Agronomic Investigation of New Microbial Isolates as Bio fertilizers ...

Greetings to All on Behalf
of My Mother Land India
Agronomic Investigation of New Microbial
Isolates as Biofertilizers in Sweet Potato
(Ipomoea batatas L. Lam) Grown in an Ultisol
of India
K. Susan John, Neetha Soma John and I.P. Anjana Devi
Central Tuber Crops Research Institute (CTCRI)
(Indian Council of Agricultural Research), Thiruvananthapuram,
Kerala, India, email: susanctcri@gmail.com

ALL INDIA NET WORK ON TUBER CROPS
Started during
1968
4
14
3
15
16
1
2
5
6 7
8
17
9
11
10
12
13
1. TNAU, Coimbatore
2. ANGRAU, Hyderabad
3. KKV, Dapoli
4. NAU, Navasari
5. IGKV, Jagadalpur
6. NDUA&T, Faizabad
7. RAU, Dholi
8. BAU, Ranchi
9. BCKV, Kalyani
10. AAU, Jorhat
11. ICAR RC NEH, Shillong
12. CARI, Port Blair
13. CAU, Imphal
14. MPUAT, Udaipur
15. UAS, Dharwad
16. CTCRI, HQ
17. CTCRI, RC

The sole institute in the world exclusively dedicated to research h on
Tropical Tuber Crops
Head quarters Thiruvananthapuram,
Kerala
Regional centre Bhubaneswar,
Orissa
Research carried out under 5 Divisions
Division of Crop Improvement Division of Crop Production
Division of Crop Protection
Section of Social Sciences
Division of Crop Utilization

Cassava( Manihot esculenta Crantz)
Sweet potato( Ipomoea batatas Lam L.)
Elephant foot yam(Amorphophallus
paeonifolius)
Yams(Dioscorea sp.)

Taro(Colocasia sp.)
(Coleus rotundifolius)
Arrowroot(Maranta
arundinaceae)
Tannia (Xanthosoma sagittifolium)

MANDATE
‣ To undertake basic, strategic and applied research for
generating technologies to enhance productivity and
utilisation potential of tuber crops viz., cassava, sweet
potato, yams, aroids (EFY, taro, tannia), coleus and yam
bean (other than potato)
‣ To act as a national repository of scientific information on
tuber crops
‣ To coordinate network research with State Agricultural
Universities for generating location specific technologies
‣ To act as a centre of human resources development for
various clientele systems involved in tuber crops research
and development
‣ To undertake transfer of tuber crops technologies through
consultancy, outreach programmes and linkage with
developmental agencies

"Greater emphasis on tuberous crops such as potato,
tapioca and sweet potato to make them available at
cheaper rates"
Dr. A P J Abdul Kalam
(Former president of India)

ALL INDIA NET WORK ON TUBER CROPS
Started during
1968
4
14
3
15
16
1
2
5
6 7
8
17
9
11
10
12
13
1. TNAU, Coimbatore
2. ANGRAU, Hyderabad
3. KKV, Dapoli
4. NAU, Navasari
5. IGKV, Jagadalpur
6. NDUA&T, Faizabad
7. RAU, Dholi
8. BAU, Ranchi
9. BCKV, Kalyani
10. AAU, Jorhat
11. ICAR RC NEH, Shillong
12. CARI, Port Blair
13. CAU, Imphal
14. MPUAT, Udaipur
15. UAS, Dharwad
16. CTCRI, HQ
17. CTCRI, RC

Introduction
Sweet potato is grown in the tropics and warm temperate regions of the
world
Globally, grown in developing countries in an area of 9 mha having a
production of 124 mt with a productivity of 13.7 t ha -1 (FAOSTAT, 2006)
China ranks first in area (4.7 m ha) and production (70 m t) with a
productivity of 14 t ha -1 (FAOSTAT, 2001)
Third most important tuber crop in India after potato and cassava
India occupies 12 th , 8 th and 5 th rank globally in terms of area, production and
productivity with an area of 0.14 mha, production 1.21 mt and productivity
8.87 t ha -1 (CMIE,2006)
In India, grown in Orissa, West Bengal, Uttar Pradesh, Bihar and Jharkand
accounting 77% of area and 82% of production
In Kerala, it is grown in an area of 505 ha with a production of 6405 t and
productivity is 12.68 t ha -1 ( Farm guide,2009)

SWEET POTATO GROWING BELT OF INDIA

Facts about sweet potato
No. of varieties released from CTCRI-27
Propagation through vine cuttings
Method of planting-Mounds, ridges, furrows and flat beds
Nutrient management-NPK@50:25:50 kg ha -1 + FYM @5 t ha -1
Major pest- Sweet potato weevil- Mass trapping of adult weevils using sex
pheromone
Post harvest utilization- Roots and leaves as human food and roots and
vines as animal feed
Processed into industrial starch, alcohol, noodles and other products viz.,
jam, jelly, pickles, squashes etc.,
Has an average protein content comparable to that of rice (1.3-10.0% on
DWB) (Purcell et al., 1972)
It is also a good source of Ca, ascorbic acid, and ß- carotene.

Some sweet potato varieties released from CTCRI
Sree Arun
Orange- fleshed sweet potato
Orange fleshed sweet potato contains β carotene and anthocyanin which are cheap
source of vitamin A and antioxidants
Orange fleshed sweet potato can combat vitamin A deficiency in developing
countries ( Harvest plus Programme)
ST-14
Sree Kanaka
β carotene 10.50 mg/100g
β carotene 8 mg/100g

Significance of the present study
N fixers, P solubilizers and K mobilizers are the most beneficial soil
microorganisms for use as biofertilizers in agriculture
The main objective of using biofertilizers is to reduce fertilizer
quantity there by reduce the cost of production and an eco-friendly
practice
Exploitation of agro-biodiversity for identifying useful
microorganisms for nutrient management as well as biocontrol is a
thrust area in the present day agriculture to substitute for chemical
fertilizers and pesticides and to maintain soil health
Objective
To screen, isolate, identify and characterize potent N fixers and P
solubilizers from the biodiversity hot spots of Western ghats of
Kerala and to agronomically evaluate their efficacy as a substitute to
chemical fertilizers in sweet potato to enhance growth and yield

Methodology
a. Microbiological work
Survey and collection of soil samples - High biodiversity hot spot
areas of South India
Microbial ( bacteria, fungi & actinomycetes) enumeration - Serial
dilution and plate counting
From the bacterial population, screening for P solubilizers -
Pikovskaya’s agar media
N fixers - Jensen’s nitrogen free solid medium
P solubilizing capacity- Vanado molybdo phosphoric yellow colour
method
N fixing capacity - Kjeldhal method
b. Preparation of biofertilizer
Mass multiplication of potent isolates
100 ml of the broth containing the isolates mixed with sand and
charred rice husk (1:4) aseptically

Sampling locations –AGASTHYAMALAI RANGES
Kulathupuzha RF
Thenmala RF
Peppara RF
Aryan Kau RF
Kollam
Kottur RF
Kottur Extension
Trivandrum
Palode RF
Kalakkad RF
Lower Kothayar RF
Kothayar RF
Pechiparai RF
Neyyar RF
Ponmudi RF
Nagarcoil

c. Molecular characterization of the biofertilizer microbes
Isolation of the genomic DNA (Sambrook et al. 1989)
Amplification of 16s rDNA - Forward 8F primer
5'AGAGTTTGATCCTGGCTCAG3' and reverse 1492R primer
5'CGGCTACCTTGTTACGACTT3’ (Babu et al. 2004)
Agarose Gel Electrophoresis (AGE) with 100 bp marker (NE
Biolabs)
The band cut, eluted and purified using the QIA quick gel
extraction kit, QIAGEN
Sequencing of the eluted product - Genei, Bangalore
Sequence analysis - National Centre of Biotechnology
Information (NCBI) database -Basic Local Alignment Search Tool
(BLAST)

• No. of treatments - 9 Replication - 3 Design – CRD
Initial status
• Organic carbon-1.36%, Available P-184 kg ha -1 , Exchangeable K-159 kg ha -1
• Quantity of N- 78%, Available P-0, Exchangeable K-83% of POP rate
(Aiyer,Nair,1985)
Treatment details of the pot experiment
Treat
d. Agronomic investigation of the bio fertilizer efficacy
1. P solubilizer
a. Controlled condition experiment in pots
Treatments
T1 Soil test based fertilizer (STBF) recommendation (NPK@ 39:0:41.5 kg ha ‐1 )
T2 Package of Practice (POP) recommendation for sweet potato (NPK @ 50:25:50 kg ha ‐1 )
T3 Phosphate solubilizing bacteria alone
T4 STBF + biofertilizer (PSB) (NPK@ 39:0:41.5 kg ha ‐1 )
T5 NK + ¼P + biofertilizer (NK @ 50:50 kgha ‐1 + P@ ¼of 25 kgha ‐1 )
T6 NK + ½P + biofertilizer (NK @ 50:50 kgha ‐1 +P@ ½of 25 kgha ‐1 )
T7 NK + ¾P +biofertilizers (NK@ 50:50 kgha ‐1 + P@ ¾of 25 kgha ‐1 )
T8 NK @ 50:50 kgha ‐1 + biofertilizer
T9 Absolute control

A View of the Pot Experiment

. Field experiment
Design-RBD Treatments -12 Replication- 2 No. of seasons- 2
Treatment details
T1-STBF T2 - POP T3 - PSB -1 T4 - PSB-2
T5 - POP+PSB-1 T6 - POP+PSB-2 T7- 25 %P+ PSB-1
T8- 25 %P+ PSB-2 T9 - 50 % P + PSB-1 T10 - 50% P + PSB-2
T11 - 75% P+PSB-1 T12 - 75% P+PSB-2
N & K @50:50 kg ha -1

A View of the Field Experiment

d. Agronomic investigation of the biofertilizer efficacy
1. N fixer
a. Controlled condition experiments in pots
• No. of treatments - 8 Replication - 3 Design - CRD
Initial status
• Organic carbon- 0.26%, Av. P- 216.16 kg ha -1 , Exch. K- 303.52 kg ha -1
• Quantity of N-117%, P- 0, K- 48% of POP rate ( Aiyer and Nair,1985)
Treat. No.
Treatments
T1 Soil test based ferlilizer (STBF) recommendation (NPK @ 58.5:0:24 kg ha -1 )
T2 POP recommendation for sweet potato (NPK@ 50:25:50 kg ha -1 )
T3 N fixer alone
T4 N fixer + STBF (NPK@58.5:0:24 kg ha -1 )
T5 N fixer + N(3/4 of STBF), P, K (STBF) i.e. (NPK @44:0:24 kg ha -1 )
T6 N fixer + N(1/2 of STBF), P, K(STBF) i.e. (NPK @29.25:0:24 kg ha -1 )
T7 N fixer + N(1/4 of STBF), P, K(STBF) i.e. (NPK @14.6:0:24 kg ha -1 )
T8 Absolute control

Results
No. of microbes isolated- 505
No. of bacteria - 341
No. of P solubilizers - 169
No. of N fixers - 194
P solubilization efficacy of the 2 potent isolates
PSB-1- 150 µg g -1 - Enterobacter sp.
PSB-2- 112.5 µg g -1 - Pantoea agglomerans
N fixing capacity of the N fixer bacteria – 4%
- Alcaligenes feacalis

Influence of P solubilizers on tuber yield of sweet potato (pot
trial)

Zonation of P solubilizers in
Pikovskayas agar medium
16s rDNA amplified product

A View of the Crop Harvest
( P solubilizer field experiment)

Treatment
Effect of P solubilizers on growth and yield of sweet potato
( Field experiment) (Mean of 2 seasons)
Tuber
yield
( t ha -1 )
Vine
yield
(t ha -1 )
Root:
shoot
ratio
Harvest
index
Soil P
(kg ha -1 )
Tuber
P (%)
Vine
P
(%)
P
solublize
rs
( x 10 3
cfu g -1
soil)
STBF 14.34 15.42 1.57 0.610 32.09 0.234 0.547 256
POP 17.59 17.13 2.01 0.665 17.67 0.264 0.553 251
PSB -1 10.71 7.50 1.96 0.630 12.15 0.262 0.484 220
PSB-2 12.30 16.00 1.47 0.595 14.21 0.278 0.382 210
POP+PSB-1 24.92 26.25 1.48 0.595 18.22 0.428 0.551 231
POP+PSB-2 9.46 14.67 1.77 0.635 9.76 0.234 0.605 175
25 %P+ PSB-1 19.88 28.08 0.95 0.480 20.09 0.219 0.484 119
25 %P+ PSB-2 16.46 7.52 3.47 0.775 8.68 0.334 0.531 194
50 % P + PSB-1 16.42 20.38 1.42 0.585 15.72 0.253 0.568 215
50% P + PSB-2 16.00 20.54 1.60 0.610 30.57 0.352 0.711 164
75% P+PSB-1 14.29 14.38 1.49 0.595 27.11 0.184 0.395 155
75% P+PSB-2 9.75 11.17 1.01 0.495 11.82 0.267 0.309 188
CD 7.097 3.339 1.016 NS 13.06 NS NS NS

Molecular characterization of N fixer

Influence of N fixer on sweet potato growth
Figure 1 Influence of N fixing bacteria on growth characters of sweet potato
200
180
160
Growth characters
140
120
100
80
60
40
20
0
T1 T2 T3 T4 T5 T6 T7 T8
Treatments
Vine length(cm) Leaf number Leaf length(cm) Leaf breadth(cm)

Effect of N fixers on tuber yield of sweet potato

Conclusions
For sweet potato, among the two P solubilizers, Enterobacter sp. was more
effective
By applying the P solubilizers, P dose can be reduced to 75% of POP
By applying N fixer, N dose can be reduced to 25% of POP
Explored the beneficial effect of new microbial isolates of N fixers and P
solubilizers as a substitute to chemical fertilizers
Reducing cost of production, improving P status of the soil
Future Thrust areas
Formulation of microbial consortium containing bio fertilizer cum bio
control agents
Agronomic investigation of the bio consortium for nutrient as well as
disease management for tropical tuber crops
Popularization and large scale production of the bio consortium as a
component of INM strategy for major crops

Acknowledgements
This paper forms a part of ICAR net work project on
‘ AMAAS ( Application of Microorganisms in Agriculture and
Allied Sectos’)
Funding from Indian Council of Agricultural Research
NBAIM ( National Beurea of Agriculturally Important
Microorganisms) for the national level coordination of the
project
Director, CTCRI, DG & DDG, ICAR for granting permission
ESA for providing an opportunity for oral presentation and
for free registration and accommodation for attending the
congress
Department of Biotechnology, Govt. of India for
international travel grant support

Thank you and Welcome to
God’s s Own Country