Elucidation of the plant growth–promoting effect of Hartmannibacter diazotrophicus on tolerance of barley to salt stress

Abstract

A novel type of methanol dehydrogenase was recently identified, this dehydrogenase is encoded by the gene xoxF and for its activity requires lanthanum (La3+) as a cofactor. The xoxF-gene was detected in the bacterium Hartmannibacter diazotrophicus which stands out by its activity as a plant growth promoter under saline stress by mechanisms that include production of ACC-deaminase, nitrogen fixation, phosphorus solubilization, among other. Methanol dehydrogenase xoxF activity of H. diazotrophicus was evaluated by its growth on methanol in a mineral medium supplemented with lanthanum and the suitable concentration of lanthanum of optimal growth was determined. Moreover, a greenhouse experiment with barley plants growing in saline soil (NaCl 3%) was reassessed and six experimental treatments were set up namely: Two treatments of barley seeds inoculated with H. diazotrophicus with/without lanthanum amendment, two treatments of seeds with addition of H. diazotrophicus death biomass with/without lanthanum amendment, one treatment of seeds with lanthanum amendment and the controls without amendments respectively. Variations in shoots and roots dry weights, changes in microbial diversity and colonization of H. diazotrophicus in roots and rhizosphere were evaluated. Inoculated plants with/without lanthanum showed significant increases of leave and root dry weights in comparison to controls. Plants growing with only La3+ also exhibited higher leave and root dry weights in contrast to treatments with death biomass addition and the controls. Plants inoculated with dead biomass of H. diazotrophicus with and without lanthanum had no significant effect in plant growth under salt stress compared to non-inoculated plants. Changes in alpha and beta bacterial diversity in communities of rhizosphere and roots were evidenced among the treatments; alpha diversity indicators showed an increase in the number of ASVs (Amplicon sequence variations) for treatments with H. diazotrophicus, whereas beta diversity analysis revealed variation in microbial communities throughout the treatments. Furthermore, for H. diazotrophicus a high colonization capacity of roots and rhizosphere was demonstrated in presence/absence of lanthanum. Furthermore, samples from natural saline environments of soil and waters were collected for bacterial enrichments in liquid mineral medium with methanol and lanthanum in order to isolate potential halotolerant bacteria with methylotrophic activity. Thirty-one bacteria were isolated and among them, one new species of Spirosoma sp. genus was identified and after polyphasic approach was proposed and accepted as Spirosoma endbachense I-24T. The draft genome of S. endbachense I-24T confirmed the position as new species and 4 clusters involved in biosynthesis of ladderane, terpene, polyketide synthese type I and III and non-ribosomal peptide synthese were identified. For Spirosoma agri KCTC 52727T and Spirosoma terrae KCTC 52035T which are the next relatives to S. endbachense I-24T, draft genome sequences were also assembled. The annotation revealed carbohydrate-active enzymes and secondary metabolite biosynthesis gene clusters as well as alkaline phosphatase, cellulose and amylase activity. The genomes contribute to the genomic knowledge of the members of the genus Spirosoma.