ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC ...

ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC 

FUNGI AND THEIR EFFECTIVENESS 

Thesis submitted to the 

University of Agricultural Sciences, Dharwad 

in partial fulfillment of the requirements for the 

Degree of 

Doctor of Philosophy 

In 

AGRICULTURAL MICROBIOLOGY 

By 

BHARATHI H. TALWAR 

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY 

COLLEGE OF AGRICULTURE, DHARWAD 

UNIVERSITY OF AGRICULTURAL SCIENCES, 

DHARWAD - 580 005 

DECEMBER, 2005

Advisory Committee 

DHARWAD (J. H. KULKARNI) 

DECEMBER, 2005 MAJOR ADVISOR 

Approved by : 

Chairman : _________________________ 

___ 

(J. H. KULKARNI) 

Members : 1. 

_________________________ 

_ 

(A. R. ALAGAWADI) 

2. 

_________________________ 

_ 

(S. LINGAPPA) 

3. 

_________________________ 

_ 

(P. U. KRISHNARAJ) 

4. 

_________________________ 

_ 

(SRIKANT KULKARNI)

Chapter 

No. 

I INTRODUCTION 

C O N T E N T S 

II REVIEW OF LITERATURE 

III MATERIAL AND METHODS 

IV EXPERIMENTAL RESULTS 

V DISCUSSION 

VI SUMMARY 

VII REFERENCES 

APPENDICES 

Title

Table 

No. 

LIST OF TABLES 

Title 

1. Occurrence and pathogenicity of entomopathogenic fungi in 

different insect hosts 

2. Details of different pesticides used in the experiments 

3. Details of food grains evaluated for mass production of 

Metarhizium anisoplias and Verticillium lecanii 

4. Details of carrier materials evaluated for survival studies 

5. Details of intensive survey conducted for incidence of 

entomopathogenic fungi in northern Karnataka 

6. List of isolates of Metarhizium anisopliae and Verticillium lecanii 

obtained during the study 

7. Morphological and cultural characters of Metarhizium anisopliae 

and Verticillium lecanii isolates 

8. Mortality caused by the isolates of Metarhizium anisopliae and 

Verticillium lecanii against Helicoverpa armigera and Brevicornia 

brassicae respectively 

9. Insect species found susceptible to Metarhizium anisopliae and 

Verticillium lecanii 

10. Total biomass production of Metarhizium anisopliae (Ma2) as 

influenced by carbon sources 

11. Total biomass production of Metarhizium anisopliae (Ma2) as 

influenced by nitrogen sources 

12. Total biomass production of Verticillium lecanii (Vl1) as influenced 

by carbon sources 

13. Total biomass production of Verticillium lecanii (Vl1) as influenced 

by nitrogen source

Contd….. 

Table 

No. 

Title 

14. Sporulation of Metarhizium anisopliae Ma2 spores on different 

grains 

15. Sporulation of Verticillium lecanii Vl2 spores on different grains 

16. Sporulation of Metarhizium anisopliae Ma2 spores on different 

agro wastes 

17. Sporulation of Verticillium lecanii Vl2 spores on agro wastes 

18. Effect of fungicides on the conidial germination of Metarhizium 

anisopliae Ma2 

19. Effect of fungicides on the conidial germination of Verticullium 

lecanii 

20. Effect of insecticides on the conidial germination of Metarhizium 

anisopliae 

21. Effect of insecticides on the conidial germination of Verticillium 

lecanii 

22. Effect of weedicides on the conidial germination of Metarihizium 

anisopliae 

23. Effect of weedicides on the conidial germination of Verticiliium 

lecanii 

24. Persistence of Metarhizium anisopliae in soil and phylloplane 

25. Persistence of Verticillium lecanii in soil and phylloplane 

26. Survival of Metarhizium anisopliae in different oil based and 

wettable powder formulations under different storage temperature 

(20 DAI) 



(45 DAI)

Contd….. 

Table 

No. 

Title 



(75 DAI) 



(90 DAI) 



(120 DAI) 



(150 DAI) 

32. Survival of Verticillium lecanii in different oil based and wettable 

powder formulations under different storage temperature (20 DAI) 








powder formulations under different storage temperature (120 

DAI) 


powder formulations under different storage temperature (150 

DAI) 

38. Per cent mortality of Brevicornia brassicae due to application of 

Verticillium lecanii under laboratory conditions

Contd….. 

Table 

No. 

Title 

39. Per cent mortality of Aleurodicus dispersus due to application of 

Verticillium lecanii under laboratory conditions 

40. Per cent reduction of nymph of Brevicornia brassicae on cabbage 

due to Verticillium lecanii (Vl1) under field conditions 





43. Per cent reduction of nymph of Aphis crassivora on cowpea due to 

Verticillium lecanii (Vl1) under field conditions 





46. Effect of wild types and derived mutants (UV) of Metarhizium 

anisopliae (Ma2) Helicoverpa armigera and Verticillium lecanii (Vl1)

Figure 

No. 

LIST OF FIGURES 

Title 

1. Mycopathogens of insect pests collected during survey in 

Northern Karnataka 

2. Conidial yield of Metarhizium anisopliae and Verticillium 

lecanii on different food grains 

3. Conidial yield of Metarhizium anisopliae and Verticillium 

lecanii on different agrowastes 

4. Effect of pesticides on the conidial germination of Metarhizium 

anisopliae 

5. Effect of pesticides on the conidial germination of Verticillium 

lecanii 

6. Survival of Metarhizium anisopliae (Ma2) and Verticillium 

lecanii (Vl1) in different formulations under different storage 

temperature (120 DAI)

Plate 

No. 

LIST OF PLATES 

Title 

1. Microconidiophore and microconidia of 

entomopathogens 

2. In vitro sporulation by different isolates of Metarhizium 

anisopliae and Verticillium lecanii 

3. Host insects of Mearhizium anisopliae 

4. Host insects of Verticillium lecanii 

5. The mortality of Helicoverpa armigera topically applied 

with Metarhizium anisopliae 

6. Inhibition of growth of Metarhizium anisopliae and 

Verticillium lecanii by pesticide 

7. Metarhizium anisopliae cultured on rice for field use

Appendix 

No. 

LIST OF APPENDICES 

Title 

I. Mean monthly weather data at Main Agricultural Research 

Station, University of Agricultural Sciences, Dharwad 

II. Survey proforma for Entomopathogenic fungi 

III. Identification report

I.INTRODUCTION 

The increased use of conventional chemical pesticides over the years has not only 

contributed to an increase in food production, but also has resulted in adverse effects on the 

environment and non-target organisms. In view of these side effects, the necessity for 

sustainable crop production through eco-friendly pest management technique is being largely 

felt in the recent times. Of the several microbial pathogens viz., bacteria, fungi, viruses, 

protozoans and entomopathogenic nematodes reported, only a few have been studied 

systematically for their usefulness. A careful evaluation of these beneficial pathogens can 

lead to gainful exploitation in microbial control programmes (Burges, 1998). 

India is bestowed with a rich biodiversity of entomopathogens and exploitation of 

these natural and renewable resource are essential in a successful biocontrol strategy. Not 

long after the discovery of fungi as the cause of some insect diseases, it was proposed to use 

fungi as biocontrol agents. Hence, a major motivation still exist for the exploitation of 

entomopathogenic fungi in the management of insect pests. 

Entomogenous fungi are potentially the most versatile biological control agents, due 

to their wide host range that often results in natural epizootics. An attractive feature of these 

fungi is that infectivity is by contact and the action is through penetration (Nadeau et al., 

1996). These fungi comprise a heterogenous group of over 100 genera with approximately 

750 species, reported from different insects. Many of these offer a great potential in pest 

management. The most important fungal pathogens are Metarhizium spp., Beauveria spp., 

Nomuraea rileyi, Verticillium lecanii and Hirsutella spp. 

In 1883, Metchinikoff initiated mass culturing of fungus and carried out the first 

experiment with two beetle pests. Metarhizium anisopliae (Metschnikoff) Sorokin is the 

second most widely exploited entomogenous fungus in biocontrol attempts. It is known to 

attack over 200 species of insects belonging to orders Coleoptera, Dermoptera, Homoptera, 

Lepidoptera and Orthoptera (Moore et al., 1996). 

Verticillium lecanii (Zimm.) popularly called the “white holo” is known to cause 

mycosis in a number of insects belonging to the insect orders Homoptera, Coleoptera and 

Lepidoptera. 

Of the over 750 species of fungi known to be pathogenic to insects, six have been 

commercialized and the cosmopolitan pathogens, such as B. bassiana, M. anisopliae are the 

best known so far. Fungi often cause spectacular epizootics with large number of pathogenic 

insects showing visible fungal outgrowth (Hall, 1982). Conservation and periodic 

enhancement of efficacy of biological control agents will help in crop protection and in 

producing agricultural commodities free from pesticide residues. There is a general feeling 

that, the development and spread of biological control will empower the resource of poor 

farmers to manage their pest problems in an eco-friendly way. This assumes importance 

since the growth rate of the use of biopesticides alone over the next 10 years has been 

forecast at 10-14 per cent per annum, in contrast to two per cent for chemical pesticides. 

The major issues involved in mass production and utilization of mycopathogens are 

selection of effective strains, development of cost effective methods for mass rearing, 

development of effective methods for storage and shipment and creation of effective 

formulation. 

Environmental factors like temperature, humidity and sunlight play a profound role on 

the field persistence of entomopathogenic fungi. One of the critical factors in the effective use 

of microbial agents as insecticides is their relatively short persistence on leaf surfaces. The 

realization of the economic potential of mycoinsecticides would benefit from advances in 

biotechnology (Miranpuri and Khachatourians, 1995). Additionally, commercial considerations 

such as identification of existing or novel isolates, quality control of product and patent 

protection (Leathers et al., 1993) would benefit the development of efficient strains. Hence, 

the following objectives were outlined for this study:

1. Systematic survey, isolation and characterization of native entomopathogenic fungi 

from infected cadavers. 

2. Improvement of fungal virulence by mutagenesis. 

3. Field evaluation of the virulent pathogens against selected insects on a test crop. 

4. Optimization of economic mass production techniques of the virulent fungi and longterm 

storage formulation. 

5. Assessment of the compatibility of selected entomopathogenic fungi with 

agrochemicals. 

6. Analysis of persistence of selected entomopathogenic fungi on phylloplane and in soil.

II. REVIEW OF LITERATURE 

The various risk factors associated with the use of chemical insecticides such as 

development of resistance, associated resurgence in insects, accumulation of pesticidal 

residues in food chain, environmental pollution, health risks and high costs have driven the 

scientist and farmers to develop alternative strategies of pest management. This necessitated 

interest on the search for biotic agents that can control destructive pests of crops. Some of 

the most significant progress in recent years has come from studies of insect pathogens, 

particularly the entomogenous fungi. 

2.1 ISOLATION AND CHARACTERIZATION OF 

ENTOMOPATHOGENIC FUNGI 

Even before the Christian Era, man has been concerned about diseased conditions in 

two of his domesticated insect species, the silk worm, Bombyx morii and the honey bee, Apis 

mellifera. The ancient Chinese silk manufacture was affected by fungus attack upon the 

silkworm. Before 1835, sericulturists thought that the diseases were caused by environmental 

or abnormal physiological conditions. Two fungi, Beauveria bassiana Bals. Vuillemin and 

Metarhizium anisopliae Metch. Sorokin are known to be pathogenic to the larval stage of the 

silkworm. Honeybees are known to be infected seriously by fungi such as Aspergillus flavus, 

Pericystis apis and Mucor hiemalis (Burges, 1981). 

Entomogenous fungi comprise a heterogenous group of cover 100 genera with 

approximately 750 species, reported from different insects and living in diverse habitats 

including fresh water, soil surfaces and aerospaces (Hajek and Ledger, 1994), many of which 

offer greater potential in pest management (Maddox, 1994). They belong to zygomycotina, 

ascomycotina, basidiomycotina and deuteromycotina. The major taxa of entompathogenic 

fungi are presented in Table 1. Several of these genera are principally or exclusively 

associated with a single family, genus or a few species of insect pests. A number of 

publications discuss insect mycosis caused by Entomopthora, Beauveria, Metarhizium and 

Aspergillus. Among the entomopathogenic fungi, Metarhizium spp. and Verticillium lecaniii 

(Zimm) have wide host range. Metarhizium spp. infect almost all insect orders (almost 7200 

insect species) inhabiting a variety of environmental niches like soil, soil surface, aerial 

location and fresh water. Verticillium lecanii on the other hand, is highly potent against various 

sucking pests (>50 insects spp.) in varied ecosystems. 

M. anisopliae has been reported to be effective in the suppression of soil borne pests 

like termites, crickets, locusts, brown plant hopper in rice, pyrilla, spittle bug in sugarcane and 

rootgrubs. The fungus as commercial product “metaquino” has been in use in Brazil. It was 

also used against coffee berry borer in Brazil and coconut leaf beetle in Taiwan. Usage of 

entomopathogenic fungi in IPM of rhinoceros beetle paid good dividend in Samoa (Ferron et 

al., 1975). 

V. lecanii has been proved to be a potent fungal pathogen in IPM of sucking pests. 

Wettable powder formulation (mycotol) caused high mortality of aphids, glasshouse whiteflies, 

thrips on tomato and ornamental crops. The organism exhibited greater promise in the 

management of coffee green scale in peninsulur India (Wilding, 1981). Other fungi pathogenic 

on insects include Coelomyces, Nomuraea, Fusarium and Hirsutella. 

The green muscardine fungus, Metarhizium anisopliae was described for the first time 

by Metschnikoff in 1879 as Entomophthora anisopliae. Tulloch (1976) changed the name to 

Metarhizium anisopliae and identified another species Metarhizium flavoviride. Metarhizium 

anisopliae has been recorded from over two hundred insect hosts belonging to Orthoptera, 

Hemiptera, Lepidoptera, Diptera, Hymenoptera and Coleoptera (Veen, 1968). 

Gams (1971) placed all hyphomycetes in the genus Cephalosporium. However, this 

broad categorization caused some confusion in the interpretation of this genus. The 

cephalosporia was re-examined and placed in a group of species containing many

entomogenous fungi into a new section. One of these, Verticillium lecanii, contains numerous 

hitherto separated species, many of which have been described as insect pathogens. 

The entomopathogenic fungus N. rileyi was described for the first time by Farlow in 

1883 as Botrytis rileyi. Later, it was transferred to Spicaria (Beauveria rileyi) by Charles in 

1936. Kish et al. (1974) changed the name of Spicaria rileyi (Farlow) Charles to Nomuraea 

rileyi (Farlow) Samson. The genus Beauveria was described in 1912 by Vuillemin Maclleod. 

Dead larvae generally mummify due to fungal infection. The cadavers show an initial white 

mycelial growth on the insect surface (except on the head capsule) and dark green (M. 

anisopliae), white (Verticillium lecanii) due to formation of conidia either in localized patches 

or over the entire surface. However, difference in conidial colour was observed. Hence, for 

confirmation of the pathogen, microscopic examination (400x) was necessary. N. rileyi 

conidiophores bear dense whorls of branches and phialides i.e. conidiogenous cells which are 

short necked, conidia are broadly ellipsoidal to cylindrical 3.5 – 4.5 x 2.3 µm. 

The conidiophores of M. anisopliae are arranged in compact to nearly stomatic 

patches, mostly, mononematous. Conidigenous cells phialides in whorls, arranged in a candle 

like fashion, clavate to cylindrical, conidia are single celled, smooth walled hyaline to slightly 

coloured, forming long chain often aggregated into prismatic columns whereas, in case of 

Verticillium, phialides usually are awl shaped conidia of various shape produced in slimy 

heads. 

2.2 SELECTION OF ISOLATES OF ENTOMOPATHOGENIC 

FUNGI 

Isolates of the fungus from different host insects have varying degrees of virulence as 

measured by per cent mortality in bio assays (Ignoffo et al., 1976), broader host range and 

time taken for spore germination. The characteristic of the isolates selected for field use 

should necessarily be virulent, specific, amenable to mass production and withstand the 

environment in which it is used. The insect however, present natural barrier to infection. 

A variety of fungicides and antibiotics have been used in selective media to isolate 

entomogenous fungi, Metarhizium spp. and Beauveria spp. from environments such as the soil. 

The first media (Veen and Ferron, 1966) contained chloramphenicol and cycloheximide 

(Actidione) and were based on a medium developed for the isolation of fungi from clinical 

specimens. Later, a simplified form of medium of Veen and Ferron, known as “Veen’s 

medium” has been successfully used in their laboratory (Milner et al., 1992) and many other 

laboratories for quantitative isolation of Metarhizium spp. from soil. Other workers, notably 

Doberski and Tribe (1980) have used similar media. 

In 1982, it was found that media incorporating the fungicides, dodine 

(N=dodecylguanidine monoacetate) successfully isolated Metarhizium anisopliae from garden 

soil while normal soil planting media did not (Beiharz et al., 1982). This lead to the 

development of Dodine based media for selective isolation of Beauveria bassiana from soil 

(Chase et al., 1986) and M. anisopliae from cockroaches. Liu et al. (1993) studied the effects 

of different concentrations of dodine on germination and colony formation by nine isolates of 

Metarhizium spp. Because of difference between isolates in their susceptibility to dodine, the 

lower concentration of 10 µg/ml was recommended for selective isolation from soil. 

M. anisopliae and B. bassiana have a world wide distribution as members of the 

natural soil flora (Zimmermann, 1993). M. anisopliae has been reported in soils from widely 

differing climatic zones and have entomopathogenic activity against a range of insect pests 

(Gillespie and Claydon, 1989 and McCoy et al., 1988). Yip et al. (1992) characterized 204 

cultures of M. anisopliae isolated from Tasmanian soil and grouped them into 16 strains. 

Margaret et al. (1991) isolated Verticillium lecanii from larvae of pear thrips and 

suggested relatively high incidence and wide distribution of V. lecanii infection in Vermount.

The entomopathogenic fungi so far isolated along with their host are mentioned in 

Table 1. 

2.3 MODE OF ENTRY AND VIRULENCE OF 


Pathogenesis is the process of chain of events in the disease development in a host 

upon infection. Differing from bacteria and viruses, fungal pathogenesis in insects occurs via 

a series of integrated, systematic events progressing upon spore attachment to germination, 

penetration, growth and proliferation within the body of the host, interaction with insect 

defense mechanism and finally re-emergence on the cadavers (Nadeau et al., 1996; Thomas 

et al., 1996). 

The infective unit in most of the entomopathogenic fungi is a conidium or spores 

which when land on a susceptible host, put forth germ tubes or infection pegs from 

appressoria. These structures secrete a complex of cuticle degrading enzymes viz., 

chitinases, proteases and lipases, which are capable of hydrolyzing corresponding cuticular 

constituents viz., chitin, protein and lipids (St Leger et al., 1992). This facilitated the germ tube 

to invade haemocoel and fat bodies. The invading vegetative hyphae consumes the contents 

of haemolymph for its growth and metamorphosis. On exhaustion of the haemolymph content, 

the host insect become moribund and the fungi sporulate after death of the host. 

Undoubtedly, many pathogen enzymes are important determinants of virulence since 

they enable the pathogen to coexist with the changing metabolic processes associated with 

the host’s diseased state. St Leger et al. (1992) cloned a gene encoding protease (Pr1) from 

M. anisopliae which solubilized cuticle protein to assist penetration and provided the nutrients 

for further growth (St Leger et al., 1988). 

The cuticle is the first barrier to infection by fungi. Hence, rapid and direct penetration 

of the cuticle is important for virulence (Pekrul and Grula, 1979). The insect procutide is 

primarily chitin fibrils embedded in a protein matrix and penetration appeared to involve both 

mechanical and enzymatic components (Charnley and St. Leger, 1989; St. Leger et al., 

1988). Penetration is a stage of infection where specificity may be determined since, many 

pathogens are virulent after being injected into the haemolymph of an otherwise nonsusceptible 

host. Virulent isolates, however, had 10-17 times more endochitinase activity and 

15-28 times more exochitinase activity than virulent isolates (El-Sayed et al., 1992). 

Metabolites of fungal pathogens are involved in the infection process. Pigments like 

bio-chrome such as bassianin and tenellin or dibenzoguinones such as oosporin are 

responsible for colour change in the insect body (Sundarababu, 1985). Many of the 

entomopathogenic fungi produce toxins which act as poisons for the insects and thereby are 

killed. 

Aerial conidia sporulated from infected or mummified cadavers are widely 

disseminated by wind. Splashing of rain also accounted for spreading but only for short 

distances. In water stagnated irrigated rice ecosystem, the fungal propagules (conidia or 

spores) are disseminated through irrigation water (Ambethgar, 1991).

Sl. 

No. 

Table 1. Occurrence and pathogenicity of entomopathogenic fungi in 

different insect hosts 

Insect species Fungi Insect order Crop plant Country References 

1. Adoryphorus couloni 

(Burm). 

2. Ancognatha 

scarabaecide 

3. Aphodius tasmaniae 

Postuare 

4. Coleomegilla 

maculate 

5. Phyllophaga anxia 

(Leconte) 

6. Popillia japonica 

Newn. 

7. Pterostichus sp. 

(Nialoe) 

8. Ferficual auricularia 

L. 

9. Acyrthosiphon 

pisum (Maskel) 

10. Nilaparvata lugens 

(Stal.) 

11. Agrotis segetum 

(D&S) 

12. Austracris guttulosa 

(Walker) 

13. Locusta magratoria 

(L.) 

14. Mahanarva postica 

(Stal.) 

15. Orinebius kanetataki 

(Finot) 

16. Cosmopolites 

sordidus (Ger.) 

17. Dicladispa armigera 

(Oliver) 

18. Leptinotarsa 

decemlineata (Say) 

19. Plocaederus 

ferrugineus (Linn.) 

Metarhizium Coleoptera - Australia Rath et al. 

(1995) 

Metarhizium Coleoptera - Columbia Rath et al. 

(1995) 


(1995) 


(1995) 

Metarhizium Coleoptera - Canada Butt et al. 

(1994) 

Metarhizium Coleoptera - Japan Butt et al. 

(1994) 

Metarhizium Coleoptera - USA Butt et al. 

(1994) 

Metarhizium Dermoptera - UK Butt et al. 

(1994) 

Metarhizium Orthoptera - UK Butt et al. 

(1994) 

Metarhizium Orthoptera Rice India, 

Srilanka 

Ambethgar 

(1997) 

Metarhizium Orthoptera - UK Butt et al. 

(1994) 

Metarhizium Orthoptera Cruciferous 

vegetables 

Australia Butt et al. 

(1994) 

Metarhizium Orthoptera Many crops Germany Kleepies and 

Zimmerman 

(1992) 

Metarhizium Orthoptera - Brazil Kleepies and 

Zimmerman 

(1992) 

Metarhizium Orthoptera - Brazil Kleepies and 

Zimmerman 

(1992) 

Beauveria spp. Coleoptera Banana Kenya Kaaya et al. 

(1991) 

Beauveria spp. Coleoptera Rice India Puzari et al. 

(1997) 

Beauveria spp. Coleoptera - USA Butt et al. 

(1994) 

Beauveria spp. Coleoptera Cashew India Ambethagar et 

al. (1998) 

20. Apis gossypii (Glov) Beauveria spp. Homoptera Beans Columbia Landa (1984) 

21. Bemisia argentifoli 

(Geog) 

22. Nilaparvata lugens 

(Stal) 

Beauveria spp. Homoptera Beans Columbia Landa (1984) 

Beauveria spp. Homoptera Rice India Ambethagar 

(1996)

Table 1. Contd….. 

Sl. 

No. 

Insect species Fungi Insect order Crop plant Country References 

23. Agrotis segetum 

(Schiff) 

24. Cnaphalocrocis 

medinalis (Guenee) 

25. Helicoverpa 

armigera (Hub.) 

Beauveria Lepidoptera Crucifers UK Butt et al. 

(1994) 

Beauveria Lepidoptera Rice India Ambethgar 

(1991) 

Beauveria Lepidoptera Chickpea 

pigeonpea 

26. Achaea janata Linn. N. rileyi Lepidoptera Castor 

bean 

27. Anticarsia 

gemmatalis (Hb.) 

28. Helicoverpa 

armigera (Hub.) 

29. Heliothis subflxa 

Guene 

30. Peridroma saucia 

(Hubner) 

31. Spodoptera exigue 

(Hbst.) 

32. Spodoptera litura 

(F.) 

33. Stenachroia 

elongella Hmps 

34. Myzus persicae 

Sulzer 

N. rileyi Lepidoptera Velvet 

bean and 

soybean 

N. rileyi Lepidoptera Potato and 

tomato 

India Gopalkrishnan 

and Narayan 

an (1988) 

India Vimladevi 

(1994) 

Brazil 

Ecuador 

Indonesia 

and Korea 

Stansly et al. 

(1990); 

Gilreath et al. 

(1986) 

Hugar and 

Hegde (1996) 

N. rileyi Lepidoptera - USA Ignoffo (1981) 

N. rileyi Lepidoptera Soybean USA Puttler et al. 

(1976) 

N. rileyi Lepidoptera Soybean India Phadke et al. 

(1978) 

N. rileyi Lepidoptera Tobacco 

chilli 

India, 

Japan 

Roa and 

Phadke (1977) 

N. rileyi Lepidoptera Sorghum India Phadke et al. 

(1978) 

V. lecanii Homoptera Salad 

crops 

- Chandler 

(1992)

2.4 ENVIRONMENT INFLUENCE ON NATURAL 

PATHOGENICITY 

Environmental factors which influence the virulence of entomopathogens must be 

considered for the successful development of the fungus as a biocontrol agent. Of all the 

ecofactors that influence epizootic of a mycopathogen, none is more critical for sporulation, 

germination and invasion of the host than high humidity (>90% RH) (Getzin, 1961; Allen et al., 

1971). Pathogenesis occurs at much lower ambient values (Ferron, 1978; Ramoska, 1982) 

probably because of high humidity in the microclimate at the insect cuticle. It is certain, 

however, that the external sporulation never occurs on the killed insect, if the relative humidity 

is too low. 

The rapidity of mycelial development and therefore, the rapidity of the evolution of 

infection depends on temperature. In general, optimum values fall between 20°C and 30°C 

(for example, 23°C for Beauveria brongiartii, 24°C for Entomophthora obscura, 25°C for 

Beauveria bassiana and Nomuraea rileyi and 27°C-28°C for Metarhizium anisopliae) with 

limits between 5° and 35°C. Temperatures lower than the optima distinctly retard the 

development of mycosis without necessarily affecting the total mortality (Ferron, 1978). 

Atmospheric temperature between 20°C and 30°C did not limit the growth and 

sporulation of N. rileyi. On the other hand, since the fungus will not grow below 15°C and 

sporulate above 30°C, long periods at these extreme limit both the initiation and development 

of epizootics (Ignoffo et al., 1976). The spore germination and colony growth of the C-3 

isolate of Verticillium lecanii had similar temperature optima. Conidia germinated more rapidly 

between 20°C and 25°C. Both germination and growth declined steeply above 25°C and 

ceased above 30°C (Burges, 1981). Gillespie and Crawford (1986) found that the infectious 

conidia of three isolates of M. anisopliae did not germinate at 93 per cent RH and that 

germination was inhibited significantly below 98 per cent RH. They reported similar results in 

the isolates of Verticillium, Beauveria and Paecilomycos, suggesting that the requirement for 

high humidity was fundamental to these fungi and strains with reduced dependence on 

humidity were unlikely to be found. Drummond et al. (1987), however, found that some 

isolates of Verticillium lecanii could tolerate lower humidities. 

Hywel-Jones and Gillespie (1990) examined spore germination of M. anisopliae and 

B. bassiana at 20-30°C and found higher germination levels (95% in 10-14 h) in M. anisopliae 

as compared to B. bassiana (95% in 14-15 h). 

The optimum temperature for the development of the fungus is not necessarily the 

same for the development of the disease. However, the influence of temperature on the host 

insect must be taken into consideration, since very short periods between moults resulting 

from a high temperature may reduce, for example, the duration of the instar to an extent that 

penetration of the fungus through the integrument is impeded. 

Survival of M. anisopliae applied to soil depend on a range of physical and biological 

factors such as soil moisture, temperature and other soil microbes (Lingg and 

Donalson, 1981). The inoculum of N. rileyi when exposed to direct sunlight on the upper leaf 

surface of cabbage reduced the half-life of spores to 3.6 h (Fargues et al., 1983). 

Rath (1992) found that the distribution and abundance of M. anisopliae and B. 

bassianas strains varied with rainfall, soil type and pH. However, there was no obvious 

relationship between temperature and the distribution of M. anispliae strains. However, the 

strains could be separated on the basis of the temperature range over which they germinated 

(McCammon and Rath, 1994). 

M. anisopliae DATF-001 spores were able to germinate on Czapeck-dox agar at all 

temperature from 2 to 25°C. Rath et al. (1995) tested the third instar larvae of Adoryphrous 

couloni with a concentration of 4.1 x 10 6 spores/g at different temperature. DATF-001 was 

pathogenic at all the temperatures and the LT50 values ranges from 36.1 days at 15°C to 

188.9 days at 5°C.

Milner et al. (2002) studied the effect of relative humidities (RH) from 90 to 100 per 

cent on germination of a termite-active isolate of M. anisopliae (isolate F125 and FI610) using 

a liquid germinating medium. Germination was increasingly delayed at water activities 

equivalent to 99, 98 and 96 per cent RH and was completely inhibited at 94, 92 and 90 per 

cent. 

Forgues and Luz (2000) studied the effect of both moisture and temperature on the 

infectivity of Beauveria bassiana and reported that the most favourable conditions were 97 

per cent RH and temperature of 20°C combined with either 75 per cent, 25°C or 43 per 

cent. Under less favourable alternating conditions (lower and higher temperature) the amount 

of inoculum required for killing 50 per cent of first instar nymph was 10 or 20 times higher. 

2.5 MASS PRODUCTION OF THE ENTOMOPATHOGENIC 

FUNGI 

2.5.1 Nutritional requirements 

The growth requirements of most entomopathogenic fungi have been poorly defined 

despite the fact that this information is essential for mass production. The choice of the 

nutrients will obviously be directly related to the nutritional requirements of the selected 

fungus. Entompathogenic fungi require oxygen, water, an organic source of carbon and 

energy, a source of inorganic or organic nitrogen and additional elements including minerals 

and growth factors. 

Many nutrient compounds including carbohydrates, organic acids, amino acids, and 

vitamins are important components of plants root exudates and soil. In addition to significant 

quantities of these substances exuded from healthy roots, more are released through cell 

lysis. The survival of fungi in a soil may be affected greatly by these compounds through their 

effects on mycelial growth, sporulation and conidial germination. Organic acids are important, 

not only because they are a source of readily available substrates for soil microorganisms, but 

also because of their secondary effects such as modification of pH in the rhizosphere and 

chelation of metals (Rovira and Wildermuth, 1981). 

Nutrients are also present on the surface of insects. Woods and Grula (1984) 

reported the presence of 17 amino acids, glucosamie, amines and peptides on the surface of 

Helicoverpa zea larvae and that these were sufficient to initiate germination and support the 

growth of Beauveria bassiana and Aspergillus niger. Smith and Grula (1983) suggested that 

nutrients on insect larval surfaces supported the conidial germination of B. bassiana. 

Mass production of B. bassiana on solid or liquid media under sterile or semi sterile 

conditions has been described (Samsinakova et al., 1981). 

According to Riba and Glander (1980), Tween-80 and higher concentration of yeast 

extract are the most essential components of the culture medium. Sutton et al. (1981) 

reported that casein at a concentration of 0.04 per cent and trehalose at 0.28 per cent 

promoted the growth of N. rileyi. However, ascorbic acid levels tested had no significant effect 

on the mycelial development. In vitro utilization of available complex proteins, trehalose and 

other nutrients govern the parasitic nature of the pathogen. 

Holdom et al. (1986) from their studies in Australia showed that N. rileyi could grow 

on an inexpensive protein based culture medium made of Brewer’s yeast, yeast hydrolysate, 

skim milk powder and whole milk powder. Various carbon sources such as soluble starch, 

corn starch or malt extract were additionally used with the protein base medium. However, 

neither the growth and conidial production were consistent. Carrot malt agar and oats malt 

agar were good for sporulation of N. rileyi (Balardin and Loch, 1989). The best combination of 

components for sporulation of the Londrina isolates was 340 ml carrot extract and 22 g malt 

meal per litre of water with 12 min sterilization. Dillon and Charnley (1990) found that soaking 

M. anisopliae conidia in distilled water could stimulate initial process in conidial germination,

and that the conidia needed carbon sources for spherical growth and germ-tube formation. 

Maltose and dextrose were found to be good carbon sources for sporulation of N. rileyi (Im et 

al., 1988; Vimaladevi, 1995). The fungus grew well on media with pH 5-8. 

Li and Holdom (1994) examined the effects of a range of carbon, nitrogen sources 

and vitamins on colony formation, mycelial growth and sporulation of two isolates (EF25 and 

EF55) and concluded that soluble starch was best among different carbon sources tested for 

growth of M. anisopliae. Interestingly, they found that nitrogen sources rich in amino acids 

showed stimulatory effect on growth and germination. 

Gopalkrishnan and Mohan (2000) tested 13 different synthetic media for sporulation 

of N. rileyi. Only SMAY, Carrot Agar Yeast (CAY), Corn Meal Agar Yeast (CMAY), Nutrient 

Agar Yeast (NAY) and Czepecks dox Agar Yeast (CAY) showed sporulation. According to 

them, enrichment of synthetic media with yeast extract was a must for mycelial growth and 

sporulation. SMAY and CAY were found suitable for production and culturing of N. rileyi. 

Though the spore yield in both cases did not differ (0.56 g/100 ml of medium) cost wise, CAY 

was cheaper. 

James (2001) tested exogenous protein and sugar sources on conidial germination of 

two pathogens, Beauveria bassiana and paecilomyces fumosoroseus. In liquid culture, sugars 

stimulated only 5-27 per cent germination of B. bassiana and less than or equal to 11 per cent 

germination of P. fumosorosus, whereas yeast extract or peptone stimulated 95-100 per cent 

germination. 

Rath et al. (1995) examined the utilization of 49 carbohydrates for 134 isolates of M. 

anisopliae and concluded that carbohydrate utilization was a useful and biologically relevant 

taxonomic criteria for the separation of Metarhizium strains and other entomogenous fungi. 

2.5.2 Natural media 

Several attempts have been made to multiply the entomogenous fungi using semisynthetic 

media and solid substrates in order to cut down the cost of production. Simple and 

cost effective mass production technology is required to make it a highly acceptable bioagent. 

It is reported the most of the deuteromycetes sporulate readily on solid media under 

aerated conditions. Semi solid fermentation provide an alternate in which the fungi grown 

primarily on the wet surface of a solid material form of processed cereal grains to which 

nutritional adjuvants were added or media of low value such as agricultural wastes. Loose 

substrates were observed to yield more conidia than solid substrates like agar (Muller, 2000). 

For mass production of conidia of M. anisopliae, a number of naturally occurring 

carrier cum growth media have been evaluated. Fogal et al. (1986) described a simple mass 

production technique for M. anisopliae using wheat bran as the carrier medium. A number of 

carrier cum growth media such as wheat bran sorghum grains, boiled rice grains, coffee husk, 

coconut water etc. have been used to assess their efficacy in promoting the growth and 

sporulation of M. anisopliae. Results showed that boiled rice was the best carrier cum growth 

medium followed by wheat bran and sorghum grain for profuse growth and abundant 

sporulation of M. anisopliae. As rice and sorghum grains are expensive the use of wheat bran 

as carrier medium is more profitable. 

Mass culturing of B. bassiana and production of conidial masses have been achieved 

using substrates such as wheat bran, whole grains, hay, straw, potatoes etc. either in plastic 

bottles, flasks or trays. McCoy and Carver (1941) described a simple method for the mass 

production of conidia of B. bassiana using wheat bran as the carrier medium. 

Grain and bagasse were shown to support the multiplication of the mycopathogen in 

a two phase conidial. The yield was significantly reduced on synthetic media but cost was low 

(Holdom et al., 1986). Silva and Loch (1987) opined that the organism could easily be 

multiplied on polished rice grains. Boiling the rice grains before sterilization resulted in higher 

spore yields. The entomopathogenic M. anisopliae was grown on several laboratory media of 

which Czapecks Dox medium was most suitable. The optimum temperature for growth was

25°C for sporulation and biomass production. Among various cereal grains tested as growth 

media, rice was the most suitable substrate. Among the agro-based material tested, puffed 

rice waste was found to be superior (Patel and Yadav, 1990). 

A method for production of the entomogenous fungus Metarhizium anisopliae on 

coarse grain rice was described (Quintela and McCoy, 1997). The production of conidia on 

coarse grain was significantly greater than on whole grain. With coarse grain, there was 

reduction in the cost of production by four times and increase in the production of conidia by 

30 per cent. 

Vimaladevi (1994) cultured the mycopathogen N. rileyi on crushed sorghum enriched 

with one per cent yeast extract. Sporulation was highest with maximum of 1.44 x 10 9 

conidia/g of crushed sorghum after 8-9 days at 25°C. Lopez et al. (1995) grew the fungus N. 

rileyi on rice sorghum and soybean and stored it for three months at 40°C. Multiplication on 

barley and also a semisynthetic medium wherein maltose was replaced by cereal extract 

were promising for cost effective production of the mycopathogen. Kulkarni (1999) studied the 

suitability of different cereal grains for mass production of N. rileyi and found that sorghum 

and rice grains were the most efficient media with an yield of 13.45 x 10 8 and 13.15 x 10 8 

respectively. 

Lopez et al. (1995) used six plant waste substrates; palm leaves (Phoenix dactylifera, 

Phoenix canariensis, Washingtonia filfera and Chamaerops humilis), Phoenix dactylifera seed 

and almond mesocarp to mass produce entomopathogens (Verticillium lecanii, Metarhizium 

anisopliae, Paecilomyces farinosus, Beauveria bassiana). Of all the plant waste substrates 

tested, only M. anisopliae grew on almond mesocarp. C. humilis leaves were excellent 

substrates for the growth and sporulation of both V. lecanii and B. bassiana, B. bassiana grew 

best on seeds of P. dactilyfera. 

2.5.3 Formulation 

Despite the wide host range of the fungi, the number of commercial products of these 

fungi has been small. Most of these products have tended to be ephemeral and it is therefore, 

difficult to specify the products available for sale at any one date. Those on sale at present 

include a few wettable powders that have been available since 1980’s e.g. Vertalac against 

aphids and mycotal against white flies, sporadically used in European greenhouses and 

containing blastospores of different strains of Verticillium lecanii. Beauveria bassiana has 

been applied on a large scale on various out door pests in Russia (Boverin), China and Brazil 

(Metarhizium anisopliae). Products including bioblast (Metarhizium anisopliae) against 

termites in the USA and Naturalis (B. bassiana) in France are being used (Burges, 1998). 

2.6 STORAGE OF THE FORMULATION OF 


Because of their lipophilic sufaces, dry and dusty conidia mix readily in oil. However, 

conidia of M. flavoviride freshly harvested by suction but not pre-dried did not survive as well 

in undried oil as in an unformulated powder. More pre-dried conidia germinated after storage 

as a dry powder with silica gel pellets than as an oil suspension (Morley-Davies et al., 1995). 

For example after 13 weeks storage at six temperatures between 10 and 50°C, the difference 

ranged between 3 and 12 per cent germination. The opposite effect was found by Moore et al. 

(1996) in their only batch of pre-dried conidia stored both as a powder (76 per cent 

germination) and in oil (91% germination). 

Studdert et al. (1990) coated dry B. bassiana conidia with a bentonite clay by mixing 

1:3 by weight, moistening a thin layer tightly with a water mist and drying. The clay increased 

the half-life of conidia in both sandy loam and peat from 207 weeks to 7-12 weeks at 30°C 

and from 12-44 weeks to 20-64 weeks at 10°C, at different combinations of soil and moisture 

content. Fargues et al. (1983) prolonged survival of B. bassiana blastopores by clay coating. 

Inglis et al. (1993) found that Beauveria bassiana conidia dispersed better in oil 

(Mycotech 9209) than in 0.05 per cent aqueous Tween-80, while substantial clumping

occurred in a 5 per cent emulsion of the oil in water even after homogenization. Conidia of 

Metarhizium flavoviride in sprays of oil and oil in water emulsion survived longer on foliage 

than those in water, which they presumed to be because, the oil component gave greater 

protection from environmental stresses (Jenkins and Thomas, 1996). 

Nutrients such as cereal flour can be formulated as the carrier in a dust on wettable 

powder. The best known example is Verticillium lecanii in the products Vertalec and Mycotal, 

used against aphid and whitefly intermittently since 1980 (Burges, 1981; Ravensberg et al., 

1990). Flour based V. lecanii products gave better pest control than sprays of spores alone 

(Kanagaratnam et al., 1982). 

Very few oils have been tested in the presence of dried silica gel. With M. flavoviride, 

there was no consistent difference between diesel oil, odourless kerosene, shellsol k, aviation 

fuel, groundnut oil or soy oil. Anti oxidants improved conidial survival in groundnut oil and in 

soy oil by 23-26 per cent (Moore et al., 1996). 

For conidial storage, mineral oils collectively, were similar to vegetable oils, some of 

which have the disadvantages of rancidity and solidification over long periods at high ambient 

temperature. Kerosene was found to be slightly better than groundnut (Stathers et al., 1993). 

Certain fatty acids from rancid oil inhibit germination of M. flavoviride conidia (Barnes and 

Moore, 1997). 

Hayler (1993) reported that the addition of rape seed oil to the fungus, V. lecanii 

increased efficiency upto 90 per cent when tested on. A. gossypii and Frankliniella 

occidentalis on cucumber in a greenhouse experiment. 

2.7 EFFICACY OF ENTOMOPATHOGENIC FUNGI AGAINST 

INSECTS 

2.7.1 Efficacy of Verticillium lecanii (Zimm.) against aphids 

Verticillium lecanii (Zimm.) (Monilales:Moniliaceae), an entomopathogenic fungus 

found world wide, has been used successfully as biological control agent against various 

species of aphids for number of years (Hall, 1982). 

The suspension of Verticillium lecanii with concentration of 16 x 10 6 spores/ml, when 

used against coffee green bug, Coccus viridis (Green) in Tamil Nadu, gave 16.6 per cent 

morality. The sprays containing surface active agents like Teepol, Triton x 100 at 0.03 per 

cent or glycerol at 0.1-0.3 per cent gave 48.9, 79.9 and 22.4 to 31.0 per cent mortality, 

respectively. Khalil et al. (1983) in Czechoslovakia, while studying on V. lecanii, found that 

when the fungus was applied at concentration of 10 8 spores/ml in sprays to plants in 

glasshouse was highly effective against the aphid species, Aphis fabae on sugar beet and 

Myzus persicae on cucumber. 

The effect of a range of humidities on the transmission and sporulation of a 

commercial preparation (Vertalec) of V. lecanii was investigated in greenhouse at 20°C on 

capsicum against aphid M. persicae by Milner and Lutton (1986). They found that at least 36 

hours is required for infection at 100 per cent relative humidity. After 96 hours of spraying, 

94.5 per cent of M. persicae were infected. 

Evaluation of the entomopathogen V. lecanii in the control of the aphid, M. persicae 

on chrysanthemum was conducted by Hincapie et al. (1990). Three strains of the fungus viz., 

VL-A, isolate from M. persicae, VL-GC isolated from Erinnyis ello and VL-MR from 

trialeurodes vaporariorum Weshw were used. VL-A caused 100 per cent mortality compared 

to 37.5 per cent for VL-GC and 30 per cent for Vl-MR. Three concentrations of VL-A was 

evaluated (1x10 4 , 1x10 6 , 1x10 8 ) and the mortality increased from 39.5 to 100 per cent.

Pathogencity of various strains/isolates of V. lecanii has been well established 

against various aphid species viz., A. fabae, D. noxia (Fegan et al., 1992), Sitobian avenae F. 

(Fegan et al., 1993; Miranpuri and Khachatourians, 1995), R. padi and M. persicae (Gardner 

et al., 1998). 

Khalil et al. (1990) studied the effectiveness of V. lecanii against the aphid M. 

persicae in laboratory, on one year old potted peach plants. The fungus was effective at 

concentrations of 10, 10 5 and 10 7 spores/ml with number of aphids alive/cm leaf area after 25 

days being 0.68, 0.61 and 0.57, respectively, compared with the control value of 1.91. 

Masuda and Kikuchi (1992) investigated pathogenicity of V. lecanii isolates on 

aphids, A. gossypii, M. persicae and whitefly T. vaporariorum. Both isolates MGV118 and 

MGV 145 were pathogenic to larvae and adults of T. vaporariorum than MGV 145. MGV 145 was 

stronger than that of MGV118 on the apterous adults of A. gossypi and adults of M. persicae. 

The mortalities caused by the two isolates were almost the same (96-100%) at higher 

concentration (10 7 and 10 8 conidia/ml). 

Two years of laboratory and field assessments using entomopathogenic fungus, V. 

lecanii against Saskatoon berry leaf aphid, Acryrthosiphon macrosiphum (Wilson) showed 70 

per cent aphid kill, three days after treatment. After five days of treatments, it showed 90 to 

100 per cent aphid kill, three days after treatments. After five days of treatment, it showed 90 

to 100 per cent aphid mortality as compared to 35 per cent in control groups. Field tests using 

two application of V. lecanii showed a significant decline in aphid population as compared to 

that on the water treated plants (Miranpuri and Khachatourians, 1995). 

2.7.2 Effect of V. lecanii against whitefly 

On the spiraling whitefly, A. disperses, no research work on the use of microbial 

pesticides has been carried out so far. 

Quinden (1984) tested the mycotal formulation of the fungus Verticillium lecanii 

applied to tomato crop against greenhouse whitefly, Trialeurodes Vaporariorum at the rate of 

5 x 10 11 spores/ha which gave significant control of the aleyrodid only 16 days after 

application. 

Suklova (1989) reported that V. lecanii + boverin (Beauveria bassiana) gave 98 per 

cent control of whitefly. Optimum condition for these fungi was 80-90 per cent relative 

humidity and a temperature of 26-28°C. 

Meade and Bruce (1991) reported on the mortality of various instars of B. tabaci and 

T. vaporariorum resulting from exposure to V. lecanii. The mortality of nymphs of all three 

instars of both species due to fungal infection was significantly higher than that in other 

treatments. 

According to Nier et al. (1993), the pathogencity of V. lecanii against nymphs of T. 

vaporariorum and B. tabaci at the concentration of 3.2 x 10 6 conidia per ml gave 91 and 100 

per cent mortality, respectively, but a suspension containing 1 x 10 7 conidia per ml resulted in 

infection rate of 78 per cent. B. tabaci was found more susceptible than T. vaporariorum. 

Application of the fungus, Paecilomyces persimilis (Wize) followed by three 

introductions of two Encarsia formosa per plant and treatment of Ashersonia species gave 

good control as did the application of V. lecanii against B. tabaci (Landa, 1984). 

Field trials were carried out in Tamil Nadu by Jayaraj (1989) to determine the 

effectiveness of V. lecanii in controlling the coffee scale Coccus viridis. The fungus caused 

73.1 per cent mortality of the pest when applied at 1.6 x 10 6 spores per ml twice at an interval 

of two weeks. The maximum mortality of 97.6 per cent was obtained when the surfactant, 

tween 20 was added to the spore suspension. High volume spray was more effective than low 

volume. The addition of 0.1 per cent glycerol enhanced the effectiveness of the fungus.

2.8 COMPATIBILITY ENTOMOPATHOGENIC FUNGI WITH 

AGROCHEMICALS 

Compatibility of entomopathogenic fungi with pesticides used in commercial crop 

protection systems is critical, if these fungi are to be utilized for insect control. Since many 

fungicides have broad spectra of activity, the suppression of entomopathogenic fungi by 

fungicides is of particular concern. Though there are many methods of evaluating fungicidal 

activity, most studies of entomopathgenic fungi, have used inhibition of growth in liquid or on 

solid media as the primary criterion (Roberts and Campbell, 1977). 

Soper et al. (1974) tested the efficacy of fungicides on growth of several species of 

entomopathogens in agar media and found that all were inhibitory. Growth of B. bassiana, a 

member of the moniliales and pathogen of several insect pests was inhibited by fungicide in 

solid and in liquid media. 

Teddeur (1981) evaluated six fungicides against the entomopathogenic fungi 

Beauveria bassiana (Balsamo) Vuilemin and Metarhizium anisopliae, both of which attack the 

pecan weevil, Curulio caryae. Triphenyltin hydroxide was the most toxic fungicide to both 

pathogens, followed by benomyl, methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate, 

zineb, zince ethylenebis (dithiocarbamats) and dodine, n-dodecylaquanidine acetate. Sulphur 

and denocap were the least toxic. 

Four fungicides used commercially for control of foliar diseases of potato were 

evaluated in vitro and under field conditions of effects on survival of spores of Beauveria 

bassiana, a pathogen of the Colorado potato beetle, Leptinotarsa decemlineata. Mancozeb, 

the most detrimental of the fungicides, substantially reduced the survival under any of the 

conditions examined (Loria et al., 1983). 

Metarhizium anisopliae and Beauveria bassiana are both compatible with many 

commonly used pesticides and are not toxic to human beings (Burges, 1981). M. anisopliae is 

soil borne fungus and infects over 200 hosts indicating a need to evaluate compatibility with 

non-targets, with pesticides and natural enemies (Gardner et al., 1998). Most importantly, 

entomopathogenic V. lecanii strains have been found non-pathogenic to plants and humans 

(Burges, 1981). 

Recommended concentrations of insecticides viz., fenitrothion, monocrotophos and 

phosphomidon and the fungicides like ziram, foltaf, dithane Z-78, chlorothalonil, captan and 

wettable sulphur were found safe to the fungus N. rileyi. Silva et al. (1993) conducted 

experiments to evaluate the effect of endosulfan (175 g a.i.), prefenophos (100 g a.i.), 

trichlorfon (400 g a.i.) and permethrin (12.5 g a.i.), on sporulation in vitro on SMAY medium. 

Sporulation was totally inhibited by profenophos and endosulfan, while trichlorfon reduced the 

conidial production. There was no significant difference between permethrin and untreated 

control. 

Influence of nine insecticides on the natural infection of A. gemmatalis by N. rileyi 

were studied by Barbosa et al. (1997). The effects of trichlorfon and chlorpyriphos did not 

differ from the untreated control. Baculovirus anticarsia, diflebenzuron, endosulfan, 

methamidophos, monocrotophos, methyl parathion and thiodicarb showed similar 

performance and caused significant decrease in the percentage of mycosed larvae. Tang and 

Hou (1998) evaluated five fungicides, eight insecticides and nine herbicides commonly used 

in maize fields, for their inhibition to conidial germination of N. rileyi by paper disc method. 

Among them, maneb and propineb (fungicides) were highly inhibitory to the fungus while, 

insecticides and herbicides examined did not affect the conidial germination significantly. 

Carbendazim was found to be most detrimental to the fungus inhibiting 75.85 per cent 

growth, while endosulfan, chlorpyriphos, carbaryl, cypermethrin and neem based insecticides 

caused 60.81, 60.25, 37.23, 22.48 and 11.23 per cent growth inhibition, respectively 

(Kulkarni, 1999).

Gopalkrishnan and Mohan (2000) tested seven insecticides and seven fungicides 

which are commonly used for the control of pests and diseases of tomato for their inhibitory 

effect on germination of conidia of N. rilei at three (low, normal and high) concentrations in 

vitro. They concluded that, monocrotophos, phosphomedon and dimethoate were safe to the 

mycopathogen at all the three concentrations. While quinalphos, carbaryl, endosulfan and 

fenvalerate were highly detrimental to the fungus at higher concentration. Among the 

fungicides, captafol, zineb, chlorothalonil, fosetyl Al and ziram were safe to the fungus at all 

the concentrations evaluated. Captan and sulphur on the other hand, though allowed conidial 

germination at low and normal concentrations caused total inhibition at higher concentrations.

III. MATERIAL AND METHODS 

The present work envisaged the isolation of entomopathogenic fungi from insect 

cadavers, their characterization, testing of efficacy against different insect pests, influence of 

different nutrient sources and influences of different environmental parameters. Studies were 

also made on mass production of chosen fungi, formulation and compatability with certain 

pesticides. Additionally the persistence of the fungi in the environment genetic improvement, 

efficacy of the strain in the field were attempted. The material used and methods followed are 

given herein. 

3.1 NATURAL INCIDENCE OF MYCOPATHOGENS AND 

COLLECTION OF SAMPLES 

Extensive and repeated survey for the occurrence of the insect mycopathogens in 

northern Karnataka covering different agro-climatic regions of agriculture and forest ecosystem 

was made during the cropping season of 2000-01. The number of visits to each 

places varied from one to five depending upon the cropping system and host insect 

availability. The details of sampling sites, crops and periods of visits are included in Table 5 

and Figure 1. 

3.2 ISOLATION AND MAINTENANCE OF 


The cadavers of the insects that appeared to be infected by fungi were collected 

during survey and brought to the laboratory and pathogens were isolated on specific media. 

To isolate the fungi, mycosed insects collected from the fields were surface sterilized with 5 

per cent sodium hypochlorite and then rinsed with sterile water several times. In a sterile 

Petridish, the diseased specimens were crushed and a small portion of infected part was 

transferred to a culture plate containing selective medium and kept under constant 

observation for the growth and development of microorganisms. After 5 days of incubation, 

the organisms were sub-cultured for purification. Slants of each culture were prepared from 

purified culture and microscopic observations such as morphological characters of mycelium 

and conidia. 

Preliminary identification of fungi was made with the help of the Atlas of 

entomopathogenic fungi (Samson et al., 1988). Later, the identity of different tentatively 

identified fungal specimens were confirmed at Agarkar Research Institute, Pune 

(Maharashtra). 

3.2.1 Maintenance of culture 

A loopful of inocula from subcultured plates of M. anisopliae and V. lecanii were 

transferred to Potato Dextrose Agar (PDA) slants and maintained as pure culture. Virulence 

was revived by passing through an insect host after 5-6 subculturing. 

For laboratory studies, the fungus was cultured on PDA medium. The medium was 

sterilized at 15 psi for 30 min in autoclave, poured to sterilized plates, cooled and inoculated 

with pure culture of the fungus under aseptic conditions. The plates were then incubated at 

room temperature (26±2°C) for ten days. After complete sporulation, conidia from the medium 

were harvested by washing them thoroughly with sterilized water containing Tween-80 (0.2%) 

for immediate use. Otherwise, spores were harvested with the help of a small sterile metal 

spatula. Harvested conidia were air dried under laminar air flow and stored in a small air tight 

screw cap vials (10 cm with 2.5 cm diameter) in refrigerator at 4°C before using for further 

studies. Colony forming units (cfu) were estimated by plating technique. Suspension of spores 

was made using distilled water with Tween-80 (0.2%) and filtered through a double layered 

muslin cloth. Spore count was made using a double rolled Neubauer’s haemocytometer after

necessary serial dilutions under phase contrast microscope. From the stock solution, further 

dilutions were made to obtain the required concentrations for further studies. 

3.3 INSECTICIDAL ACTIVITY OF Metarhizium anisopliae AND 

Verticillium lecanii AGAINST DIFFERENT INSECT PESTS 

The fungal spores from SMAY plates were mixed with distilled water and 0.2 per cent 

Tween-80 to get the spores suspension. The number of conidia were determined using a 

Neubauer haemocytometer. Finally, the spore suspension containing 1 x 10 8 conidia/ml were 

obtained for the seven individual isolates (Metarhizium anisopliae (4) and Verticillium lecanii 

(3) isolates). The spore suspensions of 1 x 10 8 conidia ml -1 of all isolates were topically 

applied on early instar of pest with a chromatography sprayer. Three replications were 

maintained and in each replication 20 larvae/nymph were tested. Another set was kept 

without addition of spores as negative control. The number of dead larvae was noted down 

from fifth day of inoculation. Finally, the per cent mortality of each isolate was computed. The 

data were subjected to statistical analysis by DMRT (Duncan’s Multiple Range Test). 

3.4 EFFECT OF NUTRITION ON THE TOTAL DRY MYCELIAL 

PRODUCTION OF ENTOMPATHOGENIC FUNGI 

The effect of different carbon and nitrogen sources on the development of biomass 

of fungi were evaluated by varying different carbon sources in the basic Czapeks medium 

with lactose, starch, fructose and dextrose at 1, 2, 3 and 4 per cent. Sources at recommended 

dose (3%) in the basal media served as the check. Similarly, different nitrogenous sources 

viz., KNO3, NaNO3, (NH4)2SO4, NH4NO3 were evaluated at 0.1, 0.15, 2.0 and 2.5 per cent. 

Initially, the fungi were cultured on SMAY plates for two weeks. The conidial 

suspension was prepared by shaking conidia from a 5 mm diameter agar plug into a test tube 

containing in 10 ml sterile water blank mixed with 0.05 per cent Tween 80. The conidial 

suspension was mixed thoroughly by shaking at 80 rpm for 10 min and the concentration of 

conidia was determined using haemocytometer. Two hundred micro tube (0.2 ml) aliquot of 

the final suspension was inoculated into 250 ml broth containing different sources and levels 

of carbon and nitrogen. The flasks were aerated on a shaker at 90 rpm under room 

temperature for 8-9 days. After complete mycelial growth, the fungal mat was taken out and 

filtered through Whatman No. 1 filter paper later dried under oven at 60°C for two days and 

dry weight was recorded. 

3.5 COMPATIBLITY OF THE FUNGI WITH PESTICIDE 

In vitro studies were under taken to assess compatibility of selected insecticides, 

fungicides and weedicides by following the poisoned food technique (Nene and Thapliyal, 

1997) and the details of the different pesticides and the concentration used are given below 

(Table 2). 

The pesticides at required concentration were added to the sterilized SMAY agar and 

poured into the petriplates after proper agitation and allowed to solidify. The conidial 

suspension of the fungi were prepared by shaking conidial from a 5 mm diameter agar plug 

(from a mat of the fungi) into a test tube containing in 10 ml sterile water blank mixed with 

0.05 per cent Tween 80. The conidial suspension was mixed thoroughly shaking at 80 rpm for 

10 min. Hundred microlitre (0.1 ml) of the suspension was spread on SMAY plates containing 

different pesticides and control (no pesticide added). All the plates were incubated at 28±1°C 

and a 12:12 light dark regime.

3.6 EVALUATION OF FOOD GRAINS FOR MASS PRODUCTION 

OF Metarhizium anisopliae (Ma2) AND Verticillium lecanii (Vl1) 

The crushed grains of sorghum, bajra, navane, maize, rice and wheat with 1 per cent 

yeast extract were assessed for their suitability as substrates for mass production of M. 

anisopliae and V. lecanii. In addition to grains, agro waste viz., crushed maize cobs, wheat 

bran, rice bran, baggase and press mud with and without molasses were also tested. To each 

of these substrates, sterile distilled water was added in order to bring the moisture content to 

50 per cent. After thorough mixing, the bottles were plugged with cotton and autoclaved at 15 

psi and 121°C for 30 minutes. Circular agar discs of 5 mm diameter were taken from the eight 

day old fungal culture grown on SMAY plates. One disc was inoculated to each bottle and 

mixed with it to disperse the inoculum. The bottles were incubated in BOD incubator at 

25±1°C. Four replications were maintained for each treatment. The spores were harvested 

from sixth day onwards at definite intervals of upto 25 days by sampling 1 g of the digested 

material. The spore suspension of each sample was made by dispersing the inoculum in 10 

ml sterile water blank with one drop of 0.02 per cent Tween-80, serially diluted and the spore 

count estimated using a haemocytometer. 

3.7 GENETIC IMPROVEMENT OF STRAINS 

The genetic improvement of the fungi was attempted for V. lecanii Vl1 and M. 

anisopliae Ma1 through mutagenesis using UV radiation. 

3.7.1 Mutation using ultra-violet rays 

Seven days old culture was taken and the conidial suspension prepared in sterile 

distilled water. The suspension was centrifuged at high speed (10000 rpm) to pellet the 

conidia. The conidial pellet was washed in 0.1M phosphate buffer (pH 7) and re-suspended in 

the same buffer. The spore concentration was later adjusted to 10 6 conidia/ml. The spores 

were then exposed to UV radiation in Petridish for 20 minutes at different heights from UV 

bulb in the UV cabinet. Later, the surviving population was analyzed by plating the 

mutagenised culture on SMAY agar. The bank of mutants which had the highest killing 

percentage were selected for further studies. 

3.8 PERSISTENCE OF SPORES OF Metarhizium aniospliae 

(Ma2) AND Verticillium lecanii (Vl1) IN SOIL AND 

PHYLLOPLANE 

Survivability of spores in soil and phylloplane in simulated field conditions (in 

glasshouse) and field conditions were studied from January 2001 to May 2002. Spores of M. 

anisopliae and V. lecanii cultured on PDA plates were mixed with soil @ 4 x 10 6 conidia per g 

of dried and sterilized soil and filled in earthen pots. In addition, the same spore load was 

sprayed on leaves one set of potted soil with cotton plant of one month old was stored under 

shade in the laboratory and the other in cage i.e., open to sunlight and rains, simulating field 

situation. Samples were drawn at monthly intervals from individual pots and composite 

sample was observed for the presence of viable conidia in soil and phylloplane. Ten gram of 

soil sample was suspended in 100 ml sterile water containing 0.02 per cent Tween-80 and 

shaken on orbital shaker (120 rpm) for 2 hours. The suspension was sterially diluted and 

plated on SMAY medium maintaining four replications per dilution and incubated at room 

temperature (26±2°C). Colonies of the fungus formed on the plate were counted after 72 h.

Sl. 

No. 

Table 2. Details of different pesticides used in the experiments 

Common name Trade 

name 

Group 0.25 

R.d 

0.5 R.d R.d 2 x R.d 

1. Endosulfan 35 EC Endocel Insecticide 0.70ml/l 1.40ml/l 2.80ml/l 5.60ml/l 

2. Quinalphos 25 EC Ekalux Insecticide 0.50ml/l 1.00ml/l 2.00ml/l 4.00ml/l 

3. Monocrotophos 36 Nuvacron Insecticide 0.62ml/l 1.25ml/l 2.50ml/l 5.00ml/l 

4. Chlorpyriphos20EC Nuchlor Insecticide 0.50ml/l 1.00ml/l 2.00ml/l 4.00ml/l 

5. Oxydemeton methyl 

25 EC 

Metasystox Insecticide 0.35ml/l 0.70ml/l 1.40ml/l 2.80ml/l 

6. Dichlorvos 78 SL Nuvan Insecticide 0.12ml/l 0.25ml/l 0.50ml/l 1.00ml/l 

7. Dicofol 20 EC Kelthane Insecticide 0.72ml/l 1.45ml/l 2.50ml/l 5.00ml/l 

8. Malathion 50 EC Malathion Insecticide 0.50ml/l 1.00ml/l 2.00ml/l 4.00ml/l 

9. Dimethoate 30 EC Rogar Insecticide 4.25ml/l 8.50ml/l 1.70ml/l 3.40ml/l 

10. Carbendazim 50WP Bavistin Fungicide 0.25g/l 0.50g/l 1.00g/l 2.00g/l 

11. Iprodione Rovrel Fungicide 0.62ml/l 1.25ml/l 2.50ml/l 5.00ml/l 

12. Chlorothalonil75WP Kavach Fungicide 0.50g/l 1.00g/l 2.00g/l 4.00g/l 

13. Propiconazole25WP Tilt Fungicide 0.25ml/l 0.50ml/l 1.00ml/l 2.00ml/l 

14. Mancozeb 75 WP Indofil M- 

45 

Fungicide 0.50g/l 1.00g/l 2.00g/l 4.00g/l 

15. Triadimefon 25 WP Bayleton Fungicide 0.25g/l 0.50g/l 1.00g/l 2.00g/l 

16. Hexaconazole Contaf Fungicide 0.25ml/l 0.50ml/l 1.00ml/l 2.00ml/l 

17. Wettable sulfer 80% Thiovit Fungicide 1.37g/l 2.75g/l 5.50g/l 11.0g/l 

18. Oxyflurfon Goal Weedicide 0.12ml/l 0.25ml/l 0.50ml/l 1.00ml/l 

19. Pendimethalin30EC Stamp Weedicide 2.00ml/l 4.00ml/l 8.00ml/l 16.0ml/l 

20. Attrazine 50% Atratof Weedicide 0.33g/l 0.66g/l 1.33g/l 2.66g/l 

21. Butahclor 5% Machete Weedicide 11.0g/l 22.0g/l 4.00g/l 8.80g/l 

22. Fluchloralin 45 EC Basalin Weedicide 0.66ml/l 1.33ml/l 2.67ml/l 5.34ml/l 

23. Glyphosate 41 SL Roundup Weedicide 1.45ml/l 2.50ml/l 5.00ml/l 10.0ml/l 

24. Alachlor 50 EC Lasso Weedicide 0.69ml/l 1.39ml/l 2.78ml/l 5.56ml/l 

R.d.: Recommended dose

Sl. 

No. 

Table 3. Details of food grains evaluated for mass production of 

Metarhizium anisoplia and Verticillium lecanii 

Common name Botanical name Plant part 

1. Sorghum Sorghum vulgare Pers Grain 

2. Rice Oryza sativa L. Grain 

3. Bajra (pearl millet) Pennisetum typhoides L. Grain 

4. Navane Setaria italica Beauv. Grain 

5. Maize Zea mays L. Grain, cobs 

6. Wheat Triticum aestivum L. Grain 

7. Sugarcane Saccaharum officinarum L. Leaves 

A) Oil 

Table 4. Details of carrier materials evaluated for survival studies 

Formulation Concentration 

1. Groundnut 

2. Mustard 

3. Sunflower 

B) Wettable powder 

1. Wheat flour 

2. Sorghum flour 

75% spore preparation + 

25% oil 


50% oil 


25% oil 


50% oil 

WP/Oil 

0.5 ml 

1.0 ml 

2.5 g 

5.0 g 

Dosage 

Spore 

preparation 

1.5 g 

1.0 g 

7.5 g 

5.0 g

3.9 EVALUATION OF DIFFERENT FORMULATIONS AND 

STORAGE METHODS 

Spores of M. anisopliae (Ma2) and V. lecanii (Vl1) were harvested from sorghum 

grains and air dried. The carrier materials initially selected in the present study are given in 

the Table 4. 

The selected oils and flour were initially sterilized in stabs and sterilizable plastic 

covers respectively. Further, inoculum (spore preparation) of M. anisopliae and V. lecanii 

were thoroughly mixed with oils and flour along with 0.1 per cent Tween-80 under sterilized 

conditions. After mixing, they were sealed and stored at different temperatures viz., 

refrigerated temperature, room temperature, in earthen pot kept on wet sand and deep 

freezer. 

The number of viable spores per g or per ml of inoculant material were determined at 

0, 20, 45, 75, 90, 120 and 150 days intervals after incubation using standard plate count 

method on SMAY media. The plates were incubated at 28°C for 7 to 10 days. Three 

replications were maintained for each treatment. 

Treatment combinations 

1. 75 per cent conidia + 25 per cent groundnut oil 

2. 50 per cent conidia + 50 per cent groundnut oil 

3. 75 per cent conidia + 25 per cent mustard oil 

4. 75 per cent conidia + 50 per cent mustard oil 

5. 75 per cent conidia + 25 per cent sunflower oil 

6. 50 per cent conidia + 50 per cent sunflower oil 

7. 75 per cent conidia + 25 per cent wheat oil 

8. 75 per cent conidia + 50 per cent wheat oil 

9. 75 per cent conidia + 25 per cent sorghum oil 

10. 75 per cent conidia + 50 per cent sorghum oil 

3.10 FIELD EFFICACY OF Verticillium lecanii AGAINST APHIDS 

(Aphis craccivora and Brevicornia brassicae) 

Field experiments were conducted during kharif 2001 to evaluate the comparative 

performance of the V. lecanii in cowpea ecosystem against aphids (Aphis craccivora) and 

cabbage aphid (Brevicornia brassicae). The experiment was conducted at the Main 

Agricultural Research Station, Dharwad and the crop was sown on June 20 th in case of 

cowpea. The experiment in cabbage ecosystem on aphids (Brevicornia brassicae) was 

conducted on farmer’s field of Madihal area of Dharwad and. The crop was sown on 19 th 

September 2001. The crop was raised following the recommended agronomic practices in 

200 m² blocks. For recording observations on the incidence of the mycopathogen, each 

treatment block was divided into four quadrants (replications) in case of cowpea and 

cabbage. The treatments were randomized completely and plants were tagged with waxed 

labels. Observations on the number of the insect infection were recorded on randomly 

selected 10 leaves per plant, a day before, 3, 7 and 14 days after imposing the treatments. 

3.11 STATISTICAL ANALYSIS 

The data obtained from the laboratory and field experiments were statistically 

analysed following standard procedures (Gomez and Gomez, 1984). Percentage values were 

transformed to arc sin values while, root transformation (√x + 0.5) was followed for larval or 

nymphal counts (both total and mycosed) wherever necessary. The data collected were 

subjected to pooled analysis of variance. Means were separated by DMRT.

IV. EXPERIMENTAL RESULTS 

4.1 EXPLORATION OF THE NATURAL OCCURRENCE OF 

INSECT MYCOPATHOGENS IN DIFFERENT ECOLOGICAL 

NICHES IN NORTHERN KARNATAKA 

Intensive and repeated survey for the occurrence of the insect mycopathogens in the 

northern districts of Karnataka covering different agro climate region of agricultural and forest 

eco-systems were made during the cropping season of 2000-01. The number of visits to each 

locality varied from one to five depending upon cropping system and host insect availability. 

The details of places, crops and periods of visits, mycopathogens and the average disease 

incidence of different fungi are presented in Table 5. The natural occurrence of different 

pathogens in Karnataka state has also been mapped in the first year (Fig. 1). Among them, 

Nomuraea rileyi was predominant organism followed by Metarhizium anisopliae, Beauveria 

bassiana, Verticillium lecanii, Cladosporium spp., Aspergillus candidus and Fusarium spp. 

The survey revealed the prevalence of disease caused by Nomuraea rileyi on Helicoverpa 

armigera, Spodoptera litura and Thysonoplusia orichalcea in different crop habitats at 

Dharwad, Hubli and Navalgund taluks of Dharwad district, Ron taluka of Gadag district, 

Belgaum, Gokak, Bailhongal and Saundatti taluks of Belgaum district. Incidence of N. rileyi on 

the Lepidoptera hosts to an extent of 88 per cent in soybean at Devalpur in Bailhongal taluka 

of Belgaum district were witnessed during August. Mycosis of S. litura to the tune of 36 per 

cent was recorded on blackgram grown on the bunds of paddy fields at Siraguppa during 

November 2000. On the same pest, ten per cent disease incidence was noticed in irrigated 

groundnut at Raichur during January and February. 

The prevalence of M. anisopliae was noticed on diamond backmoth at Hirebagewadi 

(Belgaum), coconut rhinoceros at Dharwad, brown plant hopper at Tungabhadra project area 

(Gangavati). The natural incidence of disease caused by M. anisopliae on this insect 

population was however meagre (0.5 to 3.5%). 

Verticillium lecanii prevailed only in Haveri and Uttara Kannada districts. The fungus 

had colonised jasmine aphid and teak skeletonizer. 

During the survey, the occurrence of B. bassiana was noticed on brown plant hopper 

in the Tungabadra project (TBP) area on ash weevil at Bailhongal, Belgaum district and red 

palm weevil at Bailhongal, Belgaum district and red palm weevil at Sirsi. The other 

mycopathogen of insect pests collected during survey were Cladosporium oxysporum on 

cotton, leafhopper in Dharwad and Bailhongal, Cladosporum cladosporides on castor 

leafhopper, teak skelitonizer and cotton whitefly, whereas 5-19 per cent incidence of C. 

sphaerospermum on Ligeid bug in Dharwad, Bailhongal and Bidar. 

Fusarium oxysporum was recorded only on homopterans in Raichur, Haveri and Uttar 

Kannada districts in paddy, cotton and forest ecosystem. The incidence was highest (12.39%) 

on A. gossypii followed by N. lugens (5.62%) and least (2.39%) on mealy bug. A small 

population of cotton aphids (3.89%) was mycosed by Aspergillus candidus in Dharwad district 

only. 

4.1.1 Isolation 

The cadavers of insects collected during survey were brought to the laboratory and 

the fungi isolated on Sabouruds Maltose Agar Medium fortified with 1 per cent Yeast Extract 

(SMAY).

Fig. 1. Mycopathogens of insect pests collected during 

survey in Northern Karnataka

Table 5. Details of intensive survey conducted for incidence of entomopathogenic fungi in northern Karnataka 

Month/s visited Localities Crops Surveyed 

September Bidar district 

a. Anadurwadi, Anadur, Janawad, 

Markal, Hallhalli, Dannur 

Sugarcane, Pigeonpea, Soybean, 

Sunnhemp, Groundnut, Sunflower, 

Paddy, Sorghum 

Sorghum, Pigeonpea, Sugarcane, 

Cabbage, Niger, Horsegram 

Mycosis noticed 

Crop Pest Causal agent 

Sunnhemp Ragmus 

importunitus 

Cladosporium 

sphaerospermum 

Disease 

incidence (%) 

b. Hallikhed (K), Humanabad, 

Dubalagundi, Hallikhed (B) 

- - - - 

c. Bhalki, Dhadgi Sorghum, Pigeonpea, Field Bean - - - - 

September, Gulbarga district 

November a. Kamalapur, Muttaragi, 

Pigeonpea, Bajra Niger, 

- - - - 

Gulbarga, Farhatabad, 

Firajabad 

Groundnut 

b. Chincholi, Bhemnal, Sonth, 

Narnal, Chimmanchol 

Pigeonpea, Bajra - - - - 

c. Yadgir, Gurumatkal, malkod, 

Mulkund, Aadke, Konagadda 

Pigeonpea, Bajra, Groundnut - - - - 

October Bijapur district 

a. Basavanabagewadi, Hunnigeri, Groundnut, Sunflower, Cotton, 

- - - - 

Manguli, Tikota 

Groundnut, Madiki, Niger, Grape, 

Pomegranate 

September Raichur district 

a. Sindhanoor, K. hosahalli, Paddy, Bajra, Sunflower Paddy Nilaparvata lugens Fusarium 5.38 

Gunjahalli 

oxysporum 

September, b. Yaragera, Jambaldini, 

Paddy, Castor, Cotton, Pigeonpea, Groundnut Aproerima Metarhizium 5.64 

October, Lingakandoddi, Potagal, 

Sunflower, Groundnut 

modisella Devanter anisopliae 

December Palakambdoddi, Kallur, Eklaspur, 

Gilsugur, yariginal, Yadlapur, 

Bichhalli 

February c. Lingakandoddi, rajalbanda Groundnut, Chickpea Irrigated 

groundnut 

S. litura N. rileyi 10.00 

October, March 

- : no incidence 

d. Manvi, Neermanvi Groundnut, Paddy Paddy N. lugens F. oxysporum 3.29 

5.00



September Koppal district 

a. Ginigera, Hitnal, Munirabad Groundnut, Sugarcane, Paddy, 

b. Kustagi, Kalmanagi, Tavargeri, 

Mannapur, Chikkanandihal 

Pigeonpea, Setaria, Maize, Brinjal 

Paddy, Sunflower, Pigeonpea, 

Bajra, Groundnut, Sorghum, 

Sesamum, Maize, Pundi 



Disease 

incidence (%) 

- - - - 

- - - - 

c. Yalaburga, Banapur Groundnut - - - - 

d. Gangavati, Marali, Sirampur, 

Karatagi 

Paddy, Sunflower Paddy N. lugens F. lateritium, 

M. anisopliae, 

B. bassiana 

July 

Bellary district 

a. Hadagali, Holalu, Mailar Cotton, Groundnut - - - - 

September b. Kasanakandi, Hospet, 

Pigeonpea, Sorghum, Cotton, 

- - - - 

Kurkuppa, Torangal, Kundatini, 

Bairapur 

Cabbage, Tomato, Paddy 

September c. Bellary, Bellant, Koluru, Bairapur - - - - 

October d. Hirehal, Nagasamudra, 

Siruguppa 

Groundnut, Horsegram - - - - 

October e. Siruguppa Sunflower, Paddy, Blackgram Blackgram S. litura N. rileyi 7.82 

November f. Sasmari camp, Tekkalkote 

Bagalkot district 

Blackgram Paddy Blackgram S. litura N. rileyi 36.36, 35.71 

August a. Mudhol, Sameerwadi, 

Mahalingpur, Belavadi, Vijramatti, 

Lokapur 

Sugarcane, Cotton, Maize - - - - 

October b.Badami, Maradi, Eihole, 

Amingadha 

Pigeonpea, Bajra, Sugarcane - - - - 

October c.Hungunda, Kudalasangama, 

Alimatti 

Gadag district 

Sugarcane, Sunflower, Pigeonpea - - - - 

July Gadag Greengram, Pigeonpea - - - - 

September Gadag Greengram, Sunflower, Groundnut - - - - 

October Gadag Groundnut, Sunflower Groundnut T. orichalcea N. rileyi 16.67 

March 


Lakkundi Chrysanthemum, Tomato, 

Ridgegaurd 

- - - - 

5.36 

3.62 

2.45





Disease 

incidence (%) 

September Ron Groundnut, Pigeonpea, Bajra, 

Tomato 

- - - - 

September Gajendragad 

Dharwad district 

Groundnut, Pigeonpea, Bajra, 

Tomato 

Groundnut S. litura N. rileyi 10.00 

August, a. Dharwad Groundnut, Soybean, Cotton, Groundnut S. litura 

N. rileyi 34.44 

September, 

Stylosanthus, Sorghum, 

T. orichelcea N. rileyi 35.33 

October, 

Sunflower, Niger, Chilli, Potato, 

H. armigera 


November 

Cabbage, Lucerne, Sunnhemp, Soybean S. litura 


Pigeonpea, Chickpea Etc. 

T. orichelcea N. rileyi 18.38 

H. armigera 


Sunflower S. litura 


H. armigera 


Cotton H. armigera 

N. rileyi 

9.69 

Aphis gossypi Aspergillus 

candidus 

3.89 

Amrasca devastans Cladosporium 5.63 

Bemisia tabaci oxysporium 

C. caldosporioides 1.32 

Sorghum H. armigera N. rileyi 25.91 

Potato S. litura N. rileyi 5.83 

Lucerne S. litura N. rileyi 4.76 

Greengram H. armigera N. rileyi 5.61 

Stylosanthes T. orichelcea N. rileyi 

7.54 

H. armigera 


Sunhemp T. orichelcia 

N. rileyi 

6.67 

H. armigera 


R. importunitus 

C. 

spharospermum 

18.58 

Castor E. flavescens C. cladosporioides 10.11 

Chickpea H. armigera N. rileyi 12.47 

FYM pits Oryctus rhinoceros M. anisopliae 0.56 

August, Belur Groundnut, Soybean, Pigeonpea, Groundnut S. litura N. rileyi 21.08 

September 


Cowpea, Mustard



Crop Pest 


Causal agent 

Disease 

incidence (%) 

Tegur Mustard, Soybean, Paddy, 

Stylosanthes 

Stylosanthes T. orichalcea N. rileyi 26.73 

Bachanaki Cabbage - - - - 

Kaulageri Groundnut, Chilli, Niger, Greengram Groundnut S. litura 

N. rileyi 

14.29 

H. armigera 

N. rileyi 

11.11 

Niger 

H. armigera 

N. rileyi 

18.31 

Aminabhavi Cotton, Sunflower Groundnut S. litura N. rileyi 16.31 

Timmapur Groundnut, Soybean Groundnut S. litura N. rileyi 22.44 

Kardigudda Niger, Sybean, Potato, Sesamum, 

Chrysanthemum 

Potato S. litura N. rileyi 20.99 

Garag Groundnut, Soybean, Potato Soybean 

S. litura 

N. rileyi 

30.43 

T. orichalcea 

N. rileyi 

27.27 

S. litura 

N. rileyi 

25.00 

Groundnut S. litura 

N. rileyi 

32.14 

H. armigera 

N. rileyi 

23.89 

Hebballi, Hebsur Cotton, Groundnut, Soybean, 


N. rileyi 

26.72 

Greengram, Sorghum 


N. rileyi 

18.31 

Soybean 

S. litura 

N. rileyi 

21.69 

October Salikinkop Cotton, Pigeonpea, Sorghum Sorghum H. armigera N. rileyi 18.31 

Mummigatti, Belur Cotton, Stylosanthes Stylosanthes H. armigera 

N. rileyi 

33.33 

T. orcihalcea 

N. rileyi 

77.78 

S. litura 

N. rileyi 

80.00 

December b. Hebballi, Govankop, Hebsur Cotton - - - - 

January c. Garag, Tadkod, Hubli Cotton, Chickpea - - - - 

August d. Sherewad Groundnut Groundnut S. litura N. rileyi 31.63 

Palikoppa Soybean, Groundnut Soybean T. orchichalcea N. rileyi 

18.31 

S. litura 

N. rileyi 

21.63 

September e. Hubli, Kusugal Groundnut, Soybean, Sorghum, Groundnut H. armigera 

N. rileyi 

46.67 

Cotton 

S. litura 

N. rileyi 

30.00 

Palikoppa Groundnut, Sorghum, Cotton Groundnut S. litura 

N. rileyi 

15.32 

Sorghum H. armigera 

N. rileyi 

12.31 


N. rileyi 

13.61 

f. Kalghatagi, Devikop, Tambur, Paddy, Groundnut, Forest System, Groundnut S. litura 

N. rileyi 

20.61 

Benkikonda 

Cotton 

Cotton T. orichalcea 

N. rileyi 

11.32 


H. armigera 

N. rileyi 

8.61





Disease 

incidence (%) 

g. Navalgund Groundnut - - - - 

July h. Annigeri Chilli, Onion, Groundnut Groundnut H. armigera N. rileyi 9.09 

September, i. Nalavadi Groundnut, Sorghum Groundnut S. litura 


October 

Belgaum district 


N. rileyi 

8.67 

August, a. Bailhongal, M. K. Hubli Tomato, Sugarcane, Soybean, Soybean S. litura 


September 

Bell Pepper, Mango 

T. orichalcea N. rileyi 18.61 



Nayanagar - Groundnut S. litura 




Yedalli - Soybean S. litura 





H. armigera 


Cotton H. armigera N. rileyi 40.00 

Neginhal Cotton, Groundnut, Soybean, Groundnut S. litura 


Potato 

H. armigera 

Holihosur Cotton, Soybean Soybean S. litura 




January Nayanagar, Devalapur, Bailhongal Chickpea, Sorghum, Safflower - - - - 

August Hirebagewadi Cotton, Groundnut, Cowpea, 

Cabbage 


September Pariswad Groundnut, Cotton Soybean Cabbage P. xylostella M. anisopliae 6.30 

October Belgaum Groundnut, Paddy Groundnut S. litura 


Soybean H. armigera 


Potato 

S. litura 


Hattargi Groundnut, Pigeonpea, Bhendi Groundnut H. armigera N. rileyi 14.29 

Sutagatti Sugarcane, Maize, Groundnut - - - - 

Chunchwad Paddy - - - - 

Jalikoppa Cotton, Soybean Cotton H. armigera N. rileyi 11.61 

Belavadi Soybean, Potato Potato 

S. litura 



Soybean S. litura 

N. rileyi 16.92





Disease 

incidence (%) 

Bailhongal Soybean, Groundnut, Sunnhemp Soybean S. litura 


Cotton 

H. armigera 




Myllocerus 

undecimpunctatus 

B. bassiana 1.26 

A. devastans C. oxysporum 5.31 

Sunhemp R. importunitus C. 

saperherospermu 

10.63 

Devalapur Soybean, Groundnut, Cotton, Soybean H. armigera 


Greengram, Sugarcane 

S. litura 


H. armigera 


Plusia sp. 




Greengram H. armigera N. rileyi 50.00 

September Sogal Groundnut, Soybean, Cotton Groundnut S. litura N. rileyi 27.25 

Devalapur Groundnut, Soybean, Cotton Groundnut S. litura, H. 

armigera 


Soybean S. litura, 




Khanapur Groundnut - - - - 

Nandagad Paddy, Sugarcane - - - - 

Khairavadahatti Paddy - - - - 

October Athani, Telsaga, daisayiratti, Groundnut, Pigeonpea, Turmeric, - - - - 

Muragundi, Kagawad 

Chilli, Sugarcane 

October Raibag, Ankali Chilli, Sugarcane, Soybean, Soybean S. litura 


Groundnut 

groundnut S. litura 


T. orichalcea sN. rileyi 20.61 

August Hukkeri, Sankeshwar Sugarcane, Groundnut, Soybean, Groundnut S. litura 


Sorghum 


N. rileyi 

5.81 



N. rileyi 28.02



Crop Pest 


Causal agent 

Disease 

incidence (%) 

Nippani Tobacco, Groundnut, Soybean Groundnut S. litura 

N. rileyi 

23.07 

H. armigera 

N. rileyi 

27.02 


N. rileyi 

24.07 

H. armigera 

N. rileyi 

20.02 

September Ramdurga, Shirsangi, Betkurki - - - - - 

Gokak, Yaragtti, Arabhavi Soybean Soybean S. litura N. rileyi 7.58 

August Savadatti, Hirebagewadi 

September Munvalli, Holi, Murgod, Katkol Maize, Groundnut, Soybean, 

Soybean, 

S. litura 

N. rileyi 

7.31 

Haveri district 

Greengram 

greengram H. amirgera 

N. rileyi 

5.31 

July September a. Shiggaon 

Cottons, Chilli, Groundnut Soybean, Cotton H. armigera 

N. rileyi 

9.52 

Tadas 

Groundnut, Cotton 

Cotton Aphis gossypii F. oxysporum 12.39 

Shiggaon 

Greengram, Groundnut 

- 

- 

- 

- 

b. Savanur 

Uttar Kannada district 

Jasmine Jasmine Aphids V. lecanii 6.86 

October a) Yellapur - Kiravatti Cotton, Paddy - - - - 

November Yellapur, manchikeri, Chavati Forest Eco-System Teak Teak skelitoniiser V. lecanii 

2.31 

M. anisopliae 2.50 

Teak skelitoniiser C. cladosporides 0.81 

November b. Sirsi - Bairumbe Forest Ecosystem Forest 

ecosystem 

Ants and Mealy bug F. oxysporium 2.39 

January Sirsi, Yadalli, Kansur Arecanut, Banana, Coconut Coconut Rhyncophorus 

ferrugineus 

B. bassiana 5.64 

c. Siddapur, thyagali, Kholse, 

Siddapur, Mavingudi 

Paddy, Plantation, Forest System - - - - 

November, d. Honnavar, Gerasoppa, Upponi, Forest Ecosystem, Arecanut, Paddy, - - - - 

January, March, 

April 

Hadinbal, Karki 

Groundnut 

November, e. Kumta – Bettageri, dhareshwar, Arecanut plantation, Paddy, 

Groundnut H. armigera 

N. rileyi 

10.12 

January, March, 

April 

Deevagi, bada, Aghanashini, Hegde Groundnut 

S. litura 

N. rileyi 

15.18 

November, f. Ankoka – Belse, Antravalli, Paddy, Groundnut Groundnut S. litura 

N. rileyi 

12.18 

January, March Karuvinkoppa, Hebbul, Hattikeri, 

Balekeri, bargi 

H. armigera 

N. rileyi 

9.23 

g. Haliyal – sambrani, Murkwad, Paddy, Cotton, Sugarcane, Forest 


Bagavati, Neelavani 

Ecosystem

Totally eight isolates of N. rileyi, four of Metarhizium anisopliae, three of Verticillium 

lecanii, B. bassiana, C. sphaerospermum, Fusarium oxysporum, two of C. oxysporum and 

each one of Aspergillus candidus and Fusarium lateritium. They were identified based on their 

colony characteristics and microscopic examination (Table 6 and Plate 1). Further, the identity 

of the fungi was confirmed at Agarkar Research Institute, Pune (Appendix III). 

4.2 MORPHOLOGICAL AND CULTURAL CHARACTERISTIC OF 

THE FUNGAL ISOLATES 

4.2.1 Colony characteristics 

The conidiophores of M. anisopliae appeared in compact to nearly stomatic patches, 

mononematous, conidiogenous cells with phialides in whorls, often arranged in a candle like 

fashion, clavate to cylindrical and conidia were single celled, hyaline, slightly coloured and 

forming long chain often aggregated into prismatic columns. The conidiophores of V. lecanii 

was erect and verticillate with loose whorls of phialidic conidiogenous cells. Phialides mostly 

awl-shaped and sometimes slightly inflated at the base. Conidia were single celled, hyaline, 

smooth walled and produced in slimy heads. 

Morphologically, all the isolates exhibited typical Metarhizium and Verticillium 

characteristics. Ma1 isolate from rhinoceros grub showed slight yellowish pigment with green 

coloured conidia. Ma2 isolated from brown plant hopper produced dark green coloured 

conidia with no pigmentation. Ma3 isolated from white grub showed green coloured conidia 

and Ma4 isolated from coffee berry borer produced yellowish pigment with green coloured 

conidia. In case of V. lecanii Vl3 alone, showed pinkish pigment with white coloured conidia. 

Morphological characters of all the M. anisopliae isolates revealed differences with 

respect to hyphal and conidial characters except in size of conidia. There was variation in 

conidial colour and pigmentation between the isolates. Ma2 produced dark green coloured 

spores, while the other three isolates produced green colured spores (Plate 2). Ma3 and Ma4 

required 5-6 days to initiate sporulation (Table 7). Ma1 and Ma2 accomplished this in half of 

the time. 

The hyphal and conidial characters did not vary among the isolates collected from 

different localities during survey. However, V. lecanii exhibited significant variation in number 

of days for sporulation, colony diameter, sporulation, spore yield and time taken to cover the 

given diet for mass production (Table 7). 

Among V. lecanii isolates, Vl1 from aphid recorded least spore yield (4.21 x 10 9 ) and 

took minimum time (9 days) to cover the entire diet, whereas Vl2 and Vl3 isolates isolated 

from teak skelitonizer and citrus scale respectively showed spore yield (4.34 x 10 9 to 4.98 x 

10 9 /g) and took maximum time (10 days to 14 days) to cover the entire diet. 

4.3 PATHOGENICITY OF Verticillium lecanii AND Metarhizium 

anisopliae AGAINST IMPORTANT PESTS 

The culture of insects (American bollworm and Rhinocerous beetle for Metarizhium 

and aphids, whitefly and mites for Verticillium) were raised from field collected insect 

populations on natural diet. The routine surface sterilization of rearing containers and eggs 

with 10 per cent formaldehyde was carried out to prevent fungal bacterial and viral 

contaminations of the healthy stock. Test insect of uniform age (second/third instar) and 

biomass were selected from the laboratory cultures maintained for the purpose to test the 

variability of pathogenicity of M. anisopliae Ma2 and V. lecanii Vl1 by Koch postulates. The 

pathogen was topically applied @ 1 x 10 8 conidia/l for aerial insects and mixed the conidia 

with soil (1 x 10 7 / kg soil) for soil dwelling insects.

Table 6. List of isolates of Metarhizium anisopliae and Verticillium lecanii 

obtained during the study 

Strain Source Place of collection 

Metarhizium anisopiae 

Verticilium lecanii 

Ma1 Rhinoceros grub Dharwad 

Ma2 Brown plant hopper Gangavati (TBP area) 

Ma3 Root grub Kasargod 

Ma4 Coffee berry borer Chettalli 

Vl1 Aphids Savanur 

Vl2 Teak skelitonizer Yellapur 

Vl3 Citrus scale Chettalli

Metarhizium anisopliae 

Vertucillium lecanii 

Nomuraea rileyi 

Plate 1. Microconidiopore and microconidia of entomopathogens 

a. Conidiaphore, b. Conidia

Strain 

Table 7. Morphological and cultural characters of Metarhizium anisopliae and Verticillium lecanii isolates 

No. of days for 

sporulation 

Colour of conidia 

Ma1 5 Green with slight yellowish 

pigmentation 

Colony diameter on 10 th 

DAI (mm) 

Sporulation (x10 9 

conidia/plate) 

Spore yield per 100g of 

diet (rice) (gx10 9 ) 

Time taken to 

cover the diet 

(days) 

38.60 2.92 8.93 10.30 

Ma2 5 Dark green 42.30 3.21 9.32 10.00 

Ma3 8 Green 32.30 1.53 5.62 15.60 

Ma4 5 Green with yellowish 

pigmentation 

33.19 1.62 6.03 16.30 

Vl1 5 White 52.13 1.41 4.21 9.25 

Vl2 5 White 41.10 1.34 4.34 10.10 

Vl3 5 White with pinkish 

pigmentation 

DAI – Days after incubation 

28.42 1.58 4.98 14.31

Ma 1 (Dharwad) Ma2 (Dharwad) 

Ma 3 (Kasargod) Ma 4 (Chettalli) 

Vl1 (Savanur) Vl2 (Yellapur) Vl3 (Chettalli) 

Plate 2. In vitro sporulation by different isolates of 

Metarhizium anisopliar and 

Verticillium lecanii

The pathogenicity of the mycopathogen M. anisopliae Ma2 was tested against two 

species (Helicoverpa armigera and Oryctes rhinoceros) (Plate 3) of insect pest and 

mycopathogen V. lecanii Vl1 was tested against 5 species of insect pests viz., Brevicornia 

brassicae, Aphis crassivora, Melanaphis sacchari, Polyphagotarsonemus latus, Aleurodicus 

disperses. The results are presented in Table 8 and Plate 4. 

Among the insects tested by V. lecanii Vl1, cabbage aphid B. brassicae was 

more susceptible (94.50%) followed by cowpea aphid A. crassivora (84.23%), sorghum 

aphid M. sacchari (72.42%) and chilli mite P. latus (55.10%). Spirilling whitefly, A. disperses 

recorded lowest mortality of 51.35 per cent. In case of M. anisopliae the maximum mortality 

was recorded in Rhinocerous beetle (85.10%) followed by American bollworm (82.00%) 

(Plate 5). 

The different strains of M. anisopliae and V. lecanii obtained were tested against H. 

armigera and B. brassicae. The results are presented in Table 9. The mortality of H. armigera 

larvae treated with the different isolates of M. anisopliae showed that Ma2 caused maximum 

cumulative mortality (87.50%) followed by Ma-1 (83.21%). The other two isolates Ma1 and 

Ma4 caused only 62.28 and 74.81 per cent mortality. Among the different isolates of V. lecanii 

tested against Brevicornia brassica, Vl1 recorded maximum mortality of 94.50 per cent 

followed by Vl3 (83.50%) and Vl1 (78.32%). 

4.4 EFFECT OF DIFFERENT CARBON SOURCES ON THE 

BIOMASS OF Verticillium lecanii (Vl1) AND Metarhizium 

anisopliae (Ma2) 

The effect of different carbon sources on the growth of M. anisopliae Ma2 was 

evaluated at different concentrations in the Czapecks Dox broth (Table 10). From the results, 

it was observed that the total dry matter production was significantly higher on the medium 

containing starch (6.01 g/250 ml) followed by sucrose (5.51 g/250 ml) and fructose (4.83 

g/250 ml). Lactose supported least growth with only 4.44 g/250 ml of dry matter production. 

The interaction effect of carbon source and levels on biomass production of M. 

anisopliae was maximum with starch 40 g/l (6.01 g/250 ml). As the concentration of sugar 

tested increased from 10 to 40 g/l, mean biomass also increased from 4.14 g/l at 10 g/l to 

6.01 g/l at 40 g/l. 

Among different nitrogen sources evaluated for production of M. anisopliae, KNO 3 

(5.76 g/250 ml) was found to be best source followed by NH4NO3 (5.32 g/250 ml). There was 

reduced growth in NaNO3 (4.40 g/250 ml). The optimum concentration of nitrogen was found 

to be 2 g/l (5.53) for all nitrogen sources (Table 11). 

Various concentrations of carbon and nitrogen source were evaluated against total 

dry matter production of V. lecanii. Biomass was marginal (4.219 m) at lower concentration of 

carbon source and was highest (4.66 g/250 ml) at the highest concentration (40 g/l) and was 

significantly superior to rest of the treatment. 

Among the carbon sources tested, the mean per cent biomass with sucrose (5.398 

g/250 ml) and starch (5.97 g/250 ml) were on par with each other and significantly superior to 

rest of the carbon sources tested (Table 12). 

In case of nitrogen sources, the biomass was marginal (4.66 g/ml) at lower 

concentration and it was highest at recommended dose of 2 g/l with maximum dry matter 

production of 6.70 g/l. However, there was reduction in biomass at highest concentration (2.5 

g/l). The maximum biomass was recorded when the medium was supplemented with 

(NH4)SO4 (6.77 g) followed by NaNO3 (6.61 g/250 ml) and least with KNO3 (5.51 g/ml) (Table 

13). 

Among the interaction, the highest biomass of 7.41 g/250 ml was recorded with 

(NH4)SO4 and lowest in NaNO3 which supported a total dry matter of 4.23 g/250 ml.

Table 8. Mortality caused by the isolates of Metarhizium anisopliae and 

Verticillium lecanii against Helicoverpa armigera and Brevicornia 

brassicae respectively 



Isolates Cumulative per cent mortality 

Ma1 83.21 c 

Ma2 87.50 b 

Ma3 62.28 g 

Ma4 74.84 f 

Vl1 94.50 a 

Vl2 78.32 e 

Vl3 83.50 d 

In vertical columns means followed by similar letters do not significantly different (p=0.05)

Sl. 

No. 

Table 9. Insect species found susceptible to Metarhizium anisopliae and 


Strain 

Insect 

Common name Scientific name 

Disease 

incidence (%) 

1. M. anisopliae American bollworm Helicoverpa armigera 82.00 

2. M. anisopliae Rhinoceros beetle Oryctes rhinoceros 85.10 

3. V. lecanii Cabbage aphid Brevicornia brassicae 94.50 

4. V. lecanii Cowpea aphid Aphis crassivora 84.23 

5. V. lecanii Sorghum aphid Melanaphis sacchari 72.42 

6. V. lecanii Chilli mite Polyphagotarsonemus 

latus 

55.10 

7. V. lecanii Spirilling whitefly Aleurodicus dispersus 51.35

Helicoverpa armigera 

Dimond Black Moth 

Brown plant hopper Root grub 

Plate 3. Host insets of Metarhizium anisopliae

Aphids 

Spirilling whitefly 

Spirilling whitefly 

Plate 4. Host insects of Verticillium lecanii

Plate 5. The moratality of Hlelicoverpa armigera topically applied 

with Metarhizium anisopliae 

1. Healthy larvae (8 days old larvae) 

2. Dead larvae ( 5 days after spore application) 

3. Dead larvae with thick fungal mat (7 days after spore application) 

4. Dead larvae covered with green coloured with green coloured conidia (10 days 

after spore application)

Carbon 

source 

Table 10. Total biomass production of Metarhizium anisopliae (Ma2) as 

influenced by carbon sources 

Total dry biomass content (g/ 250 ml) 

10 g/lt* 20 g/lt* 30 g/lt* 40 g/lt* Mean 

Lactose 4.00 4.47 4.31 5.00 4.44 

Starch 4.51 5.60 6.30 7.75 6.01 

Fructose 3.81 4.80 5.20 5.52 4.83 

Sucrose 4.31 5.72 5.75 6.50 5.57 

Dextrose 4.11 4.51 4.56 5.30 4.60 

Mean 4.14 5.02 5.20 6.01 5.09 

S.Em± CD (0.01) 

A (Carbon) 0.025 0.102 

B (Conc.) 0.023 0.091 

A x B 0.051 0.204 

* Concentration of carbon source in Czapeck dox medium

Table 11. Total biomass production of Metarhizium anisopliae (Ma2) as 

influenced by nitrogen sources 

Nitrogen 

source 

Total dry biomass content (g/ 250 ml) 

1 g/lt* 1.5 g/lt* 2 g/lt* 2.5 g/lt* Mean 

NaNO3 3.95 4.59 5.11 3.97 4.40 

KNO3 4.20 6.11 6.41 6.33 5.76 

(NH4)SO4 5.45 4.85 5.11 5.15 5.14 

NH4NO3 5.09 5.21 5.49 5.52 5.32 

Mean 4.67 5.19 5.53 5.24 5.15 

S.Em± CD (0.01) 

A (Nitrogen) 0.119 0.497 

B (Conc.) 0.119 0.497 

A x B 0.238 0.998 

* Concentration of nitrogen source in Czapeck dox medium

4.5 EVALUATION OF FOOD GRAINS AND AGRO WASTES FOR 

SPORULATION OF Metarhizium anisopliae (Ma2) AND V. 

lecanii (Vl1) 

4.5.1 Food grains 

Different food grains were evaluated for their suitability to support the conidial 

production of the mycopathogens based on the time taken to initiate mycelial growth, 

sporulation and conidial yield at 6, 8, 15, 20 and 25 days after inoculation (DAI). 

In general, the mycelial growth and production of conidia increased with increase in 

DAI and by 20 DAI, it covered the surface of the media almost completely. The conidial yield 

was influenced significantly by the source of grain at all intervals of observation. 

The growth of M. anisopliae was significantly higher on bajra (22.77 x 10 8 conidia/g) 

followed by sorghum (11.60 x 10 8 conidia/g), rice (8.4 10 8 conidia/g) and maize (6.76 x 10 8 

conidia/g) (Table 14 and Fig. 2). On the other hand, wheat supported least yield 3.22 x 10 8 

condiia/g, conidial production increased from 6 th to 20 th day and remained constant further in 

all the treatments. The conidial production on the 20 th day was high in bajra (24.90 x 10 8 

condia/g), whereas in navane (7.78 x 10 8 conidia/g) and wheat (3.76 x 10 8 conidia/g) grain, as 

at the previous interval faired, as poor substrate for fungal productivity. 

In case of Verticillium lecanii, rice proved superior by producing significantly higher 

spore load (24.59 x 10 8 conidia/g) followed by sorghum (17.49 x 10 8 conidia/g) bajra (10.34 x 

10 8 conidia/g). On the other hand, navane supported the least yield (3.52 x 10 8 conidia/g) and 

its suitability was no better than wheat (3.54 x 10 8 conidia/g). The conidial load was maximum 

after 20 DAI against all the treatments tested (Table 15 and Fig. 2). 

4.5.2 Agro waste 

In all, five agro wastes (viz., maize cobs, wheat bran, rice bran, baggase and press 

mud) singly and along with 10 per cent molasses were evaluated for their suitability as 

principle substrate for the mass production of fungal insect pathogens. Among the agro 

wastes, rice bran enhanced the productivity of both the fungi compared to other agro wastes. 

The conidial mass harvested per gram of dietary grain used, increased steadily with 

inoculation of molasses in both the strains. 

Among the agro waste tested with Ma-2 (Table 16 and Fig. 3), rice bran + 10 per cent 

molasses proved to be superior in producing significantly higher spore load (33.24 x 10 4 

conidia/g) followed by wheat bran + 10 per cent molasses (19.03 x 10 4 conidia/g) rice bran 

and wheat bran with a spore yield of 12.80 x 10 4 and 10.16 x 10 4 conidia/g, respectively. In 

others, the spore count was considerably less. Pressmud + 10 per cent molasses supported 

least yield 0.42 x 104 spore/g, where initiation of growth and sporutation resulted only after 20 

DAI. 

In general, mycelial growth and conidiation increased with increase in DAI upto 20 

DAI but significant reduction of conidial yield was observed at 25 DAI in some substrate viz., 

rice bran, rice bran + 10 per cent molasses and in baggasse + 10 per cent molasses. 

The growth and sporulation of V. lecanii Vl1 (Table 17 and Fig.3) was found to be 

better on rice bran + 10 per cent molasses (30.86 x 10 4 conidia/g) followed by wheat bran + 

10 per cent molasses (18.76 x 10 4 conidia/g) and rice bran (15.98 x 10 4 conidia/g). Complete 

inhibition of growth and reproduction of the fungus was noticed on bagasse and pressmud 

with 1 per cent yeast extract alone. However, growth was recorded when baggasse and 

pressmud was supplemented with 10 per cent molasses (10.88 and 7.90 conidia/g 

respectively).

Table 12. Total biomass production of Verticillium lecanii (Vl1) as influenced 

by carbon sources 

Carbon 

source 

Total dry matter content (gm/ 250 ml) 

10 g/lt* 20 g/lt* 30 g/lt* 40 g/lt* Mean 

Lactose 3.81 4.71 4.63 5.00 4.53 

Starch 4.49 5.29 6.21 7.90 5.97 

Fructose 4.05 5.05 5.38 6.09 5.14 

Sucrose 4.41 5.25 6.77 7.51 5.98 

Dextrose 4.29 4.97 5.96 7.83 5.76 

Mean 4.21 5.05 5.79 6.86 5.48 

S.Em± CD (0.01) 

A (Carbon) 0.029 0.116 

B (Conc.) 0.026 0.104 

A x B 0.057 0.232 

* Concentration of carbon source in Czapeck dox medium

Table 13. Total biomass production of Verticillium lecanii (Vl1) as influenced 

by nitrogen source 

Nitrogen 

source 

Total dry matter content (g/ 250 ml) 

1 g/lt* 1.5 g/lt* 2 g/lt* 2.5 g/lt* Mean 

NaNO3 4.23 7.37 7.52 7.33 6.61 

KNO 3 4.49 6.02 5.81 5.72 5.51 

(NH4)SO4 5.22 7.06 7.4 7.41 6.77 

NH 4NO 3 4.71 5.20 6.10 6.23 5.56 

Mean 4.66 6.41 6.70 6.67 6.11 

S.Em± CD (0.01) 

A (Nitrogen) 0.016 0.067 

B (Conc.) 0.016 0.067 

A x B 0.032 0.134 

* Concentration of nitrogen source in Czapeck dox medium

Table 14. Sporulation of Metarhizium anisopliae Ma2 spores on different grains 

Treatment 

No. of spores x 10 8 /g 

Days after inoculation 

6 8 15 20 25 Mean 

Crushed bajra + 1% of YE 17.35 23.10 24.00 24.90 24.50 22.77 

Crushed sorghum + 1% of YE 6.110 10.36 13.68 13.92 13.92 11.60 

Crushed navane + 1% of YE 2.530 4.15 6.90 7.78 7.78 5.83 

Crushed maize + 1% of YE 2.660 5.20 8.41 8.78 8.78 6.76 

Crushed rice + 1% of YE 4.090 6.56 10.55 10.55 10.55 8.46 

Crushed wheat + 1% of YE 1.905 2.92 3.76 3.76 3.76 3.22 

Mean 5.774 8.71 11.22 11.61 1.54 9.77 

YE – Yeast extract 

S.Em± CD (0.01) 

A (Food grain) 0.037 0.145 

B (Days) 0.034 0.132 

A x B 0.083 0.323

Table 15. Sporulation of Verticillium lecanii Vl2 spores on different grains 

Treatment 

No. of spores x 10 8 /g 


6 8 15 20 25 Mean 

Crushed bajra + 1% of YE 14.25 16.66 18.86 18.86 18.86 17.49 

Crushed sorghum + 1% of YE 8.37 9.75 10.26 11.66 11.66 10.34 

Crushed navane + 1% of YE 2.90 3.10 3.85 3.85 3.85 3.52 

Crushed maize + 1% of YE 3.36 4.06 5.15 5.79 5.79 4.80 

Crushed rice + 1% of YE 21.35 24.05 25.85 25.85 25.85 24.59 

Crushed wheat + 1% of YE 2.07 3.05 4.19 4.19 4.19 3.54 

Mean 8.71 10.12 11.36 11.70 11.70 10.72 


S.Em± CD (0.01) 

A (Food grain) 0.011 0.042 

B (Days) 0.010 0.038 

A x B 0.024 0.094

Mean spore yield x 10 8 /g 

25 

20 

15 

10 

5 

0 

Crushed bajra + 1% of 

YE 

Crushed sorghum + 1% 

of YE 

Metarhizium anisopliae (Ma2) 

Verticillium lecanii (Vl2) 

Crushed navane + 1% of 

YE 

Crushed maize + 1% of 

YE 

Treatments 

Crushed rice + 1% of YE Crushed wheat + 1% of 

YE 

Fig. 2. Conidial yield of Metarhizium anisopliae and Verticillium lecanii on different food grains 

Fig. 2. Conidial yield of Metarhizium anisopliae and verticillum lecanii on different food grains

Table 16. Sporulation of Metarhizium anisopliae Ma2 spores on different agro wastes 

Treatment 

No. of spores x 10 4 


10 15 20 25 Mean 

Crushed maize cobs + 1% YE 3.57 3.87 3.87 6.00 4.33 

Wheat bran +1% YE 9.25 10.25 10.25 10.90 10.16 

Rice bran +1% YE 13.40 15.15 15.15 7.50 12.80 

Baggase +1% YE 0.00 0.00 0.00 0.00 0.00 

Press mud + 1% YE 0.00 0.00 0.00 0.00 0.00 

Crushed maize cobs + 10% molasses 8.25 9.4 9.4 10.80 9.46 

Wheat bran +10% molasses 15.30 18.30 18.30 24.25 19.03 

Rice bran +10% molasses 34.37 38.25 38.25 22.10 33.24 

Baggase bran +10% molasses 7.70 9.20 9.20 4.50 7.65 

Press mud + 10% molasses 0.00 0.00 0.00 1.70 0.425 

Mean 9.185 10.442 10.442 9.140 9.803 


S.Em± CD (0.01) 

A (Agro waste) 1.662 0.353 

B (Conc.) 1.051 NS 

A x B 3.323 NS

Among the non-grain substrates, the fungus took 5 to 8 days to initiate mycelial 

growth and 8 to 10 days to produce spores. In general, mycelial growth and conidiation 

ncreased with increase in DAI only upto 15 DAI with no further conidiation. 

4.6 COMPATIBILITY OF Metarhizium anisopliae (Ma2) AND 

Verticillium lecanii (Vl1) WITH AGROCHEMICALS 

The effect of different fungicides, insecticides and weedicides were tested for their 

compatibility with M. anisopliae. The effectiveness was measured in terms of conidial 

germination of the fungus in vitro using the food poison technique. Inhibition of the conidial 

germination over untreated control was worked out for the respective concentration and 

analysed for statistical significance. The results are presented in Tables 18 to 23, Fig. 4 

and 5. 

In general, the results indicated on an average that fungicides were highly inhibitory 

and toxic (mean per cent inhibition of 73.65% and 86.45% on M. anisopliae and V. lecanii 

respectively) followed by insecticides (44.22% and 53.24%) and weedicides (23.42% and 

22.02%). 

4.6.1 Fungicides 

All the fungicides, tested for their interaction with M. anisopliae inhibited the conidial 

germination (19.90 to 100%) considerably (Table 18) at all the four concentrations tested. At 

the recommended concentration, the fungicides inhibited the germination of conidia to the 

extent of 61.31 to 100 per cent. Propiconazole and mancozeb were highly toxic inhibiting cent 

per cent of conidia from germination followed by wettable sulphur (88.35%), carbendazim 

(85.60%), chlorothalonil (69.21%) and triadimefon (74.37%). Iprodione was least inhibitory as 

it allowed maximum of 38.69 per cent conidia to germinate. The next safe fungicide was 

chlorothalonil, which inhibited 69.21 per cent conidial germination. The per cent inhibition of 

conidia increased as the dosage increased from 54.46 per cent to 88.94 per cent. 

In case of V. lecanii (Table 19), fungicides completely inhibited the conidial 

germination considerably at all the four concentrations tested (24.10 to 100%). At the 

recommended concentration, the fungicides completely inhibited the germination of condiia to 

the extent of 100 per cent in case of carbendazim, chlorothalonil, propiconazole, mancozeb 

and wettable sulphur except iprodione and triadimefom as they allowed maximum of 37.38 

and 41.62 per cent conidia to germinate respectively. 

4.6.2 Insecticides 

All the insecticides tested for their interaction with M. anisopliae in general, inhibited 

germination of the fungal conidia to the extent of 18.96, 34.10, 49.74 and 74.41 per cent at 

low, medium recommended and high dosages respectively. 

All the insecticides tested for their effect on M. anisopliae, irrespective of 

concentration tested had 34.33 to 55.89 per cent inhibition of conidial germination (Table 20). 

Among them, Dichlorvos was significantly detrimental (55.89% inhibition) than all other 

insecticides except monocrotophos in which the inhibition of conidia recorded 54.22 per cent. 

Conversely, malathion and dimethoate were signficinatly safer (34.33 to 36.99% inhibition), 

followed by endosulfan, quinolphos and chlorpyriphos (38.96 to 42.96% inhibition). At the 

recommended concentration, the insecticides inhibited the germination of conidia to the 

extent of 37.67 to 69.16 per cent (Plate 6).

Table 17. Sporulation of Verticillium lecanii Vl2 spores on agro wastes 

Treatment 

No. of spores x 10 4 


10 15 20 25 Mean 

Crushed maize cobs + 1% YE 0.00 0.00 0.00 0.00 0.00 

Wheat bran +1% YE 9.9 12.10 12.10 12.10 11.55 

Rice bran +1% YE 15.14 16.26 16.26 16.26 15.98 

Baggase +1% YE 0.00 0.00 0.00 0.00 0.00 

Press mud + 1% YE 0.00 0.00 0.00 0.00 0.00 

Crushed maize cobs + 10% molasses 9.6 10.24 10.24 10.24 10.07 

Wheat bran +10% molasses 17.92 19.05 19.05 19.05 18.76 

Rice bran +10% molasses 28.10 31.75 31.75 31.75 30.86 

Baggase +10% molasses 8.90 11.55 11.55 11.55 10.88 

Press mud + 10% molasses 7.16 8.15 8.15 8.15 7.90 

Mean 9.68 10.91 10.91 10.91 10.60 


S.Em± CD (0.01) 

A (Agro waste) 0.032 0.124 

B (Days) 0.021 0.078 

A x B 0.065 0.248

Mean spore yield x 10 4 /g 

35 

30 

25 

20 

15 

10 

5 

0 

Crushed Wheat bran 

maize cobs + 

1% YE 

+1% YE 

Rice bran 

+1% YE 

Baggase 

+1% YE 

Metarhizium anisopliae (Ma2) 

Verticillium lecanii (Vl1) 

Press mud + 

1% YE 

Treatments 

Crushed 

maize cobs + 

10% 

molasses 

Wheat bran 

+10% 

molasses 

Rice bran 

+10% 

molasses 

Baggase 

bran +10% 

molasses 

Fig. 3. Conidial yield of Metarhizium anisopliae and Verticillium lecanii on different agrowastes 

Fig. 3. Conidial yield of Mearhizium anisopliae and Vericillium lecanii on different agrowastes 

Press mud + 

10% 

molasses

Sl. 

No. 

Table 18. Effect of fungicides on the conidial germination of 

Metarhizium anisopliae Ma2 

Fungicides 

1. Carbendazim 48.48 

(45.60) 

2. Iprodione 19.90 

(26.49) 

3. Chlorothalonil 52.76 

(46.58) 

4. Propiconazole 100.00 

(90.00) 

5. Manozeb 89.27 

(70.87) 

6. Triadimefon 28.38 

(32.18) 

7. Hexaconazole 50.67 

(45.38) 

8. Wettable sulphur 46.20 

(42.82) 

Mean 54.50 

(49.99) 

Inhibition of germination of conidia over control 

(%) 

0.25 RD 0.5 RD RD 2 RD 

55.13 

(50.25) 

44.53 

(41.86) 

61.67 

(51.74) 

100.00 

(90.00) 

100.00 

(90.00) 

52.36 

(46.35) 

63.36 

(52.75) 

69.49 

(56.36) 

68.32 

(59.91) 

85.60 

(67.70) 

61.31 

(51.53) 

69.21 

(56.29) 

100.00 

(90.00) 

100.00 

(90.00) 

74.37 

(59.58) 

84.40 

(66.73) 

88.35 

(70.06) 

82.90 

(68.98) 

99.48 

(86.03) 

63.48 

(52.81) 

72.39 

(58.28) 

100.00 

(90.00) 

100.00 

(90.00) 

86.26 

(68.25) 

94.56 

(76.53) 

95.32 

(17.48) 

88.94 

(74.92) 

S.Em± CD (0.01) 

A (Fungicide) 0.094 0.352 

B (conc.) 0.066 0.249 

A x B 0.187 0.703 

Means followed by same alphabet do not differ significantly (0.01) 

Figures in the parenthesis are arc sin values 

Mean 

72.17 d 

(62.40) 

47.30 g 

(43.17) 

64.01 e 

(53.22) 

100.00 a 

(90.00) 

97.318 b 

(85.21) 

60.32 f 

(51.59) 

73.27 d 

(60.35) 

74.84 c 

(61.68) 

73.65 

(63.45)

Sl. 

No. 

Table 19. Effect of fungicides on the conidial germination of Verticullium lecanii 

Fungicides 

1. Carbendazim 100.00 

(90.00) 

2. Iprodione 44.36 

(41.78) 

3. Chlorothalonil 100.00 

(90.00) 

4. Propiconazole 100.00 

(90.00) 

5. Manozeb 100.00 

(90.00) 

6. Triadimefon 24.103 

(29.26) 

7. Hexaconazole 53.57 

(46.98) 

8. Wettable sulphur 86.28 

(68.15) 

Mean 76.03 

(68.27) 

Inhibition of germination of conidia over control 

(%) 

0.25 RD 0.5 RD RD 2 RD 

100.00 

(90.00) 

48.93 

(44.57) 

100.00 

(90.00) 

100.00 

(90.00) 

100.00 

(90.00) 

41.39 

(40.13) 

86.48 

(68.40) 

100.00 

(90.00) 

84.60 

(75.38) 

100.00 

(90.00) 

62.62 

(52.21) 

100.00 

(90.00) 

100.00 

(90.00) 

100.00 

(90.00) 

58.38 

(49.69) 

100.00 

(90.00) 

100.00 

(90.00) 

90.12 

(80.23) 

100.00 

(90.00) 

77.47 

(61.54) 

100.00 

(90.00) 

100.00 

(90.00) 

100.00 

(90.00) 

82.94 

(65.38) 

100.00 

(90.00) 

100.00 

(90.00) 

95.05 

(83.36) 

S.Em± CD (0.01) 

A (Fungicide) 0.076 0.284 

B (Conc.) 0.053 0.201 

A x B 0.151 0.567 



Mean 

100.00 a 

(90.00) 

58.34 d 

(50.02) 

100.00 a 

(90.00) 

100.00 a 

(90.00) 

100.00 a 

(90.00) 

51.70 e 

(46.11) 

85.01 c 

(73.84) 

96.57 b 

(84.53) 

86.45 

(76.81)

Per cent inhibition 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Fungicides Insecticides Weedicides 

Low Medium Recommended High 

Fig. 4. Effect of pesticides on the conidial germination of Metarihizium anisopliae 

Fig. 4. Effect of pesticides on the conidial germination of Metarihizium anisp

Per cent inhibition 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Fungicides Insecticides Weedicides 

Low Medium Recommended High 

Fig. 5. Effect of pesticides on the conidial germination of Verticillium lecanii 

Fig. 5. Effect of pesticides on the conidial germination of Verticillium lecanii

Sl. 

No. 

Table 20. Effect of insecticides on the conidial germination of Metarhizium anisopliae 

Insecticide 

1. Endosulfon 13.78 

(21.77) 

2. Quinolphos 16.77 

(24.16) 

3. Monocrotophos 28.69 

(32.37) 

4. Chlorpyriphos 18.31 

(25.15) 

5. Oxydemeton methyl 25.56 

(30.37) 

6. Dichlorvos 22.36 

(28.22) 

7. Dicofol 19.06 

(25.89) 

8. Malathion 14.74 

(22.57) 

9. Dimethoate 11.34 

(19.67) 

Mean 18.96 

(25.57) 

Inhibition of germination of conidia over control (%) 

0.25 RD 0.5 RD RD 2 RD 

33.23 

(35.20) 

34.23 

(35.80) 

39.14 

(38.72) 

29.00 

(32.58) 

38.46 

(38.33) 

41.73 

(40.24) 

38.73 

(38.46) 

28.53 

(32.28) 

23.90 

(29.26) 

34.10 

(35.65) 

44.57 

(41.88) 

37.67 

(37.85) 

65.98 

(54.30) 

49.32 

(44.62) 

41.93 

(40.36) 

60.16 

(56.15) 

54.33 

(47.48) 

39.13 

(38.72) 

42.74 

(40.82) 

49.74 

(44.69) 

64.25 

(53.27) 

68.04 

(55.57) 

83.10 

(65.72) 

75.21 

(60.13) 

79.50 

(63.08) 

90.30 

(72.02) 

84.37 

(66.71) 

65.57 

(54.07) 

59.33 

(50.37) 

74.41 

(60.10) 

S.Em± CD (0.01) 

A (Insecticide) 0.153 0.573 

B (Conc.) 0.102 0.381 

A x B 0.305 1.143 



Mean 

38.96 f 

(38.03) 

39.18 f 

(38.35) 

54.22 b 

(47.78) 

42.96 c 

(40.62) 

46.36 d 

(43.03) 

55.89 a 

(49.16) 

49.12 c 

(44.63) 

36.99 g 

(36.91) 

34.33 h 

(35.03) 

44.22 

(41.50)

Sl. 

No. 

Table 21. Effect of insecticides on the conidial germination of Verticillium lecanii 

Insecticide 

1. Endosulfan 14.49 

(22.38)* 

2. Quinolphos 20.20 

(26.70) 

3. Monocrotophos 22.33 

(28.18) 

4. Chlorpyriphos 12.24 

(20.49) 

5. Oxydemeton methyl 20.40 

(26.87) 

6. Dichlorvos 15.37 

(23.08) 

7. Dicofol 23.53 

(29.02) 

8. Malathion 32.46 

(34.73) 

9. Dimethoate 16.40 

(23.88) 

Mean 19.71 

(26.15)* 


0.25 RD 0.5 RD RD 2 RD 

26.15 

(30.74) 

67.38 

(55.18) 

47.33 

(43.47) 

31.51 

(34.14) 

45.51 

(42.42) 

25.33 

(30.22) 

54.25 

(47.42) 

67.42 

(52.22) 

30.75 

(35.50) 

43.74 

(41.26) 

49.10 

(44.48) 

79.47 

(63.05) 

57.28 

(49.16) 

51.91 

(46.09) 

62.36 

(52.14) 

51.04 

(45.59) 

73.23 

(58.84) 

81.80 

(64.75) 

55.57 

(48.19) 

62.72 

(52.48) 

59.51 

(50.47) 

100.00 

(90.00) 

75.43 

(60.28) 

81.83 

(64.77) 

90.25 

(71.82) 

89.57 

(71.15) 

100.00 

(90.00) 

100.00 

(90.00) 

87.37 

(69.18) 

87.10 

(73.07) 

S.Em± CD (0.01) 

A (Insecticide) 0.102 0.384 

B (Conc.) 0.068 0.256 

A x B 0.205 0.767 



Mean 

37.31 i 

(3.02) 

66.16 b 

(58.73) 

50.59 e 

(45.27) 

44.37 h 

(41.37) 

54.63 d 

(48.31) 

45.33g 

(42.51) 

62.75c 

(56.32) 

69.18a 

(60.42) 

48.27f 

(44.19) 

53.24 

(48.24)

1. Control 

2. 0.25 recommended dose 

3. 0.5 recommended dose 

4. Recommended dose 

5. 2 times recommended dose 

Plate 6. Inhibition of growth of Metarhizium 

Anisopliar and Verticillium lecanii 

by pesticide

Out of nine insecticides, endosulfan, quinolphos, monocrotophos, chlorpyriphos, oxydimeton 

methyl, dichlorvas, dicofol, malathion and dimethoate were included to know their effect on V. lecanii 

in vitro. Irrespective of concentration, there was 37.31 to 69.18 per cent inhibition of conidial 

germination (Table 21). Among them, malathion was significantly detrimental (69.18% inhibition) than 

all other insecticides except quinolphos (66.76%). Conversely, endosulfon and chlorpyriphos were 

signficinatly safer (37.31 to 44.37%), followed by oxydemeton methyl and dimethoate (45.33 to 

48.27% inhibition). 

4.6.3 Weedicides 

All the weedicides evaluated for their interaction with M. anisopliae in general, inhibited the 

germination of the fungal conidia to the extent of 9.01, 16.09, 26.61 and 41.95 per cent at low, 

medium recommended and high dosages, respectively (Table 22). However, the effect of all the 

weedicides on conidial germination of the mycopathogen was equal irrespective of the concentration 

tested. The inhibition ranged from 2.01 to 15.16, 9.80 to 22.41, 15.60 to 34.43 and 29.10 to 59.46 per 

cent at low, medium, recommended and higher concentration, respectively. 

Weedicides, in general, inhibited the germination of the fungus V. lecanii to the extent of 8.65, 

17.46, 25.85 and 36.12 per cent of low (one fourth and half the recommended), recommended and 

high (double the recommended) dosage respectively. Variation between the concentration was 

evident (Table 23). However, the effect of all the weedicides on conidial germination of the 

mycopathogen was equal irrespective of the concentration tested which ranged from 0.73 to 13.07, 

7.53 to 22.28, 15.51 to 34.42 and 24.30 to 48.06 per cent at low, recommended and high 

concentration, respectively. 

4.7 PERSISTENCE OF M. anisopliae (Ma2) AND V. lecanii (Vl1) ON 

PHYLLOPLANE AND SOIL 

The persistence of M. anisopliae Ma2 and V. lecanii Vl1 were evaluated on the phylloplane 

and soil of cotton under in vitro condition. The results are presented in Table 24 and 25. 

4.7.1 In soil and phylloplane 

Persistence studies of M. anisopliae and V. lecanii on soil and phylloplane were carried out in 

field and simulated field conditions over a period of 16 months. Immediately, after inoculation of M. 

anisopliae into soil, colony forming units (cfu) ranged from 25.6 x 10 9 to 35.2 x 10 5 per g of soil in field 

and simulated field conditions respectively. In the subsequent observations made at monthly interval, 

colony counts dropped gradually as the time lag increased (Table 24). The persistence of conidia was 

more under simulated field conditions i.e. upto 16 months with 2 x 10 4 cfu per g of soil whereas under 

field conditions it persisted only till 5 months. In simulated field condition, slight increase in cfu was 

encountered and continued upto declining phase. 

Phylloplane treated with aqueous suspension @ 4 x 10 6 conidia indicated that persistence in 

field condition and simulated field condition decreased from 1.31 x 10 2 and 1.04 x 10 2 cfu on the initial 

day of spray to 0.05 x 10 2 and 0.19 x 10 2 after two months. 

Initially under simulated field condition, the colony forming units (cfu) of V. lecanii accounted 

for 32.3 x 10 5 in soil and 1.47 x 10 2 on phylloplane. In the subsequent observations after every month, 

the colony counts dropped gradually to 2 x 10 4 after 360 days of inoculation in soil, whereas on 

phylloplane the mycopathogen, persisted upto 120 day of exposure with 0.02 x 10 2 cfu. 

Persistence studies of V. lecanii under field conditions revealed that the mycopathogen 

persisted, only for a period of three and two months in soil and phylloplane respectively (Table 25). 

The colony counts dropped from 20.3 x 10 9 and 1.29 x 10 2 to 0.2 x 10 2 and 0.14 x 10 2 in soil and 

phylloplane respectively.

Sl. 

No. 

Table 22. Effect of weedicides on the conidial germination of Metarihizium anisopliae 

Weedicides 

1. Oxyfluron 6.53 

(14.80) 

2. Pendimethalin 9.36 

(17.82) 

3. Atrazine 2.01 

(8.04) 

4. Alachlor 5.77 

(13.15) 

5. Butachlor 15.16 

(23.44) 

6. Fluchloralin 13.36 

(21.44) 

7. Glyphosate 10.56 

(18.97) 

Mean 9.01 

(16.88) 


0.25 RD 0.5 RD RD 2 RD 

18.00 

(25.10) 

14.43 

(22.32) 

9.80 

(18.24) 

14.30 

(21.91) 

22.41 

(28.24) 

19.28 

(26.06) 

14.43 

(22.32) 

16.09 

(23.46) 

34.43 

(35.93) 

28.33 

(32.16) 

15.60 

(23.23) 

19.66 

(26.32) 

29.67 

(33.00) 

26.33 

(30.87) 

32.26 

(34.61) 

26.61 

(30.87) 

59.46 

(50.47) 

36.99 

(37.45) 

29.10 

(32.64) 

41.36 

(40.03) 

31.20 

(38.76) 

32.43 

(34.71) 

55.13 

(47.94) 

41.95 

(40.26) 

S.Em± CD (0.01) 

A (Weedicide) 0.178 0.672 

B (Conc.) 0.135 0.508 

A x B 0.356 1.345 



Mean 

29.60 a 

(31.57) 

22.28 d 

(27.44) 

14.13 f 

(20.55) 

20.27 e 

(25.53) 

26.68 c 

(30.79) 

22.85 d 

(28.27) 

28.10 b 

(30.96) 

23.42 

(27.87)

Sl. 

No. 

Table 23. Effect of weedicides on the conidial germination of Verticiliium lecanii 

Weedicides 

1. Oxyfluron 0.73 

(4.87) 

2. Pendimethalin 21.34 

(27.51) 

3. Atrazine 1.23 

(6.35) 

4. Alachlor 13.07 

(21.18) 

5. Butachlor 11.26 

(19.61) 

6. Fluchloralin 9.78 

(18.18) 

7. Glyphosate 3.13 

(10.18) 

Mean 8.65 

(15.41) 


0.25 RD 0.5 RD RD 2 RD 

18.35 

(25.35) 

29.07 

(32.62) 

7.53 

(15.93) 

22.28 

(28.15) 

21.40 

(27.55) 

13.25 

(21.33) 

10.33 

(18.75) 

17.46 

(24.24) 

34.42 

(36.07) 

31.64 

(34.22) 

23.06 

(28.70) 

28.41 

(32.20) 

29.46 

(32.87) 

18.46 

(25.44) 

15.51 

(23.18) 

25.85 

(30.38) 

42.36 

(40.39) 

36.35 

(37.00) 

30.50 

(33.52) 

34.87 

(36.19) 

48.06 

(43.84) 

24.30 

(29.30) 

36.43 

(37.13) 

36.12 

(36.82) 

S.Em± CD (0.01) 

A (Weedicide) 0.110 0.417 

B (conc.) 0.084 0.315 

A x B 0.221 0.834 



Mean 

23.96 d 

(26.67) 

29.60 a 

(32.80) 

15.80 f 

(21.12) 

24.66 c 

(29.43) 

27.54 b 

(30.98) 

16.45 e 

(23.62) 

16.35e 

(33.31) 

22.02 

(26.71)

Days after 

storage 

Table 24. Persistence of Metarhizium anisopliae in soil and phylloplane 

Colony forming units (CFU) 

Simulated field conditions Field conditions 

Phylloplane (per 

sq.m. area) 

Soil (per g of soil 

x 10 4 ) 


sq.m. area) 

Soil (per g of soil) 

Open air temperature 

Maximum Minimum 

Rainfall (mm) 

0 104 352 131 25.6 x 10 9 

29.9 15.0 0.0 

30 69 330 48 17.2 x 10 7 

34.0 16.8 0.0 

60 19 282 5 18.4 x 10 3 

35.3 18.5 0.0 

90 - 418 - 52 35.7 22.0 52.1 

120 - 462 - 17 34.8 21.5 23.2 

150 - 345 - 2 30.3 21.3 32.5 

180 - 217 - - 26.8 21.1 33.1 

210 - 302 - - 27.2 20.9 58.1 

240 - 201 - - 30.1 20.2 53.6 

270 - 162 - - 30.1 19.9 17.0 

300 - 105 - - 31.0 17.9 0.0 

330 - 83 - - 29.6 13.7 0.0 

360 - 63 - - 30.1 16.3 0.0 

390 - 42 - - 31.9 17.7 0.0 

420 - 15 - - 36.0 20.0 62.7 

460 - 6 - - 37.2 21.7 0.0 

490 - 2 - - 34.9 23.0 67.0

Days after 

storage 

Table 25. Persistence of Verticillium lecanii in soil and phylloplane 

Colony forming units (CFU) Open air temperature Rainfall (mm) 

Simulated field conditions Field conditions 


sq.m area) 

Soil (per g of soil 

x 10 4 ) 


sq.m area) 

Soil (per g of soil) 

Maximum Minimum 

0 147 323 129 20.3 x 10 9 29.9 15.0 - 

30 105 281 14 18.6 x 10 5 34.0 16.8 - 

60 26 169 - 168 35.3 18.5 - 

90 5 210 - 24 35.7 22.0 52.1 

120 2 245 - - 34.8 21.5 23.2 

150 - 192 - - 30.3 21.3 32.5 

180 - 114 - - 26.8 21.1 33.1 

210 - 81 - - 27.2 20.9 58.1 

240 - 58 - - 30.1 20.2 53.6 

270 - 37 - - 30.1 19.9 17.0 

300 - 18 - - 31.0 17.9 0.0 

330 - 9 - - 29.6 13.7 0.0 

360 - 2 - - 30.1 16.3 0.0 

390 - - - - 31.9 17.7 0.0 

420 - - - - 36.0 20.0 62.7 

460 - - - - 37.2 21.7 0.0 

490 - - - - 34.9 23.0 67.0

4.8 SURVIVAL ABILITY OF Metarhizium anisopliae (Ma2) AND 

Verticilliumlecanii (Vl1) IN DIFFERENT OIL BASED AND 

WETTABLE POWDER FORMULATIONS UNDER 

DIFFERENT STORAGE TEMPERATURE LEVELS 

Different oil based wettable powder based formulations were evaluated for their 

suitability to support the conidial germination of M. anisopliae and V. lecanii under different 

storage temperature (room temperature, earthern pot temperature, refrigeration and deep 

freezer) at 20, 45, 75, 90 and 150 days after storage. The results are presented in Table 26 to 

37. 

4.8.1 Survival ability of Metarhizium anisopliae (Ma2) 

20 DAS 

There was a uniform germination after 20 days of storage amongst different 

formulations at all the storage temperature. 

The interaction effect of temperature and storage period was significant on conidial 

viability in room and earthen pot temperatures. Irrespective of the carrier based formulations, 

increased conidial viability was observed upto 45 days of storage at all temperatures (room, 

earthern pot, refrigerated and deep freezer storage temperature). A conidial viability above 90 

per cent was retained in case of room and earthern pot temperature. The interaction effect 

was non-significant under refrigerated and deep-freezer storage temperature at 20, 45 and 75 

days of storage. 

45 DAS 

At 45 days of storage in room temperature, 75 per cent conidia + 25 per cent 

sunflower oil formulation was able to show maximum germination of 96.91 per cent, but this 

treatment however failed to differ from 50 per cent conidia + 50 per cent sunflower oil 

(95.40%), 75 per cent conidia + 25 per cent sorghum flour (94.90%) and 50 per cent conidia + 

50 per cent sorghum flour (94.96%) at room temperature storage. Germination across all 

treatments recorded more than 90 per cent at room temperature and earthern pot after 45 

days. 

In the earthern pots, the maximum viability was found with treatment 75 per cent 

conidia + 25 per cent sunflower oil (98.36) followed by 50 per cent conidia + 50 per cent 

sunflower oil (96.26%) and 50 per cent conidia + 50 per cent sorghum flour (95.83%). 

However, the maximum reduction in germination was recorded with 50 per cent conidia + 50 

per cent groundnut oil and 50 per cent conidia + 50 per cent wheat flour at earthern pot 

storage temperature after 45 days of inoculation (Table 27). 

75 DAS 

Interaction of storage temperature, storage period on viable counts of M. anisopliae 

after 75 days of storage in case of room and earthern pot storage revealed that 75 per cent 

conidia + 25 per cent sorghum flour (85.46% and 88.73%) (Table 28) based formulation 

showed maximum viability under room and earthern pot storage respectively, which was 

significantly superior over other carrier materials. On the other hand, 50 per cent conidia + 50 

per cent sorghum flour (89.00% and 83.30%) was on par with maximum conidia i.e. 75 per 

cent conidia + 25 per cent sorghum flour under earthern pot temperature. At room 

temperature, 50 per cent conidia + 50 per cent groundnut oil (75.50%) and 50 per cent conidia + 50 

per cent mustard (75.68%) on par with each other recorded the least germination, whereas in the 

earthern pot, 50 per cent conidia + 50 per cent wheat flour (77.74%) and 50 per cent conidia 

+ 50 per cent mustard (78.13%) recorded the least viability (Table 28). After 75 days, a 

gradual decline in viability of M. anisopliae was noticed when stored at room temperature and 

earthern pot temperature, but with no significant difference in case of refrigerated and deep 

freezer storage.

Table 26. Survival of Metarhizium anisopliae in different oil based and wettable powder 

formulations under different storage temperature (20 DAI) 

Sl. 

No. 

Treatments Room 

temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

99.50 a 

(86.00) 

99.20 ab 

(84.92) 

98.76 ab 

(83.66) 

98.37 ab 

(82.66) 

98.36 ab 

(82.66) 

98.10 ab 

(82.08) 

98.53 ab 

(83.10) 

98.00 ab 

(81.81) 

98.50 b 

(82.97) 

97.83 ab 

(81.53) 

Per cent germination 

Earthern 

pots 

(22±3°C) 

99.56 a 

(86.94) 

99.46 a 

(85.89) 

98.90 a 

(84.03) 

98.98 a 

(84.19) 

98.86 a 

(83.89) 

98.73 a 

(83.56) 

98.53 a 

(83.17) 

98.73 a 

(83.73) 

98.56 a 

(83.17) 

98.03 a 

(81.94) 



Refrige 

ration 

(6°C) 

99.66 a 

(87.53) 

99.63 a 

(86.55) 

99.61 a 

(86.39) 

99.60 a 

(86.39) 

99.20 a 

(84.87) 

99.23 a 

(83.99) 

99.40 a 

(85.69) 

99.06 a 

(83.56) 

98.73 a 

(83.60) 

98.80 a 

(83.76) 

Deep freezer 

(-20°C) 

99.83 a 

(88.10) 

99.83 a 

(88.10) 

99.53 a 

(86.19) 

99.70 a 

(87.00) 

99.50 a 

(86.00) 

99.23 a 

(85.02) 

99.63 a 

(86.75) 

99.23 a 

(85.02) 

99.03 a 

(84.39) 

98.56 a 

(83.17)



Sl. 

No. 


temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

93.10 bc 

(74.77) 

90.00 d 

(71.59) 

93.40 bc 

(77.69) 

92.50 bcd 

(74.11) 

96.91 a 

(79.89) 

95.40 ab 

(77.62) 

90.90 cd 

(72.45) 

89.63 d 

(71.23) 

94.90 ab 

(79.96) 

94.96 ab 

(77.03) 


Earthern 

pots 

(22±3°C) 

93.50 c 

(75.23) 

91.23 d 

(72.77) 

94.40 c 

(76.31) 

94.33 c 

(76.23) 

98.36 a 

(82.69) 

96.26 b 

(78.19) 

93.46 c 

(75.19) 

90.90 d 

(72.45) 

94.23 c 

(76.10) 

95.83 b 

(78.25) 



Refrige 

ration 

(6°C) 

98.50 a 

(83.03) 

97.56 a 

(81.12) 

98.20 a 

(85.81) 

97.50 a 

(80.90) 

99.26 a 

(85.13) 

98.00 a 

(81.91) 

97.23 a 

(80.60) 

98.33 a 

(82.11) 

98.30 a 

(82.61) 

97.91 a 

(81.68) 

Deep freezer 

(-20°C) 

98.50 a 

(83.02) 

98.46 a 

(82.90) 

98.50 a 

(83.03) 

98.23 a 

(852.37) 

99.00 a 

(84.389) 

99.16 a 

(84.77) 

98.73 a 

(83.56) 

98.46 a 

(82.90) 

98.80 a 

(83.78) 

99.23 a 

(85.17)



Sl. 

No. 


temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

77.86 d 

(61.94) 

75.50 e 

(60.33) 

77.83 d 

(61.91) 

75.68 e 

(60.44) 

82.76 b 

(65.47) 

79.23 c 

(62.89) 

77.40 d 

(61.61) 

77.00 d 

(61.34) 

85.46 a 

(67.59) 

83.30 b 

(65.85) 


Earthern pots 

(22±3°C) 

80.23 de 

(63.60) 

79.40 f 

(63.00) 

80.63 d 

(63.89) 

78.13 g 

(62.12) 

84.63 b 

(66.92) 

82.98 c 

(65.63) 

79.56 ef 

(63.13) 

77.74 g 

(61.84) 

88.73 a 

(70.39) 

89.00 a 

(70.63) 



Refrige 

ration 

(6°C) 

96.06 a 

(78.57) 

95.43 a 

(77.67) 

95.56 a 

(77.85) 

96.56 a 

(79.37) 

96.13 a 

(78.68) 

95.41 a 

(77.66) 

86.33 a 

(79.00) 

95.83 a 

(78.25) 

96.23 a 

(78.83) 

96.00 a 

(78.48) 

Deep freezer 

(-20°C) 

97.90 a 

(81.68) 

98.13 a 

(82.18) 

97.66 a 

(81.35) 

97.86 a 

(81.35) 

98.00 a 

(81.91) 

98.50 a 

(83.03) 

97.73 a 

(81.39) 

97.56 a 

(81.12) 

97.60 a 

(81.10) 

97.73 a 

(81.49)

90 DAS 

After 90 days of storage, sharp decline in the viability of M. anisopliae was noticed 

when stored at room temperature and earthern pot, while a gradual decline in viability was 

recorded at refrigerated temperature. 

The maximum viability under room temperature was found in wettable powder based 

formulation i.e. 75 per cent conidia + 25 per cent sorghum flour (78.70%) followed by 50 per 

cent conidia + 50 per cent sorghum flour (74.94%) and 75 per cent conidia + 25 per cent 

sunflower oil (72.50%), whereas 50 per cent conidia + 50 per cent groundnut recorded least 

among the treatment carriers with 58.50 per cent germination. In case of earthern pot also 75 

per cent conidia + 25 per cent sorghum flour recorded the highest per cent germination 

(84.06%) followed by 50 per cent conidia + 50 per cent sorghum flour (79.64%), 50 per cent 

conidia + 50 per cent wheat flour (76.56%), 75 per cent conidia + 25 per cent groundnut 

(66.16%) and lowest germination with 50 per cent conidia + 50 per cent mustard (63.73%) 

and 50 per cent conidia + 50 per cent sunflower (73.66%) which were on par with each other 

(Table 29). 

At refrigerated storage, 75 per cent conidia + 25 per cent mustard (92.33%) showed 

maximum per cent germination which was on par with 50 per cent conidia + 50 per cent 

sunflower (87.90) and 75 per cent conidia + 25 per cent wheat flour (91.40%). 

120 DAS 

Metarhizium anisopliae exhibited moderate viability at both (room temperature and 

earthern pot) and per cent germination had dropped below 50 per cent after 120 days to 

storage except under 75 per cent conidia + 25 per cent sunflower oil (53.13% and 59.56%) 

and 75 per cent conidia + 25 per cent groundnut oil (51.50% and 56.73%) room temperature 

and earthern pot respectively. 

With increase in days of storage, oil formulation excelled in its ability to support the 

viability of the mycopathogen lagging behind the wettable powder formulation. Under room 

temperature, 75 per cent conidia + 25 per cent sunflower proved superior by producing 

significantly higher spore germination (53.13%) and was on par with 75 per cent conidia + 25 

per cent groundnut oil (51.50%). Mixing of 50 per cent conidia and 50 per cent mustard 

(50.33%) did not differ from 75 per cent conidia + 25 per cent groundnut (51.50%) followed by 

75 per cent conidia + 25 per cent mustard (40.33%) and 50 per cent conidia + 50 per cent 

sunflower (47.36%). In others, the spore germination was considerably less. 

At earthern pot storage temperature again, the 75 per cent conidia + 25 per cent 

sunflower proved superior with 59.56 per cent germination followed by 75 per cent conidia + 

25 per cent groundnut (56.73). However, this treatment did not differ from 75 per cent conidia 

+ 25 per cent mustard (55.90%) followed by 50 per cent conidia + 50 per cent mustard 

(54.23%) with 50 per cent conidia + 50 per cent sunflower (53.80%), whereas the least 

percentage of germination was recorded with 50 per cent conidia + 50 per cent wheat flour 

(38.43%) (Table 30 and Fig. 6). 

150 DAS 

A sharp decrease in conidial population was observed in all carrier materials at both 

temperature (room temperature and earthern pot) after 150 days and also declined at other 

storage temperature (refrigerated and deep freezer). In general, all the carrier materials had 

maximum conidial viability in case of refrigeration and deep freezer storage.

Sl. 

No. 

Table 29. Survival of Metarhizium anisopliae in different oil based and wettable 



temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

62.46 f 

(52.22) 

58.50 h 

(49.89) 

62.34 f 

(52.14) 

59.90 g 

(50.71) 

72.60 c 

(58.43) 

70.40 e 

(57.03) 

71.60 d 

(57.80) 

71.26 d 

(57.58) 

78.70 a 

(62.51) 

74.94 b 

(59.69) 


Earthern pots 

(22±3°C) 

66.16 cd 

(54.23) 

63.66 e 

(52.93) 

65.36 f 

(53.95) 

63.73 g 

(52.97) 

75.86 f 

(60.59) 

73.66 g 

(59.12) 

75.23 d 

(60.15) 

76.56 c 

(61.06) 

84.06 a 

(66.47) 

79.64 b 

(63.17) 



Refrige 

ration 

(6°C) 

90.23 e 

(71.81) 

91.90 e 

(73.46) 

92.33 a 

(73.94) 

88.90 de 

(70.55) 

80.00 cd 

(69.73) 

87.90 ab 

(69.64) 

91.40 abc 

(72.99) 

60.63 bc 

(72.18) 

89.00 de 

(70.64) 

89.06 de 

(70.69) 

Deep freezer 

(-20°C) 

98.00 a 

(81.91) 

97.23 a 

(80.47) 

98.00 a 

(81.91) 

98.33 a 

(82.71) 

97.16 a 

(80.41) 

97.03 a 

(80.12) 

98.50 a 

(83.03) 

98.03 a 

(81.97) 

98.13 a 

(82.48) 

97.66 a 

(81.30)

The maximum population of germination was recorded with 75 per cent conidia + 25 

per cent sunflower at all storage temperature i.e. 38.00 per cent (room temperature), 41.33 

per cent (earthern pot), 85.88 per cent (refrigerated), 93.73 (deep freezer) (Table 31). 

However, other carrier material failed to differ from this treatment in case of refrigerated and 

deep freezer. On the other hand, 50 per cent conidia + 50 per cent wheat flour supported 

least germination (19.00 and 22.90 per cent at room temperature and earthern pot storage) 

temperature respectively. There was no significant variation in the percentage germination 

between the different oils and wettable powder carrier material under refrigerated and deep 

freezer storage temperature. 

4.8.2 Survival ability of Verticillium lecanii (Vl1) 

20 DAS 

After 20 days of storage at room temperature and earthern pot, maximum 

germination was recorded with the formulation having 75 per cent conidia + 25 per cent 

sunflower (95.90%) which was on par with 75 per cent conidia + 25 per cent groundnut 

(96.15%). On the other hand, maximum percentage inhibition was recorded with 50 per cent 

conidia + 50 per cent wheat flour (86.40%) and 50 per cent conidia + 50 per cent mustard oil 

(86.32%) (Table 32). 

In earthern pot storage temperature, maximum viability was found in oil formulation 

i.e. 75 per cent conidia + 25 per cent sunflower and 75 per cent conidia + 25 per cent 

groundnut with per cent germination of 96.33 per cent and 96.49 respectively, which failed to 

differ from each other. The next best carrier material was 50 per cent conidia + 50 per cent 

sunflower oil (95.06%) and followed by 50 per cent conidia + 50 per cent groundnut oil 

(74.17%) and 75 per cent conidia + 25 per cent mustard oil (93.26%). The effect was nonsignificant 

under refrigerated and deep freezer storage temperature. 

45 DAS 

In general, all the oil formulation carrier material had more viability than wettable 

powder carrier material. After 45 day of storage, the conidial population of V. lecanii was 

reduced to more than 15 per cent in all the carrier materials. At room temperature, maximum 

germination of 72.76 per cent was observed with 75 per cent conidia + 25 per cent sunflower 

oil followed by 50 per cent conidia + 50 per cent groundnut (60.51%) and least germination 

was observed with 50 per cent conidia + 50 per cent wheat flour formulated carrier material 

(50.74%) (Table 33). Similar to room temperature, 75 per cent conidia + 25 per cent 

sunflower oil showed maximum viability (81.15%) under earthern pot temperature followed by 

75 per cent conidia + 25 per cent groundnut (76.32%), 50 per cent conidia + 50 per cent 

groundnut (69.98%) and 75 per cent conidia + 25 per cent sorghum flour (65.77%) (Table 33). 

Under refrigerated storage 75 per cent conidia + 25 per cent groundnut oil had 

maximum per cent germination (93.73), whereas under deep-freezer 75 per cent conidia + 25 

per cent groundnut oil and 75 per cent conidia + 25 per cent sunflower oil were on par with 

each other showing maximum per cent germination. 

75 DAS 

After 75 days of storage conidial population declined at both room temperature and 

earthern pot. The trend in reduction of conidial viability remained same at 75 days as in 45 

days of storage, under room temperature, whereas in the earthern pot storage 75 per cent 

conidia + 25 per cent groundnut oil took the upper hand lagging behind 75 per cent conidia + 

25 per cent sunflower oil and 50 per cent conidia + 50 per cent sunflower oil. However, 

maximum reduction of population was recorded with 50 per cent conidia + 50 per cent wheat 

flour formulated carrier material (Table 34).

Sl. 

No. 




temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

51.50 ab 

(45.86) 

45.90 de 

(42.64) 

48.33 c 

(44.04) 

50.33 b 

(45.19) 

53.13 a 

(47.79) 

47.36 cd 

(43.47) 

43.80 f 

(41.44) 

33.85h 

(35.56) 

45.46 ef 

(42.40) 

37.73 g 

(37.89) 


Earthern pots 

(22±3°C) 

56.73 b 

(48.87) 

48.53 d 

(44.16) 

55.90 b 

(48.38) 

54.23 c 

(47.42) 

59.56 a 

(50.51) 

53.80 c 

(47.17) 

48.10 d 

(43.91) 

38.43 g 

(38.31) 

45.33 e 

(42.32) 

41.73 f 

(40.24) 



Refrige 

ration 

(6°C) 

87.56 a 

(69.36) 

85.36 b 

(67.51) 

88.00 a 

(69.73) 

86.83 a 

(68.73) 

87.33 a 

(69.15) 

84.80 bc 

(67.05) 

87.33 a 

(69.15) 

83.83 c 

(66.29) 

87.00 a 

(68.59) 

85.16 bc 

(67.35) 

Deep freezer 

(-20°C) 

95.50 ab 

(77.76) 

94.50 bc 

(76.47) 

95.65 ab 

(77.95) 

96.50 a 

(79.23) 

95.23 ab 

(77.39) 

94.33 bc 

(76.25) 

93.60 cd 

(75.35) 

91.90 e 

(73.47) 

92.60 de 

(74.22) 

93.40 cd 

(75.13)

Sl. 

No. 




temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

31.46 c 

(34.12) 

28.06 e 

(31.99) 

27.66 e 

(31.73) 

29.73 d 

(33.04) 

38.00 a 

(38.05) 

32.90 b 

(35.00) 

26.03 f 

(30.68) 

19.00 h 

(25.84) 

27.66 e 

(31.73) 

21.06 g 

(27.31) 


Earthern 

pots 

(22±3°C) 

33.66 b 

(35.41) 

31.16 c 

(33.93) 

30.16 d 

(33.31) 

30.23 d 

(33.35) 

41.23 a 

(40.01) 

34.13 b 

(35.75) 

28.06 e 

(31.99) 

22.90 g 

(28.58) 

29.43 d 

(32.85) 

23.90 f 

(29.26) 



Refrige 

ration 

(6°C) 

85.96 ab 

(68.00) 

85.00 bcd 

(67.21) 

85.66 abc 

(67.75) 

86.50 a 

(68.45) 

85.88 ab 

(67.92) 

84.36 bc 

(66.71) 

84.00 d 

(66.43) 

82.33 e 

(65.15) 

86.33 ab 

(68.31) 

86.16 ab 

(68.18) 

Deep freezer 

(-20°C) 

92.80 bc 

(74.44) 

94.03 ab 

(75.86) 

92.23 c 

(73.84) 

92.96 bc 

(74.62) 

93.73 ab 

(75.50) 

94.70 a 

(76.69) 

93.73 ab 

(75.50) 

93.33 abc 

(75.08) 

93.00 bc 

(74.66) 

93.16 bc 

(74.86)


90 

80 

70 

60 

50 

40 

30 

20 

10 

0 



T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 

Legend 

T1 - 75% conidia + 25% groundnut oil 

T2 - 50% conidia + 50% groundnut oil 

T3 - 75% conidia + 25% mustard oil 

T4 - 50% conidia + 50% mustard oil 

T5 - 75% conidia + 25% sunflower oil 

Room temperature (28±2°C) 

T6 - 50% conidia + 50% sunflower oil 

T7 - 75% conidia + 25% wheat flour 

T8 - 50% conidia + 50% wheat flour 

T9 - 75% conidia + 25% sorghum flour 

T10 - 50% conidia + 50% sorghum flour 

Fig. 6. Survival of Metarhizium anisopliae (Ma2) and Verticillium lecanii (Vl1) in differnet formulations under different storage 

temperature (120 DAI) 

Fig. 6. Survival of Metarhizium anisopliae (Ma2) and Verticillium lecanii (Vl1) in different formulation under different storage 

Temperature (120 DAl)

Sl. 

No. 

Table 32. Survival of Verticillium lecanii in different oil based and wettable 



temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

96.15 a 

(78.60) 

93.83 c 

(75.59) 

92.02 d 

(73.57) 

86.32 f 

(68.28) 

95.90 a 

(78.36) 

94.50 b 

(76.47) 

88.20 e 

(69.91) 

86.40 f 

(68.39) 

90.03 d 

(71.60) 

87.75 e 

(69.50) 


Earthern 

pots 

(22±3°C) 

96.49 a 

(79.17) 

94.17 c 

(76.03) 

93.26 d 

(74.92) 

89.74 fg 

(71.28) 

96.33 a 

(78.96) 

95.06 b 

(77.16) 

90.26 f 

(71.82) 

89.01 g 

(70.63) 

91.90 e 

(73.46) 

90.43 f 

(71.98) 



Refrige 

ration 

(6°C) 

99.12 a 

(84.65) 

99.45 a 

(85.79) 

99.11 a 

(84.65) 

99.16 a 

(84.69) 

98.33 a 

(82.61) 

98.40 a 

(82.76) 

98.17 a 

(82.23) 

98.03 a 

(82.00) 

98.50 a 

(83.03) 

97.61 a 

(81.04) 

Deep freezer 

(-20°C) 

99.36 a 

(85.49) 

99.34 a 

(85.34) 

99.05 a 

(84.40) 

99.31 a 

(85.25) 

99.40 a 

(86.37) 

99.58 a 

(86.25) 

98.91 a 

(83.99) 

98.50 a 

(83.03) 

99.50 a 

(86.73) 

99.00 a 

(85.31)

Sl. 

No. 




temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

65.90 c 

(54.27) 

60.51 d 

(51.04) 

59.01 e 

(50.18) 

56.85 g 

(48.92) 

72.76 a 

(58.54) 

67.41 b 

(55.18) 

55.40 h 

(48.10) 

50.74l 

(45.42) 

58.26 f 

(49.76) 

55.50 h 

(48.15) 


Earthern 

pots 

(22±3°C) 

76.33 b 

(60.90) 

68.98 c 

(56.14) 

64.66 e 

(53.53) 

62.61 f 

(52.30) 

81.15 a 

(64.26) 

69.98 c 

(56.76) 

61.38 g 

(51.57) 

56.41 h 

(46.68) 

65.77 d 

(54.19) 

63.16 f 

(52.63) 



Refrige 

ration 

(6°C) 

93.73 a 

(75.59) 

91.89 b 

(73.47) 

91.50 bc 

(73.08) 

90.78 bcd 

(72.32) 

91.69 bc 

(73.22) 

91.61 bc 

(73.19) 

89.40 de 

(71.00) 

89.74 cde 

(71.32) 

91.16 bcd 

(72.73) 

88.16 e 

(69.88) 

Deep freezer 

(-20°C) 

98.00 a 

(81.91) 

94.60 b 

(76.53) 

94.00 b 

(75.83) 

94.00 b 

(75.83) 

97.45 a 

(80.91) 

93.53 bc 

(75.27) 

92.26 cd 

(73.85) 

91.33 d 

(72.88) 

94.50 b 

(76.44) 

90.83 d 

(72.38)

90 DAS 

A sharp decrease in conidia was observed in all carrier material at both temperature 

(room and earthern pot). Even in other storage temperature (refrigerated and deep freezer) 

there was decline after 90 days of storage. 

Among all the storage temperature, maximum conidial viability was recorded with 75 

per cent conidia + 25 per cent sunflower oil with per cent germination of 65.46, 87.60 and 

91.50 under room temperature, earthern pot, refrigerated and deep freezer respectively 

(Table 35). After 90 days of storage, the per cent germination had dropped below 50 per cent 

in all except 75 per cent conidia + 25 per cent sunflower (65.46%), 50 per cent conidia + 50 

per cent sunflower (59.08%) and 75 per cent conidia + 25 per cent groundnut oil (50.58%) 

stored at room temperature, whereas under earthern pot storage, 75 per cent + 25 per cent 

(65.41), 50 per cent conidia + 50 per cent sunflower (60.59%), 75 per cent conidia + 25 per 

cent groundnut (60.95), 50 per cent conidia + 50 per cent groundnut oil (51.62 per cent) and 

75 per cent conidia + 25 per cent sorghum flour recorded more than 50 per cent germination 

(Table 35). 

120 DAS 

In case of refrigerated and deep freezer storage temperature all carrier material were 

able to retain a conidial viability above 80 per cent. 

At 120 days of storage, the germination ranged from 28.60 at room temperature in 50 

per cent conidia + 50 per cent wheat flour to maximum of 88.96 per cent at deep freezer in 75 

per cent conidia + 25 per cent sunflower soil treatment (Table 36 and Fig. 6). 

The maximum viability under room temperature and earthern pot was recorded in 75 

per cent conidia + 25 per cent sunflower (62.73 and 64.26%) followed by 50 per cent conidia 

+ 50 per cent groundnut (45.28% and 50.91%) and lowest viability in 50 per cent conidia + 50 

wheat flour (28.60% and 28.73% under room temperature and earthern pot respectively). The 

conidial viability in refrigerated temperature was maximum again in 75 per cent conidia + 25 

per cent sunflower (85.32%). However, 50 per cent conidia + 50 per cent sunflower, 75 per 

cent conidia + 25 per cent sorghum flour and 75 per cent conidia + 25 per cent wheat flour 

were next in order with 84.26 per cent, 84.23 per cent and 83.66 per cent and the least 

viability was recorded in 50 per cent conidia + 50 per cent mustard (78.16 per cent 

germination) (Table 36). 

150 DAS 

Storage temperature has significant effect on the viability of V. lecanii after 150 days. 

The survival was significantly higher when stored at deep freezer, but significantly reduced 

when stored at prevailing room temperature. The per cent germination had dropped below 50 

per cent in all except 75 per cent conidia + 25 per cent sunflower oil both at room and 

earthern pot temperature condition (Table 37). 

Irrespective of the storage temperature, wettable powder formulation significantly 

affected the viability of V. lecanii at room temperature and earthen pot temperature. 

Significant variation among the various conidial protectants were observed, where in 75 per 

cent conidia + 25 per cent sunflower oil supported maximum number of conidial germination 

(54.13% and 56.46% at room and earthen pot temperature respectively) and the least in case 

of 50 per cent conidia + 50 per cent wheat flour (14.24%) at room temperature and 50 per 

cent conidia + 50 per cent sorghum flour in the earthen pot.

Sl. 

No. 




temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

54.63 c 

(47.65) 

48.73 e 

(44.25) 

46.31 f 

(42.88) 

45.16 g 

(42.22) 

64.60 a 

(53.49) 

60.83 b 

(51.25) 

46.26 f 

(42.86) 

40.50 h 

(39.52) 

51.68 d 

(45.95) 

46.26 f 

(42.86) 


Earthern pots 

(22±3°C) 

68.13 a 

(55.63) 

55.25 d 

(48.00) 

48.43 f 

(44.10) 

45.26 h 

(42.28) 

67.28 b 

(55.102) 

60.50 c 

(51.06) 

46.66 g 

(43.09) 

41.00l 

(39.81) 

53.85 e 

(47.20) 

46.50 g 

(42.99) 



Refrige 

ration 

(6°C) 

88.17 a 

(69.88) 

84.87 c 

(66.98) 

85.83 b 

(67.89) 

84.23 c 

(66.60) 

88.45 a 

(70.12) 

86.26 b 

(68.25) 

86.16 b 

(68.16) 

83.41 d 

(65.95) 

88.00 a 

(69.73) 

84.50 c 

(66.81) 

Deep freezer 

(-20°C) 

91.33 a 

(73.50) 

89.60 c 

(71.19) 

90.00 abc 

(71.57) 

88.33 c 

(70.03) 

91.61 ab 

(73.15) 

88.64 c 

(70.30) 

91.70 ab 

(73.43) 

88.41 c 

(70.09) 

89.80 bc 

(71.37) 

88.16 c 

(69.88)

Sl. 

No. 




temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

50.58 c 

(45.32) 

45.81 d 

(42.59) 

40.90 e 

(39.75) 

38.28 f 

(38.21) 

65.46 a 

(453.91) 

59.08 b 

(50.22) 

44.73 d 

(41.97) 

35.56 g 

(36.61) 

49.50 c 

(44.71) 

44.50 d 

(41.84) 


Earthern pots 

(22±3°C) 

60.95 b 

(51.31) 

51.62 c 

(45.91) 

43.25 e 

(41.11) 

40.86 f 

(39.74) 

65.41 a 

(53.97) 

60.59 b 

(51.10) 

45.83 d 

(42.61) 

36.60 g 

(37.22) 

51.26 c 

(45.72) 

45.61 d 

(42.66) 



Refrige 

ration 

(6°C) 

86.30 a 

(68.27) 

83.03 de 

(65.67) 

83.30 d 

(65.88) 

81.24 f 

(64.35) 

87.60 a 

(69.38) 

85.00 c 

(67.21) 

84.50 c 

(66.81) 

82.50 e 

(65.27) 

86.26 b 

(68.25) 

84.50 c 

(66.81) 

Deep freezer 

(-20°C) 

88.26 c 

(69.97) 

87.24 cd 

(69.07) 

87.26 cd 

(69.09) 

86.16 d 

(68.17) 

91.50 a 

(73.08) 

88.00 c 

(69.70) 

87.83 c 

(69.58) 

87.50 c 

(69.30) 

90.00 b 

(71.58) 

87.23 cd 

(69.07)

Sl. 

No. 




temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

45.28 c 

(42.28) 

42.16 e 

(40.49) 

32.53 h 

(34.77) 

33.33 g 

(35.26) 

62.73 a 

(52.37) 

56.56 b 

(48.77) 

32.50 h 

(34.75) 

28.60 l 

(32.33) 

44.28 d 

(41.70) 

41.16 f 

(39.91) 


Earthern pots 

(22±3°C) 

50.91 c 

(45.51) 

47.19 d 

(43.39) 

34.27 h 

(35.83) 

36.00 g 

(36.87) 

64.26 a 

(53.29) 

59.00 b 

(50.18) 

33.50 l 

(35.36) 

28.73 j 

(32.41) 

43.91 e 

(41.68) 

39.40 f 

(38.88) 



Refrige 

ration 

(6°C) 

81.66 e 

(64.65) 

79.50 d 

(63.06) 

79.30 d 

(62.94) 

78.16 e 

(62.14) 

85.83 a 

(67.89) 

84.26 b 

(66.63) 

83.66 b 

(66.17) 

82.33 c 

(65.15) 

84.23 b 

(66.60) 

82.29 c 

(64.82) 

Deep freezer 

(-20°C) 

87.33 bc 

(69.16) 

86.50 c 

(68.44) 

86.26 c 

(68.25) 

84.83 d 

(67.08) 

88.96 a 

(70.61) 

86.50 c 

(68.44) 

88.00 ab 

(69.74) 

787.83 ab 

(69.60) 

87.80 ab 

(69.57) 

86.26 c 

(68.25)

Sl. 

No. 




temperature 

(28±2°C) 

1. 75% conidia + 25% 

groundnut oil 

2. 50% conidia + 50% 

groundnut oil 

3. 75% conidia + 25% 

mustard oil 

4. 50% conidia + 50% 

mustard oil 

5. 75% conidia + 25% 

sunflower oil 

6. 50% conidia + 50% 

sunflower oil 

7. 75% conidia + 25% 

wheat flour 

8. 50% conidia + 50% 

wheat flour 

9. 75% conidia + 25% 

sorghum flour 

10. 50% conidia + 50% 

sorghum flour 

32.23 c 

(34.59) 

28.91 d 

(32.52) 

27.00 e 

(31.30) 

28.41 d 

(32.20) 

54.13 a 

(47.37) 

42.28 b 

(40.55) 

21.00 g 

(27.27) 

14.28l 

(22.1) 

24.06 f 

(29.37) 

15.87 h 

(23.47) 


Earthern pots 

(22±3°C) 

35.70 c 

(36.69) 

30.59 d 

(33.58) 

28.33 e 

(32.16) 

30.44 d 

(33.48) 

56.46 a 

(48.71) 

45.90 b 

(42.65) 

20.83 g 

(27.15) 

19.23h 

(26.01) 

24.00 f 

(29.35) 

18.40 l 

(25.39) 



Refrige 

ration 

(6°C) 

78.66 b 

(62.49) 

76.50 c 

(61.00) 

75.56 d 

(60.15) 

75.58 d 

(60.37) 

79.26 a 

(62.91) 

74.28 f 

(59.51) 

72.56 g 

(58.30) 

71.00h 

(57.31) 

76.60 c 

(60.91) 

75.00 e 

(60.22) 

Deep freezer 

(-20°C) 

83.23 de 

(65.82) 

83.53 cd 

(66.06) 

81.41 f 

(64.45) 

84.23 bc 

(66.60) 

85.57 a 

(67.67) 

84.51 b 

(66.81) 

84.21 bc 

(66.58) 

82.68 e 

(65.73) 

85.73 a 

(67.81) 

85.83 a 

(67.86)

4.9 PATHOGENICITY OF Verticillium lecanii ON MAJOR PESTS 

IN THE LABORATORY 

Brevicornia brassicae 

Various concentration of V. lecanii were evaluated against early instar nymph of B. 

brassicae. The mortality of nymph started after six days of treatment and was highest at 1.2x 

10 9 conidia/ml. Cumulative mortality of nymph increased with increase in concentration and 

exposure period (Table 38). The mortality of nymph on the six day was marginal (37.11%) but 

increased steadily to reach maximum of 92.30 per cent on 10 th day at the highest 

concentration and was significantly superior to the rest of the treatment at all intervals of 

observation. 

Among the concentrations tested, mean per cent mortality of 1.2 x 10 9 and 1.2 x 10 8 

conidia/ml were at par with each other and significantly superior to rest of the concentration. 

On comparison of concentrations required to cause more than 50 per cent mortality, it was 

noticed that 1.2 x 10 9 , 1.2 x 10 8 and 1.2 x 10 7 conidia/ml required eight days after spray. 

Lower dose of 1.2 x 10 5 conidia/ml was able to cause only 38.33 per cent even on 10 th day 

after spraying. Interaction studies revealed that significant difference in mortality was 

observed between all the concentration and days tested. 

Aleurodicus disperses 

The cumulative mortality increased significantly with increase in concentration and 

exposure period (Table 39). The mortality of larvae started after six days of treatment and it 

was highest at 1.2 x 10 9 conidia/ml. The mortality of the larvae on sixth day was marginal 

(29.42%) but increased steadily to reach maximum of 80.93 per cent on 10 th day at the 

highest concentration and this treatment was significantly superior to rest of the treatments at 

all intervals of observation. 

On comparison of concentration required to cause more than 50 per cent mortality, it 

was noticed that 1.2 x 10 9 , 1.2 x 10 8 and 1.2 x 10 7 conidia/ml required eight days after 

inoculation. The lowest dose of 1.2 x 10 5 conidia/ml was able to cause only 34.72 per cent 

mortality even on 10 th day after spraying. Interaction studies revealed that difference in 

mortality was observed between all the concentrations and days tested. The highest mean 

mortality of 60.25 per cent was recorded at the highest concentration of V. lecanii @ 1.2 x 10 9 

conidia/ml and mortality values decreased significantly with successive decrease in 

concentration. Similarly, between the duration, mortality increased significantly with increase 

with increase in time after treatment. 

The mortality of the mycosed nymph was significantly high at higher dose of spray 

(1.2 x 10 9 ) than the other concentrations. The mortality ranged from zero in the untreated 

check to 73.35 per cent at 1.2 x 10 9 conidia/ml at 10 days after treatment and 63.31 and 

12.88 per cent mortality at 1.2 x 10 8 and 1.2 x 10 5 conidia/ml. 


was noticed that 1.2 x 10 9 and 1.2 x 10 8 conidia/ml required eight and 10 days after spray, 

respectively. The fungus weakly exhibited its pathogenicity at 6 days of treatment with 

maximum of 27.69 per cent at 1.2 x 10 9 conidia/ml and only 9.03 per cent mortality at the 

lowest dose of 1.2 x 10 5 conidia/ml (Table 38).

Sl. 

No. 

Table 38. Per cent mortality of Brevicornia brassicae due to application of 


Dosage 

(conidia/ml) 

1. 1.2 x 10 9 

2. 1.2 x 10 8 

3. 1.2 x 10 7 

4. 1.2 x 10 6 

5. 1.2 x 10 5 

Per cent mortality 

(Days after inoculation) 

6 8 10 

Mean 

47.03 g 84.86 b 92.30 a 71.42 a 

37.11 l 77.89 o 88.79 b 71.23 a 

30.71 k 56.38 l 66.12 e 51.07 b 

18.84 m 33.97 j 41.80 l 31.53 c 

16.81 n 25.72 f 38.33 i 20.95 d 

6. Control 0.00 o 0.00 d 0.00 o 0.00 e 

Mean 25.08 c 46.47 a 54.56 a 42.03 

Means followed by same alphabet do not differ significantly (0.01)

Sl. 

No. 

Table 39. Per cent mortality of Aleurodicus dispersus due to application of 


Dosage 

(conidia/ml) 

1. 1.2 x 10 9 

2. 1.2 x 10 8 

3. 1.2 x 10 7 

4. 1.2 x 10 6 

5. 1.2 x 10 5 

Per cent mortality 

(Days after inoculation) 

6 8 10 

Mean 

29.42 h 70.50 c 80.93 a 60.28 a 

19.75 j 68.01 d 74.70 b 54.15 b 

16.70 k 56.73 e 67.88 d 47.10 c 

15.12 k 23.55 l 42.47 f 27.05 d 

9.76 l 51.04 j 34.72 g 21.84 e 

6. Control 0.00 m 0.00 m 0.00 m 0.00 f 

Mean 15.13 39.97 50.12 35.07 


Tr. 

No. 

Table 40. Per cent reduction of nymph of Brevicornia brassicae on cabbage due to Verticillium lecanii (Vl1) under field conditions 

Treatments 

I spray II spray III spray 

3 DAA 7 DAA 14 DAA 3 DAA 7 DAA 14 DAA 3 DAA 7 DAA 14 DAA 

T1 2 x 10 5 14.26 bc 21.26 d 26.67 c 14.78 d 20.14 e 28.78 c 17.99 d 23.51 c 31.48 e 

T 2 2 x 10 6 16.07 b 23.54 d 32.54 b 15.91 d 21.98 de 32.44 c 19.37 cd 26.11 c 36.80 d 

T 3 2 x 10 8 15.68 b 25.52 d 33.08 b 18.19 c 26.59 d 38.05 b 19.82 cd 28.41 c 43.01 c 

T4 2 x 10 10 15.49 b 37.41 c 31.97 b 18.86 c 42.99 c 37.54 b 21.45 c 56.28 b 48.13 b 

T5 2 x 10 12 17.88 b 43.44 b 35.68 b 22.15 b 50.37 b 39.89 b 25.31 b 61.16 b 51.68 b 

T6 

Dimethoate 30 EC 

@ 1.7 ml/litre 

50.45 a 60.74 a 53.89 a 68.25 a 76.69 a 66.35 a 78.50 a 87.53 a 81.17 a 

T7 Untreated control 10.71 e 11.59 e 14.11 d 12.49 e 11.14 f 10.50 e 13.80 e 17.03 d 17.47 f 

DAA – Days after application 


Culture grown on rice grains 

Harvest of spores 

Ready to use spore 

Plate 7. Metarhizium anisopliae cultured on rice for field use

4.11 EFFECT OF Verticillium lecanii APPLICATION ON Aphis 

crassivora UNDER FIELD CONDITION 

All the doses of the biopesticide were not significantly different from each other at 3 

(DAA). Dimethoate recorded significantly highest mortality compared to other treatments. The 

mortality ranged from 11.55 per cent in the untreated check to 85.10 per cent at chemical 

treatment at 7 day of 3 rd spraying (Table 40). 

On comparison of the biopesticide concentration required to cause more than 50 per 

cent mortality, it was noticed that 1.2 x 10 12 and 1.2 x 10 10 required seven days after second 

and third spray respectively. The fungus weakly exhibited its pathogenicity at lowest 

concentration of 1.2 x 10 5 with maximum of 32.37 per cent after third spray. The fungus was 

more effective at higher concentration and high exposure period than lower concentration and 

low exposure period. 

After first application, all the doses of biopesticide were not significantly different from 

each other at 3 DAA. This trend continued even after 3 rd spray. However, dimethoate caused 

reduction of population to over one third to stand out as significantly superior. While the 

fungus required third application to cause more than 50 per cent mortality, it was noticed that 

2 x 10 10 and 2 x 10 12 caused 57.03 and 61.80 per cent mortality after 7 DAA (Table 41). 

Verticillium lecanii even at the lowest dose was significantly superior to untreated 

control. 

There was no significant variation in nymphal population at three days after first spray 

when the mycopathogen was applied. Higher dosages of 2 x 10 10 and 2 x 10 12 performed at 

par but significantly superior to untreated control and lower dosage of V. lecanii. However, 

after third application, the fungal spray at higher dosage was significantly superior to lower 

dosage and the next best to consecutive spray was the succeeding dosage. Conversely, the 

lowest dose (T1) failed as badly as untreated check after the first spray (Table 42). 

The mortality where the mycopathogen was applied ranged from 10.74 per cent in the 

3 DAS of first spray to 66.50 per cent at higher dosage at 7 days after second spray. 

Verticillium lecanii on Brevicornia brassicae 

Treatments were imposed in the experimental area after nymphal buildup was adequate. The 

mortality of the mycosed nymph was significantly high at the highest dose of spray (2 x 10 12 ) 

compared to rest of the concentrations. The mortality ranged from 10.50 per cent in the 

untreated check after second spray to 61.16 per cent at 2 x 10 12 conidia/ml. Nevertheless, 

dicofol proved to be the most effective after each spray causing more than 50 per cent 

mortality (Table 43). 


was noticed 2 x 10 1 and 2 x 10 12 required seven days after third and second spray 

respectively. The fungus weakly exhibited its pathogenicity at lowest concentration of 1.2 x 

10 5 with maximum of 31.48 per cent mortality after third spray. 

The mycopathogen lagged behind the chemical toxicants in lowering the pest 

population due to the spray, but picked upto at 7 days after spray to prove as good as 

chemical toxicants. However, the effectiveness of fungus was dose dependent. 

Activity of aphid, B. brassicae was uniform a day before spray. However, seven days 

after imposition of treatments, the number varied significantly. Toxicity of dicofol to the 

nymphal population revealed highest mortality of 78.78 per cent after seven days of third 

spray (Table 44).

Tr. 

No. 


Treatments 



T 1 2 x 10 5 7.22 b 15.41 d 15.46 cd 11.67 de 17.52 f 19.78 d 12.16 cd 17.67 c 22.42 d 

T2 2 x 10 6 7.45 b 17.22 d 23.24 bc 13.46 cde 20.68 c 26.50 cd 12.50 cd 21.50 c 26.58 d 

T3 2 x 10 8 10.71 b 27.00 c 32.60 b 14.91 cd 25.08 d 34.08 bc 15.08 bcd 26.33 c 34.20 c 

T4 2 x 10 10 17.14 b 44.13 b 32.25 b 17.74 c 44.65 c 37.91 bc 19.83 bc 46.61 b 39.66 bc 

T5 2 x 10 12 17.03 b 45.32 b 30.37 b 23.3 b 50.65 b 41.55 b 28.58 b 52.25 b 43.83 b 

T6 



51.93 a 67.16 a 46.19 a 55.70 a 75.48 a 60.33 a 64.91 a 78.78 a 67.12 a 

T 7 Untreated control 7.03 b 11.20 e 9.54 e 11.33 e 12.91 e 10.58 e 8.83 d 9.25 d 12.41 e 



Tr. 

No. 


Treatments 



T1 2 x 10 5 3.70 b 13.42 d 13.42 c 10.17 d 16.67 f 17.67 d 9.00 cd 15.17 c 20.17 d 

T2 2 x 10 6 4.17 b 16.67 d 25.00 bc 12.50 d 19.50 e 23.50 cd 9.33 cd 19.17 c 23.67 d 

T3 2 x 10 8 7.50 b 29.17 c 32.50 b 13.50 cd 23.83 d 32.33 bcd 13.00 bc 25.50 c 31.83 c 

T4 2 x 10 10 16.04 b 47.61 b 31.73 b 17.25 c 43.50 c 36.83 bc 20.17 bc 46.50 b 40.00 b 

T 5 2 x 10 12 15.74 b 43.98 b 28.24 bc 22.17 b 50.17 b 42.40 ab 29.33 b 53.50 b 45.00 b 

T6 



52.35 a 68.05 a 43.52 a 53.33 a 75.73 a 56.00 a 63.83 a 80.00 a 67.57 a 

T7 Untreated control 3.33 b 10.74 d 7.41 d 9.83 d 9.83 g 5.17 e 2.83 d 2.83 d 9.83 e 



Tr. 

No. 

Table 43. Per cent reduction of nymph of Aphis crassivora on cowpea due to Verticillium lecanii (Vl1) under field conditions 

Treatments 



T1 2 x 10 5 17.73 bc 24.67 c 28.42 d 19.46 d 24.11 e 29.46 d 19.41 d 23.92 e 32.37 c 

T2 2 x 10 6 19.31 bc 28.79 de 32.07 cd 19.91 d 25.60 e 32.15 d 20.78 cd 28.03 de 38.53 d 

T3 2 x 10 8 19.48 bc 32.05 d 31.81 bc 20.82 cd 28.76 d 40.57 c 21.11 cd 31.89 d 44.73 c 

T4 2 x 10 10 20.00 bc 39.91 c 33.98 bcd 22.32 c 49.99 c 44.46 bc 22.83 c 55.53 c 51.03 b 

T 5 2 x 10 12 22.04 b 47.43 b 38.33 b 24.84 b 57.94 b 45.95 b 28.80 b 60.53 b 53.23 b 

T6 



61.15 a 71.01 a 62.28 a 73.53 a 80.66 a 69.33 a 76.65 a 85.10 a 78.77 a 

T7 Untreated control 16.17 c 15.27 f 16.75 e 17.34 e 13.73 f 11.55 c 17.83 d 19.50 f 20.37 f 



Tr. 

No. 


Treatments 



T1 2 x 10 5 10.79 b 17.65 c 24.13 d 10.10 d 16.17 c 28.10 c 16.57 c 23.10 c 30.60 e 

T 2 2 x 10 6 12.84 b 18.30 c 33.02 bc 11.91 d 18.37 c 32.73 bc 17.97 bc 24.20 c 35.07 d 

T3 2 x 10 8 11.89 b 19.00 c 34.35 b 15.56 c 24.43 c 35.53 b 19.53 bc 24.93 c 41.30 c 

T4 2 x 10 10 10.98 b 34.86 b 29.97 c 15.40 c 35.99 b 30.62 bc 20.08 bc 57.03 b 45.23 bc 

T5 2 x 10 12 13.73 b 39.46 b 33.03 bc 19.47 b 42.80 b 33.84 b 21.83 b 61.80 b 49.53 b 

T6 



39.75 a 50.47 a 45.50 a 62.98 a 72.73 a 63.38 a 80.35 a 89.97 a 83.57 a 

T 7 Untreated control 5.26 c 7.92 d 11.48 e 7.65 e 8.56 d 9.46 d 9.78 d 14.57 d 14.57 f 



The fungal spray at higher dosage (1.2 x 10 12 ) after 7 days of second and third spray 

only showed more than 50 per cent of mortality. The concentrations 2 x 10 12 and 2 x 10 10 

performed at par with each other and significantly superior to all doses of V. lecanii at 7 DAS 

and this trend continued at 14 DAS after the first and third spray. 

Even at the lowest dose V. lecanii was significantly superior to untreated only at 7 

and 14 DAS. After first spray there was no significant difference between all the doses of V. 

lecanii and untreated control at 3 DAS, but inferior to chemical spray became evident (Table 

45). However observations showed no difference between highest dose of V. lecanii (2 x 10 12 ) 

and succeeding dose (2 x 10 10 ). Conversely, the lowest dose (2 x 10 5 ) failed as badly as 

untreated check throughout the study period at third day after each spray (Table 45). 

Dimethoate, efficiently knocked down at the third day of first spray and continued to 

maintain its effectiveness throughout the observation period. 

4.12 CHEMICAL MUTATION 

UV radiation 

The conidial suspension of Ma2 and Vl1 exposed to UV radiation at 5 cms from UV 

bulb resulted in an optimum killing percentage of 99.97 per cent. From the stock of 

mutagenized survivors, totally twelve mutants from Vl1 and twelve mutants from Ma2 strain 

were developed. 

The test insects (H. armigera and B. brassicae) exhibited mortality to all mutants. 

However, the mortality of V. lecanii mutants values ranged from minimum of 32.59 to 

maximum of 91.97 per cent during the study (Table 46). Among Vl1 derived mutants highest 

cumulative mortality of 91.97 per cent was observed on VA2, but failed to differ statistically 

from other mutants, VA5 (90.56%), VA11 (91.96%) and VA12 (89.36%). The lowest mortality 

was recorded with VA9 with only 32.59 per cent. 

The M2 derived mutants exhibited moderate pathogenicity of H. armigera. The 

mortality ranged from 36.50 in MH12 to 91.67 in MH4 and was on par with MH2 (90.92%). 

The next best mutant was MH8 (82.75%) followed by MH5 (77.10%) and held type V1 

(73.96%).

Tr. 

No. 


Treatments 



T1 2 x 10 5 10.74 b 17.41 d 17.50 cd 19.50 c 24.00 f 29.33 e 13.57 cd 18.37 f 21.90 c 

T2 2 x 10 6 10.74 b 17.78 d 21.48 c 20.17 c 28.67 e 34.00 e 14.43 cd 21.87 e 29.50 d 

T3 2 x 10 8 13.93 b 24.83 c 32.70 b 20.73 c 34.67 d 38.83 d 16.33 c 26.33 d 35.83 c 

T4 2 x 10 10 18.24 b 40.65 b 32.78 b 31.08 b 53.17 c 46.50 c 18.28 c 45.80 c 39.00 bc 

T 5 2 x 10 12 18.33 b 46.67 b 32.50 b 32.50 b 66.50 b 54.67 b 24.60 b 51.13 b 40.70 b 

T6 



51.51 a 66.28 a 48.86 a 74.17 a 87.33 a 81.73 a 58.08 a 75.28 a 64.67 a 

T7 Untreated control 10.74 b 11.67 d 11.67 d 16.33 c 19.33 g 20.83 f 12.83 d 16.00 f 16.00 c 



Table 46. Effect of wild types and derived mutants (UV) of Metarhizium 

anisopliae (Ma2) Helicoverpa armigera and Verticillium lecanii (Vl1) 

Sl. No. Strains Per cent mortality 

1 Metarhizium anisopliae Ma2 (Wild 

type) 

73.96 ef 

Mutants 

2 MH1 64.20 g 

3 MH2 90.92 a 

4 MH3 73.81 ef 

5 MH4 91.67 a 

6 MH5 77.10 e 

7 MH6 53.77 h 

8 MH7 39.60 ij 

9 MH8 82.75 b 

10 MH9 71.75 f 

11 MH10 43.06 l 

12 MH11 65.75 g 

13 MH12 36.50 l 

14 Verticillium lecanii Vl1 (Wild type) 82.62 b 

Mutants 

15 VA1 83.77 b 

16 VA2 91.97 a 

17 VA3 64.75 g 

18 VA4 77.14 de 

19 VA5 90.56 a 

20 VA6 53.87 h 

21 VA7 81.96 be 

22 VA8 79.00 cd 

23 VA9 32.59 k 

24 VA10 83.16 b 

25 VA11 91.96 a 

26 VA12 89.36 a 


V. DISCUSSION 

Insect pathogenic fungi, often not realized by man, naturally and efficiently restrict the 

build up of insect pests without any interference. This part of the ‘Law of Natural Balance’ is 

being inadvertently destroyed by the indiscriminate use of chemical pesticides. This has led to 

the notion that, all forms of pest control should be integrated and environmentally acceptable 

to the agricultural ecosystem. It is certain that entomogenous fungi will continue to increase 

their share very rapidly in the integrated pest management. 

Mycopathogens infect insects through cuticle and are therefore, the principle 

pathogens among sucking insects which cannot ingest other pathogens that infect through 

the gut wall (Hajek and Ledger, 1994). More than 750 species of entomopathogenic fungi 

have been described (Maddox, 1994), of which only ten species have been extensively 

exploited. Current research efforts were directed at selecting native entomopathogenic fungi, 

characterizing them assessing their virulence and developing a formulation for them. 

Additionally, their compatability with certain pesticides, genetic enhancement and field 

evaluation was conducted. The results obtained are discussed herein. 

5.1 EXPLORATION OF THE NATURAL OCCURRENCE OF 

INSECT MYCOPATHOGENS IN NORTHERN KARNATAKA 

Extensive work in the area of isolation, characterization and exploitation of 

entomopathogenic fungi needs to be a continuous excercise. Understanding the ecology and 

molecular diversity of the pathogen is of immense importance in exploiting the 

biotechnological potential of the pathogens. Hence, the present work undertook an extensive 

and repeated survey covering different agro ecological niches of northern Karnataka. The 

present study focused on collection of insect cadavers that were presumed to be fungal 

infected. Extensive variation in the per cent incidence was observed. The incidence of 

mycopathogens collected during the survey is indicated in the Fig. 1. Wide spread incidence 

was observed with respect to M. anisopliae, V. lecanii, B. brassiana and F. oxysoporum. 

Amongst the different mycopathogens, N. rileyi had the highest natural incidence (30-80%) in 

the transitional belt of northern Karnataka. The natural incidence of C. sphaerospermum was 

higher in Dharwad and was observed to parasitize ligeid bug (18.58%). The natural incidence 

of M. anisopliae varied from 1.8 to 10.3 per cent and was maximum on diamond black moth, 

pest that prevailed on cabbage at Hirebagewadi. The incidence of C. cladosporides and A. 

candidus was less than 10 per cent. These results indicated a high variability of disease 

incidence due to mycopathogen attack. 

The variability observed in the disease incidence could be due to the prevalence of 

different agro climatic conditions. The incidence of M. anisopliae was seen across two 

contrasting ecological niche i.e., Dharwad which, had a relative humidity of 80-89 per cent 

and a temperature of 26.5-27.7°C and Gangavathi which had a lower relative humidity of 75- 

78 per cent and higher temperature 30-32.5°C. The ability of these to grow at varying 

temperature upto 35-37°C, indeed, has been observed (Yip et al., 1992). The present result 

also indicate the potential to use M. anisopliae as biopesticide under contrasting 

environments. 

5.2 ISOLATION AND CHARACTERIZATION OF 


The cadavers of susceptible host brought to the laboratory and those presumed to be 

infected by M. anisopliae and V. lecanii were used for isolation of the mycopathogen. A total 

of seven fungi were isolated, purified and identified based on their colony characteristic and 

microscopic examination. The origin of the isolates and the host are shown in the (Table 5 

and Fig. 1). Of the seven, four were identified to be M. anisopliae and three were assigned to 

V. lecanii. The identity of the fungi was later confirmed at Agarkar Research Institute, Pune.

The hyphal and conidial characters did not vary among the isolates collected from 

different places during survey. However, V. lecanii exhibited significant variation in the 

number of days taken for sporulation, sporulation, spore yield and time taken to cover the 

given diet for mass production. Among the different isolates of V. lecanii, Vl1 recorded highest 

colony diameter (52.13) but least spore yield (4.21x10 9 ). The Ma2 and Ma1 isolates recorded 

higher colony diameter of 42.30 mm and 38.60 mm respectively. In these two isolates, the 

sporulation commenced two to three days earlier than isolates Ma3 and Ma4. These results 

indicate variability among the isolates which need to be realised when being used for 

development of effective bioinoculant and mass production. 

5.3 TESTING OF EFFICACY OF Metarhizium anisopliae (Ma2) 

AND Verticillium lecanii (Vl1) AGAINST DIFFERENT 

INSECT PESTS 

The pathogenicity of isolates Vl1 and Ma2 was tested against various insect pest (B. 

brassiae, A. crassivora, M. sacchari, P. latus, A. disperses, H. armigera and O. rhinoceros). 

The pathogenicity was highly variable (51.35 to 94.50 per cent). Vl1 caused maximum 

pathogenicity in case of cabbage aphid B. brassica (94.50%). It also caused extensive 

damage against cowpea aphid A. crassivora (84.23%). Ma2 was highly pathogenic against H. 

armigera and O. rhinocerous. The toxicity of M. anisopliae against O. rihoceros has been 

reported by Gopalkrishnan and Narayanan (1988). These results indicate the possibility of 

using V. lecanii on aphids (B. brassica and A. crassivora) and M. anisopliae on H. armigera 

and O. rhinoceros. The wide variability observed indicates further exploration could result in 

isolation of more potent strains. It would also help to extend the host species. One of the 

ways in which the population of M. anisopliae could be enhanced in culture bank would be to 

isolate M. anisopliae from soil on a selective medium containing 10 µm/ml of dodine (Ndodecyl 

guanidine monoacetate) (Liu et al., 1993). 

In fungal cells, dodine induces the loss of 32 P (Brown and Sisler, 1960), P1, amino 

acids and UV absorbing materials and increases the permeability of the cytoplasmic 

membrane to externally added Ni 2+ (Miller and Barran, 1977). In plant cells, dodine induces 

an efflux of betacyanin and total ions. Other cationic amphiphiles (namely, cetyltrimethylammonium 

bromide, chlorohexidine and polymyxins) have been reported to cause severe 

damage to the cytoplasmic membrane in several bacterial species (Newton, 1956). Using 

dodine in the medium, thus would enhance the probability of isolating diverse and perhaps 

more effective strains. 

5.4 INFLUENCE OF DIFFERENT NUTRIENT SOURCES 

One of the key factor in enhancing the population of mycopathogens propagules in 

the inoculum is the availability the nutrients. Several attempts have been made to multiply 

these mycopathogens using semi synthetic and solid substrate primarily to cut down cost of 

production. Additionally, the availability of nutrients also influences the survival of these 

organisms in nature. Carbon and nitrogen are the most vital nutrients required for growth and 

sporulation (Campbell et al., 1983). Therefore, the effect of different carbohydrates and 

nitrogen on the growth of M. anisopliae and V. lecanii was evaluated at different 

concentrations in Czapeckdox broth. The nutritional requirement of the fungi in both species 

and strain varied. Starch supported the highest growth of M. anisopliae Ma2 (6.01 gm/250 ml) 

followed by fructose and sucrose. In case V. lecanii Vl1 too, the maximum biomass was 

obtained when grown on starch followed by sucrose. Soluble starch has been reported to be 

the best substrate tested for sporulation of M. anisopliae strain when grown on soypeptone 

based medium (Li and Holdom, 1994) and hence has been recommended to be useful carbon 

source in the production media. In earlier studies, the most effective carbon source for 

sporulation of N. rileyi were reported to be dextrose (IM et al., 1988) and Maltose (Balardrin 

and Loch, 1989). Edelstein et al. (2004) on the other hand found that media with potato 

extract induced higher growth rate.

For continuous growth and extension of the hyphae and to prevent autolysis, an 

exogenous nitrogen source is required (Smith and Grula, 1981). Hence, certain nitrogen 

source were tested. Among them KNO 3 (5.769/250 ml) was found to be the best source 

followed by NH4NO3 for biomass production of M. anisopliae Ma2. However, when NaNO3 

was used as a nitrogen source, there was reduction in the growth. Vl2 on the other hand grew 

better in the medium supplemented with (NH4)SO4 and least with medium supplemented with 

KNO3. In an earlier study KNO3 was found to be better for better growth and sporulation than 

(NH 4)SO 4 (Li and Holdom, 1994). The use of NH 4 ion is known to acidify the media much 

more than NO3. It is known that the acidity at certain levels encourages the growth of fungi 

beyond certain limits probably is responsible for the sporulation as inferred by Rath et al. 

(1995), while characterizing M. anisopliae strain on different source of organic acids as 

carbon sources. 

5.5 COST EFFECTIVE MASS MULTIPLICATION OF 


Ensuring survival and persistence of the fungi is one of the greatest challenges of 

mycoinsecticide formulation and this technology has to be optimized for individual species or 

even strains (Burges, 1998). Inexpensive culture medium is required in order to increase the 

benefit:cost ratio. Hence, several sources were tested for mass multiplication. 

Food grains 

Low cost sources of nutrients like bajra, sorghum, navane, maize, rice and wheat 

were assessed for their utility in terms of conidial yield of M. anisopliae Ma2 and V. lecanii 

Vl1. It was found that growth of Ma2 on bajra yielded the maximum conidia (22.47 x 10 8 

conidia/g of substrate) Vl1 on the other grew better and produced more conidia on rice grains 

(24.59 x 10 8 conidia/g). It has been earlier shown that fungi could be multiplied on polished 

rice grains (Silva and Loch, 1987) and crushed sorghum (Vimaladevi, 1994). The high amount 

of conidia in bajra and rice could be due to maltose released by the action of starch 

hydrolyzing enzymes present in the fungus induces sporulation. Disaccharide like maltose are 

known to induce more sporulation in unicellular fungi, Hansenula than monosaccharides 

(Crandall and Lawrence, 1980). Since chitinase and exochitinase activities are low in conidia 

and germinating seeds (Preez et al., 1985), crushing of grains is necessary to release the 

substrate for action of amylase. This indicates that rice and bajra grits as economical sources 

of mass production of M. anisopliae Ma2 and V. lecanii Vl1. The use of such grains as low 

cost and rapid technique to produce mycopathogen compared to expensive and synthetic 

SMAY medium has been suggested earlier too (Vimaladevi, 1994, 1996). 

During the present study, wheat and navane grains proved inferior for spore 

production, perhaps due to less content of starch in wheat (53%) and thus led low 

productivity. However, wheat is also known to have appreciable amounts of protein. Higher 

nitrogen has been shown to be necessary for mycelial growth of fungi (Riba and Glander, 

1980; IM et al., 1988) and thus could be reason for lower spore yield are profused mycelial 

growth. Pilot studies using substrate fermentation will help to confirm these findings. 

Agro-wastes 

Accumulation of agro-wastes, post harvest of economically important plant part 

accounts for 300-500 million ton/year (Patel and Yadav, 1990). To utilize such agro-waste 

more efficiently, their utility in supporting the growth of Ma2 and Vl1 was assessed by using 

crushed maize cobs, wheat bran, rice bran, bagasse and pressmud singly and along with 10 

per cent molasses supplementation. Rice bran supported the maximum growth of both Ma2 

and Vl1 followed by wheat bran. When supplemented with 10 per cent molasses, the conidial 

yield increased two folds. On the other hand, growth and sporulation of both Ma2 and Vl1 was 

completely absent on bagasse and pressmud. Maize cobs also did not show any growth of 

Vl1.

In an earlier experiment, bagasse was shown to support multiplication (Silva and 

Loch, 1987) of N. rileyi. When 10 per cent molasses was supplemented to N. rileyi, the spore 

yield was almost on par with wheat bran. These results indicate that, addition of molasses 

could increase the conidial yield of entomopathogenic fungi when grown on different agro 

wastes. Refined experiment using other easily available nutrient sources like corn steap liquor 

will probably yield more information on the utility of different agrowastes for production of 

mycopathogen. 

5.6 TOXICITY OF PESTICIDES TO Metarhizium anisopliae (Ma2) 

AND Verticillium lecanii (Vl1) 

Compatibility with other agents for pest control or with naturally occurring mortality 

agents are important in developing strategies for the efficient utilization of entomopathogens 

in the integrated pest management. Deleterious effects or enhanced activity resulting from 

integrating of control agents will have a major impact on the role of potential of 

entomopathogens in IPM options. 

Among the three types of pesticides studied for their compatibility with M. anisopliae 

Ma2 and V. lecanii Vl1 in the present investigation, weedicides were least inhibitory followed 

by insecticides. But, the fungicides on the other hand were highly detrimental to the 

entomopathogenic fungi and higher toxicity of fungicides to the mycopathogens as compared 

to other agro chemicals have been presented in the earlier findings (Ignoffo et al., 1975; 

Kulkarni, 1999; Patil, 2000). 

Fungicides 

All the fungicides in vitro inhibited the conidial germination of M. anisopliae Ma2 and 

V. lecanii Vl1 considerably at low, medium, recommended and high dose (table 18 and 19 

respectively). Propiconazole and mancozeb being highly detrimental prevented the conidial 

germination of M. anisopliae Ma2. In addition to these two fungicides, chlorothalonil and 

carbendazim also inhibited the germination V. lecanii Vl1 totally triadimefon and chlorothalonil 

were comparatively safe allowing 35-39 per cent conidia of M. anisopliae to germinate on the 

media and only iprodione proved to be relatively safer with V. lecanii allowing 41.50 per cent 

conidia. 

Inhibition of the fungus growth by different fungicides even at the 1/10 th of the 

recommended dose was observed by Ignoffo et al. (1975). High toxicity of carbendazim and 

mancozeb to the fungus N. rileyi has been reported by Kulkarni (1999). According to 

Gopalkrishna and Mohan (2000), chlorothalonil and wettable sulphur were completely safe to 

the fungus, but the results of the present study, contrast their study with an observed per cent 

inhibition 100 and 96.57 per cent with M. anisopliae Ma2 and 64.01 and 74.84 per cent 

inhibition in case of V. lecanii Vl1 germination respectively. In general, N. rileyi has been 

adversely affected by many fungicides (Roberts and Campbell, 1977). Teddeur (1981) 

reported Triphenyltin hydroxids to be more toxic to M. anisopliae and B. bassiana. 

Triadimefon and Iprodione were relatively safer giving an indication that these 

fungicides can be compatible with the both (M. anisopliae Ma2 and V. lecanii Vl1) 

mycopathogen in field and it may be feasible to mix the fungicides with higher loads of 

inoculum as practiced with seed coating of seed with biofertilizer and pesticides. However, it 

has to be confirmed under field situation. 

Insecticides 

Combination of synthetic insecticides and microbial insecticides have been used to 

stress the insect population thus making them more susceptible to disease (Roberts and 

Campbell, 1977). The results of the present study reveal significant variation in the toxicity of 

insecticides representing different groups to M. anisopliae Ma2 and V. lecanii Vl1. The 

detrimental effects on the mycopathogen increased with concentration.

Dichlorvos and monocrotophos were highly detrimental to the mycopathogen M. 

anisopliae Ma2 inhibiting 54.22 and 54.89 per cent of growth respectively. In case of V. lecanii 

Vl1 malathion, quinolphos, dicofol and oxydimeton methyl were highly toxic. Malathion and 

dimethoate inhibited at lesser levels in case of M. anisopliae Ma2. Other insecticides including 

endosulfon and chlorpyriphos proved less toxic to V. lecanii Vl1. The observed difference 

could be due to inherent variability in chemical properties of the two insecticides. 

Inhibitory effects of insecticides to mycopathogens under both in vitro and in vivo 

have been reported. Inhibition of fungus N. rileyi by chlorpyrifos (Ignoffo, 1981) and 

endosulfan (Silva et al., 1993) has been documented even at the lowest concentrations. 

Similar detrimental effect of thiodicarb (Barbosa et al., 1997), carboryl (Kulkarni, 1999; 

Gopalkrishnan and Mohan, 2000) and fenvalerate (Gopalkrishnan and Mohan, 2000) to the 

entomopathogen has been reported. Additionally, profenfos (Silva et al., 1993) was found 

inhibit sporulation. 

Lesser inhibition of the mycopathogen in the present study by dimethoate supports 

the observations of Gopalkrishnan and Mohan (2000) who reported dimethoate at different 

concentration were safe to N. rileyi. 

These results indicate that care is to be advised when these entomopathogens are 

applied along with the pesticides. Separation of time of application between the chemical and 

the pathogen may be advocated. However, field studies may add more information. 

Weedicides 

Weedicides are often used to suppress the weeds to the increase the availability of 

nutrients to crop plants through reduction of competition and allelopathic effects. When 

compared to insecticides and fungicides, the weedicides in general inhibited germination of 

conidia in the range of 14 to 29 per cent. Since the weedicides are applied to soil, the effect 

on plants and the persistence of mycopathogens in the agril ecosystem need to be looked 

into in a proper perspective. Since the effect as noticed in this study is not that very drastic, 

the dilution of weedicides in soil would not cause reduction in the population of these 

entomopathogens. Hence, application of weedicides in the event of a mycopathogen spray 

can be considered largely as safe. In an earlier study some of the herbicides were considered 

to be inhibitory to N. rileyi (Ignoffo et al., 1975). But largely the effects of herbicides on 

conidial germination are non detrimental (Tang and Hou, 1998). Since weedicides have been 

designed to be highly specific to certain groups of plants, it might not largely effect the fungi. 

5.7 PERSISTENCE 

Natural epizootics under favorable conditions have been documented to naturally 

control the pests. However, their subsequent use in next season needs the presence of the 

propagules of the mycopathogenic fungi in the agro systems. In the event of a natural 

epizootic in one season or external application of entomopathogenic fungi to control an insect 

outbreak, there is a release of lot of these mycelia and conidia into the environment. The 

subsequent effect of these inocula in the event of further outbreak is determined largely by 

the persistence of conidia in the environment. Soil by large are the natural niche where they 

can persist. Phylloplane also could be a niche for growth and multiplication of these fungi. To 

understand the persistence of Ma2 and Vl1, cotton leaves as well as soil beneath was 

sprayed with inoculum of Ma2 and Vl1. 

In case of Vl1, there was a continuous decrease in the population till 30 th day, after 

which the organism did not persist. In soil, however there was increase in population up till 

20 th day which later dropped down to a very low level beyond 75 days. The low persistence of 

Vl1 could be due to the inhibitory effect of sunlight. It is known that the half life of spores of N. 

rileyi had reduced to 3.6 h mainly as a result of UV radiation (Fargues et al., 1983). Since the 

soil is not a very favorable condition for rapid multiplication, it is possible that there is highly 

reduced growth of the mycopathogenes. Such reduced persistence of conidia has been 

observed in soil earlier (Vimaladevi, 1995).

M. anisopliae exhibited rapid reduction in population and dropped down to very low 

levels (8.65% and 5.51% of the original population in case of phylloplane and soil 

respectively). Thus, the results indicate that soil and phylloplane cannot be expected to 

support the growth of mycopathogens and repeated application of these mycopathogens in 

the event of insect outbreak or as a prophylactic measure is necessary. 

Formulation 

Formulation basically comprises of additives to preserve organisms, deliver them to 

targets and once there, to improve their activity. The concentration of an organism that has 

been formulated is termed as formulation. All formulations may not be useful on all the crop 

pests. Hence, its necessary to modify them by certain additives which is done normally at the 

time of application. The final formulation is termed as tank mix (Burges and Jones, 1998). A 

wide variety of approaches for formulation is available ranging from production of liquid 

suspensions to solid briquettes. 

In this study, an experiment was performed to understand the survival of M. 

anisopliae Ma2 and V. lecanii Vl1 in different oil based and wettable powder formulations. The 

influence of different storage temperature and time of storage was also studied. 

At the end of 20 days after storage, no significant difference was obtained amongst 

different oil based and wettable powder at different storage temperatures. Even at the end of 

75 days, significant difference on the survival of conidia did not exist in different oil based and 

wettable powder formation in refrigerated condition. At the end of 45 days, formulation of 75 

per cent conidia + 25 per cent sunflower oil formulation and 75 per cent conidia + 25 per cent 

sorghum flour was found to be the best and significantly different from other formulations. In 

the earthen pot however, the survival of M. anisopliae was best in the formulation, 75 per cent 

conidia + 25 per cent sunflower oil. 

At 75 days after incubation, the survival of M. anisopliae was better at both room 

temperature and earthern pot in wettable powder formulation containing 75 per cent conidia + 

25 per cent sorghum flour, than other formulations. The result was similar at 90 day after 

incubation, but at 120 and 150 days of incubation, the survival of M. anisopliae Ma2 was 

better in 75 per cent conidia + 25 per cent sunflower oil in both room temperature and 

earthern pot storage. 

In case of V. lecanii Vl1, however, significant difference was not observed at the end 

of 20 th day both at refrigerated and earthern pot storage condition. The survival was highest in 

the formulation 75 per cent conidia plus 25 per cent sunflower oil both in refrigerated and 

earthern pot storage conditions. When a higher percentage of sunflower oil (50%) was used, 

the survival ability in case of V. lecanii Vl1 reduced. The highest rate of survival in case of V. 

lecanii Vl1 was found in formulations 75 per cent conidia + 25 per cent sunflower oil at all 

days of incubation and at all storage temperature. 

The results indicate that oil formulation containing 75 per cent conidia 25 per cent 

sunflower oil was the best formulation having highest survival ability of spores. It has been 

proved that vegetable oils are preferable for developing formulations to minimize toxicity 

especially if rancidity and solidification during storage (Couch and Ignoffo, 1981). It has also 

been expressed that formulation of entomofungal pathogen in oils increase their effectiveness 

(Prior et al., 1988) probably by preventing conidial dessication, increasing adhesion, 

(Vimaladevi and Prasad, 1996). Stathers et al. (1993) found that soyabean oil maintained 

good viability for 14 weeks. Using wettable powders has its own problems which include 

avoiding settling in tanks during sprays, or adhesion of binders. This problem can overcome if 

formulations are in oil. 

Mutagenesis 

Fungi have been found to be promising biological agents. In this study, the 

insecticidal activity of M. anisopliae and V. lecanii has been studied. Genetic improvements of 

entomopathogenic fungi has been less attempted, primarily due to the limited use and the 

complexicity of the genome. However, mutagenesis has been employed as a tool to improve

the metabolic activity (Paris and Ferron, 1971). UV rays that causes alkylation and has been 

found effective in mutagenesing fungi has been employed in this study. Randomly picked 

colonies of V. lecanii and M. anisopliae that came up on SMAY agar on plating the mutant 

bank were picked up and analysed for their insecticidal activity. Four UV mutants of V. lecanii 

(VA2, VA5, VA11 and VA12) were found to be more toxic to aphids B. brassicae. The mutants 

of M. anisopliae Ma2 including MH2, MH4, MH8 and MH12 showed higher pathogencity to H. 

armigera. The highest mortality was observed in case of VA2 (91.97%) and MH4 (91.67%) on 

B. brassicae and H. armigera respectively. The mutants VA2 and MH4 showed 11.3 per cent 

and 23.94 per cent higher mortality over the wild type respectively. 

The results obtained indicate the potential of chemical and UV mutagenesis in 

improving the toxicity of entomopathogens. Mutants of M. anisopliae and Paelomyces 

farinosus have been generated earlier which were significantly more virulent. It is possible 

that more vigorous mutagenesis and wider screening will enable identification of more potent 

strains. 

Evaluation of potency of V. lecanii Vl1 against selected crop pests 

The effect of V. lecanii Vl1 was initially tested on B. brassicae and A. disperses in 

laboratory using different dosages of conidia. The per cent mortality was observed after 

different days of inoculation. The per cent mortality (92.3%) progressively increased upto 10 

days after inoculation. At 10 days, the maximum mortality of B. brassicae was observed when 

1.2 x 10 9 conidia/ml was used. The per cent mortality (80.95%) of A. disperses during the 

same period was also the highest when 1.2 x 10 9 conidia/ml inoculum was used. However, in 

an earlier study (Masuda and Kikachi, 1992), it was found that higher concentration (10 7 to 

10 8 conidia/ml) was enough to cause 96-100 per cent mortality on A. gossypii by V. lecanii. 

The results indicate variability of pathogencity of V. lecanii on the two insect tested. V. 

lecanii was found to be more toxic to B. brassicae than A. disperses. The effect of V. lecanii 

has been reported earlier on Trialeurodes vaporariorum and B. tabaci which reported a per 

cent mortality of 91 and 100 using a inoculum of 3.2 x 10 6 . The effectiveness of the fungus, V. 

lecanii has been proved against Myzus persicae (Khalil et al., 1990; Milner and Lutton, 1986). 

Having known the potential of V. lecanii to be toxic to B. brassiae, A. disperses in 

laboratory condition, a field experiment was laid out in order to assess the performance of the 

entomopathogen on B. brassicae and Apis crassivora. The mortality in the field was however, 

lower when the same dosage was used 10 days after inoculation. 

Among the three isolates, Vl1 application showed higher rate of mortality (61.16%) of 

B. brassicae followed by Vl3 (50.17%). Overall, the efficacy of fungus was more effective at 

higher concentration and high exposure period than lower concentration and low exposure 

period. 

The reduction in toxicity in the field could be due to several reasons including the 

harmful effects of UV rays on the fungal pathogen itself (Ignoffo et al., 1976), less accessibility 

of the pathogen to the insect pest and dispersal of the conidia due to wind. Hence, sprays 

with higher initial population and additional sprays are required to augment the biocontrol 

potential in field. The lower effectiveness could be also due to the low humidity prevented 

during the period of experiment. 

All the five dosages of V. lecanii proved their supremacy to uninoculated treatments. 

Comparatively, the highest dosage (2 x 10 12 conidia/ha) caused 61.6 per cent mortality of B. 

brassicae after second spray which was lesser than the mortality caused by the 

recommended spray of dimethoate 30 EC (@ 1.7 ml/l) which resulted in 87.53 per cent. 

However, the results indicated that the fungus Vl1 at higher dosage (2 x 10 12 conidia/ha) did 

not lag much. 

All the dosages of V. lecanii isolates against A. crassivora proved their superiority 

over untreated treatment. Among the three isolates, Vl3, caused highest mortality of 66.50 per 

cent after second spray of higher dosage (2 x 10 12 conidia/ha). Dimethoate maintained their 

supremacy over the mycopathogen throughout the experimental period recording significantly

higher mortality of aphids. The commercial formulation Vertalec was found to control whitefly 

and aphid effectively at 10-13 h at ≥97 per cent relative humidity in Japan (Hall, 1982). 

The lower mortality of fungus than chemicals may be due to the fact that chemical 

insecticides were quick in action and hence reduced the whitefly population considerably. The 

entomopathogen which acted slowly on the pest allowed them to live on the plant, until the 

incubation period of the disease was completed (Miranpuri and Khachatourians, 1995). 

FUTURE LINE OF WORK 

1. Extensive exploration of diverse areas and environmental niches need to be done. 

2. Mutagenesis of the isolates need to be done on more vigorous note and larger 

screening need to be done. 

3. Refinement of formulation and development of application technology to enhance the 

effectiveness of the fungus under field conditions. 

4. Genetic/molecular diversity of the isolate need to be done in order to develop an 

inoculum consortia containing diverse strains. 

5. The molecular basis of toxicity needs to analyse to attempt further exploitation of 

these mycopathogens.

VI. SUMMARY 

Investigations were carried out on isolation and characterisation of native 

entomopathogenic fungi and their effectiveness from 1999 to 2002 at the University of 

Agricultural Sciences, Dharwad. The results obtained are summarized herein. 

1. Nine insect mycopathogens, belonging to seven genera were found naturally occurring in 

the nine districts of northern Karnataka. Of these, Nomuraea rileyi was most 

predominantly found followed by Metarhizium anisopliae and Verticillum lecanii. 

2. Studies on morphological variation among the field collected isolates of M. anisopliae and 

V. lecanii for various parameters like, hyphal and conidial characters, sporulation and 

spore yield revealed that Ma2 and Ma1 proved superior to Ma3 and Ma4. In case of V. 

lecanii, V13 showed higher spore yield than Vl1 and Vl2. However, it took maximum time 

(10-14 days) to cover the entire diet. 

3. Pathogenicity of V. lecanii to aphids, whitefly and mites and M. anisopliae on American 

bollworms and Rhinocerous beetle was proved in laboratory. 

4. Among the different isolates of V. lecanii and M. anisopliae evaluated against B. 

brassicae and H. armigera, Vl1 and Ma2 recorded maximum mortality of 94.50 per cent 

and 87.50 per cent respectively. 

5. Analysis of the effect of different carbon sources on the biomass of Vl1 and Ma2 revealed 

that starch was the superior sugar source for both the mycopathogens and (NH4)SO4 and 

KNO3 as nitrogen source for Vl1 and Ma2 respectively. 

6. Evaluation of food grains for suitability for mass production of Ma2 and Vl1 revealed that 

conidial growth increased with increase in duration after inoculation. Bajra and rice grains 

served as most productive media for conidial growth of Ma2 and Vl1 with an yield of 22.77 

x 10 8 and 24.59 x 10 8 conidia per gram of media, respectively. 

7. Among the low cost agro-wastes tested for their suitability for the mass production of 

mycopathogens, rice bran with 10 per cent molasses supported better growth and 

conidial production of Ma2 and Vl2. 

8. All the fungicides tested for their interaction with Ma2 inhibited the conidial germination 

(19.90 to 100%). However, iprodione and chlorothalonil were safer with 38.69 per cent 

69.21 per cent conidial germination. 

9. In case of Vl1, complete inhibition (24.10 to 100%) of conidia was observed. However 

iprodione and triadimeton allowed maximum of 37.38 and 41.62 per cent conidia to 

germinate respectively. 

10. Among insecticides, dichlorvos (55.89%) and malathion (69.18%) were significantly 

detrimental to Ma2 and Vl1 respectively. Conversely, malathion (34.33%) and 

endosulfon (37.31%) were safer for Ma2 and Vl1 respectively. 

11. All weedicides tested for their interactions with Ma2 (9.01 to 41.95%) and Vl1 (8.65 to 

36.12%) in general, inhibited the germination of the fungal conidia. In general, fungicides 

were most detrimental to the mycopathogen than insecticides and weedicides. 

12. The persistence of mycopathogens (Ma2 and Vl1) was higher in soil (upto 16 months) 

than in phylloplane (upto 3 months). 

13. In general, all the carrier material had more conidial viability when stored in refrigerated 

and deep freezer condition and a reduction of more than 10 per cent and 8 per cent in 

refrigerated and deep freezer was observed after 150 DAS respectively.

14. Oil formulations proved better than wettable powder. Among oil formulations, sunflower 

oil proved to be best with maximum of 38 and 54.13 per cent germination at room 

temperature after 150 days of storage. 

15. Among the different concentrations of V. lecanii evaluated in laboratory against B. 

brassicae and Aleurodicus disperses, higher dosage of V. lecanii (1.2 x 10 9 conidia/ha) 

was found effective with highest mortality of 92.30 per cent and 80.93 per cent on the 

10 th day after spraying respectively. 

16. A comparative account of efficacy of different isolates of V. lecanii under field conditions 

were evaluated against B. brassicae and A. crassivora. At the highest dosage of all 

isolates (2 x 10 12 conidia/ha), Vl1 proved most potent against B. brassicae (61.16% 

mortality), whereas Vl3 showed better control against A. crassivora (66.50%). 

17. The UV radiation mutants of mycopathogen (Ma2 and Vl1) exhibited 91.67 and 91.67 

per cent higher mortality on H. armigera and B. brassicae than the respective wild type 

(73.96 and 82.62%).

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APPENDIX I 

Mean monthly weather data at Main Agricultural Research Station, University of Agricultural Sciences, Dharwad 

Jan. Feb. Mar. Apr. May June July Aug. Sep. Oct. Nov. Dec. Annual 

1999 

Maximum (°C) 29.4 32.8 36.2 36.6 32.2 28.0 26.4 27.1 28.3 28.9 29.6 28.9 30.4 

Minimum (°C) 12.4 16.9 20.2 21.1 21.3 21.0 20.8 20.4 20.0 19.8 16.0 13.4 18.6 

RH (%) 75 63 63 65 75 85 89 85 83 79 59 51 73 

RF (mm) 0.0 0.0 0.0 14.7 32.8 71.8 113.9 19.7 8.8 161.1 0.0 0.0 422.8 

No. of rainy days - - - 1 1 7 8 2 1 10 - - 30 

2000 

Maximum (°C) 30.6 32.2 35.2 37.3 33.9 29.0 26.8 27.2 29.0 29.5 30.4 28.3 30.8 

Minimum (°C) 15.1 15.7 18.5 21.5 21.1 21.3 20.4 20.2 20.4 20.2 16.9 13.4 18.7 

RH (%) 48 52 47 57 67 79 81 81 76 77 63 58 65 

RF (mm) 0.0 0.0 0.0 44.1 45.4 50.7 125.2 50.4 32.1 88.3 0.0 3.0 539.2 

No. of rainy days - - - 1 4 5 6 4 9 7 - 1 37 

2001 

Maximum (°C) 29.9 34.0 35.3 35.7 34.8 30.3 26.8 27.2 30.1 30.1 31.0 29.6 31.2 

Minimum (°C) 15.0 16.8 18.5 22.0 21.5 21.3 21.1 20.9 20.2 19.9 17.9 13.7 19.1 

RH (%) 55 50 45 55 59 75 81 81 72 66 55 55 62 

RF (mm) 0.0 0.0 0.0 52.1 23.2 32.5 33.1 58.1 53.6 17.0 0.0 Tr 269.6 

No. of rainy days - - - 4 2 5 5 5 5 3 - - 29 

2002 

Maximum (°C) 30.1 31.9 36.0 37.2 34.9 29.6 28.4 26.6 29.8 30.7 30.5 30.2 31.3 

Minimum (°C) 14.5 17.9 20.0 21.7 23.0 21.9 21.3 20.5 20.0 20.4 17.1 14.4 19.4 

RH (%) 55 50 49 53 62 80 79 82 73 67 62 47 63 

RF (mm) 0.0 62.7 0.0 67.0 57.9 60.5 15.0 48.1 6.6 103.6 7.0 0.0 428.4 

No. of rainy days - 1 - 5 4 4 0 7 1 6 1 - 29

APPENDIX II 

Survey proforma for Entomopathogenic fungi 

Date: ________________ Sample 

No.:__________________ 

I. 1. Season 

2. Place 

3. Taluka 

4. District 

5. Region/zone 

6. Ecosystem 

II. 1. Crop 

2. Cropping system 

3. Soil type 

4. Soil temperature (°C) 

5. Air temperature (°C) 

6. Relative humidity (%) 

III. Host/Pest No. of observed No. infected 

1. 

2. 

3. 

4. 

5. 

6. 

Methodology 

1. Field crops : 10 m² area/plot and 3 such plots will be observed at each location 

2. Plantations: 10 plants at random will be observed 

Infected cadavers will be collected in the vials giving sample number 

Date: Signature 

IDENTIFICATION 

Identified as: ____________________________________________________________ 

By : ____________________________________________________________ 

Date: Signature

Maharashtra Association for the Cultivation of Science 

AGHAKAR RESEARCH INSTITUTE 

(An Autonomous Grant-in-aid Institute under 

the Department of Science and Technology, Govt. of India) 

No. 3/59/2003/Adm./ 1794 

Dr. (mrs.) Alaka Pande 

Scientist E2 & In-charge 

Mycology & Plant Pathology, 

Division of Plant Sciences 

Sub: Identification repost on three fungal cultures 

Ref: Your letter No. ENT/DBT/131/02-03dt. 05/02/2003 

Dear r. Patil, 

February 19, 2003 

I am pleased o inform, you that we have completed the identification 

work of the above said material and such a brief repost of the same is being 

enclosed herewith for your kind perusal and further use. We are thankful to you 

for the payment of Rs. 210/- as identification charges for which our accounts 

section has passed on a receipt bearing No. 31992 dt. 10/02/2003. 

Encl.: a/a 

Thanking you, 

Dr. R. K. PATIL 

Entomologist (Oilseeds) 

Department of Agricultural Entomology 

University of Agricultural Sciences 

DHARWAD 580 005 

Yours faithfully 

(Alaka Pande)

Identification Repost of the fungal cultures supplied by Dr. R.K. Patil, Dept. of Agril. 

Entomology, UAS, Dharwad-580 005 dt. 15.2.01

ISOLATION AND CHARACTERIZATION OF 

ENTOMOPATHOGENIC FUNGI AND 

THEIR EFFECTIVENESS 

BHARATHI H. TALWAR 2005 Dr. J. H. KULKARNI 

MAJOR ADVISOR 

ABSTRACT 

An attempt was made to isolate and characterize native entomopathogenic fungi. 

Nine insect mycopathogens, belonging to seven genera were found naturally occurring in the 

nine districts of Northern Karnataka. Morphological variation among the field collected isolates 

of M. anisopliae (Ma2) and V. lecanii (Vl1) for various parameters like, hyphal and conidial 

characters, sporulation and spore yield were recorded. Pathogenicity of V. lecanii to aphids, 

whitefly and mites and that of M. anisopliae to American bollworms and rhinocerous beetle 

was proved in laboratory. Among different carbon sources tested, starch proved superior for 

both the mycopathogens NH4 (SO4) and KNO3 were superior as nitrogen source. Bajra and 

rice grains served as most productive media for conidial growth of Ma2 and Vl1 with an yield 

of 22.77x10 8 and 24.59x10 8 conidia per g of media, respectively. Among pesticides tested, 

fungicides showed maximum inhibition followed by insecticides and weedicides. The 

persistence of mycopathogens was higher in soil (upto 16 months) than in phylloplane (upto 3 

months). In general, all the carrier material had more conidial viability when stored in 

refrigerated and deep freezer condition and a reduction of more than 10 per cent and 8 per 

cent in refrigerated and deep fereezer was observed after 150 DAS respectively. Oil 

formulations proved better than wettable powder. 

Among the different concentrations of V. lecanii evaluated in laboratory against B. 

brassicae and Aleurodicus disperses, higher dosage of V. lecanii (1.2x10 9 conidia/ha) was 

found effective with highest mortality of 92.30 per cent and 80.93 per cent on the 10 th day 

after spraying respectively. A comparative account of efficacy of different isolates of V. lecanii 

under field conditions were evaluated against B. brassicae and A. crassivora. At the highest 

dosage of all isolates (2x10 12 conidia/ha), Vl1 proved most potent against B. brassicae 

(61.16%mortality), whereas Vl3 showed better control against A. crassivora (66.50%).

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