Endophytic bacteria from roots and leaves of rice plants were isolated and identified in order to select the diazotrophs and improve their nitrogen-fixing abilities. The nitrogen-fixing endophytes were identified by PCR amplification of the nifH gene fragment. For this purpose, two isolates, Enterobacter cloacae RCA25 and Klebsiella variicola RCA26, and two model bacteria (Herbaspirillum seropedicae z67 and Sinorhizobium fredii NGR234) were transformed to increase the biosynthesis of the main plant auxin indole-3-acetic acid (IAA). A significant increase in the production of IAA was observed for all strains. When the expression of nifH gene and the activity of the nitrogenase enzyme were analyzed in liquid cultures, we found that they were positively affected in the IAA-overproducing endophytes as compared to the wild-type ones. Rice plants inoculated with these modified strains showed a significant upregulation of the nitrogenase activity when plants infected with the wild-type strains were used as reference. Similar results were obtained too with common bean plants infected with the S. fredii NGR234 strain. These findings suggest that IAA overproduction improves nitrogen-fixing apparatus of endophytic bacteria both in liquid cultures and in inoculated host plants. The present study highlights new perspectives to enhance nitrogen-fixing ability in non-legume crops. These strains could be used as bioinoculants to improve the growth and the yield of agricultural crops, offering an alternative to the use of chemical nitrogen fertilizers.

The drought–stress response in plant involves the cross-talk between abscisic acid (ABA) and other phytohormones, such as jasmonates and ethylene. The auxin indole-3-acetic acid (IAA) plays an integral part in plant adaptation to drought stress. Investigation was made to see how the main auxin IAA interacted with other plant hormones under water stress, applied through two different growth conditions (solid and hydroponic). Medicago sativa plants nodulated by the Ensifer meliloti wild type 1021 (Ms-1021) and its IAA-overproducing RD64 derivative strains (Ms-RD64) were subjected to drought stress, comparing their response. When the expression of nifH gene and the activity of the nitrogenase enzyme were measured after stress treatments, Ms-RD64 plants recorded a significantly weaker damage. These results were correlated with a lower biomass reduction, and a higher Rubisco protein level measured for the Ms-RD64-stressed plants as compared to the Ms-1021-stressed ones. It has been verified that the stress response observed for Ms-RD64-stressed plants was related to the production of greater amount of low-molecular-weight osmolytes, such as proline and pinitol, measured in these plants. For the Ms-RD64 plants the immunoblotting analysis of thylakoid membrane proteins showed that some of the photosystem proteins increased after the stress. An increased non-photochemical quenching after the stress was also observed for these plants. The reduced wilting signs observed for these plants were also connected to the significant down-regulation of the MtAA03 gene involved in the ABA biosynthesis, and with the unchanged expression of the two genes (Mt-2g006330 and Mt-8g095330) of ABA signaling. When the expression level of the ethylene-signaling genes was evaluated by qPCR analysis no significant alteration of the key positive regulators was recorded for Ms-RD64-stressed plants. Coherently, these plants accumulated 40% less ethylene as compared to Ms-1021-stressed ones. The results presented herein indicate that the variations in endogenous IAA levels, triggered by the overproduction of rhizobial IAA inside root nodules, positively affected drought stress response in nodulated alfalfa plants.

Indole-3-acetic acid (IAA) is the main auxin acting as a phytohormone in many plant developmental processes. The ability to synthesize IAA is widely associated with plant growth-promoting rhizobacteria (PGPR). Several studies have been published on the potential application of PGPR to improve plant growth through the enhancement of their main metabolic processes. In this study, the IAA-overproducing Ensifer meliloti strain RD64 and its parental strain 1021 were used to inoculate Medicago sativa plants. After verifying that the endogenous biosynthesis of IAA did not lead to genomic changes during the initial phases of the symbiotic process, we analyzed whether the overproduction of bacterial IAA inside root nodules influenced, in a coordinated manner, the activity of the nitrogen-fixing apparatus and the photosynthetic function, which are the two processes playing a key role in legume plant growth and productivity. Higher nitrogen-fixing activity and a greater amount of total nitrogen (N), carbon (C), Rubisco, nitrogen-rich amino acids, soluble sugars, and organic acids were measured for RD64-nodulated plants compared to the plants nodulated by the wild-type strain 1021. Furthermore, the RD64-nodulated plants showed a biomass increase over time, with the highest increment (more than 60%) being reached at six weeks after infection. Our findings show that the RD64-nodulated plants need more substrate derived from photosynthesis to generate the ATP required for their increased nitrogenase activity. This high carbohydrate demand further stimulates the photosynthetic function with the production of molecules that can be used to promote plant growth. We thus speculate that the use of PGPR able to stimulate both C and N metabolism with a balanced C/N ratio represents an efficient strategy to obtain substantial gains in plant productivity.

Summary Several important bacterial characteristics, such as biological nitrogen fixation, phosphate solubilization, 1‐aminocyclopropane‐1‐carboxylate deaminase activity and production of siderophores and phytohormones, can be assessed as plant growth promotion traits. Our aim was to evaluate the effects of nitrogen fixing and indole‐3‐acetic acid (IAA) producing endophytes in two Oryza sativa cultivars (Baldo and Vialone Nano). Three bacteria, Herbaspirillum huttiense RCA24, Enterobacter asburiae RCA23 and Staphylococcus sp. 377, producing different IAA levels, were tested for their ability to enhance nifH gene expression and nitrogenase activity in Enterobacter cloacae RCA25. Results showed that H. huttiense RCA24 performed best. Improvement in nitrogen fixation and changes in physiological parameters such as chlorophyll, nitrogen content and shoot dry weight were observed for plants co‐inoculated with strains RCA25 and RCA24 in a 10:1 ratio. Based on confocal laser scanning microscopy analysis, strain RCA24 was the best colonizer of the root interior and the only IAA producer located in the same root niche occupied by RCA25 cells. This work shows that the choice of a bio‐inoculum having the right composition is one of the key aspects to be considered for the inoculation of a specific host plant cultivar with microbial consortia.