Xiaoyun Liu

In order to improve the quality of fresh alfalfa silage, we investigated the effects of jujube powder (JP) addition on the ensiling process of high-moisture alfalfa (75% moisture content) over fermented periods of 1, 5, 15, 30, 45 and 60 days. We evaluated chemical composition, bacterial and fungal community dynamics and diversity, co-occurrence networks, microbial functionality and the ability to restrict pathogenic contamination. Results showed that JP addition optimized key fermentation parameters (pH, lactic acid, volatile fatty acids, and NH ₃ -N) in high-moisture alfalfa, achieving levels comparable to those of traditional wilted alfalfa qualified silage. JP also increased the abundance of beneficial bacteria while suppressing undesirable organisms. A dominant lactic acid bacteria (LAB) combination— Lactobacillus-Pediococcus-Lactococcus was observed, showing strong positive correlations with silage quality indicators (higher qualified LAB counts and NH ₃ -N content, and lower butyric and propionic acid levels). Metabolic pathway analysis revealed that JP supplementation effectively restricted the amino acid metabolism in harmful bacteria while significantly enhancing key carbohydrate-utilization pathways. Notably, D-alanine was unregulated in JP-treated, supporting the survival and function of LAB as the primary fermentation agents. In contrast, the biosynthesis and degradation of branched-amino acids (valine, leucine and isoleucine) remained stable, contributing to better protein preservationl. Furthermore, JP addition helped control plant and animal pathogens and limited saprotrophic activity. In conclusion, by adding JP, the energy- and labor-intensive pre-wilting procedure could be replaced for optimizing the high-moisture alfalfa silage. This work also identifies Lactobacillus-Pediococcus-Lactococcus as a promising microbial combination for future inoculant development.

Four pigment-producing rhizobial strains nodulating Sesbania cannabina (Retz.) Poir. formed a unique group in genus Microvirga in the phylogeny of a 16S rRNA gene and five housekeeping genes (gyrB, recA, dnaK, glnA, and atpD) in a genome analysis, phenotypic characteristics analysis, and chemotaxonomic analysis. These four strains shared as high as 99.3% similarity with Microvirga tunisiensis LmiM8^T in the 16S rRNA gene sequence and, in an MLSA, were subdivided into two clusters, ANI (genome average nucleotide) and dDDH (digital DNA–DNA hybridization) which shared sequence similarities lower than the species thresholds with each other and with the reference strains for related Microvirga species. The polar lipids elucidated that phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin were the main components for strain SWF67558^T and for strain HBU65207^T, with the exception of PC. SWF67558^T and HBU65207^T strains had similar predominant cellular fatty acids, including C16:0, C18:0, summed feature 2, and summed feature8, but with different contents. In addition, all the four novel strains produced pink-pigment, and the main coloring material extract from strain SWF67558^T was identified as zeaxanthin, which presented antioxidant ability and reduction power. With all the phylogenetic and phenotypic divergency, we proposed these pink-pigmented symbiotic bacteria as two novel species, named Microvirga sesbaniae sp. nov. and Microvirga yunnanensis sp. nov., with SWF67558^T (=KCTC82331^T=GDMCC1.2024^T) and HBU65207^T (=KCTC92125^T=GDMCC1.2023^T) as the type strains, respectively.

The double selection of environment adaptation and host specificity forced the diversification of rhizobia in nature. In the tropical region of China, Medicago polymorpha and Medicago lupulina are widely distributed, particularly in purple soil. However, the local distribution and diversity of rhizobia associated with these legumes has not been systematically investigated. To this end, root nodules of M. polymorpha and M. lupulina grown in purple soil at seven locations in Yunnan Province of China were collected for rhizobial isolation. The obtained rhizobia were characterized by RFLP of 16S–23S rRNA intergenic spacer, BOXAIR fingerprinting, and phylogeny of housekeeping and symbiosis genes. As result, a total of 91 rhizobial strains were classified into species Sinorhizobium medicae and S. meliloti, while three nodC gene types were identified among them. S. medicae containing nodC of type I was dominant in farmlands associated with M. polymorpha; while S. meliloti harboring nodC of type III was dominant in wild land nodulated by M. lupulina. For both rhizobial species, greater genetic diversity was detected in the populations isolated from their preferred host plant. A high level of genetic differentiation was observed between the two Sinorhizobium species, and gene flow was evident within the populations of the same species derived from different soil types, indicating that rhizobial evolution is likely associated with the soil features. To examine the effects of environmental features on rhizobial distribution, soil physicochemical traits and rhizobial genotypes were applied for constrained analysis of principle coordinates, which demonstrated that soil features like pH, nitrogen and sodium were the principle factors governing the rhizobial geographical distribution. Altogether, both S. medicae and S. meliloti strains could naturally nodulate with M. polymorpha and M. lupulina, but the rhizobium-legume symbiosis compatibility determined by both the host species and soil factors was also highlighted.

Aims The nitrogen-fixing rhizobia associated with Medicago polymorpha and M. lupulina in Yunnan, China have been poorly documented. This study aims to analyze the diversity of rhizobia isolated from these two Medicago species and investigate the impact of abiotic (soil properties) and biotic (plant hosts) factors on Medicago-associated rhizobia in this region. Methods 91 rhizobial isolates were characterized by RFLP of 16S rDNA and 16S–23S IGS, BOX-PCR fingerprinting, nodulation assays and phylogeny analyses based on housekeeping and symbiosis genes. The genetic diversity of the rhizobial isolates was assessed by the BOX AIR pattern and Shannon index. Additionally, the correlation of soil properties and rhizobial distribution was determined by the constrained analysis of principle coordinates (CAP) based on Bray-Curtis distance of presence/absence (PA) transformed species data. Results All the tested strains fell in the genus Ensifer and divided into two species, E. medicae and E. meliloti. Both host plants and soil properties contributed to the rhizobial diversity. For either E. meliloti or E. medicae, isolates from native host plants tended to be more genetically diverse than those of the same species from non-native hosts. The soil edaphic factor analysis elucidated that nitrogen, organic matter as well as Ca²⁺ and Na⁺ are the key factors to shape the biogeographical distribution of rhizobia. Conclusions This study evidenced the microsymbiont preference of M. polymorpha to E. medicae and M. lupulina to E. meliloti, but also revealed the considerable impacts of both plant hosts and soil factors on the rhizobial diversity and biodistribution.

In order to investigated diversity and geographic distribitution of rhizobia associated with invasive Mimosa species, Mimosa nodules and soils around the plants were sampled from five provinces in southern China. In total, 361 isolates were obtained from Mimosa pudica and Mimosa diplotricha in 25 locations. A multi-locus sequence analysis (MLSA) including 16S rRNA, atpD, dnaK, glnA, gyrB, and recA identified the isolates into eight genospecies corresponding to Paraburkhleria mimosarum, Paraburkholderia phymatum, Paraburkholeria carbensis, Cupriavidus taiwanensis, Cupriavidus sp., Rhizobium altiplani, Rhizobium mesoamericanum, and Rhizobium etli. The majority of the isolates were Cupriavidus (62.6%), followed by Paraburkholderia (33.5%) and Rhizobium (2.9%). Cupriavidus strains were more predominant in nodules of M. diplotricha (76.2) than in M. pudica (59.9%), and the distribution of P. phymatum in those two plant species was reverse (3.4:18.2%). Four symbiotypes were defined among the isolates based upon the phylogeny of nodA-nifH genes, represented by P. mimosarum, P. phymatum–P. caribensis, Cupriavidus spp., and Rhizobium spp. The species affiliation and the symbiotype division among the isolates demonstrated the multiple origins of Mimosa rhizobia in China: most were similar to those found in the original centers of Mimosa plants, but Cupriavidus sp. might have a local origin. The unbalanced distribution of symbionts between the two Mimosa species might be related to the soil pH, organic matter and available nitrogen; Cupriavidus spp. generally dominated most of the soils colonized by Mimosa in this study, but it had a particular preference for neutral-alkaline soils with low fertility whereas. While Paraburkholderia spp. preferred more acidic and fertile soils. The Rhizobium spp. tended to prefer neutral–acidic soils with high fertility soils.

Rhizobia were isolated from invasive Mimosa spp. (M. diplotricha and M. pudica) in Dehong district of the province of Yunnan in subtropical southern

In order to investigate the genetic diversity of rhizobia associated with various exotic and invasive species in tropical mainland China, 116 bacterial isolates were obtained from Mimosa root nodules collected from Sishuangbanna and Yuanjiang districts of Yunnan province. Isolated rhizobia were characterized by RFLP analysis of 16S rRNA genes, SDS-PAGE of whole-cell proteins and BOX-PCR. Most of the isolated strains were identified as β-rhizobia belonging to diverse populations of Burkholderia and Cupriavidus, and the phylogenetic relationships of their 16S rRNA gene sequences showed that they were closely related to one of four β-rhizobia species: Burkholderia phymatum, B. mimosarum, B. caribensis or Cupriavidus taiwanensis. Additionally, among the 116 isolates, 53 different whole-cell SDS-PAGE profiles and 30 distinct BOX-PCR genotypic patterns were detected, which demonstrated the genetic and phenotypic diversity found within these Burkholderia and Cupriavidus strains. To the best of our knowledge, this is the first report that β-rhizobia are extant and possibly widespread on the Chinese mainland and nodulate easily with Mimosa plants. We also find it especially interesting that this appears to be the first report from mainland China of Cupriavidus symbionts of Mimosa. These records enrich our knowledge and understanding of the geographical distribution and diversity of these bacteria.

The terms alpha- and beta-rhizobia were proposed to distinguish the rhizobial alpha- and beta-Proteorhizobia respectively. In this review we present recently development of rhizobial taxonomy about beta-rhizobia in the genus Burkholeria, Cupriavidus isolated from Mimosa and alpha-Proteorhizobia belonging to Methylobacterium, Devosia, Ochrobactrum, Phyllobacterium and Blastobacter lineage. Furthermore, we also introduced the evolution of symbiosis related genes such as nodA and nifH, and posed some questions in detecting rhizobia. Finally, we discuss the prospects of research on rhizoiba in future.