Long-term vegetation restoration promotes lignin phenol preservation and microbial anabolism in forest plantations: Implications for soil organic carbon dynamics.

Vegetation restoration contributes to soil organic carbon (C; SOC) sequestration through the accumulation of plant and microbial residues, but the mechanisms underlying this microbially mediated process are not well resolved. To depict the dynamics of plant- and microbial-derived C in restored forest ecosystems, soil samples were collected from Robinia pseudoacacia plantations of different stand ages (15, 25, 35, 45 years old) established on degraded wheat fields. The results showed that the degree of lignin phenol oxidation decreased with increasing stand age (P < 0.05), and hemicellulose-degrading genes were detected at higher relative abundances than other functional gene categories, indicating selective preservation of recalcitrant lignin phenols. Despite both glucosamine (R2 = 0.61, P < 0.001) and muramic acid (R2 = 0.37, P < 0.001) contents trending upward over time, fungal residual C accounted for a greater proportion of SOC compared with bacterial residual C. Accordingly, fungal residual C, which exhibited a similar response pattern as total microbial residual C to vegetation restoration, was considered a major contributor to the SOC pool. These results provided evidence that long-term vegetation restoration enhanced SOC sequestration in R. pseudoacacia forest by promoting the preservation of plant-derived lignin phenols and concomitant microbial anabolism. Partial least squares-discriminant analysis identified two important ecological clusters (i.e., modules) in the fungal network that profoundly influenced lignin phenol oxidation (P < 0.05) and microbial residual C accumulation (P < 0.01). Among the dominant taxa in microbial networks, the bacterial phyla Proteobacteria and Acidobacteriota had potential to degrade recalcitrant C compounds (e.g., cellulose, lignin), whereas the fungal phylum Ascomycota could outcompete for labile C fractions (e.g., dissolved organic C). Findings of this study can enable a mechanistic understanding of SOC stability driven by microbial turnover in restored forest ecosystems.

Broad environmental adaptation of abundant microbial taxa in Robinia pseudoacacia forests during long-term vegetation restoration.

Vegetation restoration has significant impacts on ecosystems, and a comprehensive understanding of microbial environmental adaptability could facilitate coping with ecological challenges such as environmental change and biodiversity loss. Here, abundant and rare soil bacterial and fungal communities were characterized along a 15-45-year chronosequence of forest vegetation restoration in the Loess Plateau region. Phylogenetic-bin-based null model analysis (iCAMP), niche breadth index, and co-occurrence network analysis were used to assess microbial community assembly and environmental adaptation of a Robinia pseudoacacia plantation under long-term vegetation restoration. The drift process governed community assembly of abundant and rare soil fungi and bacteria. With increasing soil total phosphorus content, the relative importance of drift increased, while dispersal limitation and heterogeneous selection exhibited opposite trends for abundant and rare fungi. Rare soil fungal composition dissimilarities were dominated by species replacement processes. Abundant microbial taxa had higher ecological niche width and contribution to ecosystem multifunctionality than rare taxa. Node property values (e.g., degree and betweenness) of abundant microbial taxa were substantially higher than those of rare microbial taxa, indicating abundant species occupied a central position in the network. This study provides insights into the diversity and stability of microbial communities during vegetation restoration in Loess Plateau. The findings highlight that abundant soil fungi and bacteria have broad environmental adaptation and major implications for soil multifunctionality under long-term vegetation restoration.

Temporal shifts in root exudates driven by vegetation restoration alter rhizosphere microbiota in Robinia pseudoacacia plantations.

Plant-soil-microbiota interactions mediated by root exudates regulate plant growth and drive rhizosphere microbial feedbacks. It remains unknown how root exudates affect rhizosphere microbiota and soil functions in the course of forest plantation restoration. The metabolic profiles of tree root exudates are expected to shift with stand age, leading to variation in rhizosphere microbiota structure, and in turn, potentially altering soil functions. To unravel the effects of root exudates, a multi-omics study was conducted using untargeted metabonomic profiling, high-throughput microbiome sequencing and functional gene array. The interactions among root exudates, rhizosphere microbiota and nutrient cycling-related functional genes were explored under 15- to 45-year-old Robinia pseudoacacia plantations in the Loess Plateau region of China. Root exudate metabolic profiles, rather than chemodiversity, markedly changed with an increase in stand age. A total of 138 age-related metabolites were extracted from a key module of root exudates. The relative contents of six biomarker metabolites, such as glucose-1-phosphate, gluconic acid and N-acetylneuraminic acid, increased distinctly over time. The biomarker taxa (16 classes) of rhizosphere microbiota varied in a time-sensitive manner, which played potential roles in nutrient cycling and plant health. Nitrospira, Alphaproteobacteria and Acidobacteria were enriched in the rhizosphere of older stands. Key root exudates influenced functional gene abundances in the rhizosphere via direct effects or indirectly through biomarker microbial taxa (e.g., Nitrososphaeria). Overall, root exudates and rhizosphere microbiota are essential for soil function maintenance in R. pseudoacacia plantation restoration.

New Insight into the Evolution of Symbiotic Genes in Black Locust-Associated Rhizobia.

Nitrogen fixation in legumes occurs via symbiosis with rhizobia. This process involves packages of symbiotic genes on mobile genetic elements that are readily transferred within or between rhizobial species, furnishing the recipient with the ability to interact with plant hosts. However, it remains elusive whether plant host migration has played a role in shaping the current distribution of genetic variation in symbiotic genes. Herein, we examined the genetic structure and phylogeographic pattern of symbiotic genes in 286 symbiotic strains of Mesorhizobium nodulating black locust (Robinia pseudoacacia), a cross-continental invasive legume species that is native to North America. We conducted detailed phylogeographic analysis and approximate Bayesian computation to unravel the complex demographic history of five key symbiotic genes. The sequencing results indicate an origin of symbiotic genes in Germany rather than North America. Our findings provide strong evidence of prehistoric lineage splitting and spatial expansion events resulting in multiple radiations of descendent clones from founding sequence types worldwide. Estimates of the timescale of divergence in North American and Chinese subclades suggest that black locust-specific symbiotic genes have been present in these continent many thousands of years before recent migration of plant host. Although numerous crop plants, including legumes, have found their centers of origin as centers of evolution and diversity, the number of legume-specific symbiotic genes with a known geographic origin is limited. This work sheds light on the coevolution of legumes and rhizobia.

Isolation, characterization, and selection of heavy metal-resistant and plant growth-promoting endophytic bacteria from root nodules of Robinia pseudoacacia in a Pb/Zn mining area.

Multiple heavy metals (HMs) commonly coexist in mining areas, which highlights the necessity to select multiple HM-resistant plant growth-promoting bacteria for improving phytoremediation efficiency. In this study, we isolated and characterized 82 endophytic bacteria from the root nodules of black locust (Robinia pseudoacacia) grown in a Pb-Zn mining area. There were 80 isolates showing resistance to four HMs, 0.01-18.0 mM/L for Cd, 0.2-40.0 mM/L for Zn, 0.3-2.2 mM/L for Pb, and 0.2-1.4 mM/L for Cu. Indole-3-acetic acid production, siderophore production, and 1-aminocyclopropane-1-carboxylate deaminase activity were detected in 43, 50, and 17 isolates, respectively. Two symbiotic isolates selected with the highest potential for HM resistance and PGP traits, designated Mesorhizobium loti HZ76 and Agrobacterium radiobacter HZ6, were evaluated for promotion of plant growth and metal uptake by R. pseudoacacia seedlings grown in pots containing different levels of Cd, Zn, Pb, or Cu. HZ76 significantly increased plant shoot biomass, while HZ6 did not, compared with non-inoculated controls. The results indicate that inoculation with HZ76 or HZ6 relieved HM stress in the plants, depending on the type and concentration of HM in the treatment. Mesorhizobium loti HZ76 may be a better candidate for application in phytoremediation than A. radiobacter HZ6. The microsymbiosis between HM-resistant rhizobia and R. pseudoacacia is an interesting mutualistic system for phytoremediation in mining areas contaminated with multiple HMs.

Herbaspirillum robiniae sp. nov., isolated from root nodules of Robinia pseudoacacia in a lead-zinc mine.

A novel endophytic bacterium, designated strain HZ10T, was isolated from root nodules of Robinia pseudoacacia growing in a lead-zinc mine in Mianxian County, Shaanxi Province, China. The bacterium was Gram-stain-negative, aerobic, motile, slightly curved- and rod-shaped, methyl red-negative, catalase-positive, and did not produce H2S. Strain HZ10T grew at 4-45 °C (optimum, 25-30 °C), pH 5-9 (optimum, pH 7-8) and 0-1 % (w/v) NaCl. The major fatty acids were identified as C16 : 0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), and the quinone type was Q-8. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of the genomic DNA was 64.9 mol% based on the whole genome sequence. According to the 16S rRNA gene sequence analysis, the closest phylogenetic relative to strain HZ10T is Herbaspirillum chlorophenolicum CPW301T (98.72 % sequence identity). Genome relatedness of the type strains H. chlorophenolicum CPW301T, Herbaspirillum seropedicae Z67T and Herbaspirillum aquaticum IEH 4430T, was quantified by using the average nucleotide identity (86.9-88.0 %) and a genome-to-genome distance analysis (26.6 %-29.3 %), with both strongly supporting the notion that strain HZ10T belongs to the genus Herbaspirillum as a novel species. Based on the results from phylogenetic, chemotaxonomic and physiological analyses, strain HZ10T represents a novel Herbaspirillum species, for which the name Herbaspirillum robiniae sp. nov. is proposed. The type strain is HZ10T (=JCM 31754T=CCTCC AB 2014352T).

Enhanced phytoremdiation of Robinia pseudoacacia in heavy metal-contaminated soils with rhizobia and the associated bacterial community structure and function.

Heavy metals can cause serious contamination of soils, especially in mining regions. A detailed understanding of the effects of heavy metals on plants and root-associated microbial communities could help to improve phytoremediation systems. In this study, black locust (Robinia pseudoacacia) seedlings with or without rhizobial inoculation were planted in soils contaminated with different levels of heavy metals. Bacterial communities in rhizosphere and bulk soil samples were analyzed using 16S rRNA gene sequencing on the Illumina MiSeq platform and shotgun metagenome sequencing on the Illumina HiSeq platform. Soil bacterial communities varied significantly depending on the level of soil contamination, and planting also had some influence. Although inoculation of Mesorhizobium loti HZ76 (a natural microsymbiont of R. pseudoacacia) was a relatively minor factor, it did influence the soil bacterial community. Under the selective pressure, plant growth promotion-related biomarkers in the rhizosphere increased after inoculation compared with non-inoculated controls, especially those associated with Mesorhizobium, Variovorax, Streptomyces, and Rhodococcus genera. Genes encoding ATP-binding cassette transporters were up-regulated in the rhizosphere after inoculation compared with genes related to sulfur/nitrogen metabolism. These results provide insight into soil bacterial communities and their functions in the R. pseudoacacia rhizosphere in response to rhizobial inoculation and heavy metal contamination. This knowledge may prove useful for improving phytoremediation of metal-contaminated soils.

Mitsuaria noduli sp. nov., isolated from the root nodules of Robinia pseudoacacia in a lead-zinc mine.

A novel endophytic bacterium, designated strain HZ7T, was isolated from the root nodules of Robinia pseudoacacia growing in a lead-zinc mine in Mianxian County, Shaanxi Province, China. Cells were Gram-reaction-negative, aerobic, motile, rod-shaped, methyl-red-negative, catalase-positive, positive for chitosan-degrading activity and did not produce H2S. Strain HZ7T grew at 4-45 °C (optimum 25-30 °C), at pH 5-9 (optimum pH 7-8) and with 0-1 % (w/v) NaCl. The quinone type was ubiquinone 8 (UQ-8). The major fatty acids were identified as C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The G+C content of the genomic DNA was 68.5 mol% by whole genome sequencing. According to 16S rRNA gene sequence analysis, the closest phylogenetic relative was Mitsuaria chitosanitabida 3001T (99.05 % similarity). Genome relatedness was computed using average nucleotide identity and genome-to-genome distance analysis, both of which strongly supported strain HZ7Tas belonging to the genus Mitsuaria as a representative of a novel species. On the basis of phylogenetic analysis, chemotaxonomic data and physiological characteristics, strain HZ7T represents a novel species of the genus Mitsuaria, for which the name Mitsuaria noduli sp. nov. is proposed. The type strain is HZ7T (=JCM 31671T=CCTCC AB 2014353T).

Interactions of plant growth-promoting rhizobacteria and soil factors in two leguminous plants.

Although the rhizomicrobiome has been extensively studied, little is known about the interactions between soil properties and the assemblage of plant growth-promoting microbes in the rhizosphere. Herein, we analysed the composition and structure of rhizomicrobiomes associated with soybean and alfalfa plants growing in different soil types using deep Illumina 16S rRNA sequencing. Soil pH, P and K significantly affected the composition of the soybean rhizomicrobiome, whereas soil pH and N had a significant effect on the alfalfa rhizomicrobiome. Plant biomass was influenced by plant species, the composition of the rhizomicrobiome, soil pH, N, P and plant growth stage. The beta diversity of the rhizomicrobiome was the second most influential factor on plant growth (biomass). Rhizomicrobes associated with plant biomass were identified and divided into four groups: (1) positively associated with soybean biomass; (2) negatively associated with soybean biomass; (3) positively associated with alfalfa biomass; and (4) negatively associated with alfalfa biomass. Genera assemblages among the four groups differentially responded to soil properties; Group 1 and Group 2 were significantly correlated with soil pH and P, whereas Group 3 and Group 4 were significantly correlated with soil N, K and C. The influence of soil properties on the relative abundance of plant biomass-associated rhizomicrobes differed between soybean and alfalfa. The results suggest the rhizomicrobiome has a pronounced influence on plant growth, and the rhizomicrobiome assemblage and plant growth-associated microbes are differentially structured by soil properties and leguminous plant species.

Microbial communities in riparian soils of a settling pond for mine drainage treatment.

Mine drainage leads to serious contamination of soil. To assess the effects of mine drainage on microbial communities in riparian soils, we used an Illumina MiSeq platform to explore the soil microbial composition and diversity along a settling pond used for mine drainage treatment. Non-metric multidimensional scaling analysis showed that the microbial communities differed significantly among the four sampling zones (influent, upstream, downstream and effluent), but not seasonally. Constrained analysis of principal coordinates indicated heavy metals (zinc, lead and copper), total sulphur, pH and available potassium significantly influenced the microbial community compositions. Heavy metals were the key determinants separating the influent zone from the other three zones. Lower diversity indices were observed in the influent zone. However, more potential indicator species, related to sulphur and organic matter metabolism were found there, such as the sulphur-oxidizing genera Acidiferrobacter, Thermithiobacillus, Limnobacter, Thioprofundum and Thiovirga, and the sulphur-reducing genera Desulfotomaculum and Desulfobulbus; the organic matter degrading genera, Porphyrobacter and Paucimonas, were also identified. The results indicated that more microorganisms related to sulphur- and carbon-cycles may exist in soils heavily contaminated by mine drainage.

Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China.

A total of 155 nodule isolates that originated from seven sites in Northwest China were characterized by PCR-RFLP of the 16S rRNA gene and sequence analysis of multiple core genes (16S rRNA, recA, atpD, and glnII) in order to investigate the diversity and biogeography of Glycine soja-nodulating rhizobia. Among the isolates, 80 were Ensifer fredii, 19 were Ensifer morelense, 49 were Rhizobium radiobacter, and 7 were putative novel Rhizobium species. The phylogenies of E. fredii and E. morelense isolates in a concatenate tree (assembly of all housekeeping genes) were generally consistent with those in individual gene trees. However, incongruence was found in the phylogenies of the different genes of Rhizobium isolates, indicating that lateral transfer or recombination possibly occurred in these gene loci. Despite their species identity, all the isolates in this study formed a single lineage related to E. fredii in nodAand nifH gene phylogenies, which also indicated that the symbiotic genes were laterally transferred between different species. Biogeographic patterns were found at the species and strain genomic type levels, as revealed by BOXA1R fingerprinting, demonstrating that the evolution of rhizobial populations in different geographic locations was related to soil types, altitude and spatial effects. This study is the first to report that E. morelense, R. radiobacter, and Rhizobium sp. are microsymbionts of G. soja, as well as showing that the diversity of G. soja rhizobia is enhanced and new rhizobia have evolved in Northwest China.