M. 1990. Neem in nematode control. In: Nematode biocontrol
aspects and prospects (eds. Jairajpuri, M. S., Alam, M. M.
and Ahmad, I.) CBS publishers and distributors, Delhi, India, pp. 51-
55.
Ali, S. S. 2003. Integrated management of plant nematodes/ soil
pathogens in pulse based cropping system. Annual report of the
NATP project, IIPR, Kanpur. pp. 85.
Baker, K. R. and Cook, R. J. 1974. Biological control of plant pathogens.
W. H. Freeman and company, San Francisco.
Haque, S. E., Sultana, V., Abid, M. Ara, J. and Ghaffar, A. 1997. Use of
Calotropis procera and microbial antagonist in the control of
Meloidogyne root-knot nematode on mungbean. Pakistan J.
phytopathology, 9 : 2, 108–110.
Latif, Z.H., Ahmad, R. and Inam-ul-Haq, M. 1999. Effect of seed
treatments with neem cake, neem oil and latex of aak on the
germination of Cowpea and its vulnerability to root-knot nematode
(Meloidogyne incognita). Pakistan J. Phytopathology, 11: 52-55.
Maqbool, M. A., Hashmi, S. and Ghaffar, A. 1987. Effect of latex
extract from Euphorbia caducifolia and Calotropis procera on
root-knot nematode Meloidogyne incognita infesting tomato and
egg plant. Pakistan J. Nematology, 5 (1): 43-47.
Mojitahedi, H., Santo, G. S. and Ingham, R. E. 1993. Suppression of
Meloidogyne chitwoodi with Sudan grass cultivars as green manure.
J. Nematology, 25 : 303–311.
Mojumdar, V., Kamra, A. and Dureja, P. 2002. Effect of neem extracts
on activity and mortality of second-stage juveniles of Meloidogyne
incognita. Nematol. medit., 30 : 83-84.
Sasser, J. N. and Carter, C. C. 1985. An advanced treatise on Meloidogyne,
vol. I, II, North California State University Graphics, Releigh.
Upadhyay, K. D., Dwivedi, K. and Uttam, S. K. 2003. Effect of some
plant extracts on the mortality and hatching of Meloidogyne
incognita and Heterodera cajani infested pigeonpea. Nematol.
medit., 31: 29-31.
Viaene, N. M. and Abawi, G. S. 1998. Management of Meloidogyne
hapla on lettuce in organic soil with Sudan grass as a cover crops.
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Received on 30-10-2009 Accepted on 15-11-2009

Trends in Biosciences 2 (2): 45-47, 2009
Heterosis Estimates of Garden Pea Crosses (Pisum sativum L. hortense)
SAUMYA AWASTHI, G. ROOPA LAVANYA AND RASHMI JAIN
Department of Genetics and Plant Breeding
Allahabad Agricultural Institute Deemed University, Allahabad 211 007
e-mail: lavanya.roopa@gmail.com
ABSTRACT
An experiment was conducted to study heterosis in garden pea
for different quantitative characters. Analysis of variance for
heterosis showed significant differences among all crosses for
all characters, indicating that there is ample scope for selection
of promising crosses from present gene pool for yield
improvement in garden pea. Cross EC 328758 x Swarnamer
showed significant heterosis, heterobeltiosis and standard
heterosis for pods per plant and seed yield whereas EC 269396
x Pusa Pragati exhibited negative significant heterosis and
heterobeltiosis for days to maturity, indicating earliness.
Key words
Heterosis, heterobeltiosis, standard heterosis,
Pea (Pisum sativum L.) is among the four important
cultivated legumes next to soybean, groundnut and bean
(Hulse, 1994). The pea occupies an area of 2.0 to 2.5 million
hectare with an annual production of 17.65 million tones
(Masood Ali and Shiv Kumar, 2008). In India, productivity of
this crop is remained stagnant and area is shrinking due to
competition from irrigated wheat and more remunerative
pulses. The estimates of heterosis may be utilized for
determining the potential for production of transgressive
segregants in segregating generation. The present experiment
was undertaken to study heterosis and character association
among different yield contributing characters and with seed
yield in garden pea.
MATERIALS AND METHODS
The experiment was carried out during rabi, 2007-08 at
Field Experimentation Centre, Allahabad Agriculture
University, Naini, Allahabad. The experimental material for the
present study comprised 34 entries (16 parents, 17 crosses
and a check), planted in randomized block design with three
replications in two row plot of 4 m length. Recommended
agronomic practices were followed to grow a healthy crop.
Observations were recorded on 10 randomly selected plants
for eight characters viz., days to 50% flowering, days to
maturity, plant height (cm), no. of primary branches per plant,
pod length, no. of pods per plant, 100-seed weight (g) and
seed yield per plant (g). Per cent heterosis was estimated as
deviation from the mid parent (heterosis), from better parent
(heterobeltiosis) and high yielding check (standard heterosis)
and were calculated using a standard procedure (Fonseca
and Patterson, 1968).
RESULTS AND DISCUSSION
Analysis of variance for heterosis showed significant
differences among all entries, indicating the presence of
adequate genetic variability among them for all characters
under study. Mean performance for days to 50% flowering
ranged from E 6 (61.66) to EC 269396 (81.33). Among crosses
VRP 36 x Arkel (56.00cm) recorded minimum days whereas
VRP 318 x Swarnamer (86.67cm) recorded more number of
days to 50% flowering. Plant height ranged from 42.75 cm for
Ageta to 144.20 cm for EC 328758. But among crosses, P 6 x
Arkel recorded 47.70 cm height whereas E 6 x Ageta registered
maximum plant height (146.53 cm).
High mean for primary branches per plant was obtained
for EC 328758 (3.33) and low mean (1.00) was recorded for five
parents viz., EC 269396, P 6, Azad, Pusa Pragati and
Swarnamer. Among crosses, JP 585 x Arkel (2.33) and JP 585
x Azad (2.33) were found to be superior. More number of pods
per plant were registered for JP 585 x and less pods for VRP 36
(8.66) while EC 328758 x Swarnamer had maximum number of
pods (57.33) among crosses. Pod length was not varied much
among the parents as well as among the hybrids.
Garden / green peas are eaten cooked as vegetable and
marketed fresh, canned and frozen. The genotypes matured
earlier are better suited to the market to fetch high price. The
parent genotypes E 6 (92.66) took less number of days to
maturity while cross EC 2269396 x Pusa Pragati (92.33) matured
earlier than check variety, AP 3 (93.66). However, Pusa Pragati
(20.33g) had low 100 seed weight due to small seed size and
EC 328758 recorded maximum 100 seed weight (28.80g) whereas
VRP 21 x Ageta registered 22.32 g seed weight but lower than
AP 3 (25.81g). Parent genotype EC 328758 (57.49g) showed
maximum seed yield and interestingly the cross which include
it as one of the parents i.e., EC 328758 x Swarnamer (80.02g)
also recorded maximum seed yield.
Among the crosses, EC 328758 x Swarnamer showed
positive significant heterosis for seed yield (76.41**) (Ram, et
al., 1986; Tyagi and Srivastava, 1999; Kumar, et al., 2000 and
Sharma, et al., 2007), heterobeltiosis for seed yield (39.20**)
and pods per plant (14.67**) (Sanjay, et al., 2000; Tyagi and
Srivastava, 2001 and Kaur, et al., 2003). and positive significant
standard heterosis for seed yield (631.70**) and pods per
plant (473.33**) (Table 1). Another cross, EC 269396 x Pusa

4 6 Trends in Biosciences 2 (2), 2009
Table 1.
Estimates of heterosis (H), heterobeltiosis (HB) and standard heterosis (SH) for yield and yield contributing characters
in garden pea crosses
Cross
No.
Parentage
1. EC 2269396 x Azad
2. EC 2269396 x Pusa Pragati
3. NDVP 10 x Azad
4. VRP 36 x Arkel
5. VRP 318 x Arkel
6. VRP 318 x Azad
7. VRP 318 x Swarnamer
8. VRPE 55 x Ageta
9. JP 585 X Arkel
10. JP 585 X Azad
11. VRP 21 x Arkel
12. VRP 21 x Ageta
13. EC 328758 x Swarnamer
14. E 6 x Ageta
15. P 6 x Arkel
16. P 6 x Pusa Pragati
17. VRP 33 x Azad
Days to
50%
flowering
Plant
height
(cm)
* and **significant at 5% and 1% level of significance, respectively
No. of
primary
branches
plant -1
No. of pods
plant -1
Pod
length
(cm)
Days to
maturity
100-seed
weight
(g)
Seed yield
plant -1
(g)
H -2.51 30.84** 33.33 -4.88 4.85 -2.33 -19.69** -19.29
HB -12.30** 21.05 33.33 -15.22 -5.01 -6.09** -22.40** -27.75
SH 2.39 46.57** -20.00 30.00- -5.01 4.27* -24.34** 53.09
H -5.67 43.32** 66.67* -16.88 0.55 -9.18** -8.58** -23.55
HB -14.75** 35.49** 66.67* -21.95 -1.63 -11.22** -14.48** -42.84
SH -0.48 56.61** 0.00 6.67 -1.63 -1.42 -22.25** 21.12
H 6.35* -0.64 -40.00* -51.22* -5.29 8.09** -19.61** -57.40*
HB -1.74 -3.15 -57.14** -56.52* 5.33 7.17** -21.36** -57.93
SH 8.13** 17.27 -40.00* -33.33 -5.33 11.74** -19.84** -27.77
H -12.95** -15.44 -45.45** -63.46** -4.83 8.67** -1.22 -68.99**
HB -14.29** -25.33* -50.00** -75.64** -9.65 1.99 -2.12* -78.27**
SH -19.62** -10.22 -40.00* -36.67 -9.65 9.25** -18.35** -38.65
H 11.00** 3.27 9.09 -50.00** -3.56 6.80** -8.31** -56.56**
HB 10.71** -3.35 0.00 -57.69** -4.81 4.32* -15.53** -59.62**
SH 3.83 33.29* 20.00 10.00 -4.81 11.74** -17.89** 32.73
H 18.97** 18.67* 11.11 20.00 5.22 7.13** -17.87** 10.32
HB 18.97** 11.43 -16.67 11.11 2.37 6.94** 17.99** -16.74
SH 11.00** 53.68** 0.00 100.00* 2.37 9.61** -20.04** 173.70**
H 22.07** -4.17 33.33 -27.27 2.33 -0.17 -24.88** -30.89
HB 12.55** -21.07* 0.00 -33.33 -1.15 -2.65 -29.15** -53.55**
SH 24.40** 8.85 20.00 20.00 -1.15 4.63* -31.12** 52.70
H 2.95 36.89** -42.86** -27.08 7.26 4.40* -27.58** 27.76
HB 2.67 19.91 -50.00** -41.67* 3.08 2.52 -32.39** 8.49
SH -8.13** 27.49* -20.00 16.67 3.08 1.42 -35.94** 52.70
H 21.83** -12.44 16.66667 -66.54** 3.33 3.75* -8.47** -12.18
HB 21.21** -16.80 0.00 -75.98** -2.59 1.00 -12.04** -35.74
SH 14.83** 11.10 40.00* 43.33 -2.59 8.19** -21.88** 81.44
H 25.19* -2.50 40.00* -53.78** 0.08 5.06** -12.14** 56.21
HB 24.24** -7.05 0.00 -70.95** -4.32 4.51* -16.06** 39.13
SH 17.70** 24.13 40.00* 73.33 -4.32 7.12** -18.16** 133.01*
H 14.43** 8.02 11.11 -12.00 4.93 1.37 -29.22** 34.35
HB 13.57** 2.16 0.00 -15.38 -0.71 -1.33 -31.98** -1.69
SH 8.13** 22.82 0.00 120.00** -0.71 5.69** -39.58** 177.57**
H 11.69** 12.61 -40.00* -40.74* -4.51 9.95** 1.19 31.34
HB 8.04** -1.69 -50.00** -55.56** -8.16 6.67** -2.60** 15.00
SH 2.87 5.36 -40.00* 6.67 -8.16 8.19** -13.50** 50.47
H 4.14 52.70** 0.00 76.41** -8.46 2.20 -11.54** 129.06**
HB -7.36** 1.62 -33.33** 14.67** -14.02** 0.00 -20.81** 39.20**
SH 2.39 174.06** 20.00 473.33** -14.02** 7.47** -13.69** 631.70**
H 12.13** 10.82 -50.00** 2.78 -2.73 9.47** -11.64** 62.32
HB 11.83** 10.06 -50.00** 2.78 -4.42 7.84** -17.79** 60.50
SH -0.48 -10.79 -40.00* 23.33 -4.42 2.85 -21.53** 61.38
H 7.20** 32.62** -25.00 0.00 0.00 3.39 -29.28** 32.32
HB 4.35 18.91 40.00* -24.36 -1.33 1.33 -35.44** -11.87
SH 3.35 42.96** -40.00* 96.67* -1.33 8.54** -35.98** 148.83**
H 9.41** 27.62* 100.00** -30.86 -0.67 2.56 -33.96** 1.99
HB 6.76* 16.45 100.00** -31.71 -2.56 1.01 -40.61** -3.46
SH 5.74* 34.60** 20.00 -6.67 -2.56 7.12** -41.11** 1.34
H 7.18** -5.92 -11.11 -44.06* 9.31 4.14 -20.29** -29.77
HB 7.18** -9.31 -33.33* -58.76** 4.77 3.42 -26.53** -49.17**
SH 0.00 18.33 -20.00 33.33 4.77 7.47** -28.37** 90.22

AWASTHI et al., Heterosis Estimates of Garden Pea Crosses (Pisum sativum L. hortense) 4 7
Pragati exhibited negative significant heterosis (-9.18**) and
heterobeltiosis (-11.22**) for days to maturity. These results
further in concordance with the findings of Misra,1998, Gupta,
et al., 1998 and Pathak and Jamval, 2002 (Table 1).
The high values of heterotic effects further indicate that
parents used for study were of diverse origin. Considerable
high and low heterosis revealed that nature of gene action
varied with genetic architecture of the parents. The magnitude
of heterosis depicted by crosses would be useful in identifying
superior cross combinations.
From the above results, it could be concluded that cross
EC 269396 x Pusa Pragati exhibited negative significant
heterosis for days to maturity governed by dominant gene
with negative effect, while cross EC 328758 x Swarnamer
exhibited positive significant heterosis for pods per plant and
seed yield per plant were governed by genes with positive
effect. Thus cross EC 328758 x Swarnamer is the best cross
combination for seed yield and pods per plant and EC 269396
x Pusa Pragati for early maturity.
LITERATURE CITED
Fonseca, S. and Patterson, F. L. 1968. Hybrid vigour in seven parents
diallel cross in common wheat (Triticum aestivum L.), Crop Science,
8: 85-88.
Gupta, M. K., Singh, J. P. and Mishra, V. K. 1998. Heritability, genetic
advance and correlation analysis in pea (Pisum sativum L.). Crop
Research, 16(2): 202-204.
Hulse, J. H. 1994. Nature, composition and utilization of food legumes,
In: Expanding the production and use of cool season food legumes,
pp. 77-97.
Kaur, R., Singh, M., Kaur, S., Brar, P. S. and Dhillon, T. S. 2003.
Heterosis and correlation studies in pea (Pisum sativum L.).
Environment and Ecology, 21(1): 11-15.
Kumar, S., Singh, K. P. and Panda, P. K. 2000. Heterosis for pod yield
and its components in garden pea. Haryana Journal of Horticultural
Science, 29(1&2): 99-101.
Massod, A. and Shiv Kumar, 2008. India and global food security, The
Hindu Survey of Indian Agriculture. pp. 43-46.
Misra, S. K. 1998. Heterosis for yield and yield components in pea.
Indian Journal of Pulses Research, 11: 11-15.
Pathak, S. and Jamval, R. S. 2002. Variability and correlations for
economic traits in powdery mildew resistant genotypes of garden
pea (Pisum sativum L.). Himachal Journal of Agricultural
Research, 28(1/2): 34-39.
Ram, R. A., Chauhan, Y. S. Srivastava, R. L. and Singh, I. B. 1986.
Heterosis in peas. Farm Science Journal, 1(1-2): 42-47.
Sanjay, K., Singh, K. P., Panda, P. K. and Kumar, S. 2000. Heterosis for
green pod yield and its components in garden pea. Haryana Journal
of Horticultural Science, 29(1-2): 99-101.
Sharma, A., Singh, G., Sharma, S. and Sood, S. 2007. Combining ability
and heterosis for pod yield and its related horticultural traits in
garden pea under mid-hill sub temperate and high-hill dry temperate
conditions of Himachal Pradesh. Indian Journal of Genetics and
Plant Breeding, 67(1): 47-50.
Tyagi, M. K. and Srivastava, C. P. 2001. Analysis of gene effects in
pea. Legume Research, 24(2): 71-76.
Tyagi, M. K. and Srivastava, C. P. 1999. Heterosis and inbreeding
depression in pea. Annals of Agriculture and Biological Research,
4(1): 71-74.
Recieved on 23-10-2009 Acceted on 15-11-2009

4Trends 8 in Biosciences 2 (2): 48-49, 2009
Trends in Biosciences 2 (2), 2009
Estimation of Unavoidable Yield Losses in Certain Rabi Pulse Crops Due to the
Root-Knot Nematode, Meloidogyne javanica
S.S. ALI
Indian Institute of Pulses Research, Kanpur 208 024
email: ss_ali@rediffmail.com
ABSTRACT
Field trials were laid down to estimate unavoidable yield losses
in chickpea, fieldpea and lentil crops due to infestation of rootknot
nematode, Meloidogyne javanica at Indian Institute of
Pulses Research Kanpur. Paired plot treatment method was
employed to know the extent of yield losses of these crops
infested with root-knot nematode in sandy loam soils.
Carbofuran and phorate @ 2 kg a.i./ha were used to check the
root-knot nematode population in the infested fields of
chickpea, fieldpea and lentil respectively. Results indicated
that root-knot nematode had incurred unavoidable yield losses
to the tune of 25.6% in chickpea and 15% each in pea and
lentil.
Key words
Chickpea, lentil, fieldpea, yield loss, root- knot
nematode
Chickpea (Cicer arietinum L.), fieldpea (Pisum sativum
L.) and lentil (Lens culinaris Medik) are premier rabi pulse
crops (post rainy season winter crops) of India and cultivated
in 7.50, 0.78 and 1.50 million hectares and accounts for 5.91,
0.71 and 0.95 million tones respectively (Masood Ali and Shiv
Kumar, 2009). These crops being an important source of protein
in human food animal feed with an advantage to help in
management of soil fertility in subsistence farming system.
They are mainly grown as rainfed on residual moisture,
conserved from the preceding monsoon. However with the
extension of irrigation in the country a sizeable area of these
crops have been brought under irrigation.
On the basis of limited surveys, root knot nematodes
are considered as one of the biotic constraint in the cultivation
of these crops in major pulse growing areas of Uttar Pradesh,
Rajasthan, Bihar, Andhra Pradesh, Madhya Pradesh (Ali, 1995)
and causing damage to roots, reducing grain quality and
quantity, suppress rhizobium nodulation and thereby cutting
a part of nitrogen availability to plant and soil and increases
the severity of many soil borne diseases. Being cultivated on
marginal lands mostly as sole crops, year after year without
any kind of plant protection measures, provide an ideal and
congenital habitate for multiplication of the root-knot
nematodes.
Pathogenic effect of Meloidogyne javanica on chickpea
(Ahmad and Husain, 1988: Dhangar and Gupta, 1983:
Srivastava, et al., 1974) on pea (Paruthi, et al., 1987) were
described as significant reduction in growth parameter and
vigour. However studies on the extent of yield losses incurred
due to M. javanica are meager in chickpea while there is scanty
or no information available on the yield losses in fieldpea and
lentil crops respectively. Therefore an attempt was made to
assess the yield losses in chickpea, fieldpea and lentil crops
due to M. javanica under field conditions.
MATERIALS AND METHODS
For the assessment of unavoidable yield losses in
chickpea, fieldpea and lentil crops, field trials were conducted
at Indian Institute of Pulses Research Kanpur in sandy loam
soils, naturally infested with M. javanica. During the month
of October, seeds of chickpea cv. BG 256, fieldpea cv. Pant P-
5 and lentil cv. K-75 (All susceptible to M. javanica) were
sown in 5x2 m plot size. Paired plot treatment method was
designed for the experiments and each replicated seven times.
Carbofuran 3G @ 2kg a.i./ha was applied in furrows and
thoroughly mixed in the soil before sowing chickpea seeds,
while adjacent plot left untreated to serve as control. Phorate
10G @ 2kg a.i./ha was used as soil application under crop row
in case of fieldpea and lentil trials along with an untreated
control. Fertilizer application, weed management and other
cultural practices were carried out as per recommendation for
each crop in both untreated and treated plots. The initial
nematode population of each plot was 215±10 juveniles of M.
javanica per 250 ml soil. At maturity of these crops root-knot
indices were recorded (Table 1). Grain yield of treated and
untreated plot were recorded after harvest of the respective
crops.
RESULTS AND DISCUSSION
Analysis of data indicated that there was significant
reduction in the root knot indices in the plants protected either
with carbofuran in chickpea or with phorate granules @ 2 kg
a.i./ha in the case of fieldpea and lentil over unprotected plants
(Table 1) . Root knot nematode disease was reduced to 41, 49
and 45% in chickpea, fieldpea and lentil crops respectively in
treated plots while untreated plants exhibited heavy galling
on roots, stunting early senescence and reduced plant vigour.
The per cent avoidable losses in the yield of chickpea
cv 256 and fieldpea cv. Pant P-5 were recorded 25.6 and 15.2
respectively due to infesting of M.javanica (Table 1). Yield
loss due to M.javanica in chickpea cv. Gaurav was reported
9.2 per cent in light textured soil. (Gupta and Verma, 1989).

S.S. ALI, Estimation of Unavoidable Yield Losses in Certain Rabi Pulse Crops Due to the Root-Knot Nematode 4 9
Table 1.
Unavoidable yield loss assessment in chickpea,
fieldpea and lentil due to infestation of
Meloidogyne javanica.
S.No. Crop
Mean root-knot
index
Mean yield
(kg/ha)
% yield
loss
Treated Untreated Treated Untreated
1 Chickpea 2.3* 3.9 2719.8** 2023.5 25.6
2 Fieldpea 1.9* 3.7 2594.8** 2261.5 15.2
3 Lentil 1.7** 3.1 2237.7** 1892.5 15.4
* Significant (P= 0.05) differences from untreated plants according to
‘t’ test for paired comparison.
** Significant (P= 0.01) differences from untreated plants according to
‘t’ test for paired comparison.
While yield loss due to M. incognita in chickpea cv. T-3 was
reported 40% in sandy loam soil (Upadhyay and Diwivedi,
1987). In case of fieldpea cv. Bonneville, the avoidable loss in
yield incurred due the mixture of M. incognita and M.javanica
in light sandy loam was 50.6 per cent (Sharma, 1989), while in
another variety of fieldpea cv. T-163 it was 33.3% due to M.
incognita in sandy loam soil (Upadhyaya and Dwivedi, 1987).
However present study indicated that yield losses varies with
genotype, species of root-knot nematode involved as well as
soil type in case of chickpea and fieldpea.
The per cent grain yield of lentil due to M. javanica was
recorded 15.4 (Table 1). The unprotected plants exhibited small
galls and roots along with considerable reduction in bacterial
galls as compared to plants protected with phorate. The
pathogenic effects of M.incognita on lentil cv. Pant L-209
and Lens 830 was adverse on growth parameters and bacterial
nodulation (Fazal, et al.,1991). However for the first time it is
reported that M. javanica is a serious nematode disease of
lentil and incurred economic yield losses. To minimize the
yield losses in rabi pulses there is a need to develop cropping
system based on tolerant and resistant cultivars when no
chemical soil treatment are economically feasible in the
country.
LITERATURE CITED
Ahmad, S. and Husain, S.I. 1988. Effect of root knot nematode on
qualitative and quantitative characters of chickpea. Int, Nematol.
Network Newsl., 5(1): 12-13
Ali, S. S. 1995. Nematodes Problem in chickpea. Indian Institute of
Pulses Research Kanpur 208 024, pp. 1-184
Dhangar, D.S. and Gupta, D.C. 1983. Pathogenecity of Meloidogyne
javanica to chickpea (Cicer arietinum) in relation to soil types,
Rhizobium treatment, size of plots and time interval. Indian J.
Nematology, 13(2): 161-170
Fazal, M., Siddiqui M.R and Husain S.I. 1991. Pathogenic effect of
Meloidogyne incognita (Kofoid and white, 1919) Chitwood, 1949,
on lentil. Current Nematology, 2(1): 51-52.
Gupta, D.C. and Verma, K.K. 1989. Reaction of germplasm and studies
on avoidable yield losses in chickpea due to root knot nematode,
Meloidogyne incognita. Haryana agric. Univ. J.Res., 19(4): 318-
320.
Paruthi, I.J., Jain, R.K. and Gupta, D.C. 1987. Paththogenic effect of
Meloidogyne incognita on pea (Pisum sativum). Haryana agric.
Univ. J. Res., 17(2): 187-188.
Masood Ali and Shiv Kumar 2009. Major technological advances in
pulses; Indian Scenario. In: Mile stones in Food Legume Research
(eds. Masood Ali and Shiv Kumar). Indian Institute of Pulses
Resaerch, Kanpur. pp. 1-20
Sharma, G.L. 1989. Estimated losses due to root knot nematodes,
Meloidogyne incognita and M. javanica in pea crops. Int. Nematol.
Network Newsl., 6(1): 28-29
Srivastava, A.S., Upadyaya, K.D. and Singh, G. 1974. Effect of root
knot nematode Meloidogyne javanica on gram crops. Indian J.
Nematology, 4: 248-251
Upadhyaya, K.D. and Dwivedi, K. 1987. Analysis of crop losses in pea
and gram due to Meloidogyne incognita. Int. Nematol. Network
Newsl., 4(4): 6-7
Received on 15-07-2009 Accepted on 30-11-2009

5Trends 0 in Biosciences 2 (2): 50-52, 2009
Trends in Biosciences 2 (2), 2009
Efect of Intercrops on Quantitative and Qualitative Leaf Yield of Mulberry (Morus
Alba L.)
K.A. MURUGESH
Department of Sericulture, Tamil Nadu Agricultural University, Coimbaotre 641 003
email: murugeshka2002@yahoo.co.in
ABSTRACT
A study was undertaken to assess the influence of various
intercrops on biometric and yield parameters of mulberry.
Among the various intercrops, significantly higher biometric
parameters were recorded in clusterbean and cowpea
intercropped situations than mulberry without intercrop.
Significantly higher N, P and K were registered in mulberry
leaf harvested from cowpea and clusterbean intercropped
situations. Clusterbean and cowpea enhanced the mulberry
leaf production by 13.40 and 11.25 per cent, respectively.
Statistically superior yield traits viz., mulberry leaf yield and
leaf equivalent yield of intercrops were also registered in
clusterbean (5820 and 6736 kg/ ha) intercropped situation.
Key words
Mulberry, intercrops, clusterbean, cowpea, leaf yield
Mulberry (Morus alba L.), the sole food plant for
domesticated silkworm, Bombyx mori L. is cultivated with
wider spacing which provides a niche for intercropping.
Kusiwar, 1989 worked out the possibilities of raising intercrops
in mulberry and revealed that leaf yield, shoot length, number
of branches and leaves were increased when mulberry was
intercropped with peanut. Tikader, 1992 reported that short
term high yielding and fast growing leguminous crops could
be used for intercropping with mulberry. Studies have already
been carried out towards supplementing the nitrogen
requirement of mulberry through intercropping of different
legumes (Ahsan and Dhar, 1989). Keeping this in mind, an
attempt was made by effectively utilizing the available
interspaces in mulberry garden to obtain higher mulberry leaf
yield.
MATERIALS AND METHODS
A field trial was conducted during 2005 – 2008 using
three years old mulberry garden (Variety MR2) with spacing
of (150 x 90) + 60 cm. The mulberry was pruned at two feet
height and the seeds of intercrops were sown after first and
fourth pruning. The green manure crop (sunnhemp) was sown
after third and sixth pruning and incorporated into the soil.
Cultural operations for mulberry were carried out
(Krishnaswami, 1978) without additional fertilizer application
for intercrops. The experiment was laid out in a randomized
complete block design with three replications each of the
following treatments
T1- Mulberry + Clusterbean, T2- Mulberry + Cowpea,
T3- Mulberry + Greengram T4- Mulberry + Soyabean, T5-
Mulberry + Gingelly, T6- Mulberry + Coriander, T7- Mulberry
alone
Mulberry leaf was harvested 60 days after basal pruning
and the economic parts of intercrops were harvested at their
maturity stage. The observations on various biometric and
yield related parameters of mulberry were recorded. The yield
of intercrops was also recorded and mulberry leaf equivalent
yield was computed based on the prevailing price of intercrop
produce.The nutritional status of mulberry leaf viz., nitrogen
(Humphries, 1956), phosphorus
(Olsen, et al., 1954) and potassium (Hanway and Heidal,
1952) were also estimated. The moisture content was calculated
by gravimetric method and moisture retention capacity was
estimated after 6 hours of leaf harvest (Anonymous, 2000).
The data were analyzed as per the statistical procedure (Panse
and Sukhatme, 1985).
RESULTS AND DISCUSSION
Biometric parameters of mulberry
The biometric parameters of mulberry were presented in
Table 1. Maximum average shoot length (112.12 and 107.00
cm) and number of branches per plant (14.00 and 12.25) were
registered in clusterbean and cowpea intercropped situation
and were found to be statistically superior over all other
intercropped situations, similar type of findings has also been
reported by Shankar, et al., 1994. The maximum number of
mulberry leaves per plant (297.50) was recorded in mulberry
clusterbean intercropping system was significantly differed
from all other treatments. Similar observations were recorded
by Shankar, et al., 1994, in intercropping of pulses with
mulberry. The leaf area index was also higher in intercropping
of mulberry with clusterbean (4.28) followed by greengram
(4.20). Higher leaf area index under mulberry + soybean
intercropping has also been reported by Sridhar Babu, 1994.
Nutritional status of mulberry leaf
Chemical analysis of leaf samples revealed positive
impact of various intercrops on the nutritional status of
mulberry. Significantly higher nitrogen content was recorded
in mulberry leaf intercropped with clusterbean (3.84 %) and
cowpea (3.81 %) over the mulberry grown without intercrops
(3.65 %) (Table 1). Sridhar Babu, 1994 and Bongale, et al., 1998

MURUGESH, Efect of Intercrops on Quantitative and Qualitative Leaf Yield of Mulberry (Morus Alba L.) 5 1
also reported an increased nitrogen content in the leaf
harvested from mulberry garden intercropped with leguminous
crops.
The higher level of phosphorous and potassium was
also recorded in mulberry leaf grown with clusterbean (2.97
and 2.23 % respectively) followed by cowpea (2.94 and 2.20 %
respectively) than mulberry without intercrops (2.71 and 2.02
% respectively) (Table 1). This may be due to the lack of
competition for nutrient between main crop as well as intercrops
and nitrogen fixation by intercrops which led to improvement
in nutrient content of mulberry leaf. This view was
strengthened by the findings of Tikader, 1992 and Hadimani,
2003, who recorded significantly higher quantity of
phosphorus and potasium in mulberry raised with pulses.
Moisture content and moisture retention capacity of
mulberry leaf
Maximum moisture content over sole mulberry crop was
recorded in the mulberry intercropped with clusterbean
(74.50%) followed by cowpea (73.34 %) and were found to be
statistically significant to all other treatments (Table 2).
Similarly the moisture retention capacity was also higher in
the same treatment and with cluster bean followed by cowpea
(85.70%). This observation was strengthened by the findings
of Ahsan and Dhar, 1989 who recorded higher moisture
content in mulberry grown with pulses as intercrop. The leaf
of mulberry intercropped with clusterbean recorded highest
moisture retention capacity (87.40 %) followed by cowpea
(85.70 %) and were found to be statistically superior over the
leaf harvested from mulberry raised without intercrop (73.40
%) (Table 2). These studies can be corroborated with the
observation of Bongale, et al., 1998 and Anonymous, 2000
and Ahsan and Dhar, 1989.
Yield parameters
Among the different intercropped, clusterbean recorded
maximum leaf yield (5820 kg/ ha) followed by greengram (5712
kg/ ha) and least leaf yield was recorded in mulbery without
intercrop (5134 kg/ ha). This might be due to the beneficial
effects of legumes crops in terms of atmospheric nitrogen
fixation and improvement the available nutrients. Tikader,
1992 reported that mulberry intercropped with field pea
increased leaf yield then mulberry alone. Among the various
intercrops, clusterbean (6736 kg/ ha) recorded highest
Table 1.
Biometric parameters of mulberry 60 days after pruning in different intercropping situations and N, P and K content
of mulberry leaf.
Intercrops
Clusterbean
Cowpea
Greengram
Soybean
Gingelly
Coriander
Mulberry alone
Average shoot
length (cm)
112.12
107.00
105.40
101.15
99.50
94.05
97.00
Average number
of branches/ plant
14.00
12.25
10.05
10.60
8.55
9.80
9.35
Average number
of leaves/ plant
297.50
228.00
217.68
204.40
166.04
180.00
181.60
Leaf area index
(LAI)
Nitrogen
(%)
Phosphorus
(%)
‘F’ test * * * * * * *
4.28
4.13
4.20
4.17
4.05
3.95
4.00
3.84
3.81
3.75
3.68
3.71
3.68
3.65
2.97
2.94
2.88
2.79
2.83
2.75
2.71
Potassium
(%)
S.Em± 2.16 0.72 7.31 0.07 0.02 0.01 0.01
CD (p=0.05) 4.25 1.13 12.30 0.18 0.04 0.03 0.03
2.23
2.20
2.15
2.09
2.14
2.00
2.02
Table 2.
Intercrops
Clusterbean
Cowpea
Greengram
Soybean
Gingelly
Coriander
Mulberry alone
Moisture content and moisture retention capacity, yield of mulberry leaf and leaf equivalent yield of intercrops.
Moisture content
(%)
74.50
73.34
71.00
68.28
68.15
63.32
62.71
Moisture retention
capacity (%)
87.40
85.70
80.15
74.52
76.90
76.24
73.41
Mulberry leaf yield
(kg/ ha)
5820
5712
5685
5310
5195
5270
5134
Mulberry leaf equivalent yield
(kg/ha)
‘F’ test * * * ---
S.Em± 1.20 1.58 7.12 __
CD (p=0.05) 2.50 3.75 18.00 ---
6736
6570
5568
5232
5961
5499
---

5 2 Trends in Biosciences 2 (2), 2009
mulberry leaf equivalent yield and was followed by greengram
(6570 kg/ ha) (Table 2). This study can be corroborated with
the observations of Sinha, et al,. 1987 and Hadimani, 2003
who recorded higher leaf equivalent yield when mulberry was
raised with greengram and blackgram as intercrops.
It is clear from the study that intercropping of
clusterbean and cowpea in mulberry garden could be practiced
for maximizing quantitative and qualitative yield of mulberry
and increasing the productivity from the system.
LITERATURE CITED
Ahsan, M.M and Dhar, K.L. 1989. Studies on intercropping of short
duration crops with mulberry. Indian J. Seric., 28(2): 194-199.
Anonymous. 2000. Handbook of Sericulture Technologies. (eds. Dandin,
S.B., Jayant Jayaswal and Giridar, G.), Central Silk Board, Bangalore,
pp.287.
Bongale, U.D., Shivaprakash, R.M. and Veeresh, M. 1998. Studies on
suitability of soybean as an intercrop in mulberry under irrigated
condition. Sericologia, 38(1): 167-170.
Hadimani, D.K. 2003. Impact of legume intercropping in paired row of
mulberry on silkworm. M.Sc. (Seri) Thesis, GKVK, University of
Agricultural Sciences, Bangalore, pp.98.
Hanway, J. and Heidal, H. 1952. Soil analysis methods as used in Iowa
State College Soil Testing Laboratory, Iowa State College Agric.
Bull., 57: 1–13.
Humphries, E. C. 1956. Mineral components and ash analysis in modern
method of plant analysis. Springer- Verlag, Berlin., pp.502
Krishnaswami, S. 1978. Packages of practices for mulberry cultivation
in South India. Central Sericultural Research and Training Institute,
Mysore. pp.19.
Kusiwar, E. 1989. Growth and leaf production of mulberry (Morus alba
L.) pure and mixed with soybean and peanut. Bul. Peuetitian Hutan
O. 510: 1–8.
Olsen, S.R., Cole, C.V., Watanabe, F.S. and Dean. D.A. 1954. Estimation
of available phosphorus in soils by extraction with sodium
bicarbonate. US Dept. Agric. Cir., pp.939.
Panse, V.G. and Sukhatme, P.V. 1985. Statistical methods for agricultural
sciences. Indian Council of Agricultural Sciences, New Delhi, pp.327
Shankar, M.A., Ravi, K., Puttasamy, S. and Devaiah, M.C. 1994. Minor
millets and pulses in mulberry garden, Indian silk, 33(5): 13-14.
Sinha, A.C., Sarkar, A. and Das, A.C. 1987. Technology for intercropping
in mulberry. Indian farming, 36(11): 11-12.
Sridhar Babu, D.K. 1994. Effect of nitrogen and intercropping on
mulberry varieties. M.Sc. (Seri.) Thesis, Tami Nadu Agricultural
University, Coimbatore, pp. 73.
Tikader, A. 1992. Intercropping with legumes to increase mulberry
production. Indian silk, 30(11): 41-42.
Recieved on 9-9-2009 Acceted on 9-10-2009

Trends in Biosciences 2 (2): 53-58, 2009
Description of Some Aphid Species (Homoptera: Aphididae: Aphidinae) Belonging
to Tribe Macrosiphini Recorded from Aligarh Region
M.K. USMANI AND UZMA RAFI
Section of Entomology, Department of Zoology, Aligarh Muslim University, Aligarh 202 002
email: usmanikamil94@gmail.com
ABSTRACT
Six species of Aphids belonging to tribe Macrosiphini are
described from Aligarh district viz., Acyrthosiphon pisum (Harris)
(Alatoid nymph.), Brevicoryne brassicae (Linnaeus) (Aptera),
Lipaphis erysimi (Kaltenbach) (Alata), Macrosiphoniella
sanborni (Gillete) Alata), Macrosiphum (Sitobion) rosaeformis
Das (Alata), Myzus persicae (Sulzer)(Aptera and Alata). Their
distribution, host plants and seasonal occurrence are given.
Name of their Natural enemies are also included.
Key words
Homoptera, aphididae, macrosiphini, Aligarh
Aphids are the small, soft bodied insects belonging to
the Family Aphididae of Order Hemiptera, and Suborder
Homoptera. About 4702 species of aphids are known world
wide and about 653 species are known from Indian region
(Rajendran, 2002). They are sap sucker insects commonly
known as ‘Plant Lice’. They feed on different parts of plant
and cause direct damage by sucking cell sap from their host
plant and hence they are serious agricultural pests. They prefer
to feed on tender parts of plants like young leaves and shoots,
and some species may infest roots as well. Aphids act as a
vector of plant viruses and thus assume more economic
importance. About 300 species of aphids have been tested as
vector of 300 different viruses in about the same number of
plants. In India there about 45000 plants species, out of which
about 1800 plant spp. are of significant medical importance
and are used in preparing different therapeutic formulations
in the country. Out of them, nearly 1250 plant species belong
to the 700 genera and 175 plant families which are used as
food plants by 653 species representing 208 genera of aphids
in India. The perusal of literature survey demonstrated that
among them, 428 species/subspecies of aphids infest 530
species of medicinal plants belonging to 117 plant families in
India. (Eastop, 1979).
A survey of aphid on agricultural crops production areas
in different localities of Aligarh and I.A.R.I. New Delhi was
conducted during 2006-2008. The survey yielded a good
number of specimens, which served as a basis for the present
study. This is the first systematic collection of Aphids from
Aligarh region. Further, it has revealed interesting observations
on the distribution of aphid species in different regions.
In the present study the author upholds recent workers
in classifying aphids with a few generally accepted changes.
Brief diagnosis of family, subfamily, tribe and genera is given.
Keys to genera and species where ever necessary are given.
Most of the genera are represented by single species. The
species represented in this region are briefly described and
illustrated. Notes on coloration are also given.
MATERIALS AND METHODS
Aphids were collected from their host plants with a soft
brush soaked in alcohol and fixed either in 96% alcohol or in
fluid containing of 2 vols. of 96% alcohol and 1vols of 75 % of
lactic acid. They were mounted on slides according to standard
method for detailed study.
The prepared slides were studied under light microscope.
The identification of species was done on the basis of
morphological characters of material. Measurements of the
specimens were done by using ocular micrometer and drawings
were also made by camera Lucida. All measurement are in mm.
Abbreviations
F.T.C.- First tarsal chaetotaxy; h.t.2- Second tarsal
segment of hind tibia; p.t.- Processus terminalis; u.r.s.- Ultimate
rostral segment; I, II…..VI- Antennal segment I, II, III…..VI;
1,2,3……8- Abdominal segment 1, 2, 3……8.
RESULTS AND DISCUSSION
Superfamily: Aphidoidea
Tribe Macrosiphini is represented by six genera from
this region.
Key to the Genera of Tribe Macrosiphini.
1. Some of the secondary hairs on u.r.s. as long as or longer
than primary hairs…………………..Macrosiphoniella.
Secondary hairs on u.r.s. usually much shorter than
primary hairs……………………………..……….2.
2. Siphunculi reticulated (Cells either isodiametrical or
transversely elongated)
.………………..……………. Macrosiphum.
Siphunculi not reticulated but sometimes a few
interconnecting striae present
near the apical flange………………………..........……..2.
3. Apterae without any median frontal prominence……..….
Acyrthosiphon.

5 4 Trends in Biosciences 2 (2), 2009
Apterae with median frontal prominence……………….3.
4. Dorsum of head in apterae usually smooth or may be
slightly wrinkled…………………………………….4.
Dorsum of head in apterae spinulose or warty……….
Myzus passerini.
5. Siphunculi shorter than cauda……………..Brevicoryne
Siphunculi longer than cauda………….……Lipaphis
Genus Acyrthosiphon Mordvilko
Acyrthosiphon Mordvilko, 1914. Fauna Russie, 1:75.
Type-species: Aphis pisi Kaltenbach, 1843 (=Aphis pisum
Harris, 1776).
This genus represented by single species from this
region.
1. Acyrthosiphone pisum (Harris) (Fig. 1)
Aphis pisum Harris, 1776, Exposit. English Insect,
London, 66.
Material examined
4 Alatoid nymphs on Pisum sativum from Aligarh.
Diagnostic characters
They are pale yellow or pale brown in colour. Head,
distal portions of antennae, Siphunculi and legs are dark
brown. Lateral frontal tubercles well developed, frontal sinus
almost V-shaped in aptera while slightly raised in alatae.
Antennae 6 segmented, longer than body. Processus terminalis
longer than the base of last antennal segment. Dark sclerotic
cones present at the base of Siphunculi. Cauda broad and
bears 6 hairs. F.T.C. 3,3,3.
Aptera: (From Lit.) length of body 4.20, width 2.01;
antenna 4.20, segments III:IV:V:VI 1.01:0.82:0.71:(0.28+1.05);
u.r.s. 0.11; h.t.2 0.17; siphunculus 0.97; cauda 0.61.
Alata: (From Lit.) length of body 3.70, width 1.29; antenna
4.40, segments III:IV:V:VI 1.01:0.85:0.78:(0.30+1.15), u.r.s. 0.11;
h.t.2 0.17; siphunculus 0.83; cauda 0.49.
Host plants
Distribution
Pisum sativum, Pisum sp., Vicia faba (Papilionaceae).
Cosmopolitan.
Seasonal occurrence
Almost throughout the year, more prevalent during July-
September
Natural enemies
Coleoptera: Coccinellidae Cheilomenes sexmaculata
(F.), Coccinella transversalis F.
Genus Bravicoryne Van der Goot
Brevicoryne Van der Goot, 1915, Beitr. Kennt. Holland.
Blattlause, 245.
Type-species: Aphis brassicae Linnaeus, 1758.
This genus represented by single species from this
region.
2. Bravicoryne brassicae (Linnaeus) (Fig. 2)
Aphis brassicae Linnaeus, 1758, Syst. Nat., (10 ed), 1:452.
Material examined
3 alatoid nymphs and 2 apterous adults on Brassica
oleracea capitate from I.A.R.I. and Aligarh.
Diagnostic characters
Apterae medium-sized, grayish-green in colour, with dark
head, body covered with greyish-white wax. Alatae with dark
head and thorax and black transverse bars on dorsal abdomen.
Antennae shorter than the body length. U.r.s. reaches upto
mid coxae, shorter than ht.2 . Prothorax in apterae with marginal
brown patches; pigmented patches present on lateral side of
abdominal segment 2-5. Siphunculi barrel shaped, dark and
longer than cauda. Cauda short and triangular shaped. F.T.C.
3,3,3.
Aptera: Length of body 1.7, width 0.91; antenna 0.85,
segments III:IV:V:VI 0.22:0.12:0.11:(0.08+0.20); u.r.s. 0.11; h.t.2
0.13; siphunculus 0.16; cauda 0.12.
Alata: (From Lit.) Length of body 2.28, width 1.03;
antenna 1.96, segments III:IV:V:VI 0.67:0.14:0.25(0.13+0.51);
u.r.s 0.10; h.t.2 0.19; siphunculus 0.13, Cauda 0.16.
Host Plants
Beta vulgaris (Chenopodiaceae), Brassica compestris,
Brassica nepus, Brassica oleracea, Capsella bursapestoris,
Cardamine hirsute and Raphanus sativa (Cruciferae).
Distribution
India: West Bengal; virtually cosmopolitan.
Seasonal occurrence
December - March
Natural enemies
Coleoptera: Coccinellidae: Cheilomenes sexmaculata
(F.), Coccinella septempunctata L., C. transversalis F.
Hymenoptera: Braconidae: Aphidiinae: Diaeretiella rapae
(M’Intosh),
Genus Lipaphis Mordvilko
Lipaphis Mordvilko, 1928, Keys to the Russian Insects,
200 (in Russian).
Type-species: Aphis erysimi Kaltenbach, 1848.
This genus represented by single species from this region

USMANI AND RAFI, Description of Some Aphid Species (Homoptera: Aphididae: Aphidinae) Belonging 5 5
3. Lipaphis erysimi (Kaltenbach) (Fig. 3)
Aphis erysimi, 1843, Mon. fam. Pfl., 99.
Material examined
2 alatae and 15 Alatoid nymphs on Brassica oleracea,
Brassica compestris and Solenum melongena from Aligarh. 1
alatae on Brassica compestris from I.A.R.I.
Diagnostic characters
Aptarae small to medium-sized, yellowish green in colour.
Alatae have a dusky green abdomen with dark lateral sclerites,
and dusky wing veins. Head wrinkled and pigmented with
low developed lateral frontal tubercles and distinctly
developed median frontal prominence. Antenna 6 segmented,
shorter than body. Secondary rhinaria found only in alatae on
segment III and IV. Rostrum short, hardly reached to mid coxae.
U.r.s. shorter than ht.2. In apterae abdominal dorsum pale while
in alatae pigmented patches present. Siphunculi
subcylindrical, dark and slightly constricted at middle with
distinct small apical flange. Cauda dusky, bears 6 hairs and
smaller than siphunculi. F.T.C. 3,3,3.
Aptera: (From Lit.) Length of body 1.52, width 0.97;
antenna 0.93, segments III:IV:V:VI 0.27:0.10: 0.11:(0.10+0.21);
u.r.s. 0.09; h.t.2 0.11; siphunculus 0.18; Cauda 0.15.
Alata: Length of body 1.58, width 0.73; antenna 1.25,
segments III:IV:V:VI 0.37:0.18: 0.17:(0.12+0.29); u.r.s 0.09; h.t.2
0.14; siphunculus 0.18; Cauda 0.16.
Host plants
Drymeria cardata (Caryophyllaceae); Calendula sp.,
Emilia sonchifolia, Lactusa sativa (Compositae); Brassica
sp., Iberis amara, Nasturtium indicum, Raphanus sativus
(Cruciferae) and Tropeolum majus (Tropeolaceae).
Distribution
India: Arunachal Pradesh, Assam, Bihar, Himachal
Pradesh, Manipur, Meghalaya, Nagaland, Rajasthan, Sikkim,
South India, Tripura, Uttar Pradesh, West Bengal; Bhutan,
Nepal, and virtually cosmopolitan.
Seasonal occurrence
September - March
Natural enemies
Coleoptera: Coccinellidae: Coccinella septumpunctata
was recorded from Aligarh on Lipaphis erysimi. It is reddish
brown colored beetle having 7 black round spots on elytra. It
can be seen through out the year, but more abundant and
active from December to March and then from July to
September. Both larvae and adults are voracious.
Ischiodon scutellaris (Diptera : Syrphidae) was also
recorded from Aligarh. Widely distributed in India. Only larvae
feed on aphids. Adult females lay their eggs near aphid colony,
when larvae hatched out from eggs, start feeding on aphids.
Diaeretiella rapae (M’Intosh) (Hymenoptera :
Aphidiidae) was recorded from this aphid species from Aligarh.
Adult female lay their eggs inside the body of aphid, when
eggs hatched, the larvae start feeding on haemolymph and
tissues of aphids.
Genus Macrosiphoniella de Guercio
Microsiphoniella del Guercio, 1911, Redia, 7:331.
Type-species: Siphonora atra Ferrari, 1872.
This genus represented by single species from this region
4. Macrosiphoniella sanborni (Gillete) (Fig. 4)
Macrosiphoniella sanborni Gillete, 1908, Can. Ent.,
40:65.
Material examined
4 Alatae on Chrysanthemum sp. from Aligarh.
Diagnostic characters
Apterae shiny blackish brown, body broadly spindle
shaped, with black, thick siphunculi, and cauda. Alatae similarly
pigmented. Head pale brown, smooth with moderately well
developed diverging lateral frontal tubercles without median
prominence; 3 pairs of dorsal cephalic hairs present. Antennae
6 segmented and almost equal to body length. Processus
terminalis three to five times longer than the base of last
antennal segment. Antennal segment III possesses 24 and
segment IV possesses 3 secondary rhinaria. Abdominal
dorsum in alatae with well developed marginal patches, similar
patches also present behind the base of siphunculi. Siphunculi
dark, imbricated and with distinct apical flange. Cauda always
longer than siphunculi, dark and bears 10-20 hairs.
Aptera : (From Lit.) Length of the body : 2.07 Width :
0.94, Antenna 2.0 Segments III:IV:V:IV 0.51:0.29:0.29:
(0.13+0.54); u.r.s 0.13 ; h.t.2 0.13, Siphunculus 0.27; cauda
0.39.
Alata: Length of body 1.9, width 0.8; antenna 1.2,
segments III:IV:V:VI 0.28:0.22:0.18:(0.12+0.28); u.r.s. 0.12; h.t.2
0.11; siphunculus 0.2; cauda 0.22.
Host plants
Artemisia sp; Chrysanthemum sp; C. coronarium and
C. indicum (Compositae).
Seasonal Occurrence
October – March.
Natural enemies
Coleoptera: Coccinellidae: Cheilomenes sexmaculata
(F.), Hymenoptera: Braconidae: Aphidiinae: Lysaphidus
quadrii Shuja Uddin,
Distribution
India: All over; and virtually Cosmopolitan.

5 6 Trends in Biosciences 2 (2), 2009
Genus Macrosiphum Passerini
Macrosiphum Passerini, 1860, Gli. Afidi. Parma, 27
Type-species: Aphis rosae Linnaeus, 1758
This genus represented by single species from this region
5. Macrosiphum (Sitobion) rosaeiformis Das (Fig. 5)
Macrosiphum rosaeiformis Das, 1918, Mem. Indian Mus.,
6(4):158.
Material examined
1 Alatae and 4 Apterous on Rosa indica from Aligarh.
Diagnostic characters
Medium to large sized aphids, spindle-shaped, green to
reddish brown. Head smooth with few fine hairs, well
developed diverging lateral frontal tubercle and indistinct
median frontal prominence. Antennae equal to or less than
body length, sometimes larger than body. Processus terminalis
longer than the base of last antennal segment. Segment III
about equal to processus terminalis. Non protuberant
secondary rhinaria present on III antennal segment in both
alate and apterae. Siphunculi are basally pale and apically
dark in apterae, wholly dark in alatae, almost twice in length of
that of cauda, apical side distinctly reticulated. Cauda
elongated, dark and with nine hairs.
Population I
Aptera: Length of body 3.36, width 1.48; Antenna 3.58,
Segments III:IV:V:VI 1.06:0.73:0.61:(0.17+0.76); u.r.s. 0.14; h.t.2.
0.16: Siphunculus 1.24; cauda 0.62.
Alata: Length of body 3.47, width 1.28; antenna 4.00
Segments III:IV:V:VI 1.08:0.78:0.74:(0.16+0.76); u.r.s. 0.14; h.t.2.
0.15; Siphunculus 1.84; cauda 0.52.
Population II:
Aptera: Length of body 2.22, width 1.08; Antenna 2.33,
Segments III:IV:V:VI 0.52:0.41:0.40:(0.14+0.62); u.r.s. 0.13; h.t.2.
0.14: Siphunculus 0.8; cauda 0.26.
Host plants
Rosa cania, Rosa macrophylla, Rosa moschata, Rosa
Sp. Spirarea corymbosa (Rosaceae).
Seasonal occurrence
September-October.
Natural enemies
Not found in this region.
Distribution
India: Arunachal Pradesh, Assam, Bihar, Himachal
Pradesh, Manipur, Meghalaya, Nagaland, Sikkim, south India,
Uttar Pradesh, West Bengal; Nepal & Pakistan.
105.
Genus Myzus Passerini
Myzus Passerini, 1860, Gli. Afidi, Parma, 27.
Type-species: Aphis cerasi Fabricius, 1785.
This genus represented by single species from this region
6. Myzus persicae (Sulzer) (Fig. 6)
Aphis persicae Sulzer, 1776, Abgekurzte Gesch. Insekten,
Material examined
2 Alatae on Morus alba, 1 alatae, 2 Apterous on
Echinocloa Colona, Solenum tuberosum from Aligarh and 2
Apterous on Brassica oleracea capitat from Delhi.
Diagnostic characters
Apterae small to medium in size, pale yellow-green,
sometime pink, or red ; Alatae have a black central patch on
abdominal dorsum. Head dorsally spinulose with well
developed median frontal prominence and lateral frontal
tubercles with inner margin converging; dorsal cephalic hairs
short. Antennae 6 segmented, often slightly shorter than body
in case of apterae while longer than body in case of alate. In
alatae forms Secondary rhinaria present on III segment. U.r.s.
usually equal to ht.2. abdominal dorsum in aptera sparsely
spinulose and without scattered patches while in alatae a dark
large central patch present. Siphunculi cylindrical without
distinct apical flange and longer than cauda. Wing venation
normal.
Population I: (Brassica oleracea)
Aptera: Length of body 2.12, width 1.24; antenna 1.78,
segments III:IV:V:VI 0.42:0.33:0.26:(0.13+0.46); u.r.s 0.12; h.t.2
0.12; siphunculus 0.57; cauda 0.22.
Population II: (Echinocloa colona)
Aptera: Length of body 1.79, width 1.00; antenna 1.66,
segments III:IV:V:VI 0.40:0.29:0.24:(0.13+0.44); u.r.s 0.11; h.t.2
0.11; siphunculus 0.49; cauda 0.19.
Alata: Length of body 1.82, width 0.96; antenna 2.39,
segments III:IV:V:VI 0.54:0.48:0.35:(0.13+0.69); u.r.s 0.12; h.t.2
0.12; siphunculus 0.42; cauda 0.21.
Host plants
Extremely polyphagous. Morus alba, Echinoloa
colona, Brassica oleracea capitata, Brassica oleracea
botrytis, Solenum tuberosum
Seasonal occurrence
All through the year, with heavy infestation during winter
and early summer.
Natural enemies
Coleoptera: Coccinellidae: Cheilomenes sexmaculata
(F.), Coccinella transversalis F.

USMANI AND RAFI, Description of Some Aphid Species (Homoptera: Aphididae: Aphidinae) Belonging 5 7

5 8 Trends in Biosciences 2 (2), 2009
Distribution
India: All over; and virtually cosmopolitan
Out of the 4702 aphid species recorded world over, 1015
are known to be in the oriental region with 13.8% of them
being large number of race and endemic species. About 400
aphid species are found in eastern and northeastern
Himalayas. Subfamily Aphidinae shows the highest frequency
in Subtropical India (64%) while 30% species are in the tropical
regions of the country.
There are about 36 genera and 58 species of aphids
recorded from U.P. Most of them were recorded from hill
regions now i.e. Uttarakhand. As far as the plain areas are
concerned, very few species were recorded and large number
of areas remain untouched.
This is the first consolidated work from Aligarh region.
We have recorded six genera and six species belonging to
tribe Macrosiphini from Aligarh and its remote areas.
Experience has shown that control of agricultural pests
is made easier when their taxonomy and biological
observations have been placed on a sound basis.
A compilation of insect pests known to occur in an area
or locality has value when information is needed promptly
about the distribution, seasonal occurrence, host-plant
relationships, and economic importance of a species.
ACKNOWLEDGEMENT
Authors are greatly thankful to Prof. A.M. Khan,
Chairman, Deptt. of Zoology, A.M.U Aligarh for providing
necessary facilities. Authors are also thankful to Prof. Samiran
Chakraborti, Deptt. of Zoology, University of Kalyani, Kalyani
(W.B.) for helping us in identification of Aphids. Second auther
is thankful to University Grant Commission for providing
financial support during tenure of the work.
LITERATURE CITED
Basu, R.C. and Raychaudhari, D.N. 1976. Studies on the Aphids
(Homoptera: Aphididae) from India XXV. The Genus Myzus with
five new species from Eastern India. Oriental Insect, 10(1): 93-
112.
Behura, B.K. 1997. Bio-Ecology of Indian Aphids : A Review, J. Aphidol.,
11(2): 147-154.
Bhattacharya, D.K. and Dey, S.R. 1996. A new species and a new record
of aphids (Homoptera: Aphididae) from Garhwal Range of Western
Himalayas, India. Entomon, 21(3&4): 285-289.
Blackman, R.L. and Eastop, V.F. 1984. Aphids on the world’s crop. In:
An Identification And Information Guide, Vol. 1. Wiley Chichester.
Blackman, R.L. and Eastop, V.F. 2000. Aphids on the World’s Crops.
In: An Identification and Information Guide. 2 nd edition. John Wiley
& Sons, New York, pp.466
Chakrabarty, S. and Sarkar, A. 2001. A Supplement to the Food-Plant
Catalogue of Indian Aphididae (Homoptera: Aphididae) J. Aphidol.,
15: 9-62.
Eastop, V.F. and Hille Ris Lambers, D. 1976. Survey of the World’s
Aphids. The Hague: Junk, pp.573
Eastop, V.F. 1979. Key to the genera of sub tribe Aphidina (Homoptera),
Systematic Ent., 4: 379-388.
Ghosh, A.K. 1969. On a collection of Aphids (Homoptera: Aphididae)
from Uttar Pradesh, India. Sci. Cult., 35: 493.
Ghosh, M.R. and Raychaudhari, D.N. 1981. Aphids (Homoptera:
Aphididae) Infesting Rosaceous fruit plants in Darjeeling District
of WB and Sikkim. Entomon, 6(1): 61-68.
Ghosh, L.K. and Basu, R.C. 1994. Insecta : Hemiptera : Aphididae,
State fauna series 3; Fauna of West Bengal, part 5: 125-318.
Ghosh, L.K., Biswas, B., Chakrabarti, S.P. and Sen, G.C. 1989. Insecta:
Hemiptera, State Fauna Series No.1; Fauna of Orissa, Part 2: 181-
185.
Krishnamurty, B. 1930. Aphididae of Mysore. J. Bombay nat. Hist.
Soc., 34(2): 411–419.
Kulkarni, P.P. 2006. Insecta: Aphidoidea: Aphididae, Zool. Surv. India.
Fauna of Sanjay Gandhi National Park (Invertebrates) Conservation
area series, 26: 51-63.
Kulkarni, P.P. 2005. Insecta: Homoptera: Aphidoidea: Aphids Zool.
Surv. India, Fauna of Melghat Tiger Reserve, Conservation Area
Series, 24: 359-368.
Raychaudhari, 1980. Aphids of Northeast India and Bhutan. The
Zoological Society Calcutta. pp. 1-521.
Rajendran, T. P. 2002. Ecology and diversity of aphids (Homoptera :
Aphididae) in the Indian region – A Thesis for future investigation,
J. Aphidol., 16: 203-208.
Singh, R., Agarwal, R.and Pandey, S. 2003. Records of the Aphids (Insecta:
Homoptera: Aphididae) infesting Medicinal Plants of India, J.
Aphidol., 17: 1-58.
Verma, K.D. and Chandla, V. K. 1999. Potato Aphids and their
Management, Technical Bulletin no. 26 (Revised), Central Potato
Research Institute (ICAR) Simla, H.P.
Recieved on 8-11-2009 Acceted on 30-11-2009

Trends in Biosciences 2 (2): 59-60, 2009
Nitrogenase Activity in Cyanobacterial Biofilm
P. PARAMESWARAN*, S. PALANI AND D.VENKATESAN
Anna Bioresearch Foundation, Department of Biotechnology, Arunai Engineering College,
Tiruvannamalai 606 603. e-mail : ppbiotech@gmail.com
ABSTRACT
The nitrogenase activity could be detected in cyanobacteria
only when there is a special or temporal separation of
photosynthesis and nitrogen fixation. We could induce the
activity of nitrogenase in a few isolates of Oscillatoria, O.sancata
is best in forming cyanobacterial biofilm with other
heterocystous cyanobacteria. The activity of nitrogenase was
detected when nitrate grown cells were starved for combined
nitrogen for 1to 15 days and grown in low light intensity of
6.022 x 10 17 photons s -1 m -2 . Therefore it appears that nonheterocystous
cyanobacteria could very well participate in rice
field N 2
fixation. Perhaps it is necessary to have an indepth
study of non-heterocystous forms to select ideal strains for use
as biofertilizer mixed with heterocystous forms to make biofilm
for higher nitrogen fixation in biofertilizer programme in wet
land crops.
Key words
Non-heterocystous cyanobacteria, nitrogen fixation,
biofertilization ,photons s -1 m 2
Cyanobacteria abundantly inhabit rice field ecosphere
and contribute to the balancing of the soil nitrogen status.
Indian rice fields harbour large number of unicellular and nonheterocystous
filamentous organisms along with
heterocystous filamentous forms. Recent evidence on nitrogen
fixation by unicellular forms like Gloeothece, filamentous
Oscillatoria and Lyngbya enhance the value of cyanobacterial
potential as biofertilizer. Recently we screened a number of
strains of non hetrocystous filamentous forms for their
capacity to fix nitrogen and found that light/dark modulation
nitrogenase exists in them. Strains of Oscillatoria, Lyngbya,
Phormidium and Chlorogloea fix nitrogen and grow in
chambers with light/dark regimes in medium without any
combined nitrogen. Field experiments also suggested a
positive role for such organisms. Oscillatoria sancta a nonheterocystous
filamentous organisms is observed good in
nitrogenase activity under low light intensity (Parameswaran
and Anand, 2007) is mixed with five other better heterocystous
strains to form cyanobacterial biofilms observed higher rate
of nitrogen fixation for biofertilizer programme
In nature, non heterocystous forms such as Oscillatoria
spp. are found to fix nitrogen aerobially when they are present
as components of microbial mats ( Parameswaran and Anand,
2007) Also, diffuse light is known to enhance nitrogenase
activity in Oscillatoria sancta (Parameswaran and
Shanmugasundaram,1996). Trichodesmium, a nonheterocystous,
marine filamentous cyanobacterium has
evolved a new method of fixing nitrogen in light, parallel with
photosynthesis by producing the enzyme nitrogenase only
in few special cells in a colony where it is protected from
oxygen (Fredriksson and Bergman, 1995).
Non-heterocystous cyanobacteria like Oscillatoria,
Plectonema, Phormidium and Schizothrix are often found in
south indian rice fields and are rather considered as weeds.
The reason is their inability to express nitrogenase in light
and poor activity in dark can also participate in the biofertilizer
programme.
MATERIALS AND METHODS
Cyanobacterial Cultures used
The cyanobacterial strains were isolated from south
indian rice fields viz., Oscillatoria sp. (10 isolates), Anabaena
sp. (1 isolate), Aulosera sp. (1 isolate), Nostoc sp. (1 isolate),
Oscillatoria+5 heterocystous cyanobacterial mix. and axenic
cultures were maintained in BG-11 medium (Rippka, et al.,
1970) with 30 uE m -2 s -1 illumination for heterocystous
cynobacteria,1uEs-1m-2 for non heterocystous cyanobacteria.
After the preliminary screnning of 10 Oscillatoria sp, one
strain O. sancta was selected for further study.
Nitrate grown cells were starved for combined nitrogen
from 1 to 15 days and grown in low light intensity of 6.022 10 17
1uE s -1 m -2 . Growth was monitored interms of total protein
(Lowry, et al., 1951) and dry weight. Acetylene Reduction
Assay (Stewart, et al., 1967) was carried out for estimating
nitrogense activity for a period of 15 days at 5days interval.
heterocystous forms tested individually, 5 mixed strains, 5 mix
with one Oscillatoria sp to form cyanobacterial biofilm (1-4)
synthetically and tested for its nitrogenase activity.
RESULTS AND DISCUSSION
Almost all strains showed maximum growth on the 15th
day and highest nitrogenase activity on the 10th day.
Oscillatoria 184 was found to be the highest nitrogen fixer
(180.0 nmoles C 2
H 4
released nig protein -1 ) among the isolates
tested (Table 1)
Nitrogenase activity is known to be present in
unicellular, colonial and non-heterocystous filamentous
cyanobacteria in the absence of photosynthetic O 2
liberation.
In the present work the cyanobacterial nitrogenase activity
was studied under low light intensity of 6.022. 10 17 photon s -
1
m -2 . There was no O 2
liberation however, O 2
consumption

6 0 Trends in Biosciences 2 (2), 2009
Table 1.
Growth and nitrogenase activity in non- hetrocystous cyanobacteria in N free BG 11
medium
Isolates N-free
n moles of C2H4h-1 released Protein (mg) Dry weight (mg)
5 day 10 day 15 day 5 day 10 day 15 day 5 day 10 day 15 day
Oscillatoria sancta 104.136 171.076 90.714 11.1 31.0 33.9 2.0 4.2 5.0
Oscillatoria sp. 030.882 140.030 100.016 4.0 30.3 31.3 1.0 4.0 4.6
Oscillatoria sp. 070.844 88.024 127.000 11.0 25.5 30.0 2.0 3.8 4.0
Oscillatoria sp 112.000 170.108 120.828 11.5 33.0 33.0 1.6 4.7 5.0
Oscillatoria sp. 120.028 165.050 133.924 15.0 30. 32.0 1.7 4.5 4.8
Oscillatoria sp. 110.914 160.826 130.832 15.0 30.0 25.0 1.8 3.3 3.5
Oscillatoria sp. 070.012 098.442 080.006 2.5 22.0 24.0 1.0 3.2 3.2
Oscillatoria sp. 022.824 070.716 040.428 9.0 20.0 23.0 1.0 3.0 3.1
Oscillatoria sp. 084.624 083.834 060.234 10.0 20.0 33.0 1.0 2.9 3.5
Oscillatoria sp. 043.922 046.704 030.568 12.0 27.0 30.0 1.0 3.3 4.0
Table 2.
Growth and nitrogenase activity in hetrocystous cyanobacteria and biofilm in N free BG 11
medium
Isolates N-free
n moles of C2H4h-1 released protein (mg) dry weight (mg)
5 day 10 day 15 day 5 day 10 day 15 day 5 day 10 day 15 day
Anabaena sp. 5.8 4.9 3.0 11.1 31.0 33.9 2.0 4.2 5.0
Aulozera sp. 5.6 5.0 3.2 4.0 30.3 31.3 1.0 4.0 4.6
Nostoc sp. 2.8 2.5 2.0 11.0 25.5 30.0 2.0 3.8 4.0
Tolypothrix sp. 3.7 3.0 2.5 11.5 33.0 33.0 1.6 4.7 5.0
Westillopsis sp. 3.9 3.1 2.6 15.0 30.0 32.0 1.7 4.5 4.8
5 mix strains 22.7 20.0 16.7 15.0 30.0 25.0 2.5 5.3 5.5
Oscillatoria+ mix strains 27.1 25.1 20.0 2.5 22.0 24.0 3.0 6.0 6.2
was recorded at such low light intensity which was congenial
for nitrogenase activity. From the results it is clear that for
every n.mole of O 2
consumed in the absence of O 2
liberation,
mere is atleast 6 n.moles of C 2
H 4
formed and this ratio (1:6) is
recorded in all isolates tested. 5 individual and mixed strains
of heterocystous cyanobacteria tested showed maximum on
5 th day, 5mix with non heterocystous cyanobacteria showed
better than 5 individual mix (Table2). In Oscillatoria sp. oxygen
protection mechanism exists that allows maximum nitrogenase
activity at oxygen concentrations upto 0.15 atm (Stal and
Krumbein, 1985).
This study in non-heterocystous cyanobacteria as
biofertilizer for rice production has acentered and new area on
importance of biofertilizer development in our country. These
organisms are considered as weeds and therefore not chosen
as microbes for use as inoculants in low input agriculture.
These results have stimulated strong interest in the concept
of non-heterocystous cyanobacteria can be a member of
biofertilizer programme for rice production in India. In
cyanobacteria, endogenous reserve play a critical role during
nitrogen fixation in dark. However, in low light, the mechanism
must be quite different and it needs further study. The present
report further proves the fact that non-hetero-cystous
cyanobacteria could undoubtedly participate in rice field
nitrogen fixation during dawn and dusk period and also once
the rice seedlings are grown to cover the field.
ACKNOWLEDGEMENT
Authors thanks the Chairman, Arunai Engineering
College, Anna Bioresearch Foundation, Tiruvannamalai for
providing lab. facilities and preparation of this manuscript
LITERATURE CITED
Fredriksson, C and B.Bergman, 1995. Nitrogenase quantity varies
diurnally in a subset of cells within colonies of the non- heterocystous
cyanobacteria Trichodes-mium spp. Microbiology, 141:2471-2478.
Lowry, D.L., Rossbrough, N.J., Fair, A.L. and Randall, R.J., 1951. Protein
measurement with folinphenol reagent. J.Biol.Chem., 193:265-
275.
Parameswaran, P. and Shanmugasundaram, S. 1996. Nirogenase activity
in non-hetrrocystous cyanobacteria, III Asia-Pacific Conference
on Agricultural Biotech., Thailand., vol (13). pp.15-17.
Parameswaran, P and Anand, N. 2007. The effect of light intensity on
nitrogen fixation of fresh water cyanobacterium Oscillatoria sancta.
J. Biol., 21: 271-284
Rippka, R., Deruelles, J., Waterbury, J.R., Herdman, M. and Stanier,
R.Y. 1970. Generic assignments, strain histories and properties of
pure cultures of cyanobacteria. J. gen. microbiol., 111: 1-61.
Stal, L.J. and Krumbein, W.E., 1985. Oxygen protection of nitrogenase
in the aerobically nitrogen fixing non-heterocystous cyanobacterium
Oscillatoria sp. Arch. Microbiol., 143: 72-76.
Stewart, W.D.P., Fitzgerald, G.P. and Burris, R.H. 1967. In situ studies
on nitrogen fixation using the acetylene techniques. Proc. Natn.
Acad. Sci. USA, 58: 2071-2078.
Received on 27-11-2009 Accepted on 02-12-2009

Trends in Biosciences 2 (2): 61-63, 2009
Morphological Traits as Descriptors for Characterization of Important Composites
of Maize in Kashmir Valley
F.A. NEHVI., AJAZ, A. LONE., VASEEM YOUSAF., M.I. MAKHDOOMI AND S.A. DAR
Karewa Damodor Research Station, Sher-e-Kashmir University of Agricultural Sciences & Technology, Kashmir,
Post Box No. 905, GPO Srinagar, Kashmir, J & K, email: ajaz999@gmail.com
ABSTRACT
The objective of the study was to analyze the variation in
morphological characteristics of important composites of maize
grown in Jammu and Kashmir. Identification key for
characterizing the maize genotypes was based on different
morphological traits and significant differences were observed
among the genotypes and all the genotypes could be identified
on the basis of these particular morphological traits. There
was consistency of the results from the replicated plots
indicating reliability of these traits as a tool for genotypes
identification and their subsequent characterization.
Morphological traits are widely accepted in plant variety
protection, registration, and inscription and patenting as
descriptors that are capable of showing identity, uniformness
and distinctness.
Key words Characterization, morphological traits, maize
Variety registration is an important area of plant genetic
resource characterization and utilization and the prerequisite
for this is that there should be proper description of the
varieties based on their various morphological, biochemical
or molecular traits. Accurate description of the varieties will
be beneficial for agriculture in general and plant breeding in
particular. The “Plant Variety Chain” covers all the steps from
plant genetic resources to the end product. Beginning with
promising genetic resources, the breeder develops the new
variety by either using this germplasm directly or by
incorporating it into existing varieties. The varieties
developed have to be tested for DUS, whereby the
breeder can request Plant Variety Protection (PVP) or utility
patents. It is commonly known that morphology can be of
dubious taxonomic reliability because of environmental
interaction and largely unknown mechanisms of genetic
control of these traits (Camussi, et al., 1985). However,
problems associated with the interpretations of morphological
descriptions can be minimized by measuring the traits carefully
and by limiting comparisons to those traits for which effects
of environmental interactions are minimal (Goodman and
Patemiani, 1969) Accurate morphological descriptions of
cultivars have provided the basis of assurance to farmers and
the dealers that they are being offered specific varieties or
classes of product to certain minimum standards of quality
and purity. Besides, continued usages of morphological
features to describe varieties indicate the popularity as
descriptors. Maize being model crop for plant breeders and
crop improvement programmes have resulted in manifold
addition to the diversification of varietal profile and the huge
task ahead is to preserve the genetic treasure loaded in these
varieties. In the present study, an attempt was made to
characterize some important maize composites of maize
cultivated widely in Jammu and Kashmir so as to develop
reliable and easily observable identification catalogue.
MATERIALS AND METHODS
The present study was conducted at Karewa Damodor
Research Station, a lead centre for maize research in Jammu
and Kashmir during kharif seasons of 2007 and 2008. The
experimental material consisted of eight promising composites
of maize viz., C 6, C 8, C 14, C 15, Super 1, Shalimar KG 1,
Shalimar KG 2 and KDM 438. These genotypes were planted
in three replications and recommended package of practice
was adopted in terms of plant geometry, nutrient inputs and
management practices. The genotypes were studied for twenty
seven morphological markers as suggested in DUS testing
protocol of Directorate of Maize Research (DMR), New Delhi.
The characters studied comprised of leaf traits, flowering traits,
kernel traits and general plant traits which included all peculiar
morphological features associated with a particular genotype
for its identification. Data was recorded carefully on ten
randomly selected plants from each plot to end up with the
interpretations.
RESULTS AND DISCUSSION
Plant morphological traits for all the maize composites
are presented in Table 1. All the maize composites under study
showed less variation in terms of leaf angle between blade
and stem. All the genotypes were showing small leaf angle,
straight attitude of blade except KDM 438 were it was drooping
and there was no anthocyanin colouration of the sheath in all
the genotypes. Composites C 8, Shalimar KG 1 and Shalimar
KG 2 possessed narrow width of leaf blade and for rest of the
genotypes it was medium. Anthocyanin colouration at the
base of the glumes was present only in C 14 and C 15, while
colouration of glumes excluding base was absent in all the
composites. Time of anthesis and silk emergence ranged from
early to medium in all the composites. Anthocyanin colouration
of the anthers was found only in composite C 14. Coloured
brace root development was observed only in composite

6 2 Trends in Biosciences 2 (2), 2009
Table 1.
Summary of different plant morphological traits of Maize genotypes
Characteristics
Genotypes
C 6 C 8 C 14 C 15 Super 1 Shalimar
KG 1
Shalimar
KG2
Leaf : angle between blade and stem Small Small Small Small Small Small Small Small
KDM 438
Leaf attitude of blade Straight Straight Straight Straight Straight Straight Straight Drooping
Anthocyanin colouration of brace roots Absent Absent Absent Absent present Absent Absent Absent
Time of anthesis Medium Medium Early Early Medium Early Early Early
Anthocyanin colouration of base of glumes Absent Absent Present Present Absent Absent Absent Absent
Anthocyanin colouration of glumes excluding base Absent Absent Absent Absent Absent Absent Absent Absent
Anthocyanin colouration of anthers Absent Absent Present Absent Absent Absent Absent Absent
Density of spikelets Sparse Sparse Sparse Sparse Dense Sparse Sparse Sparse
Angle between main axis and lateral branches Wide Wide Wide Wide Wide Wide Wide Narrow
Tassel attitude of lateral branches Curved Curved Curved Wide Wide Strongly
curved
Strongly
curved
Time of silk emergence Medium Medium Early Medium Medium Early Early Medium
Anthocyanin colouration of silks Absent Absent Present Absent Absent Absent Absent Absent
Anthocyanin colouration of sheath Absent Absent Absent Absent Absent Absent Absent Absent
Tassel length of main axis above lowest side branch Medium Medium Medium Medium Medium Short Short Medium
Plant length Medium Long Medium Medium Medium short short Long
Ear placement Medium Medium Medium Medium Medium Low Low Medium
Width of leaf blade Medium Narrow Medium Medium Medium Narrow Narrow Medium
Ear length without husk Long Long Long Long Long Medium Medium Medium
Ear diameter Large Large Medium Large Large Large Large Medium
Ear shape
Conico
cylindrical
Conical Conical Conical Conico
cylindrical
Conico
cylindrical
Conico
cylindrical
Grain rows per ear Medium Many Medium Many Many Medium Medium Many
Curved
Conico
cylindrical
Type of grain Semi flint Dent Flint Dent Flint Flint Flint Semi Flint
Colour of top of grain Orange White Orange Yellow
with cap
Colouration of glumes of cob White White Light
purple
White
Yellow Yellow Yellow Yellow
Light
purple
White White White
Kernel row arrangement Straight Straight Straight Straight Straight Straight Straight Straight
Kernel shape Round Indented Indented Indented Toothed Round Round Round
1000 kernel weight (g) 224 264 271 210 252 201 198 250
Super-1. Anthocyanin colouration of the different floral parts
and brace roots remained constant over years and locations
under optimal growth conditions. However, little variation was
observed in the colouration pattern when the crop faced
certain stress conditions like drought or excess soil moisture
conditions (Zaidi, et al.,2007). This signifies the importance
of these traits to be used as morphological markers for diverse
breeding objectives.
Spikelet density was found to be dense only in
composite Super 1 making it good pollen shedder and can be
used as a male parent in crop improvement programme, rest of
the composites had sparsely placed spikelets. Plant height
was found to be long in KDM 438 and C 8, where it ranged
between 180-210 cm. Even though their plant height was on
the higher side but still their ear placement was medium as in
most of the genotypes proving their ability to withstand
lodging caused by higher wind speeds or torrential rains. On
the basis of grain type the composites were categorized into
three classes, KDM 438 and C 6 had semi flint grain type,
Super 1, Shalimar KG 1, Shalimar KG 2 and C 14 had flint
type of grain while as C 8 and C 15 had dent type of grain.
Only C 8 composite possessed white colouration of the grain
and C 6 and C 14 has orange type of kernel. Kernel row
arrangement was found to be straight in all the composites.
1000 kernel weight ranged from 198 g in Shalimar KG 1 to
271g in C 14.

NEHVI et al., Morphological Traits as Descriptors for Characterization of Important Composites of Maize in Kashmir Valley 6 3
The morphological differences observed in these eight
composites on the basis of distinct plant traits can be used as
a clear cut identification index of these composites and testing
their genetic purity. However there are certain morphological
traits like leaf angle, attitude of blade, colouration of brace
roots, colouration of glumes excluding base, anthocyanin
colouration of the sheath etc. which could not discriminate
the genotypes and the possible reason for it may be attributed
to lower inherent genetic variability in these particular traits
and increased number of genetically related releases by plant
breeders that has made unique identification especially by
morphological markers is more difficult to achieve (Smith and
Smith, 1989). The problem of unique identification becomes
more acute when convergent selection towards similar
morphologies is practiced (Wagner and McDonald, 1981)
The morphological traits can be reliable descriptors at
least of phenotype, provided that data is collected with highest
care from replicated plots across years and locations (Smith
and Smith, 1992). Further the traits which lack correlated
response or which are independent of each other should be
used as descriptors.
LITERATURE CITED
Camussi, A., Maviano, E. O., Camnski, T. and Kagzmarik, Z. 1985.
Genetic distances based on quantitative traits. Genetics, 111:945-
962
Goodman, M. M. and Patemiani, E. 1969. The races of maize III.
Choices of appropriate characters for racial classification. Eco.
Bot., 23: 265-271.
Zaidi, P.H., Maniselvan, P., Sultana, R., Yadav, M., Singh, R.P., Singh,
S.B., Dass, S. and Srinivasan, G. 2007. Importance of secondary
traits in improvement of maize (Zea mays L.) for enhancing
tolerance to excessive soil moisture stress. Cereal Research
Communication, 35(3): 1427-1435
Smith, J.S.C. and Smith, O. S. 1989. The description and assessment of
distance between inbred lines of maize I. the use of morphological
traits as descriptors. Maydica, 34: 141-150.
Smith, J.S.C. and Smith, O. S. 1992. Fingerprinting crop varieties. Adv.
Agron., 47: 85-140
Wagner, C.K. and McDonald, M.B.1981. Identification of soybean
(Glycine max L.) cultivars using rapid laboratory techniques Agri.
Res. Dev. Res. Bull., pp. 1133
Recieved on 5-10-2009 Accepted on 30-10-2009

6Trends 4 in Biosciences 2 (2): 64-65, 2009
Trends in Biosciences 2 (2), 2009
Avoidable Yield Loss Due to Meloidogyne incognita and Other Parasitic Nematode
in Fieldpea (Pisum sativum) in Field Condition
M.G. HAIDAR, T.H.ASKARY* AND S.S.ALI*
Rajendra Agricultural University, Pusa, Samastipur 848 125
*Indian Institute of Pulses Research, Kanpur 208 024
ABSTRACT
Avoidable yield losses due to infestation of Meloidogyne
incognita and other plant parasitic nematodes on fieldpea cv.
Bonnevilla was studied under field condition for three
consecutive years. Paired plot technique was employed using
carbofuran @ 2 kg/a.i./ha along with untreated control,
replicated six times. Yield of fieldpea was declined 18.32% in
untreated plots as compared to treated plots. There was an
increase of 52.06 % in total population of plant parasitic
nematodes in untreated plots while a decrease of 60.5 % over
initial nematode population was recorded in treated plots.
There was a significant reduction in rhizobium nodulation in
nematode infested plots than to treated plots.
Kew words
Meloidogyne incognita, fieldpea, yield losses, plant
parasitic nematodes
Fieldpea (Pisum sativum) is grown on 0.78 mh area with
production of 0.71 mt and mostly used as vegetable and also
in Indian diet. Fletcher as early as 1914 observed root-knot
nematode infestation and injuries to fieldpea plants while
Zenmeyer, 1962 considered fieldpea as important host of
different species of root-knot nematodes. Parvatha Reddy and
Singh, 1965 recognized Meloidogyne incognita as serious
problem for fieldpea causing considerable losses of pod yield.
Pathogenicity of M. incognita in fieldpea was established by
Bhagwati and Phunkan, 1991 that significant reduction started
at and above 1000 larvae/plant.
MATERIALS AND METHODS
In view of the importance of root-knot nematodes in
particular and other plant parasitic nematodes in general on
field pea an experiment was conducted in infested field to
estimate the yield losses caused by this pest. Fieldpea cv.
Bonnevilla was sown in nematode infested microplots. Paired
plot technique was employed with two treatments, treated
with carbofuran @ 2 kg a. i.ha. and untreated control, replicated
six times in a plot size 2 x 2.5 qm. and repeated for three
consecutive years. Nematicide was applied at the time of
sowing along with basal doses of fertilizers. Normal
agronomical operations were done as and when required. After
harvest yields were recorded from each plot. Pre-treatment
and post harvest nematode population was also estimated.
RESULTS AND DISCUSSION
Data were pooled for three years, indicated that there
was an increase of 52.06 % in total population of parasitic
nematodes in untreated plots while a decrease of 60.5 % over
initial population was observed in treated plots. On the other
hand, the yield of the fieldpea was declined to the tune of
18.32 % in untreated plots as compared to treated plots.
Besides losses in yield there was significant reduction in
rhizobium nodulation in infested plots as compared to treated
plots (Table 1).
It is well established that nematode caused yield losses
to different pulse crops including fieldpea. Sharma, 1989,
estimated 20-33 % reduction in yield of fieldpea due to
Meloidogyne spp. Dalal and Rajesh, 1999), estimated loses
in yield of 15.8 % to fieldpea due to Rotylenchulus reniformis
under field condition. Bhagwati and Phunkan, 1999, Rajesh
Table 1.
Avoidable yield loss due to Meloidogyne incognita
and other plant parasitic nematodes in fieldpea.
Treatment Nematode
population/
100 g soil
Initial Final
Treated 1003.4 396.83
(-60.5)*
Root-knot
index
Bacterial
nodule
Yield q/ha
2.5 131 26.4
Untreated 1003.4 1525.83* 4.0 19.8
(-84.9)**
19.93
(-18.32)**
CD at 5% - 363.793 - 27.432 3.608
* - Figures in prenthesis indicate % decrease (-) or increase (+) over
initial population.
** - Figures in prenthesis indicate % reduction over treated plots.
Table 2.
Nematode
Plant parasitic nematode population/100 g soil
associated with fieldpea crop.
Nematode population/100g soil
Initial Treated Untreated
Meloidogyne incognita 276.7 143.3 461.0
Rotylenchulus reniformis 200.0 94.0 390.0
Pratylenchus zeae 36.7 7.3 80.0
Helicotylenchus indicus 55.0 5.0 51.7
Tylenchorhynchus mashhoodi 386.7 140.63 488.13
Hoplolaimus indicus 48.3 6.6 55.0
Total parasitic nematodes 1003.4 396.83
(-60.5)*
1525.83
(52.06)*
Saprozoic nematodes 706.7 423.3 481.7
Total 1710.1 820.13 2007.53

HAIDAR et al., Avoidable Yield Loss Due to Meloidogyne incognita and Other Parasitic Nematode in Fieldpea 6 5
and Dalal, 1998 observed that M. incognita and reduced the
rhizobium nodulation in fieldpea.
Developing cultivars with resistant to root-knot, Lesion
and Reniform nematodes are required in view of heavy yield
losses incurred in fieldpea by these nematodes. Emphasis
needs to be placed on the use of integrated nematode
management (INM) based on judicious nematicide use and
biological nematode control through appropriate cropping
system and using fieldpea resistant varieties to get more yields.
LITERATURE CITED
Bhagwati, B and Phunkan. 1993. Development of root-knot nematode
Meloidogyne incognita on pea seedlings of different ages. Journal
of the Agricultural Science Society of North East India. 6: 81-82.
Dalal, M.R. and Rajesh, V. 1998. Estimation of loss in pea (Pisum
sativum) due to Rotylenchulus reniformis. In: Nematology:
Challenges and Opportunities in 21st century. Proceedings of the
3rd International Symposium of Afro-Asian Society of
Nematologist. Sugarcane Breeding Institute, Coimbatore, India,
April, 16-19, pp. 8-9.
Fletcher, T. B. C. 1914. Some south Indian insects and other animals of
importance. Madras Agril. Dept. Bull., pp. 45.
Parvatha Reddy, and Singh, R. 1981. Assessment of yield loss in okra,
brinjal, french bean, cowpea and pea due to root-knot nematode.
Proc. Third Int. Symp. P. Path., New Delhi, pp. 93-94.
Rajesh, V. and Dalal, M.R. 1998. Interrelation between Rotylenchulus
reniformis and Rhizobuim leguminosarum on Pisum sativum. In:
Nematology: Challenges and Opportunities in 21 st century.
Proceedings of the 3 rd International Symposium of Afro Asian
Society of Nematologists, Sugarcane Breeding Institute, Coimbatore,
India, April, 16-19, pp. 98-101.
Sharma, G. 1998. Estimated loses due to root-knot nematodes,
Meloidogyne incognita and Meloidogyne javanica in pea. Int.
Nematol., Newsl., 6 (1): 26-29.
Zenmeyer, W.J.C. 1962. Pea diseases, U.S. Deptt. Agric. Handbook.,
pp. 1-30.
Received on 07-10-2009 Accepted on 30-11-2009

6Trends 6 in Biosciences 2 (2): 66-67, 2009
Trends in Biosciences 2 (2), 2009
Management of Shoot and Capsule Borer, Conogathes punctifaralis L in Castor by
Intercropping
B.S.PATEL AND I.S.PATEL
Main Castor and Mustard Research Station, Main pulses Research Station,
S.D.Agricultural University, Sardarkrushinagar 385 506;
email:dr.ispatel@gmai.com
ABSTRACT
An experiment on impact of intercropping on the incidence of
shoot and capsule borer Conogathes punctifaralis L in castor
crop was carried out at Main Castor and Mustard Research
Station, Sardarkrushinagar Dantiwada Agricultural University,
Sardarkrushinagar during 2007-08. Results revealed that lowest
(34.18 %) infestation of capsule borer was recorded in castor
inter cropped with cow pea (1:2). Maximum seed yield of castor
was obtained from green gram (1:1) inter crop but it was not
differed from sesame (1:1) or (1:2) inter crop. Overall results
revealed that green gram and sesame were profitable by
achieving higher seed yield and reducing the infestation of
capsule borer.
Key words
Ricinus communis, Conogathes punctifaralis,
intercropping, castor
Castor (Ricinus communis) is an important industrial oil
seed crop cultivated in India. Castor shoot and capsule borer,
Conogathes punctifaralis is the one of the important pest of
castor in India. In the traditional area of Gujarat, it is grown
mostly under irrigated condition where there are several
production constraints. Among them, arthropod pests are of
greater importance. In India more than 107 species of insects
and 6 species of mites are recorded on castor at different
phenological stages of the crop. Several studies indicated
that diversification practices such as inter cropping in castor
are beneficial because of reduced pest damage (Songa, et al.,
2007).Keeping these consideration in view, we attempted to
examine how the incidence of insect pests differ in a
intercropping system as compared to a sole castor crop
situation
MATERIALS AND METHODS
A field trial was conducted to evaluate the impact of
intercrops on the incidence of shoot and capsule borer in
castor at Main Castor and Mustard Research Station,
Sardarkrushinagar Dantiwada Agricultural University,
Sardarkrushinagar during 2007-08. The experiment was carried
out with eight treatments in three replications (Table 1). All
intercropping systems were followed in 1:1 and 1:2 ratios except
in case of intercropping with cow pea and castor where row of
castor and cowpea was 1:2. Castor and intercrops were drilled
in furrows opened in the experimental field. Castor rows were
spaced at 90 cm and sowing distance between plant to plant
was kept 60 cm. Intercrops were sown in between rows of
castor in an additive manner so as to keep the population of
castor plants constant across the different cropping systems.
All packages of practices were applied as per
recommendations. No pest control measures were applied
during the entire period of experiment. Observations on
damaged capsule and total capsule were recorded from five
selected and tagged plants in each treatment at spike order
wise picking and thus per cent infestation of pest damage
was worked out. Seed yield was also recorded from each net
plot at every picking and thus average seed yield per hectare
was computed.
Table 1.
Infestation of castor capsule borer in castor
intercropped with different crops
Treatments Per cent infestation Seed yield (kg/ha)
1. Sole castor 47.40 (53.67)* 2541
2. Green gram (1:1) 53.36 (63.85) 2906
3. Green gram (1:2) 36.43 (34.89) 2634
4. Sesame (1:1) 40.10 (41.45) 2635
5. Sesame (1:2) 38.80 (39.17) 2108
6. Moth beam (1:1) 51.39 (60.44) 2213
7. Moth beam (1:2) 49.86 (57.85) 2290
8. Cowpea (1:2) 35.67 (34.18) 1948
C.D (0.05) 12.02 399
Figures in parenthesis are original value and outside arcsine transformed
value
RESULTS AND DISCUSSION
Differences among different combinations of
intercropping with respect to the infestation of shoot and
capsule borer were significant. Among all the combination,
cowpea intercropped with castor (1:2) had lowest infestation
of capsule borer (34.18) and was not differed from green gram
(1:2), sesame (1:2) sesame (1:1) and sole crop. Significantly
maximum infestation of capsule borer was recorded in the
intercrop of green gram with castor (1:1) which was at par
with moth bean (1:1) and (1:2). So far as yield is concerned,

PATEL & PATEL, Management of Shoot and Capsule Borer, Conogathes punctifaralis L in Castor by Intercropping 6 7
maximum yield was obtained in inter crop of green gram (1:1)
with castor (2906 kg/ha) but it was not differed from green
gram (1:2 ) and sesame (1:1). Similar results were also reported
by Srinivasan Rao, 2009 who reported some impact of
intercropping systems on the incidence of capsule borer in
castor crop. It is concluded that castor crop intercropped with
green gram either (1:1) or, (1:2) and sesame 1:1 were found
profitable and could gave higher seed yield and low infestation
of capsule borer.
LITERATURE CITED
Songa, J.M., Jiang, N., Schulthess, F. and Omega, C. 2007. The role of
intercropping of different cereal species in controlling lepidopteron
stem borer in maize in Kenya. Journal of Applied Entomology,
131: 40
Srinivasan Rao, M., Ram Rao, C.A.; Srinivas, K.G., Pratibha, G., Vani,
Sree. and Venkateswardu, B. 2009. Intercropping as a tools of
Integrated Pest Management in castor, J.Oil Seed Research, 26
(Sp.): 509-511
Received on 09-11-2009 Accepted on 30-11-2009

6Trends 8 in Biosciences 2 (2): 68-69, 2009
Trends in Biosciences 2 (2), 2009
Seed Vigour Studies in Relation to Position of Pod in Pea
PRABHA SHANKAR SHUKLA AND RAJENDRA PRASAD
Department of Seed Science & Technology, GBPUA&T, Hill Campus, Ranichauri 249 199, Tehri Garhwal,
Uttarakhand; email: ps.shukla@rediffmail.com
ABSTRACT
An experiment was conducted on pea (Pisum sativum L.) cv. VL-
1 to evaluate the effect of position of pod on the plant on seed
vigour under controlled conditions during rabi season of 2004-
05. Hundred plants were selected randomly in four replications
and divided into three equal parts, i.e. upper, middle and lower
at the time of harvesting. Pods were picked and threshed by
hand, dried in sun and kept in plastic jars. The seeds harvested
and threshed from rest of the plants without partitioning formed
the control treatment. The method employed for vigour
measurements consisted 1000 seed weight, first count, standard
germination, seedling length, seedling fresh weight, seedling
dry weight, vigour index I st and II nd , electrical conductivity of
seed leachate, leachate absorbance at 480 nm, topographic
tetrazolium test and quantitative T z.
All the four treatments of
pea under study were significantly different from each other
with respect to most of the vigour tests employed, except 1000-
seed weight and topographic T z
test, while no significant
differences were observed in the result of control and seeds
received from middle portion with respect to their seed vigour.
The seeds from upper portion exhibited excellent performance
while those from middle portion and control treatment showed
moderate performance. Seed from lower portion exhibited
significantly poor performance in majority of the tests.
Key words
Position – pod, pea, seed vigour, germination,
seedling.
Seed quality depends on a number of factors. Seed
maturity and time of harvest are among the major
considerations in deciding the quality of seed. Seed quality
may be impaired while the seeds are still on the mother plant
(Pollock, 1972). Physico-morphological and physiological
changes might set in, if the seeds are retained on the mother
plant for a longer duration beyond physiological maturity
(Ovcharov and Kizilova, 1966). This is particularly shown
under inimical environmental conditions which would lead to
the development of either hard seeds or off colour seeds in
pulses (Dharmalingam and Ramakrishnan, 1978). Indeterminate
flowering of pulse crops leads to differential maturity of seeds
resulting in great difference in seed quality. This poses
challenging problems to seed researchers in their experimental
results. The present study was conducted to evaluate the
quality of seeds in relation to their position of pod on the
plant in Pisum sativum L. variety VL-1.
MATERIALS AND METHODS
Seed quality differences in relation to pods on plant
were studied at Hill Campus, Ranichauri during rabi season.
The crop was sown in the month of November in four
replications. The data were recorded on randomly selected
hundred plants of similar height and plants were divided into
three equal parts i.e. upper, middle and lower with the help of
scissors at the time of harvesting. Pods of each equal part
were picked and threshed by hand and kept in plastic jars.
The seeds received from rest of the plants without partitioning
used as the control. Seed quality attributes studies for vigour
measurements included 1000 seed weight, first count, standard
germination, seedling length, seedling fresh weight, seedling
dry weight, vigour index, topographic tetrazolium test,
electrical conductivity of seed leachate, leachate absorbance
at 480 nm and quantitative topographic tetrazolium test.
The mean 1000 seed weight of each treatment was taken
by electronic balance. Seeds were kept for germination in BP
(Between Paper) method at 20 0 C in germinator. Normal
seedlings were counted at 5 th day for first count and 8 th day
for standard germination. Out of total normal seedlings, ten
were selected randomly for measuring mean length of seedling
and mean fresh weight. Seedlings taken for fresh weight were
kept in an oven at 80 0 C for 24 hours to determine the mean dry
weight. Vigour index were calculated by two ways i.e. standard
germination x mean length of seedling and standard
germination x dry weight of seedlings. In topographic
tetrazolium test, seeds were soaked in fresh water for four
hours then seed coats were removed and kept in 0.2%
tetrazolium solution for complete staining. Viable and non
viable seeds were counted as per standard. For leachate test,
replications of 5 g seeds were taken in different beakers for
each treatment. Seeds were surface sterilized with 0.1 %
mercuric chloride solution then washed thoroughly with
distilled water and soaked in 25ml of distilled water for 12
hours. The electrical conductivity was measured in milimohose
/cm with the help of conductivity meter at room temperature.
(Leachate absorbent and quantitative tetrazolium test were
measured at 480 nm of wave length through
spectrophotometer).
RESULTS AND DISCUSSION
Evaluation of position of pod on plant provides a better
understanding of seed quality as well as causes for
heterogeneity in seed lot. The seeds within a plant on lower,
middle and upper position showed significant differences in
various studies of seed vigour test i.e. first count, standard
germination, seedling length, seedling fresh and dry weight,

SHUKLA AND PRASAD, Seed Vigour Studies in Relation to Position of Pod in Pea 6 9
Table 1.
Mean value of different seed vigour tests with respect to position of pod in pea variety VL-1.
Characters
Position of pod
Lower Middle Upper Control CD at 5%
1000 - seed weight (g) 197.915 201.927 204.36 198.515 11.711
First count (% germination) 62.50 77.50 79.25 75.75 5.022
Standard germination (%) 83.75 96.25 97.25 94.00 4.363
Seedling length (cm) 11.783 16.725 20.025 16.965 3.555
Seedling fresh weight (g) 5.410 6.625 6.930 6.420 1.010
Seedling dry weight (g) 1.5130 1.6795 1.7925 1.6217 0.165
Vigour index I 986.83 1609.78 1947.43 1594.71 285.030
Vigour index II 126.71 161.65 174.32 152.44 20.703
T z test (% viability) 94.75 95.00 95.50 95.25 1.484
Electrical conductivity (milimohos /cm) 0.334 0.199 0.154 0.180 0.066
Quantitative T z test (Transmitant at 480 nm) 56.95 48.58 34.73 47.43 3.862
Leachate absorbent (480 nm) 0.129 0.124 0.072 0.117 0.049
vigour index I and II, electrical conductivity of seed leachate,
quantitative T z
test and leachate absorbent except 1000 seeds
weight and topographic tetrazolium test.
There was no significant difference with respect to 1000
seed weight among all treatments. In first count and standard
germination, significantly maximum germination was observed
in upper portion of pod seed while lowest values were
recorded in lower portion of pod. No significant difference
was observed between middle portion of pod seeds and
control. Average seedling length, fresh weight and dry weight
of seedling were found significantly higher in seeds of upper
portion of pods and lowest in seeds of lower portion of pods
while seeds received from middle portion of plant pod and
control were insignificant in moderate performance. In vigour
index, i.e. standard germination x seedling length and standard
germination x dry weight of seedlings were significantly more
in upper portion of pod and lowest in lower portion of pod
while no significant difference was recorded between seed
received from middle portion and control and results were
also at par with upper portion of pod seed. No significant
differences were observed with respect to topographic
tetrazolium test among all treatments. Electrical conductivity
is adversely proportional to seed vigour. Hence, maximum
significant value was observed in seeds lower portion of pods
and the least value was observed in upper portion and results
of middle portion and control were at par with upper portion
of pods. In quantitative tetrazolium test, transmitant at 480 nm
was also significantly higher in lower portion of pods, while
significantly least observation was recorded in seeds of upper
portion of pods among all the treatments and the transmitant
of middle portion pod seed and control was non significant.
In vigour evaluation with respect to seed leachate absorbent
at 480 nm was also significantly least in seeds of upper portion
of pods over lower and middle portion of pod seed and results
at par with control. The result of seeds from lower portion
and middle portion of pods along with control were non
significant. 1000- seed weight did not differ significantly in
any treatment indicating that all seeds received from different
portion of plant were physiologically matured before
harvesting. In most of the vigour tests i.e. first count, standard
germination, length, dry and fresh weight and vigour index of
seedling were observed significantly higher in seeds received
from upper portion of the plant. It is very likely that the seeds
received from lower and middle portion were stored for a long
period on the plant after seed gained physiological maturity
than the seeds from upper portion of pods similar findings
were also reported by Adenhinju, 1971 and Abdul Babi, 1960
in cocao beans . It also indicates that seeds received from
lower and middle portion and control were as viable as that
from upper portion of pods but not so in vigour as that from
upper portion.
The topographic tetrazolium test did not show in any
significant differences among all the treatments. It was
indicated that all seeds were viable but not as vigourous as
that from upper portion. Electrical conductivity, quantitative
tetrazolium test (transmitant at 480nm) and seed leachate
absorbent at 480 nm is negatively correlated with seed vigour.
It can be concluded that result of above test were significantly
higher in seeds from upper portion of pods than seeds received
from lower portion of the plant. Farmers can very well save
upper portion of pods for seeds for the next season crop.
Vigorous seed may also be increase the yield of per unit area.
LITERATURE CITED
Abdul Babi. 1960. Effect of maturity and ageing on activities of glucose
utilization germination, growth and O 2
uptakes: Principle of seed
science and Tech. kalyani Publishers, pp. 38.
Adenhinju.1971. Effect of maturity in cocao beans (Theobroma cacao).
Principles of seed technology, Kalyani Publishers, pp. 38.
Dharmalingam, C. and Ramakrishnan, V. 1978. Seed quality in relation
to seed size and seed coat colour variation in black gram (Vigna
mungo L. Hepper ), Seed Res., 6:101-109.
Ovcharov, K.E. and Kizilova, K.G. 1966. Differences in seed quality
and plant productivity. In: physiological basis of seed germination,
Amerind Publishing, New Delhi. pp. 54-140
Pollock, B.M. 1972. Effect of environment after sowing on viability.
Viability of seeds (ed. E.H. Roberts), Chapmans Hill Ltd., London
EC-4. pp. 150-171
Recieved on 27-11-2009 Accepted 4-12-2009

7Trends 0 in Biosciences 2 (2): 70-73, 2009
Trends in Biosciences 2 (2), 2009
Infectivity of Spodoptera litura (F.) (Lepidoptera: Noctuidae) by Certain Native
Entomopathogenic Nematodes and Their Penetration in Test Insect and in vivo
Production
RASHID PERVEZ* AND S. S. ALI
Indian Institute of Pulses Research, Kanpur 208 024
*Indian Institute of Spices Research, Calicut 673 012
Email: rashid_pervez@rediffmail.com, ss_ali@rediffmail.com
ABSTRACT
A study was conducted to assess the susceptibility of tobacco
caterpillar, Spodoptera litura (F.) to Steinernema mushtaqi, S.
masoodi, S. seemae and S. carpocapsae (Lucknow strain)and in
vivo production of these species carried out during the year
2007. On exposure of the insect pest at a dose of 100 IJs/ml
each of S. masoodi and S. mushtaqi were found lethal against S.
litura, and cent per cent mortality after 72 h post exposure. The
rest of test EPN species brought cent per cent to insect mortality
after 96 h. Observation on in vivo production on tobacco
catterpillar showed that, highest yield of S. mushtaqi (0.79 X
10 5 IJs/ cadaver was obtained, followed by S. seemae, (0.72 X 10 5
IJs/ cadaver), S. carpocapsae, (0.67 X 10 5 IJs/cadaver) and S.
masoodi, (0.51 X 10 5 IJs/ cadaver). The rate of penetration of
these species inside the S. litura larvae was also evaluated.
Among these, S. masoodi was found highest value (14.41±2.99;
CV = 20.74 %), followed by S. mushtaqi (11.25±4.82; CV = 42.84
%). This is the first report on infectivity of tobacco caterpillar
by four native species of EPNs and using the insect as a host for
culturing these nematodes.
Key words
Entomopathogenic nematodes, Steinernema spp.,
infectivity, Spodoptera litura, in vivo production
Entomopathogenic nematodes are potent candidate that
can be used as biopesticides against insect pests and can
replace the harmful chemicals. Third stage infective juveniles
contain cells of mutual enterobacteria in their intestine. As
the IJs penetrate the host’s natural openings and enter the
haemocoel, they release the bacteria which eventually and kill
the host by septicaemia and death of the insect within 24 to 72
h (Kaya and Gaugler, 1993). Steinernema spp. have a great
potential as biological control agents against insect pests
because of their wide host range.
EPN can be mass produced in vivo where the insect
serves as a small biological reactor. Greater wax moth, Galleria
mellonella L. has been widely used for in vivo mass
production of EPN. Other insects like Chilo sacchariphagus
indicus (Karunakar, et al., 1992 and 1999) Helicoverpa
armigera, G. mellonella and Corcyra cephalonica (Ali, et
al., 2005a and 2008) and Athalia proxima and Nezara viridula
(Pervez, et al., 2007 and 2008b) have been used for
multiplication of various species of Steinernema and
Heterorhabditis. They have recorded varying yield of infective
juveniles depend upon size of larvae of test insects.
Spodoptera litura is also known as the Oriental leaf
worm moth, Cluster caterpillar, Cotton leafworm, Tobacco
cutworm, Tropical armyworm, Taro caterpillar, Tobacco
budworm, Rice cutworm, and Cotton Cutworm. It has a very
wide host range of over 120 plant species, including pulses,
vegetables, plantation and horticultural crops etc. This pest
is considered to be of concern from a regulatory perspective.
It is believed to have potentially high economic impact in
terms of its direct pest damage and trade implications.
Present study was carried out to test the comparative
efficacy and penetration of four native species of EPN, namely,
Steinernema masoodi (Ali, et al., 2005b), S. mushtaqi (Pervez
et al., 2009b), S. seemae (Ali, et al., 2005b) and S. carpocapsae
[(Weiser, 1955); Wouts, Mráèek, Gerdin and Bedding, 1982]
against larvae of tobacco caterpillar S. litura. Studies on in
vivo production of the entomopathogenic nematodes from
test insect were also undertaken.
MATERIALS AND METHODS
Nematode and insect cultures
The culture of Steinernema mushtaqi, S. masoodi, S.
seemae and S. carpocapsae (Lucknow strain) were taken from
EPN laboratory of this institute which were earlier obtained
by baiting of soil samples from Lalitpur, Kanpur, Hamirpur
and Lucknow districts of Uttar Pradesh. All of these EPNs
were cultured on fully grown Galleria mellonella larvae as
per the procedure described by Woodring and Kaya, 1988.
Freshly emerged infective juveniles (IJs) were surface sterilised
in 0.1% Hyamine solution and stored in distilled water. The
test insect, S. litura larvae were collected from standing
chickpea crop from the Indian Institute of Pulses Research
Experimental farm and CSAU & T field and maintain the
population on plant leaves. All the insect cultures were
maintained at room temperature. The larvae were sorted out
and those of same size were taken for present study.

PERVEZ AND ALI, Infectivity of Spodoptera litura (F.) (Lepidoptera: Noctuidae) by Certain Native Entomopathogenic 7 1
Bioassay studies
To test the mortality of S. litura and mass production of
EPNs
In 6 well plate, one larvae of S. litura was kept in each
well and 100 infective juveniles (IJs) each of S. mushtaqi, S.
masoodi, S. seemae and S. carpocapsae were inoculated and
observation on their mortality were recorded after 24 h interval.
Each species of EPN tested separately and singly. The
experiment was conducted at 28 ±°C under BOD incubator
and replicated twelve times along with control. EPN infected
dead larvae of test insects were removed from each well and
kept on to white trap (White, 1927) for the emergence of EPN
from the body of insect and were collected daily, till the
emergence of IJs was stopped in about 15 days. From this
collection, the total emerged populations of EPN was counted
thrice under a Leica MS 5 stereoscopic binocular microscope
with the help of Syracuse counting dish and mean values
were worked out.
Rate of penetration of IJs into host
All test species of EPN as mentioned above have been
used in this study against final instar larvae of S. litura. The
assay was done in six well plate lined by Whatman No.1 filter
paper. One larvae of S. litura was kept in each well and each
EPN species at a concentration of 100 IJs/ml was sprayed
over the filter paper at the bottom of the well plate. The well
plate was kept at 28+2 0 C for 72 h or until a 100% morality
occurred and replicated six times. After that, the enzymatic
digestion of host larvae was done using 3 mL pepsin in a tube
and kept in a shaker incubator at 28 0 C, 120 rpm for 1 h. The
tubes were then shaken well for 10 s and returned to the shaker
for another 20 min after which 7 ml of tween 80 were added to
each tube and shaken very well and kept at 5 0 C for 48 h or
until the nematode count was done. Counting was done under
Leica MS 5 stereoscopic binocular microscope. The
penetration rate was calculated according to following formula
PR = N 1
x100/N 2
PR - penetration rate, N 1
- mean nematode number found
within host and N 2
- original nematode number used.
RESULTS AND DISCUSSION
Among these four test species of EPN, S. masoodi and
S. mushtaqi were found more pathogenic to S. litura as it
brought about cent per cent mortality within 72 h post exposure
followed by S. seemae and S. carpocapsae, where they gave
89 per cent mortalities, respectively after 72 h post exposure.
Then a cent per cent mortality was obtained at 96 h, post
exposure. No mortality was observed in control (Fig.
1).Infectivity of S. litura by these species was first time
reported.
In case of production of infective juveniles of test EPN
species from this insect showed that, highest yield of S.
mushtaqi which was 0.79 X 10 5 IJs/ cadaver, followed by S.
seemae, 0.72 X 10 5 IJs/ cadaver, S. carpocapsae, 0.67 X 10 5
IJs/cadaver and S. masoodi, 0.51 X 10 5 IJs/ cadaver (Fig. 2).
Data showed (Table 1) that, the rate of penetration of
the IJs of S. masoodi was the highest against S. litura after 72
h of exposure (14.41±2.99; CL = 1.90 at 95 %; SV = 8.99),
followed by S. mushtaqi (11.25±4.82; CL = 3.06 at 95 %; SV =
Table 1.
Penetration rate of different species of EPN at dose
of 100 IJs/larvae.
S. EPN species Mean± SD, CV (%),
No.
(Range); SE
1. Steinernema mushtaqi 11.25± 4.82, 42.84,
(5-19);1.39
2. S. masoodi 14.41± 2.99, 20.74,
(9-19);0.86
3. S. seemae 9.50± 2.90, 30.52,
(5-15);0.83
4. S. carpocapsae 7.25± 3.36, 46.34,
(3-14);0.97
Confidence level
(95 %); SV
3.06 (23.29)
1.90 (8.99)
1.84 (8.45)
2.13 (11.29)
Fig. 1.
Mortality of S. litura by the four entomopathogenic
nematodes.
Fig. 2.
In vivo production of infective juveniles of the four
entomopathogenic nematodes on the S. litura.
(Sm= S. mushtaqi, Sma= S. masoodi, Ss= S. seemae,
Sc= S. carpocapsae)

7 2 Trends in Biosciences 2 (2), 2009
23.29). The lowest rate of penetration was observed in the S.
seemae (9.50±2.90; CV = 30.52 %; SE = 0.83; CL = 1.84 at 95 %;
SV = 8.45) and S. carpocapsae (7.25±3.36; CV = 46.34 %; SE =
0.97; CL = 2.13 at 95 %; SV = 11.29), respectively.
These results indicated that S. litura is susceptible to
four EPN tested, viz., S. mushtaqi, S. masoodi, S. seemae, and
S. carpocapsae. Similarly, Abbas and Saleh, 1998 reported
that pathogenicity of S. abbasi against 4 th instar larvae of S.
littoralis with the highest mortality (91.7 %) in 3 days posttreatment.
However, Heterorhabditis sp. (ELG), H. indica,
Heterorhabditis sp. (ELB) against S. littoralis recorded 100%
mortality within 24 h (Abdel-Razek and Abd-Elgawad, 2007).
These results are in confirmity with many other studies
showing that the nematode penetration were in accordance
with nematode insecticidal activity based on host mortality
(Kondo and Ishibashi, 1986; Mannion and Ansson, 1993;
Shannag, et al., 1994).
The present study revealed that the rate of penetration
could be used as a real measure of host infection. This could
be real too if we use standard methods of measurements.
Dunphy and Webster, 1988 and 1991 reported that, the
difference in the toxicity of bacterial symbionts is also related
to the difference in protinacious substances in their cell wall,
which may have led at the end to the relative destruction of
host hemocytes and finally the death of the host. The variation
in efficiency of the different nematodes under investigations
may be due to the difference in the bacterial symbionts (Forst,
et al., 1997; Boemare and Givaudan, 1998; Boemare, 2002).
It is to be concluded that, S. masoodi and S. mushtaqi
was found more fatal than other species of entomopathogenic
nematodes to the larvae of S. litura and provide a strong
possibility to use them as potential biopesticide for field
evaluation. The larvae of tobacco caterpillar was found
suitable, host for the multiplication of S. mushtaqi, and S.
seemae and this insect can be selected as an alternate host for
in vivo production of IJs of EPN under laboratory condition.
Further studies are required on the efficacy of these EPNs to
be used as a potential biopesticide for the management of
insect pest. Also to know the exact behaviour, pathogenicity,
mode of action and multiplication of these EPNs on cadaver
and its associated bacteria on S. litura larvae.
ACKNOWLEDGEMENT
The authors express their gratitude to Dr. Masood Ali,
Director, Indian Institute of Pulses Research, Kanpur, for
providing all the facilities for this study. First author is thankful
to Department of Science and Technology (DST), Ministry of
Science & Technology, Government of India, New Delhi, for
providing financial support.
LITERATURE CITED
Abbas, M. S. T. and Saleh, M. M. E. 1998. Comparative pathogenicity
of Steinernema abbasi and S. riobravae to Spodoptera littoralis
(Lepidoptera: Noctuidae). International Journal of Nematology,
8(1): 43-45.
Abdel-Razek, A. S. and Abd-Elgawad, M. M. 2007. Investigations on
the efficacy of entomopathogenic nematodes against Spodoptera
littoralis (Biosd.) and Galleria mellonella (L.). Archives of
Phytopathology and Plant Protection, 40(6): 414–422
Ali, S. S., Ahmad, R., Hussain, M. A. and Pervez, R. 2005a. Pest
management of pulses through entomopathogenic nematodes.
Indian Institute of Pulses Research, Kanpur, pp. 59.
Ali, S. S., Pervez, R., Hussain, M.A. and Ahmad, R. 2008. Susceptibility
of three lepidopteran pest to five entomopathogenic nematodes
and in vivo mass production of these nematodes. Archieves of
Phytopathology and Plant Protection, 41(4): 300–304.
Ali, S. S., Shaheen, A., Pervez, R. and Hussain, M.A. 2005b. Steinernema
masoodi sp. n. and Steinernema seemae sp. n. (Rhabditida:
Steinernematidae) from Uttar Pradesh, India. International Journal
of Nematology, 15(1): 89–99.
Boemare, N, Givaudan, A. 1998. Pathogenicity of the symbionts. In:
Pathogenicity of entomopathogenic nematodes versus insect
defense mechanisms: impact on selection of virulent strains (eds:
Simoes, Boemare, N. and Ehalers R-U). Luxembourg: European
Commission. pp. 3–7.
Boemare, N. 2002. Biology, taxonomy and systematics of
Photorhabdus and Xenorhabdus. In: Entomopathogenic
Nematology (ed. Gaugler R,), UK: CABI. pp. 35–56.
Dunphy, G. B. and Webster, R. B. 1988. Lipopolysaccharides of
Xenorhabdus nematophilus (Insecta: Lepidoptera) larvae. Journal
of G. Microbiology, 134: 1017–1028.
Dunphy, G. B. and Webster, R. B. 1991. Antihaemocytic surface
components of Xenorhabdus nematophilus var. dutki and their
modification by serum of non-immune larvae of Galleria mellonella.
Journal of Invertebrate Pathology, 58: 40–51.
Forst, S., Dowds, B., Boemare, N. and Stackbrandt, E. 1997. Xenorhabdus
and Photorhabdus spp: Buges that kill buge. Ann. Rev. Microbiology,
51: 47–72.
Karunakar G., David, H. and Easwaramoorthy, S. 1992. Influence of
temperature on infectivity, penetration and multiplication of
Steinernema feltiae, S. glaseri and Heterorhabditis indicus on
mortality of the host and multiplication of infective juveniles in
sugarcane internode borer, Chilo sacchariphagus indicus. Journal
of Biological Control, 6: 26-28.
Karunakar, G., Easwaramoorthy, S. and David, H. 1999. Susceptibility
of nine lepidopteran insects to Steinernema glaseri, S. feltiae and
Heterorhabditis indicus infection. International Journal of
Nematology, 9: 68-71.
Kaya, H.K. and Gaugler, R. 1993. Entomopathogenic nematodes. Annual
Review of Entomology, 38: 181-206.
Kondo, E. and Ishibashi, N. 1986. Frequency and propagation of
entomopathogenic nematode, Steinernema carpocapsae spp., on
the common cutworm, Spodoptera litura (Lepidoptera : Noctuidae).
Applied Entomological Zoologie, 21: 95–108.
Mannion, C. M. and Jansson, R. K. 1993. Infectivity of five
entomopathogenic nematodes to the sweet potato weevil, Cylas
formicarius (F.) (Coleoptera : Apionidae), in three experimental
arena. Journal of Invertebrate Pathology, 62: 29–36.

PERVEZ AND ALI, Infectivity of Spodoptera litura (F.) (Lepidoptera: Noctuidae) by Certain Native Entomopathogenic 7 3
Pervez, R., Ali, S. S. and Ahmad, R. 2007. Efficacy of some
entomopathogenic nematodes against mustard saw fly and in vivo
production of these nematodes. International Journal of
Nematology,17(1): 55-58.
Pervez, R., Ali, S. S. and Ahmad, R. 2008b. Efficacy of
entomopathogenic nematodes against green bug, Nezara viridula
(L.) and their in vivo mass production. Trends in Biosciences, 1(1,2):
49-51.
Pervez, R., Ali, S. S. and Asif, M. 2009b. A new species of
entomopathogenic nematodes Steinernema mushtaqi sp. n.
(Nematoda : Rhabditidae : Steinernematidae) from chickpea
rhizosphere. In: International Conference on Legumes (ICGL), at
Indian Institute of Pulses Research, Kanpur on 14-16’ Feb.
Shannag, H. K. , Web, S. E. and Capinera, J. L. 1994. Entomopathogenic
nematode effect on pickleworm (Lepidotera :Pyralidae) under
laboratory and field conditions. Journal of Economic Entomology,
87: 1205–1212.
Weiser, J. 1955. Neoaplectana carpocapsae n. sp. (Anguillulata,
Steinernematidae) novy cizopasnic housenik obalece jablecneho,
Carpocapsa pomonella L. Vestnik Cesk. Zool. Spolecnosti, 19:
44-52.
White, G. F., 1927. A method for obtaining infective nematode larvae
from cultures. Science, 66: 302-303.
Woodring, J.L. and Kaya, H.K. 1988. Steinernematid and heterorhabditid
nematodes: a handbook of biology and techniques. Southern
Cooperative Series Bulletin 331, Arkansas Agricultural Experiment
Station, Arkansas, Fayetteville, pp. 28.
Wouts, W. M., Mracek, Z., Gerdin, S. & Bedding, R. A. 1982.
Neoaplectana Steiner, 1929 a junior synonym of Steinernema
Travassos, 1927 (Nematoda: Rhabditida). Systematic Parasitology,
4 (2): 147-154.
Recieved on 12-7-2009 Acceted on 10-10-2009

7Trends 4 in Biosciences 2 (2): 74-75, 2009
Trends in Biosciences 2 (2), 2009
Seasonal Prevalence of Oesophagostomum sp. in Goats and Sheep from Kanpur
SIDDIQUA BANO 1 , N. SULTANA 2 AND VED PRAKASH3
1,2
Dept. of Zoology, A.N.D. College, Harsh Nagar Kanpur 208 012
3
Department of Animal Husbandry and Dairying, C.S.A. University of Agriculture and Technology,
Kanpur 208 002 ; e-mail: siddi.bano18@gmail.com
ABSTRACT
A total of 27948 goats and 5028 sheep were studied during the
year 2000-2005 in different areas of Kanpur. On conducting
microscopic examination of faecal samples and post-mortem
examination of these animals aged between 1-2 years and
materials collected from the butcher houses, meat and mutton
shops; two different species of Oesophagostomum
(Oesophagostomum colombianum and O. venulosum) were
recorded in 22 goats and 31 sheep. Year wise infection of these
species were also recorded. Seasonal infection and severity of
infection both were found higher in rainy season in comparison
with winter and summer season. Infection of Oesophagostomum
colombianum was reported 0.05% in goats and 0.39% in sheep,
whereas for Oesophagostomum venulosum it was 0.028% in goats
and 0.218% in sheep.
Key words
Prevalence, Oesophagostomum spp., goats, sheep,
infection
Sheep and goats are important livestock species for small
and marginal farmers and landless labours. It helps to increase
economic status of the rural poors. Due to improper care,
unhygienic environment, extreme climate and close contact
with infected animals they get infected with a variety of
parasites. Parasitism in sheep and goat is substantial problem
across the country. Amongst the parasitic diseases,
endoparasites are of greatest importance in sheep and goats.
Endoparasites of sheep and goat include Oesophagostomum,
round worms, tape worms and liver flukes (Bagley, 1997).
Oesophagostomum being nodular worms, commonly live in
the colon of the sheep, goats and a number of wild antelopes.
Although such parasites are not blood suckers and do not
attach themselves to the mucosa, but according to Wetzel,1931
these parasites irritate the mucosa, probably through
secretions of their oesophageal or cephalic glands, which
results mucous inflammatory products which in turn serve as
their food. The worms undoubtedly secrete a substance which
is absorbed by the host and has a toxic action on the body
and animal shows progressive emaciation, general weakness
and diarrhoea. The characteristic symptoms of chronic
oesophagostomiasis in sheep and goats is that of extreme
emaciation and cachexia with atrophy of the muscles, resulting
in complete prostration for 1-3 days and consequently
occurrence of death.
MATERIALS AND METHODS
During the study period large intestine and colon from
27948 slaughtered goats and 5028 slaughtered sheep aged
between 1 to 2 years collected randomly from the butcher
houses, meat and mutton shops of different areas of Kanpur
were examined in the laboratory and processed for worms and
eggs recovery. Intestinal contents and mucosal digester were
washed with luke warm water on 300-mesh sieve. And some
of them were fixed in the hot alcohol, glycerol (95:5) and
mounted in lacto-phenol for temporary preparation for their
identification. Faecal samples from slaughtered goats and
sheep were also collected randomly and examined. Counting
of eggs was done by Mc. Master egg counting technique
described by Kelly, 1974. Seasonal prevalence were also
observed throughout the study dividing into four seasons
viz., spring (Feb. to April), summer (May to July), rainy (Aug.
to Oct.) and winter (Nov. to Jan.).
RESULTS AND DISCUSSION
Total postmortem examination of 27948 goats and 5028
sheep were conducted from 2000 to 2005, of which 22 (0.078%)
goats and 31 (0.616%) sheep were found to be positive for
two species of Oesophagostomum (Oesophagostomum
colombianum and O. venulosum). The prevalence of
Oesophagostomum sp. was reported 30.68% in goats in Durg
district, Chhattisgarh state by Pathak and Pal, 2008. While, in
Assam, the prevalence of Oesophagostomum sp. was found
to be fairly high about 54.87% in goats (Rajkhowa and Hazarika,
2001). This difference indicates the adoptability according to
the climatic conditions existing in different geographical areas.
In the present study seasonal infection and severity of
infection both were found higher in rainy season in
comparison with winter and summer season. Climatic factors
also influence the rate of larval movements. During the
investigation, the overall percentage of infection for O.
colombianum was noted 0.05% in goats and 0.39% in sheep,
whereas for O. venulosum it was observed 0.028% in goats
and 0.218% in sheep. The present study indicated that the
sheep living in herd and dirty places appeared to be more
prone of infection than goats heavily infected than goats.
Sheep look much sensitive to pick up the infection early than
goats.

BANO et al., Seasonal Prevalence of Oesophagostomum sp. in Goats and Sheep from Kanpur 7 5
In the present study, the lowest prevalence of
Oesophagostomum sp. in goats and sheep was observed in
winter season. It could be attributed to low temperature which
also helps in arresting development in host and environment.
In addition, in winter the grazing hours of the animals are
reduced which naturally reduces the chance of contact
between the host and parasite. This is in agreement with the
observations made by Rajkhowa and Hazarika, 2001, who
recorded lowest prevalence of intestinal helminthes of goats
during winter season. The highest percentage of infection
during rainy seasons might be due to more availability of
grazing grass with infective eggs and larvae. In this season,
higher temperature and humidity provides favourable
condition for propagation of parasites resulting higher
parasitic burden among sheep and goats. Hence, the present
results require much attention during rainy season.
ACKNOWLEDGEMENT
The authors are thankful to the Officer-in-charge,
Slaughter House, Bazaria, Kanpur for the help rendered in
supplying the material and to Deptt. of Zoology, A.N.D.
College. Kanpur for providing necessary research facilities in
the laboratory.
LITERATURE CITED
Bagley 1997. Clell V. Extension Veterinarian Internal Parasites. Utah
State University Extension.
Kelly, W. R. 1974. In: Veterinary clinical diagnosis, Bailliere Tindall
Co. London, U. K. pp. 200-201.
Pathak, A. K. and Pal, S. 2008. Seasonal prevalence of gastrointestinal
parasites in goats from Durg district of Chhattisgarh. Veterinary
World, 1(5): 136-137.
Rajkhowa, S. and Hazarika, G. C. 2001. Effect of age, sex and season on
the prevalence of helminthic and coccidian infection in goats.
Indian Veterinary Medical Journal, 25: 367-369.
Wetzel, R. 1931. N. Amer. Vet., 12(9): 25-29.
Recieved on 3-8-2009 Acceted on 30-10-2009

7Trends 6 in Biosciences 2 (2): 76-78, 2009
Trends in Biosciences 2 (2), 2009
Evaluation of Relatively Eco-friendly Insecticides Against Pigeon Pea Podfly
Melanagromyza obtusa (Malloch) in Late Pigeon Pea.
MANZOOR HUSSAIN DAR, P.Q.RIZVI AND HEM SAXENA*
Department of Plant Protection, Faculty of Agricultural Sciences, AMU, Aligarh 202 002
*
Division of Crop Protection, Indian Institute of Pulses Research, Kanpur 208 024
email: m.dar@irri.org
ABSTRACT
Relative performance of most widely used relatively ecofriendly
insecticides against podfly was evaluated. It was found
that two sprays of dimethoate, profenophos and quinalphos
were superior. Maximum yield (1660 to 2046 kg/ha) and
minimum grain damage (13.5 to 21.6%) was recorded in these
treatments. The other treatments also showed a significant
reduction in grain damage and increase in grain yield as
compared to untreated check (38.4 to 44.8).
Key words
Melanagromyza obtusa, efficacy, dimethoate,
profenophos, quinalphos
The major constraint for low yield in pigeon pea is due
to heavy infestation of an array of insect pest complex during
reproductive phase of the crop. Pest complex commonly
occurring on this include; lycanid borer, Lampides boeticus
Linnaeus, plume moth, Exelastis atomosa Walsingham, gram
pod borer, Helicoverpa armigera Hubner, podfly
Melanagromyza obtusa Malloch, bruchid borer
Callosobruchus sp., brown bug, Clavigralla gibossa, spinola
and thrips. Among these, the podfly, Melanagromyza obtusa
Malloch is the most predominant and major insect pest of
pigeon pea in north India. Crop losses due to podfly have
been estimated from 10 to 95 per cent (Ahmad, 1938; Gangrade,
1963 ; Bindra and Jakhmola, 1967; Srivastava, et al., 1971 and
Kooner, et al., 1972). In order to provide an effective control
of podfly M. obtusa, an integrated approach involving use of
insecticides, host plant resistance and biological control have
been recommended. Till date, chemicals are the only available
efficient strategy against M. obtusa yet it involves several
limitations. With this point of view an experiment to test the
relatively eco-friendly insecticides against pigeon pea podfly
was carried out in the cropping season for two consecutive
years at experimental fields of Indian Institute of Pulses
Research, Kanpur.
MATERIALS AND METHODS
To determine relative efficacy of different eco-friendly
insecticides against podfly, an experiment was laid down in a
randomized block design for two successive years under field
conditions. Each plot was kept 5 m apart, replicated thrice.
NSKE commonly used by the farmers was also taken as a
check apart from the unsprayed check. Two sprays of each
treatment were given, one at 50 % pod primordial stage
followed by other after 15 days. The population of eggs and
maggots was recorded 15 days after each spray from 500
randomly collected pods from each replicate. The other
parameters like pod and grain damage were recorded at the
time of crop maturity by randomly collecting 500 pods from
each replicate. The yield was recorded at the harvest of the
crop. Details of the experiment were as follows
1 st spray—2 nd spray
T1 Acephate 0.07%— Acephate 0.07%, T2 Quinalphos
0.05% —Quinalphos 0.05%, T3 Lamdacyhalothrin0.002% —
Lamdacyhalothrin 0.002%, T4 Profenophos 0.1%—
Profenophos 0.1%, T5 Dimethoate 0.03%—Dimethoate 0.03%,
T6 Imidacloprid 0.04%—Imidacloprid 0.04%, T7 NSKE 5%
Check— NSKE 5% Check, T8 Profenophos 0.1%—
Dimethoate0.03%, T9 Profenophos 0.1%— Monocrotophos
.04%, T10 Profenophos 0.1%— Garlic extract 2%, T11
Profenophos 0.1%— NSKE 5%, T12 Profenophos 0.1%—
Onion extract 2%, T13 Endosulfan 0.07%— Endosulfan 0.07%,
T14 Unsprayed.
RESULTS AND DISCUSSION
Egg and Larval Population
All the insecticides significantly reduced the egg and
larval population. On the basis of mean of two observations
in first year the minimum egg population (0.05 eggs/pod) was
recorded in the treatment of twice sprayed dimethoate (Table
1). This was however, at par with the treatments of two sprays
of lamdacyhalothrin, imidacloprid, profenophos, endosulfan
and quinalphos. A significant reduction in maggot population
was observed (0.17 maggots/pod) in the treatment of first
spray of profenophos and second of monocrotophos at par
with two sprays of dimethoate, profenophos, imidacloprid and
quinalphos (0.18 maggots/pod). (Table 1)
In second year the lowest egg population was recorded
in the treatments of two sprays of dimethoate (0.04 eggs/pod)
and profenophos, quinalphos and imidacloprid (0.06 eggs/
pod) followed by subsequent sprays of profenophos and
monocrotophos, profenophos and dimethoate and
profenophos and NSKE (0.07 eggs/pod) against the control
(0.26 eggs/pod). Minimum larval population (0.16 maggots/
pod) was in the treatments of profenophos and imidacloprid

DAR et al., Evaluation of Relatively Eco-friendly Insecticides Against Pigeon Pea Podfly Melanagromyza obtusa 7 7
Table 1.
Effect of relatively eco-friendly insecticides on pod/grain damage and yield of pigeon pea in 1 st year and 2 nd year.
Treatments
Pod damage*
(%)
Profenophos? Dimethoate 38.0(38.1)**
28.0 (32.0)
Acephate 2 sprays 34.5(36.0)
26.0(30.7)
Profenophos? monocrotophos 37.3(37.6)
26.0(30.7)
Profenophos 2 sprays 34.4(35.9)
22.0(28.0)
Dimethoate 2 sprays 32.2(34.6)
19.3(26.1)
Profenophos? Garlic 32.5(34.8)
27.3(31.5)
Profenophos? NSKE 37.6(37.8)
28.6(32.3)
Profenophos? Onion 37.6 (37.8)
31.5(34.1)
Endosulfan 2 sprays 38.3 (38.2)
28.6(32.3)
Quinalphos 2 sprays 33.8(35.6)
26.3(30.9)
Lamdacyhalothrin 2 sprays 37.6 (37.8)
28.2(32.1)
Imidacloprid 2 sprays 34.8 (36.2)
23.4(28.9)
NSKE 2 sprays 43.3(41.2)
32.6(34.8)
Control (Untreated) 48.2(44.0)
38.0(38.1)
CD at 5% 2.8
7.0
Grain damage*
(%)
22.5(28.3)
17.3 (24.6)
23.0(28.7)
17.5 (24.7)
18.3(25.3)
15.1(22.9)
21.6(27.7)
14.6 (22.5)
14.6(22.5)
13.5 (21.6)
19.1(25.9)
26.6 (31.0)
20.9(27.2)
17.7 (24.9)
22.3(28.2)
20.0 (26.6)
27.0(31.2)
22.2 (28.1)
16.4 (23.9)
17.6 (24.8)
20.2 (26.7)
17.0 (24.4)
18.5 (25.5)
16.5 (24.0)
28.2(32.4)
27.4 (31.6)
44.8(42.0)
38.4 (38.3)
5.8
7.9
* Upper figures are data of 1 st year followed by lower figures are data for 2 nd year
** Data in parenthesis are mean values.
Avoidable
losses*
(%)
49.8
55.0
48.7
54.5
59.2
60.7
51.8
62.0
67.5
64.9
57.4
30.8
53.4
54.0
50.3
15.2
39.8
42.2
63.4
54.2
55.0
55.8
58.8
57.1
37.1
28.7
Yield Mean population*
kg/ha* E M
1746
1675
1595
1591
1725
1710
2046
1933
1750
1730
1646
1560
1733
1590
1513
1490
1410
1435
1740
1660
1630
1648
1720
1696
1277
1240
- 929
1076
- 10.5
1.42
0.08
0.07
0.8
0.8
0.08
0.07
0.07
0.06
0.05
0.04
0.10
0.09
0,10
0.07
0.09
0.11
0.07
0.12
0.07
0.06
0.06
0.08
0.06
0.06
0.10
0.13
0.30
0.26
0.13
0.19
0.22
0.21
0.22
0.19
0.17
0.16
0.18
0.16
0.18
0.17
0.33
0.32
0.30
0.26
0.32
0.31
0.32
0.29
0.18
0.17
0.21
0.19
0.18
0.16
0.28
0.33
0.05
0.05
0.09
0.32
(two sprays each) and subsequent sprays of profenophos
and monocrotophos followed by dimethoate and quinalphos
two sprays each (0.17 maggots/pod). (Table 1)
Pod and grain damage
The pod damage, among the treatments, varied
significantly (32.3 to 38.3%) over control (48.2%) as well as
NSKE (43.3%) during first year. As far as grain damage was
concerned, the minimum was recorded in dimethoate (14.6%)
followed by profenophos (16.4%) sprayed twice each. All other
treatments were superior to control (44.8%). In second year,
all the treatments significantly reduced the pod damage except
two sprays of NSKE (32.6%) and subsequent sprays of as
compared to untreated control (38.0%). The lowest grain
damage was recorded in dimethoate (13.5%) followed by
profenophos (14.6).
Yield
In the 1 st year maximum grain yield was obtained in two
sprays of profenophos (2046 kg/ha) followed by dimethoate
(1750 kg/ha), subsequent sprays of profenophos and
dimethoate (1746 kg/ha) and quinalphos (1740 kg/ha). In the
following year, the maximum grain yield of 1933 kg/ha was
obtained in two sprays of profenophos followed by 1730 kg/
ha (dimethoate), 1710 kg/ha (subsequent sprays of
profenophos and monocrotophos). (Table I).
In the present study, a number of insecticides viz.,
dimethoate, profenophos, quinalphos and acephate showed

7 8 Trends in Biosciences 2 (2), 2009
significantly better control of podfly when sprayed twice and
also in interchanging combinations. Similar findings have been
reported earlier by Mishra and Saxena, 1985, Patel and
Patel,1989, Patil, et al. 1990, and Bhandari and Ujagir, 2002.
Dimethoate 0.03% at the time of 50% pod primordial stage and
next 15 days after first spray, gave effective control against
podfly. These findings are in conformity with earlier work (Patil,
et al., 1990, Lal and Yadava, 1988 and Singh, et al., 2001.
LITERATURE CITED
Ahmad, T. 1938. The tur podfly, Agromyza obtusa Malloch. A pest of
Cajanus cajan. Indian J. of Agric. Sci., 8: 53-76.
Bhandari, P. and Ujagir, R. 2002. Bioefficacy of certain insecticides
against pod borer complex of early pigeonpea, Cajanus cajan (L.)
Millsp. Ann. Pl. Prot. Sci., 10 (2): 225-229.
Bindra, O.S. and Jakhmola, S.S. 1967. Incidence and losses caused by
some pod infesting insects in different varieties of arhar. Ind. J.
Agri. Sci., 37: 177-196.
Gangrade, G.A. 1963. Assessment of damage to tur (Cajanus cajan) in
Madhya Pradesh by the tur podfly, Agromyza obtusa. Ind. J. Agri.
Sci., 33: 17-20.
Kooner, B.S., Singh, Harcharan and Singh, K.B. 1972. Relative
susceptibility of germplasm of pigeonpea against tur-podfly
Melanagromyza obtusa M. under field conditions. Pl. Prot. Bull.
24 (1-2): 7-18.
Lal, S.S. and Yadava, C.P. 1988. Efficacy of certain insecticides against
pod borers infesting pigeonpea. Pesticides, 22 (1-2): 30-35.
Mishra, P.N. and Saxena, H.P. 1985. Dissipation of quinalphos in
pigeonpea, Cajanus cajan (L.) Millsp. Var. Prabhat. Pesticides, 19
(5): 26-27.
Patel, P.S. and Patel, J.R. 1989. Efficacy of emulsifiable concentrates
and dust formulations of fenvalerate in comparison to other
insecticides against Heloithis armigera Hobber and Melanagromyza
obtusa Malloch infesting pigeonpea. Ind. J. Pl. Prot., 17: 2, 223-
226.
Patil, C.S., Khare, V.M. and Mote, U.N. 1990. Comparative performance
of different insecticides against pigeonpea pod borer complex on
short duration pigeonpea. J. Mah. Agri. Uni., 15 (3): 337-339.
Singh, D.P, Singh, R.P, Singh, S.K and Kumar, A. 2001. Evaluation of
certain insecticides against major insect pest of pigeonpea Ann.
P1. Prot. Sci., 9 (2): 313-14.
Srivastava, A.S., Katiyar, S.S.L. and Srivastava, K.M. 1971. Damage of
Agromyza obtusa Malloch. (Diptera: Agromyzidae) to Cajanus
cajan Linn. Crop. Laldev J. Sci. and Tech., 9: 71-73.
Recieved on 18-10-2009 Acceted on 30-11-2009

Trends in Biosciences 2 (2): 79-80, 2009
Studies on the Anamorph Characters and Management of Powdery Mildew of
Mustard
SHABBIR ASHRAF AND BHAWANA YADAV
Department of Plant Protection, Faculty of Agricultural Sciences, A.M.U., Aligarh 202 002
e-mail:shabbiragri@yahoo.co.uk
ABSTRACT
Experiments were conducted to study the effect of pathogen on
five cultivars of mustard viz. Varuna, Alankar, Kranti, RH-30,
and Pusa Bahar obtained from NRCRM, Bharatpur. It has been
observed that the conidia of Erysiphe cruciferarum were ellipsoid
to cylindrical in shape, measuring 27.5-35.0µm x 12.5-17.5µm
without fibrosin bodies. The conidia germinated by the
formation of straight but two types of germ tubes i.e. short but
slightly lobed appressoria and long unlobed appressoria. The
length of germ tube ranged 20-30µm. It was observed that the
optimum temperature for the germination of conidia was
between 20-25°C± 2°C. The most effective fungicide was found
to be Hexaconazole for the management of powdery mildew.
Key words
Powdery mildew, conidia, fungicides
Rapeseed mustard Brassica juncea the second most
important oilseed crop after groundnut, contributing nearly
30% of the total oilseed production in India. The crop suffers
from many diseases but the important among them are
powdery mildew Erysiphe cruciferarum, Opiz. Ex Junell, downy
mildew, white rust, Alternaria blight (Mukerji, et al., 1999).
Powdery mildew, is wide spread and cause considerable yield
loss especially when slightly high temperature prevails during
flowering stage. The reduction in grain yield of mustard due
to powdery mildew disease is 17.40 per cent (Saharan and
Kaushik, 1981). Powdery mildew first appears on the upper
surface in the lowermost (oldest) leaves as small (4-5 cm dia
m), scattered, white almost circular colonies which eventually
coalesce as the colonies grow further eventually covering the
entire leaf surface under favourable environmental conditions
(Singh, 2000a). This disease is most commonly observed on
the upper sides of the leaves. Infected buds may fail to open.
Powdery mildew is severe in warm climates. This is because
the fungus does not need the presence of water on the leaf
surface for the infection to occur. However, the relative
humidity of the air does need to be high for spore germination.
Therefore, the disease is common in crowded plantings where
air circulation is poor and in damp, shaded areas. Powdery
mildew of mustard affects not only foliage but also developing
green siliquae as well as growth plant (Enright and Cipollini,
2007).
MATERIALS AND METHODS
Experiments were conducted in 2007-08 and 2008-09
(Table 1). Seeds of five different cultivars obtained from
National Research Centre for Rapeseed and Mustard
(NRCRM). Bharatpur were sown on 25 th November, 2007 and
2008 in gross plot size of 5.0 x 2.25m in randomized block
design with three replications. Row spacing of 45cm was also
maintained. The infected leaves of plants were collected in
polyethylene bags and dusted on a dry clean slide. Ten conidia
from each variety were measured to determine the length and
breadth of the conidia and the average was taken. For studying
the type of germ tube, conidia were dusted over dry clean
slides and then placed in petri dishes containing double
distilled water at the bottom to make a humid environment
and kept for incubation in B.O.D. incubator at 25±2°C for 24
hs. The humid chamber was kept in B.O.D. incubator at the
temperature of 5±2°C and the germination was checked after
24, 48, 72 hrs. and per cent germination was recorded. This
procedure was repeated at 20°, 25°, 30°, and 35°C. To study
the effect of fungicides on the development of disease an
experiment was conducted only on cultivar Varuna being most
susceptible cultivar. The plants were sprayed at the appearance
of disease (40-45 days after sowing). The second and third
sprays were given at 20 days interval. Observations on disease
intensity were recorded on 15 days of last spraying using 0-5
scale.
RESULTS AND DISCUSSION
The conidia obtained from infected mustard leaves were
found to be ellipsoid to cylindrical in shape measuring 27.5-
35µm x 12.5-17.5 µm in size without fibrosin bodies (Table 1).
The conidia germinated by the formation of straight but two
types of germ tube i.e. short but slightly lobed appressoria
Table 1.
S. Cultivars
No
Measurement of conidia and germ tube of Erysiphe
cruciferarum
Conidia
(µm)*
* Each value is an average of ten replicates
Length of germ tube at 24 ºC at
different intervals (µm)
Length Width 24 hrs 48 hrs 72 hrs Mean
1 Alankar 30.0 15.0 12 28 32 24
2 Kranti 30.0 15.0 12 38 32 24
3 Pusa bahar 27.5 12.5 10 23 27 20
4 RH-30 27.5 12.5 10 23 27 20
5 Varuna 35.0 17.5 15 35 40 30

8 0 Trends in Biosciences 2 (2), 2009
Table 2.
Germination of conidia (%) of Erysiphe
cruciferarum at different temperatures and time
intervals
Temperature 12 hrs 24 hrs 48 hrs 72 hrs
(°C)
5 N T T T
20 22.3 32.1 40.7 47.3
25 21.8 30.2 34.6 40.3
30 22.0 26.3 D D
35 N D D D
N = No germination, T = Germination in traces, D = Conidia deformed
and long unlobed appressoria. Simple and forked germ tube
emerged apically and basally with or without appressorium.
The presence of conidia prove that the fungus belongs to be
genus Erysiphe (Khan and Sharma, 1995). The germination of
conidia was observed at 24±2°C after intervals of 24, 48 and
72 hrs. of germination. It was found that after 24 hrs. the length
of germ tube ranged 10-15 µm; while after 48 hrs., it ranged
between 23-35µm and after 72 hrs. it was 27-40 µm. The average
length of germ tube was found to be 20-30 µm. It was found
that at 5°C temperature, after time interval of 12 hrs., there was
no germination, while after 24, 48 and 72 hrs. there was
germination in traces. The maximum germination was observed
at 20°C after 72 hrs. of germination. The conidia germinated
very well at temperatures between 20-25 ± 2°C up to 72 hrs. of
germination. The minimum germination was observed up to
12 hrs. and after 72 hrs. the conidia started getting deformed
(Table 3). Once symptoms began to appear, mildly diseased
plants were sprayed with fungicides in an attempt to minimize
disease severity. Fungicides were carefully sprayed only on
aerial plant parts of the target plant with minimal over spraying
on to the soil to minimize fungicide effects on soil borne fungi
of neighbouring plants. There was no significant difference
in mildew severity among the plots prior to or following the
first fungicide application. After three fungicide applications,
all the treatments significantly reduced the severity of mustard
powdery mildew. This indicates that the optimum temperature
for the growth of powdery mildew is between 20-25°C at wide
range of relative humidity as reported by Singh and Chauhan,
1998. All the tested fungicides reduced the disease incidence
as compared to control. Hexaconazole proved to be the most
effective as compared to other fungicides. The minimum
Table 3.
Effect of different fungicides on powdery mildew
of mustard
Treatments Concentration Disease
intensity
(%)
disease intensity was recorded in Hexaconazole treated plants
followed by Calixin, Wet-Sulphur, Bavistin, Blitox-50, Sulphur
dust and Topsin-M respectively. The mustard grain yield was
maximum in treatment with Hexaconazole and similar results
were found with other fungicides but less effective (Table 3).
Present findings confirm the observations of Singh and
Chauhan, 1998 and Patel and Patel, 2008 who have reported
similar effect of various fungicides against powdery mildew
of mustard
LITERATURE CITED
Disease
reduction over
control (%)
Calixin 0.2 33.4 62.88
Hexaconazole 0.2 30.6 66.0
Bavistin 0.1 46.5 48.3
Wet-sulphur 0.2 40.3 55.2
Topsin-M 0.2 63.7 29.2
Blitox-50 0.2 49.9 44.5
Control - 100.0 -
C.D.at 0.05% - 0.69 -
Enright, S. M. and Cipollini, D. 2007. Infection of powdery mildew
Erysiphe cruciferarum (Erysiphaceae) strongly affects growth and
fitness of Alliaria petiolata (Brassicaceae). American journal of
Botany. 94: 1813-1820.
Khan, M. W. and Sharma, G.K. 1995. Taxonomic evaluation of
anamorph characters in identification of powdery mildew fungi on
cucurbits. Indian Phytopath., 48: 314-324.
Mukerji, K. G., Upadhyay, R. K., Saharan, G. S., Sokhi, S. S. and Khangura,
R. K. 1999. IPM system in agriculture, vol.5’ (eds. R.K. Upadhyay,
K.G. Mukerji and R.L. Jalak), Aditya books Pvt. Ltd., New Delhi.
pp.91-135.
Patel, J. S. and Patel, S. J. 2008. Seed health status of mustard seeds as
influenced by foliar sprays against powdery mildew disease. Indian
J. Agri. Sci., 21(3): 464-465.
Saharan, G.S. and Kaushik, J.C. 1981. Occurrance of epidemiology of
Powdery mildew of Brassica. Indian Phytopath., 34: 54.
Singh, U.P. 2000a. Pea powdery mildew-an ideal pathosystem. Indian
Phytopath., 52: 1-9.
Singh, V. K. and Chauhan, V. B. 1998. Management of powdery mildew
of mustard. Ann. Pl. Protec. Sci., 6(1): 93-94.
Recieved on 27-11-2009 Acceted on 10-12-2009

Trends in Biosciences 2 (2): 81-82, 2009
SHORT COMMUNICATION
Resupination and Flower Structure: the Adaptation for Pollination in Impatiens L.?
R. RAMASUBBU, A.K. SREEKALA AND A.G. PANDURANGAN
Plant Systematics and Evolutionary Science Division, Tropical Botanic Garden and Research Institute, Palode,
Thiruvananthapuram, Kerala; e-mail: racprabha@yahoo.com
Impatiens L.? are beautiful plants bearing variously
coloured flowers with peculiar floral structure. Majority of the
Impatiens have great horticultural potential. It is distributed
in tropics and sub-tropics of the old world, but several species
also occur in temperate Eurasia and North America. In India,
the genus represented by 205 species is mainly distributed in
three major centres of diversity i.e. western Himalayas, Hills
of north eastern states and western Ghats. Out of 92 species
available in peninsular India, more than 80 are endemic and
confined to western Ghats (Pandurangan, 1996). The present
research work focused mainly on flower structure,
development and resupination of flower buds for pollination
in wild Impatiens species distributed on the southern western
Ghats.
Flowers of Impatiens are basically 5-merous, though
various modifications of structures are evident. Roper, 1830
was the first author who stated that, the perianth of Impatiens
consists of a calyx and a corolla that both are pentamerous.
The flowers are always zygomorphic and this had led to an
enormous diversity in development of floral structures. Floral
parts such as anterior petal, the lateral petals and especially
the spurred sepal are extremely variable not only in colour but
also in shape and structure within the same species (Impatiens
balsamina, I. chinensis, I. diversifolia and I. tomentosa).
During the development of floral buds, initially the free sepals
and petals increase their zygomorphic structure with increase
in the size of the lower sepal and upper petal causing a
subsequent decrease in size and displacement of lateral sepals.
The greater enlargement of the lower sepal and the upper
petal causing the disappearance of the upper pair of lateral
sepal and subsequent displacement and uniting of the lateral
petals into pairs. The anthers lie closely together and adjacent
anther lobes fuse. The four sporangia that are involved merge
to form a common space, which contains the pollen of both
thecae. The pollen is released through a slit at the top of this
common space via the pressure created by the swollen cells
in the connective region of the anther. The gynoecium is
always considered to be five carpellate. Only after the pollen
has been released and the androecia shed will the star shaped
stigmas ripen. The coherent stigma is commonly spread and
the star shaped receptive surface exposed (I. coelotropis, I.
phoenicea and I. platyadena).
The rich variation in the floral structure may be linked to
adaptation with pollination. There is a relationship between
shape and structure of spur and different set of pollinators.
Selection for successful visitation based on pollination
behaviour might be much more important with respect to
evolution of floral structure than adaptation to improve the
mechanical fit between pollinators and flower. The
zygomorphic structure and the elaboration of the lower petals
in Impatiens have three fold function such as, to attract the
pollinators, to act as a platform for the pollinators to land and
to provide an access point to the lower sepal (lip) and spur
where nectar is stored. The labellum (standard/ upper petal)
also provides guide to visiting pollinators in the flowers (most
of the species). The extra floral nectarines found in the lower/
adjacent to the reproductive part are helpful to the pollination
and further mechanism. The nectarines tissues carries
important role in the stigma touches of the pollinators during
forage time. The nectarines were observed to secrete both
cane and grape sugars which act as an attractant to pollinators.
In the majority of balsams buds are positioned with the
standard uppermost and the lip below them. However, some
flowers are born with lip above the standard petals which are
lower most. The reversal of position of flower buds occurs as
a result of a process called resupination which takes place
before 2-3 days of anthesis (I. coelotropis, I. phoenicea and I.
platyadena). In the most of the species, the buds turn only to
the extent necessary to place standard lower most which is
usually 180 0 , but depending on the position of inflorescence
the turning can be more or less; some species do not
resupinate at all and their flowers are often described as being
borne upside down.
Darvin suggested that, resupination facilitates
pollination because the labellum assumes the position of a
lower petal, so that insects can easily visit the flower.
Resupination usually occurs 2-3 days prior to anthesis. Once
the flowers are fully open, they can no longer resupinate.
However, the flowers of some species of Orchids deresupinate
following pollination. Another interesting
characteristic of resupination is that in some species (I. grandis,
I. maculata and I. flaccida) and hybrids, the buds alternate in
resupinating clock (CL) and counter clock (CL) wise.
Resupination may also provide flowers with space to open
because in many balsams the buds are arranged tightly on the
developing inflorescence. In Orchids, the resupination may
also be influenced by the weight of the flower on a slender
inflorescence. In this context, the resupination is a key factor

8 2 Trends in Biosciences 2 (2), 2009
in the evolution of lower sepals and spur otherwise, it would
be difficult to visualize the development of such a wide range
of bulky structure if resupination did not occur. Therefore,
the resupination is closely associated with pollinator/
pollination evolution.
Zygomorphy and the accompanying resupination in
Impatiens is clearly an evolutionary progress towards highly
specialized pollinations, a trend that often seems to lead a
high degree of speciation. Possible selection process on such
systems permits rapid speciation, often in small areas
geographically and may help to account for the high degree
of endemism (Grey Wilson, 1980). Joseph Arditti, 2003
suggested that the synthetic cytokinin (BA) can also reestablish
resupination to some extent, but it probably acts
through its auxin sparing effect. These findings suggested
that resupination is a garvitropic phenomenon which confirms
to the cholodny-Went hypothesis. Though the experiments
with auxin transport inhibitors, an anti-auxin and a calcium
chelator suggested that resupination may be controlled by
auxins the mechanism of resupination is however not
understood clearly so far. On resupination, little studies have
been carried out to understand its effect on the flower and its
evolutionary significance as far as pollination and pollinators
are concerned. Therefore, work on the physiological aspects
and the mechanism involved would yield a valuable and
interesting data that ought to interpret as many groups as
possible in which resupination is known to occur.
LITERATURE CITED
Grey Wilson, C. 1980. Impatiens of Africa. Balkema, A. A., Rotterdam,
Nether lands.
Joseph Arditti. 2003. Resupination. Lankesteriana, 7: 95-96.
Pandurangan, A. G. 1996. Systematic studies on Balsaminaceae in
Peninsular India, Final Project report submitted to Kerala Forest
Department, Thiruvananthapuram, Kerala.
Roper, J.A.C. 1830. De floribus et affinitatibus Balsaminearum, Basel,
Switzerland.
Recieved on 5-9-2009 Accepted on 29-10-2009

Trends in Biosciences 2 (2): 83-84, 2009
SHORT COMMUNICATION
Comparative Efficacy of Ranide and Fasinex Against Fascioliasis in Sheep
SIDDIQUA BANO, N. SULTANA AND VED PRAKASH*
Deptartment of Zoology, A.N.D. College, Harsh Nagar Kanpur 208 012,
*Department of Animal Husbandry and Dairying C.S. Azad University of Agriculture and Technology,
Kanpur 208 002; e-mail: siddi.bano18@gmail.com
Fascioliasis caused by Fasciola gigantica is
predominantly a disease of all domestic ruminants, especially
in the tropical region of the world, includiong India (Gupta, et
al., 2003). Fascioliasis is a serious impediment to sheep farming
and causes enormous economic losses all over the world and
these losses are due to reduction in milk, meat and wool
production. In Bareilly the losses were calculated to be Rs.
4.178 millions due to rejection of livers (Arora, 1967 and Boray,
1985) estimated the economic losses due to fascioliasis to be
about U.S.$ 2000 million per annum globally. According to
Soulsby, 1982 disease has immense economic importance,
often resulting in chronicity and subclinical infection in cattle.
Commonly fascioliasis of ruminant is characterized by
diarrhoea, anemia, bottle jaw, stunted growth, loss of weight,
wool and milk production, bloody discharge from anus and
even caused sudden death (Basith, 2002). Treatment of worm
disease by herbal drugs is the oldest tradition, however, some
of these drugs are still in use mostly in rural area, such as male
fern, arecanut, oil of chenopodium or oil of worm wood and
seed. Since the ancient time many drugs have been discovered
as anthelmintics such as carbon tetrachloride,
tetrachlorethylene, penothiazine etc. Thus many new effective
drugs have been introduced against parasitic disease. Modern
broad spectrum, flukicides like Albendazol (Analgon, Albomar,
Labenzole etc.) Distodin (Pfizer), Flukin (Arex), Distonex
(Nesparic), Fasinex (Ciba geigy), Zanil (ICI)Ranide (Refoxanide
M.S.D.), Talzan (Intervet), Trodax (Rhone P.), Exinot (cadila)
and closantel 15% are effective against all stage of flukes. Out
of these, Ranide and Fasinex are broad spectrum flukicide
having potential to kill majority of flukes of sheep and goats,
being used extensively for parasite control due to the cheapest
cost is Rs. 5-8 per dose at present in Kanpur. The present
study on efficacy of Ranide and Fasinex against liver fluker of
sheep have been taken up for low control of these disease.
Experiments were carried out on nine positive sheep
aged above 1 to 2 years, naturally infected with fascioliasis
were selected on the basis of faecal examination for
administration of the drugs. The faecal samples were examined
by direct smear, sedimentation technique and zinc sulphate
flotation technique for the presence of fluke eggs as per the
standard procedure described by Sastry, 2000. The trematode
eggs were identified on the basis of morphological details
described for F. gingantica eggs by Yamaguti, 1975. Counting
of eggs was done by Mc. Master eggs counting technique,
Kelly, 1974. For efficacy of drugs, sheep were randomly divided
into three groups A, B and C of three sheep each. Group A
sheep were given Ranide suspension (10 mg/kg body weight)
through oral route. Sheep of group B received Fasinex
suspension as per the dose and route mentioned for Ranide.
Group-C sheep were kept as untreated control. Supportive
therapy with electrolytes drip and live extract with vitamin B
complex intramuscularly was also given to the sheep of both
group A and B for 2-3 days. On zero to 21 days post treatment,
faecal samples were again collected and examined with the
same methods stated above.
FECR (%) = (FECbt – FEC at) x 100/FEC bt
Where, FECR (%) denotes per cent faecal egg count
reduction, FECbt and FEC at, stand for egg count before and
after treatment, respectively.
Three times examination of the individual faecal samples
of selected sheep on 21 days treatment revealed 95.91% and
90.69% reduction in faecal egg count in treatment group A
and B respectively, indicating 95.91% efficacy of the Ranide
and 90.69% of Fasinex (Table 1). Group C (untreated) sheep
were found to be positive for ova of F. gigantica till 21 days
post treatment. The body coat and general health of all treated
sheep started improving after three days of treatment. The
drug did not produce any side effect.
The data revealed that Ranide is 96% effective to cure
trematode fluke infection in sheep by interfaring with energy
metabolism by inhibition of polymerization microtubules of
parasites. Fasinex is a broad spectrum flukicide and it acts by
Table 1.
Pre and post-treatment observation on the EPG of
sheep infected with Fasciola gigantica
Groups Treatments
(mg/kg orally)
Mean EPG
0 DPT 21 DPT
FECR
%
A Ranide @ 10 mg/kg 98 04 95.91
B Fasinex @ 10 mg/kg 86 08 90.69
C Untreated control 80 110 -
EPG=Eggs per gram of faeces, 0 DPT=Zero day post treatment
21 DPT =21 days post treatment FECR % = Faecal egg count reduction
percentage

8 4 Trends in Biosciences 2 (2), 2009
inhibiting the fumerate reductase enzyme of the parasite
(Brander, et al. 1991). On the basis of results obtained, it can
be suggested that Ranide suspension was proved to be a
better effective control than Fasinex on fascioliasis in sheep.
ACKNOWLEDGEMENT
Authors are thankful to Department of Zoology, A.N.D.
College, Kanpur and Department of Animal Husbandry and
Dairying, C.S.A. University of Agriculture and Technology,
Kanpur for providing necessary research facilities.
LITERATURE CITED
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Recieved on 3-10-2009 Acceted on 30-11-2009

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