Plant-microbe interaction in wheat (Triticum aestivum L.) under different crop rotations

Abstract

Present study was conducted on plant-microbe interactions in wheat (Triticum aestivum L.) under wheat-rice and wheat-cotton rotations. Soil samples were collected from rhizosphere of wheat and used for isolation of bacteria on LB medium as well as on selective media for phosphate solubilizers and nitrogen fixers. A total of 29 isolates were obtained and identified on the basis of 16S rRNA gene sequence analysis as Azospirillum (2 strains), Acinetobacter (2 strains), Actinobacteria (1 strain), Arthrobacter (3 strains), Bacillus (5 strains), Enterobacter (3 strains), Microbacterium (2 strains), Pantoea (one strain), Pseudomonas (4 strains) and one strain each of the genera Sphingobacteria, Terribacillus and Xanthomonas. In the present study, pqqE (a gene known to have a role in P-solubilization) was PCR amplified and sequenced. Sequence analysis of pqqE gene amplified from Arthrobacter sp. WP-2, Pseudomonas spp. T-27 and NN-4 showed maximum (80-81%) sequence similarity with that of Pseudomonas putida and that of Pantoea sp. WP-5 with Klebsiella (84%). Phosphate solubilization (a plant beneficial trait) activity was detected in 12 bacterial strains. Among the tested strains, high P- solubilization activity (207-311 μg/mL) was detected in the pure cultures of Arthrobacter sp. WP-2, Azospirillum sp. WS-1, Bacillus sp. T-34, Enterobacter spp. T-41 & T-42, Pantoea sp. WP-5 and Pseudomonas sp. T-27. The most efficient indol-3-acetic acid producing (11-31 μg/mL ) strains among the isolates were Bacillus sp. T-34, Enterobacter sp. T-41, Pseudomonas sp. WP-1, Arthrobacter sp. WP-2 and Azospirillum sp. WS-1. Selected bacterial isolates were tested as inocula for wheat grown in sterelized sand, in earthen pots (filled with non-sterelized soil) and finally evaluated in field trials under wheat-rice and wheat-cotton crop rotation. Maximum increase in grain yield over control was noted in plants inoculated with Azospirillum spp. WS-1 & WB-3 and Bacillus sp. T-34 under wheat-rice rotation (10-15%) and under wheat-cotton rotation (7-14%). Bacterial population determined at different growth stages of inoculated plants indicated maximum number of bacteria (108-109 cfu/g dry soil) at booting stage. Organic acids (acetic acid, citric acid, malic acid and oxalic acid) and sugars (sucrose and glucose) were detected in rhizosheaths of wheat grown under both crop rotations. Diversity of bacteria in the rhizosheath of wheat under both crop rotations was studied through direct soil DNA analysis of 16S rRNA using barcoded pyrosequencing. From the soil samples a total XIV of 46,971 cleaned sequences were obtained with read length of 319 bp. Among these sequences, 48.8% sequences were obtained from wheat-cotton rotation while 51.2% from wheat-rice crop rotation. Out of total 46,971 sequences, 11,729 (24.97%) showed 97% similarity with phylotypes having PGPR activity. The results showed that in wheat-cotton and wheat-rice rotation, Proteobacteria were dominant (25.1% and 35.7%, respectively), followed by un-classified bacteria (20.5% and 17.1%, respectively) and Actinobacteria (17.7% and 13.6%). However, Chloroflexi, Firmicutes, Acidobacteria, Planctomyctes, Bacteriodetes, Cyanobacteria, Verrucomicrobia and Nitrospora were also present. It was also observed that out of 495 different phylotypes detected, 280 phylotypes were common in both the crop rotations while 96 were only abundant in wheat-rice rotation and 41 were only present in wheat-cotton rotation system. Diversity of diazotrophs was determined in rhizosheath of wheat under both crop rotations by nifH sequence analysis amplified from soil DNA. A total of 41,287 nifH sequences were obtained with read length of 339-345 bp. The results showed that in wheat-rice and wheat-cotton cropping system, Proteobacteria were dominant (61.3% and 59.9%, respectively). However, sequences related to Cyanobacteria, Chlorobi, Firmicutes, Verrucomicrobia, Euryarchaeota, Spirochaetes, Actinobacteria and Fibrobacteres were also detected. Sequence analysis indicated presence of nifH sequences belonging to 150 different nitrogen fixing genera. Among these 150 genera, 22.6% genera were present only in wheat-rice rotation, 12.6% genera were found in wheat-cotton rotaion and 40.6% genera were present in both the cropping systems.