Diversity and Plant Growth Promotion Abilities of Actinomyeetes in Wheat Rhizosphere of Gujarat Region

Abstract

Actinomycetes, the ubiquitously present, high GC, filamentous bacteria are extensively studied for antibiotics and related secondary metabolites. This group also plays a vital role in soil nutrient cycling and biocontrol activities. However, little is known about their role in plant rhizosphere. The present study aims at exploring actinomycete diversity and its plant growth promotion (PGP) activities from wheat rhizosphere. Gujarat state is divided in four major wheat producing region (Mehsana, Bhal, North Gujarat, Saurashtra) depending on agro-climatic zones, type of soils and wheat grown. Rhizosphere and bulk soil samples were collected by composite sampling from all the zones to isolate rare as well as common actinomycetes using prescribed pre-treatment and enrichment techniques on different typical actinomycete isolation media. A total of over 1000 actinomycete isolates were obtained from wheat rhizosphere across the state and characterized by classical, chemo-taxonomic and molecular methods. After the hylogenetic analysis (16S rDNA amplification), over 100 morphologically and genotypically distinct actinomycetes belonging to Streptomyces,Nocardia, Arthrobacter, Rhodococcus, Brevibacillus, Cellulosimicrobium, Microbacterium, etc were obtained. The identified cultures were deposited in ‘Bank a bug’ repository under Gujarat Genomics Inititiative (GGi, GSBTM) while culture nucleotide sequences were submitted to NCBI (GeneBank). Diversity indices that indicate actinomycete species richness and evenness were calculated for each sector and correlated to soil types and soil nutrients.The actinomycete diversity in wheat rhizosphere of Gujrat region was also assessed through metagenomic approach. The community DNA from soil samples was isolated, amplified and analyzed using denaturation gradient gel electrophoresis (DGGE). The dominant and unusual bands for each region were eluted, purified and sequenced whilst phylogeny was inferred by NJ and UPGMA methods. Actinomycete richness and distribution was correlated to carbon source, nitrogen and soil pH using PCA (principal component analysis) values on the basis of Eigen score.The isolated and identified cultures were further screened qualitatively and quantitatively for PGP activities that included phosphate solubilization, IAA, siderophore and soil enzymes production. The best phosphate solubilizing actinomycete was addressed for mechanism for organic acid production by gene expression and enzyme assays. The antifungal isolates were screened for chitinases and other small antifungal compounds. The low molecular weight antifungal compounds in most effective isolates inhibiting fungal pathogens were produced,extracted, purified and identified through GC-MS and 1H-NMR. Similarly, isolates were screened for production of soil fertility marker enzymes for carbon, nitrogen and phosphorous. Promising isolates were explored for soil enzyme production under soil state fermentation using soil extract as nutrient source. The rhizosphere abundant isolates with excellent PGP activities (including biocontrol) were inoculated for in-vivo plant experiments (wheat) in controlled (hydroponics) and pot assays with normal, pathogen challenged and nutrient stress conditions. Actinomycete inoculation significantly increased number of roots,shoots, root length, shoot length and biomass of one month old plants. The project findings have been very promising and minimum 3 significant PGP Streptomyces capable of surviving draught, salinity and pathogens were isolated that enhanced plant (wheat, chickpea) growth and biomass by 2 folds under non-axenic conditions in green house experiments. Future studies may include scaling up of these isolates as potential bio-inoculants and bio-control agents. The future experiments may be intended to study localization of endophytic PGP actinomycete in plant roots by fluorescent tagging using confocal microscopy.