Effect sea-water irrigation and inoculations with Azospirillum brasilense on the bacterial diversity in the rhizosphere of two wheat cultivars

Soil salinization is a serious threat to agricultural productivity in semi-arid ecosystems. Several strategies are followed to attenuate stress of salinization and allow productive crop cultivation. This includes primarily the breeding of salt-tolerant cultivars but there is also a potential to use microbial inoculants. Inoculations with microorganisms can provide plant growth promoting properties, e.g., nitrogen fixation, and at the same time they may protect roots and thus increase salt-tolerance. Several decades of research have already indicated that members of the genus Azospirillum have a potential to stimulate under specific environmental conditions the growth of crops, i.e., wheat, but its potential to also provide salt-tolerance has not yet thoroughly been explored. Here we report on results of an experimental field study which was conducted to elucidate the response of the wheat rhizosphere inhabiting bacterial community to irrigation with diluted seawater and, in addition, to inoculations with Azospirillum brasilense. The field was located in the Southern Sonoran Desert near La Paz, Baja California Sur, Mexico. It included two wheat cultivars, Opata, and Panter. In preceding studies, Opata exhibited stronger tolerance to salinity. A. brasilense was inoculated with seeds, or, alternatively, the bacteria were supplied with the irrigation water on a weekly basis. Control treatments included irrigation with non-saline water. One hundred and twenty days after sowing, the wheat plants were harvested, and their yield was determined. Total community DNA was extracted from rhizosphere microbial cells and PCR amplified, partial 16S rRNA genes were analyzed by both DGGE and high-throughput, ultra-deep Illumina amplicon sequencing. The experimental design considered four independent replicates for each treatment (cultivar, irrigation, inoculations). DGGE analysis of Alphaproteobacteria revealed that the inoculated A. brasilense was not abundant in the rhizosphere of both wheat cultivars, independent of whether irrigation was done with diluted seawater or non-saline control water. However, the DGGE patterns also demonstrated, similarly for both wheat cultivars, significant seawater effects on the diversity of the Alphaproteobacteria. While control water irrigated plots showed Alphaproteobacteria fingerprints of all treatments with high similarity, the fingerprints from the seawater-treatments resulted in a separate clustering of the weekly-inoculated wheat plants, but not for the seed inoculated, from the non-inoculated controls. By Illumina sequencing, a total of more than 4 million quality filtered 16S rRNA gene amplicon sequences, spanning the V3 and V4 region, were obtained. The average number of sequences for each sample was 85,000 sequences, and it included a total of 20 phyla, and approx. 50 classes, 90 orders, 200 families and 500 different genera. The bacterial diversity expressed as Shannon index based on operational taxonomic units (OTUs) did not significantly differ between samples from different irrigation regimes or between the cultivars. However, for treatments irrigated with saline water, a significantly higher diversity was observed for samples from the control and the seed inoculated treatments compared to the weekly inoculation. The number of OTUs was significantly higher for irrigation with control water compared irrigation with the diluted seawater, but no cultivar or treatment differences could be detected. Statistical analyses of similarities (ANOSIM) revealed highly significant differences at all taxonomic ranks for samples from seawater compared to control water irrigations. The R-values increased consistently with taxonomic resolution, i.e, from phylum to genus level. Non-metric multidimensional scaling (NMDS) showed highly significant effects of seawater irrigation compared to control water for both Opata and Panter at the phylum level. The higher R-values of Opata indicated that this cultivar responded stronger to the salinity than Panter. Thus, the stronger responses were possibly linked to higher levels of salt tolerance. As revealed by NMDS, the effect of seawater irrigation was for both cultivars even more pronounced at the genus level. Cultivar-related differences on bacterial diversity were also detected at the genus level, and, again, these effects were more pronounced with control water than with the diluted seawater. Effects of seed and weekly inoculation with A. brasilense were stronger with seawater and for Opata compared to Panter. At the OTU level, all treatments had significant effects. Several OTUs which decreased in their abundance due to seawater irrigation were identified, among them different Arthrobacter and Rhizobium species. A strong increase in relative abundance was observed for OTUs related to Thalassospira sp., one species, Ensifer adhaerens, and others. The phylogenetic assignments of several OTUs increasing in their abundance, suggests that they originated from the seawater itself. Other OTUs which were, among others, related to Pseudomonas, Streptomyces, or Devosia, were apparently not affected in their relative abundance by the seawater irrigation. For inoculation with A. brasilense, only minor changes in bacterial diversity were detected. The taxa differed in treatments with and without seawater irrigation. Less than 0.05% of all sequences detected in this study were affiliated to the genus Azospirillum, including more than 20 different OTUs. But only two of those were affiliated to A. brasilense. There was no difference in the relative abundance among control, seed inoculated and weekly inoculated. One of the two OTU appeared to be sensitive to seawater irrigation. Overall this study demonstrates the profound changes of irrigation with diluted seawater on the rhizosphere bacterial communities. The ultra-deep amplicon sequencing gives insights into the suppression and stimulation of certain bacterial taxa. This information should be highly useful for understanding the adaptation of wheat plants to salinity stress and for developing efficient microbial inoculants for promoting wheat cultivation under saline conditions.