Bacterial Endophytes from Legumes Native to Arid Environments Are Promising Tools to Improve Mesorhizobium-Chickpea Symbiosis under Salinity

Abstract

Symbiotic nitrogen fixation is a major contributor of N in agricultural ecosystems, but the establishment of legume-rhizobium symbiosis is highly affected by soil salinity. Our interest is focused on the use of non-rhizobial endophytes to assist the symbiosis between chickpea and its microsymbiont under salinity to avoid loss of production and fertility. Our aims were (1) to investigate the impact of salinity on both symbiotic partners; including on early events of the Mesorhizobium-chickpea symbiosis, and (2) to evaluate the potential of four non-rhizobial endophytes isolated from legumes native to arid regions (Phyllobacterium salinisoli, P. ifriqiyense, Xanthomonas translucens, and Cupriavidus respiraculi) to promote chickpea growth and nodulation under salinity. Our results show a significant reduction in chickpea seed germination rate and in the microsymbiont Mesorhizobium ciceri LMS-1 growth under different levels of salinity. The composition of phenolic compounds in chickpea root exudates significantly changed when the plants were subjected to salinity, which in turn affected the nod genes expression in LMS-1. Furthermore, the LMS-1 response to root exudate stimuli was suppressed by the presence of salinity (250 mM NaCl). On the contrary, a significant upregulation of exoY and otsA genes, which are involved in exopolysaccharide and trehalose biosynthesis, respectively, was registered in salt-stressed LMS-1 cells. In addition, chickpea co-inoculation with LMS-1 along with the consortium containing two non-rhizobial bacterial endophytes, P. salinisoli and X. translucens, resulted in significant improvement of the chickpea growth and the symbiotic performance of LMS-1 under salinity. These results indicate that this non-rhizobial endophytic consortium may be an appropriate ecological and safe tool to improve chickpea growth and its adaptation to salt-degraded soils.

Keywords: abiotic stress; endophytic bacteria; grain legume; inoculants; root exudates; salt-stress.

Figure 1

Transcription levels of early symbiotic genes and salt tolerance genes in LMS-1 cells exposed to salt stress and/or to chickpea root exudates. (A–D) correspond to the expression levels, determined by qRT-PCR, of nodD, nodC, exoY, and otsA genes, respectively. R + salt: LMS-1 exposed to salt (250 mM NaCl); R + NRE: LMS-1 exposed to normal root exudates; R + SRE: LMS-1 exposed to root exudates collected under salinity; R + salt + NRE: LMS-1 exposed to both salt (250 mM NaCl) and normal root exudates. Data are presented as the mean and standard error values of six independent technical replicates. Means followed by the same letter do not differ significantly by Duncan test (p > 0.05).

Figure 2

Effect of non-rhizobial endophytes co-inoculation on chickpea growth parameters under salinity: (A) shoot dry weight (SDW) and (B) root dry weight (RDW). PC + salt: uninoculated plants supplemented with synthetic nitrogen; LMS-1 (R): inoculated plants with LMS-1 alone; R + P1 + X: inoculated plants with LMS-1 + Phyllobacterium salinisoli + Xanthomonas translucens; R + P1 + P2 + X: inoculated plants with LMS-1 + P. salinisoli + P. ifriqiyense + X. translucens; R + P2 + C: inoculated plants with LMS-1 + P. ifriqiyense + Cupriavidus respiraculi; R + P2 + C + X: inoculated plants with LMS-1 + P. ifriqiyense + C. respiraculi + X. translucens. Error bars represent the standard error. Different letters correspond to statistically significant differences by Duncan test (p < 0.05).

Figure 3

Effect of co-inoculation with non-rhizobial bacterial endophytes isolated from legumes grown on arid environments on Mesorhizobium symbiotic performance under salt stress: (A) number of nodules, (B) nodule dry weight (NDW), (C) average weight per nodule, and (D) total nitrogen content. PC + salt: uninoculated plants supplemented with nitrogen; LMS-1 (R): inoculated plants with LMS-1 alone; R + P1 + X: inoculated plants with LMS-1 + P. salinisoli + X. translucens; R + P1 + P2 + X: inoculated plants with LMS-1 + P. salinisoli + P. ifriqiyense + X. translucens; R + P2 + C: inoculated plants with LMS-1 + P. ifriqiyense + C. respiraculi; R + P2 + C + X: inoculated plants with LMS-1 + P. ifriqiyense + C. respiraculi + X. translucens. Data correspond to the mean and standard error values of 5 biological replicates. Different letters correspond to statistically significant differences by Duncan test (p < 0.05).

Figure 4

Effect of co-inoculation with endophytes from an arid environment on rhizobia nodules development under salt stress. Bright field micrographs of nodules histological sections. (A–C) Nodules from rhizobia alone, (D–F) nodules from R + P1 + X, (G) nodules from R + P1 + P2 + X, (H) nodules from R + P2 + C, and (I) nodules from R + P2 + C+X. Me: meristematic zone, In: infection zone, and Fi: fixation zone. Magnification in (A,D,E,G–I) is 40×; (B,C,F) is 63×. The red arrows in (F) indicate differentiated bacteroids, and the yellow arrowheads indicate uninfected cells.