Summary: Nitrogen‐fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N₂‐fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen‐fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane‐bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species‐rich pantropical legume clade.

The objective of this research was to evaluate the PGPR effect on nodulation and nitrogen-fixing efficiency of soybean (Glycine max (L.) Merr.) by co-inoculation with Bradyrhizobium diazoefficiens USDA110. Co-inoculation of Bacillus velezensis S141 with USDA110 into soybean resulted in enhanced nodulation and N2-fixing efficiency by producing larger nodules. To understand the role of S141 on soybean and USDA110 symbiosis, putative genes related to IAA biosynthesis were disrupted, suggesting that co-inoculation of USDA110 with S141ΔyhcX reduces the number of large size nodules. It was revealed that yhcX may play a major role in IAA biosynthesis in S141 as well as provide a major impact on soybean growth promotion. The disruption of genes related to cytokinin biosynthesis and co-inoculation of USDA110 with S141ΔIPI reduced the number of very large size nodules, and it appears that IPI might play an important role in nodule size of soybean–Bradyrhizobium symbiosis. However, it was possible that not only IAA and cytokinin but also some other substances secreted from S141 facilitate Bradyrhizobium to trigger bigger nodule formation, resulting in enhanced N2-fixation. Therefore, the ability of S141 with Bradyrhizobium co-inoculation to enhance soybean N2-fixation strategy could be further developed for supreme soybean inoculants.

Japan's largest platform for academic e-journals: J-STAGE is a full text database for reviewed academic papers published by Japanese societies

Plant colonization by bradyrhizobia is found not only in leguminous plants but also in nonleguminous species such as rice. To understand the evolution of the endophytic symbiosis of bradyrhizobia, the effect of the ecosystems of rice plantations on their associations was investigated. Samples were collected from various rice ( Oryza sativa ) tissues and crop rotational systems. The rice endophytic bradyrhizobia were isolated on the basis of oligotrophic properties, selective medium, and nodulation on siratro ( Macroptilium atropurpureum ). Six bradyrhizobial strains were obtained exclusively from rice grown in a crop rotational system. The isolates were separated into photosynthetic bradyrhizobia (PB) and nonphotosynthetic bradyrhizobia (non-PB). Thai bradyrhizobial strains promoted rice growth of Thai rice cultivars better than the Japanese bradyrhizobial strains. This implies that the rice cultivars possess characteristics that govern rice-bacterium associations. To examine whether leguminous plants in a rice plantation system support the persistence of rice endophytic bradyrhizobia, isolates were tested for legume nodulation. All PB strains formed symbioses with Aeschynomene indica and Aeschynomene evenia . On the other hand, non-PB strains were able to nodulate Aeschynomene americana , Vigna radiata , and M. atropurpureum but unable to nodulate either A. indica or A. evenia . Interestingly, the nodABC genes of all of these bradyrhizobial strains seem to exhibit low levels of similarity to those of Bradyrhizobium diazoefficiens USDA110 and Bradyrhizobium sp. strain ORS285. From these results, we discuss the evolution of the plant-bradyrhizobium association, including nonlegumes, in terms of photosynthetic lifestyle and nod -independent interactions.

Ethylene is synthesized at accelerated rate in response to stress conditions, and regarded as a stress hormone that inhibits overall plant growth. Apparently, the amount of ethylene can be reduced by using silver thiosulfate (STS) or bacteria containing 1-amino-cyclopropane-1-carboxylate (ACC) deaminase. In this study, we examined whether different concentrations of STS differently prevent legume growth under various environment stress conditions, and whether co-inoculation of mungbean (Vigna radiata (L.) R. Wilczek) by Bradyrhizobium and rhizobacteria containing ACC deaminase also alleviates legume stress. Mungbean is one of the most responsive plants among tested legume species, and different varieties of mungbean also respond differently to STS. Stress conditions and inappropriate concentrations of STS affect plant growth and symbiosis, while a suitable concentration of STS supports plant growth under stress conditions. Three isolates of bacteria containing ACC deaminase, Enterobacter sp. ACC1, Enterobacter sp. ACC2, and Chryseobacterium sp. ACC3, were selected according to their ACC deaminase activity and resistance abilities to high temperature, drought, and salt stress conditions. Interestingly, the gene encoding ACC deaminase, acdS, of selected ACC deaminase bacteria was highly induced when the cell culture was exposed to stress conditions. It coincided with co-inoculation of plants by Bradyrhizobium and rhizobacteria containing ACC deaminase. This co-inoculation obviously alleviated the stress of plants growing under stress conditions. These results revealed the role of bacteria containing ACC deaminase that can adjust its expression of ACC deaminase to different levels of stress conditions, which will be useful in agriculture under global warming.

The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif and T3SS genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (ΩrhcN) to nodulate legumes belonging to Dalbergioid, Millettioid and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice.