Ph.D

Biofertilizers are the living microorganisms that promote plant growth by increasing nutrient availability in soil. They are renewable, ecofriendly and reduce dependency on chemical fertilizers. The consortium of biofertilizers is a combination of various microorganisms which act together as a community that contributes to plant growth. Nutmeg, an important spice crop of India, is widely cultivated in Kerala. The nutrient exhaustive crop is used in this study to reduce the dependency on chemical fertilizers. Wayanad, an agrarian district, is known for its organic production of spices. The present investigation titled “Development of a native microbial consortium of biofertilizers for plant growth promotion in nutmeg” was carried out with the objective of isolation of native nitrogen fixers, phosphate solubilizers, and potassium solubilizers from the rhizosphere soils of nutmeg of Wayanad district and to develop microbial consortia and evaluate microbial consortium for plant growth promotion of nutmeg under pot culture and field evaluation in the Wayanad district. Nutmeg rhizosphere soil samples were collected from 10 different locations of Wayanad district in Kerala. A total of 55 isolates were obtained comprising of 21 nitrogen fixing bacteria, 18 phosphorus solubilizing bacteria and 16 potassium solubilizing bacteria. These beneficial bacterial isolates were screened for plant growth promoting activities viz., nitrogen fixation, phosphorus solubilization, potassium solubilization and IAA production under in vitro condition. The nitrogen fixation by the bacterial isolates ranged from 0.70 to 4.90 mg g-1 of carbon source. The phosphorus solubilization ranged from 12.98 µg P ml-1 to 71.65 µg P ml-1. The potassium solubilization values by the bacterial isolates ranged from 6.66 mg K ml 1 to 8.30 mg K ml-1. IAA production was qualitatively and quantitatively estimated for all 55 bacterial isolates. IAA production of nitrogen fixing bacteria ranged from 0.46 µg ml-1 to 11.09 µg ml-1. IAA production of phosphorus solubilizing bacteria ranged from 0.07 µg ml-1 to 1.85 µg ml-1. IAA production of potassium solubilizing bacterial isolates ranged from 0.63 µg ml-1 to 9.77 µg ml-1. Based on their functional efficiency, 15 most promising bacterial isolates were characterized for the morphological, cultural, and biochemical characters. Ten of the bacterial isolates were Gram positive and five identified as Gram negative. Ten of the isolates were rod shaped while the other five cocci shaped. Fifteen isolates were further screened for abiotic stress tolerance. The moisture stress tolerance was studied at 10 %, 20 %, 30 %, and 40 % using PEG 6000 in Tryptone Soy broth for 48 h at 28 ± 2 ℃. Similarly, temperature stress tolerance was screened at temperatures of 30 ℃, 35 ℃, 40 ℃ and 45 ℃ for 48 h. All the 15 bacterial isolates were found to be tolerant to the maximum stress of 40 % moisture stress and 45 ℃ temperature stress. Compatibility of the most promising 15 isolates were assessed. All 14 bacterial isolates were compatible among themselves. One potassium solubilizing bacterial isolate viz., KKK 2 was incompatible with 5 bacterial isolates (nitrogen fixing bacteria, phosphorus solubilizing bacteria). Based on the plant growth promoting activities, abiotic stress tolerance and compatibility studies, 9 most promising bacterial isolates were selected. Molecular characterization of the nine bacterial isolates were done using 16S rRNA gene sequencing and identified as Staphylococcus equorum VTN 2, Staphylococcus equorum KPN 3, Enterobacter sp. KPN 1, Staphylococcus pasteuri PPP, Bacillus velezensis KKP 1, Pseudomonas aeruginosa MKP 2, Sphingobacteriumanhuiense MPK, Staphylococcus epidermidis NMK 2 and Pseudomonas veronii MHK. Based on the ranking of the bacterial isolates, the 3 best nitrogen fixing bacteria (Staphylococcus equorum VTN 2, Staphylococcus equorum KPN 3 and Enterobacter sp. KPN 1), 3 phosphorus solubilizing bacteria (Staphylococcus pasteuri PPP, Bacillus velezensis KKP 1, and Pseudomonas aeruginosa MKP 2) and 3 potassium solubilizing bacteria (Sphingobacteriumanhuiense MPK, Staphylococcus epidermidis NMK 2 and Pseudomonas veronii MHK) were selected for developing 3 distinct formulations of liquid biofertilizer consortia. The liquid biofertilizer consortia was evaluated under pot culture at Block 10, RARS, Ambalavayal, Wayanad with 6-month-old nutmeg seedlings. Combined application of liquid biofertilizer with 2 levels (50 % and 75 % of N, P, K) of recommended dose of fertilizers, commercial formulation PGPR Mix 1, inorganic POP, organic POP, control were the treatments. Pot culture evaluation results revealed that liquid biofertilizer consortium C2 (Staphylococcus equorum KPN 3 + Bacillus velezensis KKP 1 + Staphylococcus epidermidis NMK 2) with 50 % RDF was the most promising among the treatments, resulting in 50% reduction of chemical fertilizer dependency. Based on the plant growth parameters, the 2 best consortia (Consortium 2 (Staphylococcus equorum KPN 3 + Bacillus velezensis KKP 1 + Staphylococcus epidermidis NMK 2) and Consortium 1 (Staphylococcus equorum VTN2, Staphylococcus pasteuri PPP, Sphingobacteriumanhuiense MPK)) were selected for further field evaluation. The 2 best biofertilizer consortia obtained from pot culture experiment in Wayanad district were evaluated under field condition at Block 5, RARS, Ambalavayal, Wayanad. Plant growth parameters revealed that application of consortium 2 (Staphylococcus equorum KPN 3 + Bacillus velezensis KKP 1 + Staphylococcus epidermidis NMK 2) with 50 % RDF was the most promising treatment. Metagenomics analysis of the soil samples before and after application of biofertilizer consortia revealed that there was no change in the predominant phyla level classification, except for its composition percentage. Proteobacteria, Actinobacteria and Acidobacteria dominated at the phylum level composition. There were changes at the species level. At the species level composition, Aciditerrimonasferrireducens (12.94 %) dominated in the initial soil sample and Sphingomonaslimnosediminicola (31.18 %) dominated in the case of the best biofertilizer consortia application (Staphylococcus equorum KPN 3 + Bacillus velezensis KKP 1 + Staphylococcus epidermidis NMK 2 + 50 % RDF of N, P, K). Field level application of liquid biofertilizer consortia has resulted in changes to the rhizosphere bacteria of nutmeg plants, which in turn has enhanced their growth and development. Hence, it can be concluded that liquid biofertilizer consortium 2 (Staphylococcus equorum KPN 3 + Bacillus velezensis KKP 1 + Staphylococcus epidermidis NMK 2) increased nutrient availability to the plants and promoted nutmeg growth. Native biofertilizer consortia reduced 50 % dependency of chemical fertilizers and increased nutmeg seedling growth. These results indicated the importance of exploiting native bacterial isolates rather than a non-native consortium. It is an ecofriendly, cost effective and sustainable method compared to synthetic fertilizers. Multilocation trials and risk assessment of the biofertilizer need to be assessed before commercialization of the liquid biofertilizer consortium.