4. CROP AND NATURAL RESOURCE MANAGEMENT FOR SUSTAINABLE ENVIRONMENT
4.7 MICROBIOLOGY
contained 25% quinoa and 10% pearl millet flour along with rice and potato. The baked snack has 13.27% fat and 10.5% protein. The snack had 6.4 mg/ 100g iron, 2.85 mg 100 g-1 zinc and 453 mg 100 g-1 calcium. Shelf life studies of the snack upon packaging in metallized laminated packages have revealed its shelf life to be 4 months.
4.6.13 Development of roasted onion flakes
Three thermal methods viz. single pass tray drying, single pass microwave oven heating and a two stage heating including first tray drying followed by microwave oven drying were applied for development of roasted onion flakes. Heating in both single passes and two stage processes significantly influenced the textural and physic-chemical properties of onion flakes.
Two stage heating (1st stage heating in tray dryer at 60°C up to 30% moisture and 2nd stage heating in microwave oven up to 2-3% moisture) was found better in terms of improving crunchy texture and reducing time of heating. Pusa White Round and Pusa White Flat were found better for making crunchy onion flakes.
4.7.3 Bio-control agents (BCA) and their impact on rhizospheric microbial communities under soybean-wheat cropping system
The effect of bacterial inoculants, host genotypes and growth stages of the host on rhizospheric microbial community in terms of soil enzymes and microbial biomass C in soybean and wheat crops were evaluated.
Impact of different BCA on soybean rhizospheric microbial community was evaluated through qPCR using 16S rDNA, nosZ and nifH genes as biological markers. It revealed that BCA inoculants had significant impact on the functional genes of soybean. Fluorescent pseudomonads isolated from the rhizospheres of BCA inoculated soybean varied in their antagonistic potential, as well as in other PGP traits.
4.7.4 Conservation Agriculture (CA) influences spore inoculum level of arbuscular mycorrhizal fungi (AMF)
Inoculum level of indigenous AMF in IARI field under CA was measured in order to rate the nutrient mobilizing potential of soil. In general, CA recorded higher number of AMF spores than conventional till (CT) wheat. Per cent increase in spore population was 88-140% and 45% in CA and CT within a span of 68 days of crop growth respectively. Spore inoculum density of AMF under zero till direct-seeded rice with wheat residue were highest (11 spores per gram of dry soil) and statistically significant (P ≤ 0.05). The native AMF isolates were categorized into three morphotypes on the basis of spore diameter and designated as ‘A’
(> 250 µm), ‘B’ (106- 250 µm) and ‘C’ (53-106 µm).
Population of spore morphotype ‘C’ was maximum followed by ‘B’ and ‘A'.
4.7.5 Characterization of EPS for enhancing water holding capacity (WHC) of soil through Anabaena and Nostoc strains
The exopolysaccharides (EPSs) produced by cyanobacteria are important constituents for the development of biofilm through association of microbial communities in various habitats. They are formed on solid surface and act as a reservoir of water and nutrients, which can severely diminish
water infiltration by inducing surface sealing and pore clogging and increase nutrient availability. Anabaena sp. CCC 745 produced two types, namely capsular polysaccharide (CPS) and released polysaccharide (RPS). These were purified using Size Exclusion Chromatography (SEC). Molecular weight of CPS and RPS were 30.29 and 19.57 KDa, respectively.
The carbohydrate was the main component for both EPSs, which are heteropolysaccharide and composed of glucose, xylose, rhamnose, and glucuronic acid.
The backbone of both EPSs was made of [→4)-Glcp- (1→] and →4)-Rhap-(1→ linkage. The charges of both EPSs were found negative. The aqueous solution of both EPSs exhibited classically pseudoplastic fluid behaviour. EPS and RPS from Anabaena sp CCC-746 was also characterized in similar fashion.
Mole fraction of monosaccharide present in CPS and RPS after SEC
S.N. Monosaccharide mol (%)
CPS RPS
1 Glucose 32.53 32.87
2 Xylose 32.58 32.52
3 Glucuronic acid 11.48 16.97
4 Rhamnose 22.24 16.32
5 Others 1.17 1.32
6 Total 100.00 100.00
4.7.6 Enhancing microbe mediated nutrient cycling under non-flooded (aerobic) and flooded (anaerobic) conditions for improved productivity in rice-wheat cropping sequence
Soil application of zinc and inorganic N fertilizer with and without biofertilizers were examined for microbial activities in two rice cultivars (Pusa Basmati 1509 and DRR Dhan 45). Fe reduction varied from 90 to 160 µg Fe2+ g-1 soil d-1. The rhizospheres of DRR Dhan 45 had higher concentrations of reduced Fe due to soil application of zinc with or without biofertilizers such as B1-4, and biofilmed inoculants at 60 days after transplanting. The composition of microbial communities based on the taxonomic groups (16S rRNA genes of Alphaproteobacteria, Betaproteobacteria,
Actinobacteria, Firmicutes, Bacteroidetes and Acidobacteria) was determined by the qPCR assays. The gene copies of 16S rRNA Betaproteobacteria were higher in the ranges of 106 to 108 g-1 soil, compared to other phyla examined. The application of N as RDF led to highest gene copies of 2.9 × 108 g-1 soil. Both the application of zinc and bioinoculants led to characteristic changes in the gene copies of Betaproteobacteria in the rhizospheres of cv. Pusa Basmati 1509 and cv. DRR Dhan 45. The abundances of 16S rRNA gene copies of Actinobacteria ranged from 102 to 105 g-1 soil, with highest abundance (8.6 × 105 g-1 soil) in RDF. The compositions of microbial communities in terms of total bacterial-, archaeal, and cyanobacterial 16S rRNA gene copies were characteristically different from that with other fertilizer management practices in both the cultivars.
4.7.7 Microbes mediated water stress alleviation in crops
The effect of inoculation of moisture stress tolerant Bacillus megaterium on the growth and physiology of the turmeric (Curcuma longa) exposed to moisture stress, imposed by withholding water supply) was studied. Inoculation improved plant height and fresh rhizome weight, chlorophyll, phenol contents, ascorbate peroxidase and auxins. A positive impact of the Bacillus megaterium inoculation on the plant metabolism enabled it to tide over the moisture stress under field conditions.
4.7.8 Bacterial endophytes for plant growth promotion
Iron deficiency is a serious problem in calcareous and alkaline soils (pH > 7.5) where iron forms oxyhydroxide polymers of very low solubility.
Microbial siderophores are known to influence the Fe availability as it chelates ferric ions and make it available for itself and the plant system.
Two bacteria producing different chemotypes of siderophores were used for raising wheat crop under field conditions. They were Sid 1:catecholate type siderophore producing Pantoea agglomerans with Fe chelation efficiency-3.3ngFe59mg-1 siderophore;
and Sid 2: hydroxymate type siderophore producing Pseudomonas plecoglosida with Fe chelation efficiency- 2.9ngFe59mg-1 siderophore. Fe content in wheat shoot and grain was increased by 29.9 and 83.5%, respectively due to Sid 1, and by 38.2 and 48.6%, respectively due to Sid 2 over control treatment. A liquid BioIron formulation has been developed using a consortium (1:1) of both Sid 1 and Sid 2 isolates.
Effeect of Sid-1 and Sid-2 on Fe contant in wheat spoty
4.7.9 Exploring functional diversity of Azospirillum for application in rainfed pearl millet
A second year field trial was conducted in kharif 2019 with pearl millet variety RHB173 with 75% RDN and bacterial inoculations. Treatment with Azospirillum spp strains resulted in higher shoot fresh biomass (2- 14% increase over unioculated control with 75% RDN), root biomass (up to 36% increase over unioculated control with 75% RDN). Bacterial inoculation, with strain Azo38 performing best in terms of overall plant growth and grain yield (8% increase over control with 100% RDF), indicating its potential application in rainfed pearlmillet.
4.7.10 Azolla biomass as feed supplement and value added product for improving nutritional quality of lactating Murrah buffaloes
Green and red Azolla consist of 73.5% and 67.4%
saturated fatty acids, respectively. A feeding trial was conducted for assessing the effect of incorporating
sun-dried powder of green aquatic fern Azolla (Azolla microphylla) in diet of lactating Murrah buffaloes.
After three weeks of preliminary feeding a digestion test was conducted for assessing nutrient utilization.
Results indicated positive response on digestibility of dry matter (P<0.05), organic matter (P<0.05), neutral detergent fiber (P=0.01) and acid detergent fiber (P=0.015), while the digestibility of crude protein and ether extract remained unaffected (P>0.05).
Improvement in digestibility of different nutrients without influencing the intake of nutrients indicated the importance of dried Azolla in the ration of lactating Murrah buffaloes.
4.7.11 Utilization of agri-residues for production of industrially important biopolymer poly-β-hydroxyalkanoates by microorganisms
Two potential halophilic PHB producing isolates, Halomonas sp LB7 and Bacillus cereus 2S4R1 were selected, growth media using saccharified paddy straw leachate was formulated with optimized C: N and batch fermentation parameters were standardized.
Optimized fermentation parameters for maximum PHB recovery were found to be pH 7.0, DO 100%, air flow rate 1.0 vvm, temperature 30°C and 20% carbon source concentration. The isolate produced 8.71 g L-1 CDM (cell dry mass) and 6.24 g L-1 PHB. FTIR analysis of the produced polymer also confirmed the presence of characteristic marker ester carbonyl band for polyhydroxyalkanoates.
4.7.12. Production of biofuels/value added products from lignocellulosic wastes rice straw
Rice straw is highly recalcitrant due to lignin- carbohydrate complex and high ash. It has to be deconstructed to access sugars, which can be converted to high value products. An improvised NaOH pretreatment coupled with acidified waterwash was applied to retrieve high cellulose and lignin. More than 80% cellulose was recovered in pretreated solids and >65% lignins recovered from acidification of alkali prehydrolysates/washwaters. Enzymatic hydrolysis of solids with commercial cellulases yielded ~5.5% and
3.3% sugar syrups which can be fermented to value added chemicals. Saccharomyces cerevisiae LN produced 0.508 g g-1 and 0.403 g g-1 ethanol (fermentation efficiency 77-97%) from hydrolysates within 24 h consuming all glucose while xylose was unutilised. It showed that this process can potentially yield 130 L ethanol and ~100 kg lignin from 1.0 tonne rice straw with limited water.
Lignin recovered was under-valued and underutilized byproduct. Therefore, lignin degrading organisms (bacteria, fungi, actinomycetes) were isolated through enrichment culture (minimal medium containing 0.1%
lignin and 0.25% glucose; pH 5.0 and 8.0) at different temperatures. Four different strains of bacteria and three different fungi were purified and tested for production of lignin degrading enzymes (Laccases and Lignin peroxidases Lip). All bacteria showed very good zones showing LiP production. Two fungal strains showed high laccase production.
4.7.13 Limited nitrogen application with microbial inoculation can accelerate in situ degradation of paddy straw
A consortium of two lignocellulolytic fungi, namely Coprinopsis cinerea LA2 and Cyathus stercoreus ITCC 3745 were applied in field for in situ degradation of rice straw. Highest population of bacteria (8.46 log cfu/g soil), fungi (4.97 log cfu/g soil), phosphate solubilizing bacteria (5.12 log cfu/g soil), cellulose degrading microorganisms (4.92 log cfu/g soil), Nitrosomonas and Nitrobacter (3.3 and 0.65 log cfu/g soil) were observed with the microbial consortium applied with limited N (i.e., straw + microbial inoculation @ 3 kg ha-1 + urea
@ 30 kg ha-1)at 30 days. This treatment also registered significantly higher activities of extracellular hydrolytic enzymes (cellulase, xylanase, β-glucosidase). Again, soil microbial activity in terms of dehydrogenase, FDA hydrolase, alkaline phosphatase, peroxidase, urease, and microbial biomass carbon (562.7 mg/kg soil) was also highest in this treatment.
4.7.14 On-farm demonstration of Pusa decomposer and microbial inoculants
In-situ application of Pusa decomposer on paddy residue was demonstrated in farmers’ fields of villages Badwasni (Sonepat), Thakurdwara (Mukerian),
Nangalbhoor (Pathankot), Bhangwa (Gurdaspur), Bal
& Kohali (Amritsar), and Pheowa (Kurushetra). The liquid culture was inoculated @ 10 litres per acre and field was ploughed with rotavator and wheat was sown after 15 days. Demonstrations were also carried out at KVK Panipat, Mohali, Ujwa and Saharanpur for farmers. In collaboration with MNREGA a large scale demonstration was held at village Puranpur, Pilibhit for pit composting of various residues.
Farmers’ participatory on-farm trials (OFT) on carrier based as well as liquid formulation bio- fertilizers, namely, blue green algae (BGA), Rhizobium, Azotobacter, Azospirillum, phosphate solubilizing bacteria (PSB), AM fungi and Azolla were conducted in farmers’ fields in collaboration with 21 KVKs located in 12 states (J&K, Punjab, Bihar, Jharkhand, Gujarat, Rajasthan, Chhatishgarh, Odisha, Madhya Pradesh, Uttar Pradesh, Uttarakhand and West Bengal). Crops included food, pulse, oilseed, vegetable, plantation crops. The feedback received from the farmers depicts that integrated application of BGA and other biofertilizers are cost-effective, eco-friendly and easy to use. Average increase in rice grain yield across the locations due to application of BGA and PSB varied between 3.4-8.5% over the farmers’ practices.
Biofertilizer applied plots had bolder and shiny rice grains, and less lodging. BGA application led to marked improvement in soil physical condition and saved 10-25 kg N/ ha. In pulses, increase in yield due to Rhizobium inoculation was 11.4-16.8% in green gram, 10.6-15.4% in chickpea and 10.8-16.7% in field pea.
Also considerable increment in yield (3.2-7.6%) was found in brinjal due to biofertilizer inoculation. Liquid formulations of Azotobacter, Azospirillum, ZnSB and KSB were found user-friendly, effective and potential sources of crop nutrition in rice, brinjal and other crops at different locations in India. Inoculation of these liquid formulations increased wheat grain yield by 4.8- 12.5%, 3.4-10.2%, 3.5-7.2% and 2.4-5.6%, respectively over the farmers’ practices across the locations.
4.7.15 Bioresources
Whole genome sequence of Azotobacter chroococcum W5 (MTCC 25045) an indigenous free living diazotroph
has been deposited at DDBJ/EMBL/GenBank under Bioproject ID: PRJNA610299. This bacterium has been isolated from wheat rhizosphere long back and has been recommended as a Biofertilizer for many crops, including vegetables, agronomically tested and evaluated. The strain has been formulated as liquid formulation with a shelf life of more than 2 years, and the technology has been commercialized and applied for patent. Seven oleaginous yeasts have been submitted to NBAIM, Mau, viz:
Trichosporon mycotoxinivorans S2 (NAIMCC-F-03902);
Candida tropicalis Pe1 (NAIMCC-F-03903); Candida dubliniensis Pe2(NAIMCC-F-03904); Meyerozyma caribbica Pe3 (NAIMCC-F-03905); Candida tropicalis Po2 (NAIMCC-F-03906); Candida tropicalis L2 (NAIMCC-F-03907); Candida quercitrusa L3 (NAIMCC-F-03908). 16S rRNA sequences of fifteen siderophore chemotyped bacterial isolates (Accession No. MK253238- MK253251) and 10 heavy metal remediating bacterial isolates (Accession No.
MN524169-MN524178) have been deposited in NCBI.
Besides, partially characterized and identified 30 unialgal freshwater cyanobacteria cultures belonging to 12 genera have been deposited at BGA Germplasm, CCUBGA, IARI, New Delhi, which is repository and service centre for fresh water blue green algae maintained in unialgal condition.