Bradyrhizobium sp. strain SUTN9‐2 was confirmed as rice endophytic bacteria and also as rice growth promotion agent. SUTN9‐2 showed the capability of plant growth promotion characteristics, such as indole‐3‐acetic acid (IAA) and 1‐amino‐cyclopropane‐1‐carboxylic acid (ACC) deaminase productions and nitrogen fixation. In this study, the ability of SUTN9‐2 to stimulate rice growth was investigated at different stages with N‐free and NH4NO3 under in vivo condition. The rice dry weight and chlorophyll content could be enhanced when SUTN9‐2 was inoculated in N‐free, especially at seedling stage (7 and 14 dai). The rice dry weight was also increased when SUTN9‐2 was inoculated with NH4NO3 at 7 and14 dai. The results of quantitative analysis of IAA and ACC deaminase were inconsistent with the expression of genes involved in IAA (nit) and ACC deaminase (acdS) productions. This inconsistently could implied that IAA and ACC deaminase produced from SUTN9‐2 do not directly affect rice growth, but other factors resulting from the production of IAA and ACC deaminase could be involved. Moreover, the expression of genes involved in nitrogen fixation (nifH and nifV) of SUTN9‐2 was also induced in rice tissues. This finding suggested that rice growth promotion may be supported by NH4NO3 together with nitrogen fixation by SUTN9‐2. Significance and Impact of the Study Indole‐3‐acetic acid, 1‐amino‐cyclopropane‐1‐carboxylic acid deaminase productions and nitrogen fixation may play important roles in rice growth promotion by endophytic SUTN9‐2, especially at early rice seedling growth stage, which has the potential to be used as rice seedling growth promoter in the system of rice intensification.

Bacteria exhibiting 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, which inhibits the biosynthesis of ethylene in higher plants, promote plant growth through the degradation of ethylene precursors, such as ACC. ACC deaminase activity in Bradyrhizobium sp. SUTN9-2 was enhanced by genetic engineering and adaptive laboratory evolution (ALE)-based methods. The transferal of a plasmid containing the acdR and acdS genes into SUTN9-2 was genetic engineering improved, while the ALE method was performed based on the accumulation of an adaptive bacterial population that continuously grew under specified growth conditions for a long time. ACC deaminase enzyme activity was 8.9-fold higher in SUTN9-2:pMG103::acdRS and 1.4-fold higher in SUTN9-2 (ACCDadap) than in the wild-type strain. The effects of increased activity were examined in the host plant (Vigna radiata (L.) R.Wilczek SUT1). The improved strains enhanced nodulation in early stage of plant growth. SUTN9-2:pMG103::acdRS also maintained nitrogen fixation under water deficit conditions and increased the plant biomass after rehydration. Changes in nucleotides and amino acids in the AcdS protein of SUTN9-2 (ACCDadap) were then investigated. Some nucleotides predicted to be located in the ACC-binding site were mutated. These mutations may have increased ACC deaminase activity, which enhanced both symbiotic interactions and drought tolerance and promoted recovery after rehydration more than lower ACC deaminase activity. Adaptive evolution represents a promising strategy for further applications in the field.

Bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase is a key factor for alleviating the plant ethylene biosynthesis, which is induced by stress. The ACC deaminase-improved strains of the rice endophytic Bradyrhizobium sp. SUTN9-2, SUTN9-2 (ACCDadap) and SUTN9-2:pMG103::acdRS, exhibit 1.4-and 8.9-fold higher ACC deaminase activity than the wild type, respectively (Sarapat S, Songwattana P, Longtonglang A, Umnajkitikorn K, Girdthai T, Tittabutr P, Boonkerd N, Teaumroong N. 2020. Effects of Increased 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity in Bradyrhizobium sp. SUTN9-2 on Mung Bean Symbiosis under Water Deficit Conditions. Microbes Environ. 35). The effects of these on rice growth under water deficit conditions were evaluated. The bacterial inoculations reduced ethylene synthesis, leading to a reduction in membrane destruction and the chlorophyll content of rice. Furthermore, the bacterial inoculations improved the leaf relative water content, survival, recovery rates, and improved the crop yield in field conditions. Therefore, the water deficit tolerance of rice was improved by controlling ethylene biosynthesis by improving ACC deaminase activity with endophytic SUTN9-2. Moreover, the SUTN9-2 (ACCDadap) strain can be used as a bio-inoculant under field conditions to enhance rice growth, grain yield, and enhance drought tolerance.