Mosambi
3.11 MICROBIOLOGY
3.11.2 Exploitation of Microorganisms for Crop Production
3.11.2.1 Identification of efficient microorganisms for mass production of biofertilizer and their protocol development
Screening of released varieties of soybean for nodulation with native rhizobia under rainfed conditions.
The nodulation status of the eight varieties of soybean (Pusa 16, Pusa 20, Pusa 22, Pusa 24, Pusa 37, Pusa 40, DS 9712
Effect of organic amendments on grain yield and soil properties under rice cultivation
Treatment pH EC Organic Available Yield
(mS cm-1) C (%) N (%) (kg/4.77 m2)
Control 8.6 0.300 0.7 0.007 -
N120 P60 8.6 0.225 0.7 0.012 0.820
(Full recommended dose)
Compost @ 6 t/ha 8.6 0.225 1.0 0.012 0.800
Poultry manure 8.3 0.225 1.1 0.010 1.270
3 t/ha
N60 P30+compost 8.7 0.250 1.2 0.008 1.470 3 t/ha
N60 P30+poultry 8.5 0.250 0.7 0.010 0.800 manure 1.5 t/ha
CD 5% NS NS 0.1 0.002 0.82
Integrated use of micronutrients and B. japonicum inoculation for nodulation and yield of soybean. A field experiment was carried out to assess the effect of integrated use of micronutrients (B, Zn andMo) and inoculation with Bradyrhizobium on nodulation and yield of soybean (DS 9814). The observations on number of nodules, dry weight of nodule and shoot, N concentration in dry matter at 50%
flowering stage, and grain yield were recorded at harvest.
Response in nodule dry weight was significant but with other parameters it was marginal. On an average, the number of nodules ranged from 16.8/plant (Zn alone) to 37.3 (B.
japonicum alone). The number of nodules was the highest with control (~45.8/plant). The dry weights of nodules ranged from 39 to 98 mg/plant. Nodule mass was the highest in control (98 mg/plant), followed by B. japonicum inoculation amended with borax @ 5 kg boron /ha. Maximum dry weight of roots was observed with control and B. japonicum inoculation with Zn amendment. Even though increase in grain yield was marginal, B. japonicum inoculation with boron amendment gave a maximum yield of 1850 kg/ha which was 52% higher than that of absolute control, and 59 % higher than that of B.
japonicum alone.
Evaluation of soybean germplasm lines for nodulation by native rhizobia. Thirty germplasm lines of soybean were and DS 9814) released by the breeders under All India
Coordinated Research Programme was examined. Seeds were sown on ridges, and 3 ridges per plot (4.0 m x 2.75 m) were maintained. The nodulation was observed at 50% flowering of each variety and the yield was taken at harvest.
On an average, 33-41 nodules per plant were observed. The number of nodules per plant was maximum with var. Pusa 20, while the dry weight of shoot was maximum with var. Pusa 37 (4.3 g/plant). The grain yield ranged from 767 to 1783 kg/ha. The maximum grain yield was observed with DS 9712 (1783 kg/ha) followed by DS 9814 (1493 kg/ha).
Screening of released varieties for nodulation with native homologous rhizobia
Variety Nodules/plant Dry weight Yield (kg/ha) of shoot/plant (g)
Pusa 16 32.6 3.8 1183
Pusa 20 40.7 3.8 1200
Pusa 22 34.4 4.03 867
Pusa 24 37.0 3.4 950
Pusa 37 32.7 4.3 767
Pusa 40 40.2 3.9 867
DS 9712 37.0 2.95 1783
DS 9814 33.0 3.6 1493
Isolations of rhizobia were made from 20 individual nodules from six plants of each variety. A total of 46 rhizobial isolates (8, Pusa 16; 12, Pusa 20; 6, Pusa 22 and 20, Pusa 40) were purified and examined for colony morphology and growth on CRYEMA medium. Most of these isolates were found slow growers, with round and smooth colony morphologies. All isolates were found ketolactose negative.
The PCR amplification of genes indicated the presence of RS α molecular marker fragment in 20 out of 25 isolates of rhizobia. This conserved sequence of ~900 bp was found in all soybean root nodulating bradyrhizobia.
Nodulation and yield of soybean with B. japonicum inoculation amended with micronutrients
Treatment Nodules/ Nodule Root Shoot Yield plant dry weight/ dry dry (kg/ha)
plant (mg) weight/ weight/
plant (g) plant (g)
Control 45.8 98 5.0 12.12 1216
B. japonicum 37.3 74 1.12 10.99 1160
Local check 20.3 39 0.58 6.12 1383
B. japonicum + B 27.9 81 0.81 7.2 1850
@ 5 kg/ha
B. japonicum + Zn 22.1 60 5.0 7.58 1350
@ 5 kg /ha
B. japonicum + Mo 34.6 67 1.12 11.52 1367
@ 4 g/kg
Boron @ 5 kg/ha 28.9 64 1.1 11.33 1250
Zinc @ 5kg/ha 16.8 40 0.94 10.05 1150
Molybdenum 36.1 70 0.74 7.81 1350
@ 4g/kg seed as seed treatment
B. japonicum + 7 + 24.7 55 0.96 9.3 1217
8 + 9
CD (0.05) NS 16 NS NS NS
PCR amplification of RSα fragment in slow growing root nodulating isolates of soybean varieties. Lanes 1-4: Pusa 16; lanes 5-8: Pusa 20;
lanes 9-12: Pusa 22; and lanes 13-18: Pusa 40
screened for nodule number, dry weights of nodule and shoot.
Nodule number ranged from 3.0 to 35.6/plant, roots weight from 0.73 to 1.67 g/plant, and shoot dry weight from 6.82 to 14.06 g/plant. The germplasm line GP 06-22 gave the highest nodules per plant (38.0), while the line GP 06-10 gave the highest root weight (1.67 g/plant) and shoot dry weight (14.06 g/plant).
Selection of Azotobacter strains for enhancement in seed germination. The influence of A. chroococcum strains (JL 17, JL18, JL 104, A 41, JMS 100b) capable of utilizing complex carbon sources was assessed on seed germination, length of radicle and weight of seedling of wheat.
Interestingly, response in seed germination of wheat was considerable with A 41 and JMS 100b. Maximum increase in the fresh weight of seedlings with JL 17 was followed by JMS 100b, JL 18, and JL 104. Response to inoculation in radical length was significant with JL 18 and JMS 100b.
Response in wheat seed germination with Azotobacter inoculation A. chroococcum Germination Weight of Length of
strains (%) seedlings(g) radicle(cm)
Uninoculated control 71(22.24) 1.81(1.544) 1.40(1.378)
JL 17 75(22.75) 1.93(1.560) 1.52(1.399)
JL 18 80(23.57) 1.89(1.546) 1.66(1.476)
JL 104 78(23.22) 1.87(1.539) 1.47(1.401)
A 41 86(24.60) 1.84(1.531) 1.43(1.389)
JMS 100b 85(23.34) 1.90(1.552) 1.60(1.459)
S. Em.(±) (0.604) (0.006) (0.028)
CD (0.05) (2.39) (0.028) (0.124)
Figures in the parentheses are angular transformations of the original value
Screening of rifampicin resistant mutants of A. chroococcum strains for IAA production and nitrogen fixation
A.chroococcum IAA (µg/mg protein) ARA (n moles/mg protein/hr) strain JL 17 JL 104 RJ 1 JL 17 JL 104 RJ 1
Wild type 1.30 1.51 2.66 21.29 54.75 28.48
M 1 3.68 4.37 10.81 3.63 0.71 6.27
M 2 0.85 0.93 12.09 0.96 1.39 30.91
M 3 3.25 2.59 3.36 19.31 1.96 39.79
M 4 11.10 3.71 0.71 1.41 7.05 30.34
M 5 1.38 9.73 2.89 21.69 5.85 0.85
M 6 2.72 1.21 4.32 2.95 60.77 24.35
M 7 3.41 2.77 16.31 1.37 4.79 2.09
M 8 2.99 2.93 2.24 2.87 49.75 2.07
S Em ± 0.31 0.29 0.59 4.76 3.78 3.15
CD (0.05) 0.93 0.88 1.78 14.45 11.46 9.54 Figures are the angular transformation of the original value
Characterization of liquid formulation
Recipes pH Emulsion stability test
Separation Oily Creamy Suspension Sedimentation of solids layer layer
A 6.4 No No Yes Good Yes
C 5.9 No No No Good Yes
O 6.0 No No No Good Yes
P 6.2 No Yes No Good Yes
Q 6.2 No No Yes Good Yes
R 6.4 No No Yes Good Yes
S 6.4 No No Yes Best Yes
T 6.1 No No Yes Good Yes
U 6.5 No No Yes Best Yes
V 5.9 No No Yes Good Yes
W 6.5 No No No Best Yes
X 6.3 No Yes No Good Yes
Y 7.0 No No Yes Good Yes
Z 6.8 No No No Good Yes
Selection of mutants of A. chroococcum for plant growth promotion and nitrogen fixation. Mutants of A.
chroococcum strains sensitive to rifampicin (JL 17, JL 104 and RJ 1) were generated and compared with their wild types for nitrogen fixation and plant growth promoting activities.
It was observed that mutants JL 17 (2,5), JL 104 (2,6) and RJ 1 (3,5,6) were isogenic with respect to IAA production.
However, mutants were found to very in nitrogen fixation (ARA activity) and 3,5 of JL 17, 6 and 8 of JL 104, and 2,4 and 6 of RJ 1 were isogenic.
Development of liquid bioinoculants for Azotobacter chroococcum with longer shelf life. The effect of glycerol, methyl crotonate, mineral oil and butanol on survival of A.
chroococcum was evaluated on wheat seed. Glycerol and
methyl crotonate were found to support the viability of A.
chroococcum on seed. The highest viability of A. chroococcum on seed was obtained when methyl crotonate was used as a seed additive. Butanol and mineral oil had deleterious effect on the viability of Azotobacter, and at the end of three days of incubation, a drastic reduction in the viability of A.
chroococcum was observed on seed.
Twenty-seven liquid formulations of A. chroococcum were evaluated for stability at ambient temperature. No
separation of solids and oily layer appeared in any of the selected recipes.
3.11.2.2 Microorganisms as biocontrol agents Biochemical characterization of anti-fungal compound against Rhizoctonia bataticola. Isolate HKA-15 was found to control R. bataticola, the causative pathogen for charcoal rot in soybean. The anti-fungal metabolite produced by the isolate HKA-15 was found to be very resilient in nature. It neither lost its anti-fungal activity during autoclaving (1210C for 30 min) nor on treatment with protease enzyme.
The broth culture of the isolate HKA-15 was extracted with n-Butanol, concentrated to dryness, dissolved in minimum volume of methanol and spotted on TLC plates.
Partial purification of the crude metabolite was carried out by exclusion chromatography. Anti-fungal activity was assessed in vitro using purified fraction of this metabolite.
The root colonizing ability of isolates HKA-15 and HKA- 121 was tested by examining their growth on soybean roots.
HKA-15 was found to be a better colonizer than HKA-121.
Identification of the biocide producing microorganism isolated from leaf compost. The isolate (4A) earlier identified as Streptomyces purpeofuscus (MTCC- 8377) on the basis of their morphological and physiological characteristics was subjected to DNA sequencing. Its 16S
rDNA fragment was amplified through PCR using universal primers. The amplified product was 1.5 kb size which was purified using microcon filters. The purified PCR product was cloned into pGEMT-Esay vector, checked for the presence of the insert and got it sequenced. The organism was BLAST searched and on the basis of similarity (~99%), it was designated as Streptomyces albus. The complete sequence of the 16S rDNA fragment of Streptomyces albus (MTCC-8377) submitted to NCBI was allotted an accession- No.EU-523135.
Fungicidal activity of the metabolites extracted from culture filtrate of Streptomyces albus. For testing antagonism amongst S. albus against Alternaria solani and Macrophomina phaseolina, the metabolites were extracted from culture filtrate of S. albus with ethyl acetate and butanol. The ethyl acetate extracted fraction showed 25%
growth inhibition of A. solani at 200 ppm, whereas butanol fraction showed similar inhibition at 800 ppm.
Macrophomina phaseolina was found to be more sensitive than A. solani, showing 50% growth inhibition at 10 ppm of ethyl extract fraction and at 200 ppm of butanol fraction.
Growth inhibition of Macrophomina phaseolina by the natural metabolite of Streptomyces albus
Ethyl acetate extracted metabolite (ppm)
0 0.62 1.25 2.50 5.00 10.00 Colony diameter (cm) 9.0 6.9 5.9 5.8 5.1 4.4 Growth inhibition (%) 0.0 23.3 34.4 35.5 43.3 51.1
Butanol extracted metabolite (ppm)
0 12.5 25.0 50.0 100.0 200.0 Colony diameter (cm) 9.0 5.8 5.7 5.1 5.4 4.1 Growth inhibition (%) 0.0 35.5 36.6 43.3 40.0 54.4 Growth inhibition of Alternaria solani by the natural metabolite of Streptomyces albus
Ethyl acetate extracted metabolite (ppm)
0 25 50 100 200 400 800
Colony diameter (cm) 5.7 5.5 4.5 4.5 4.3 4.4 4.6 Growth inhibition (%) 0.0 4.34 21.7 21.7 25.2 23.4 15.6
Butanol extracted metabolite (ppm)
0 25 50 100 200 400 800
Colony diameter (cm) 5.7 5.0 4.7 4.5 4.6 4.4 4.3 Growth inhibition (%) 0.0 13.0 18.2 21.7 15.6 23.4 25.2 16S rDNA sequence of Streptomyces albus
