Microbial community structure and functional genes drive soil priming effect following afforestation.

Afforestation substantially modifies native soil organic carbon (SOC) decomposition via plant carbon inputs (the priming effect), and in turn, triggers vital biogeochemical processes that influence the regulation of soil carbon dynamics. Soil microbes are crucial in regulating the direction and magnitude of the priming effect. In the present study, we performed metagenomic sequencing and 13C-glucose labeling analyses of microbial communities and priming effects across a Robinia pseudoacacia afforestation chronosequence (14-, 20-, 30-, and 45-year-old stands) in the Loess Plateau in China, with adjacent farmland being selected as a control. Our results revealed that the cumulative priming effect across five sites along the afforestation chronosequence initially increased and approached a peak value in the 20-year-old stand, after which it declined. The priming effect was predominantly driven by the microbial community structure (i.e., the fungal-to-bacterial ratios and relative abundances of Proteobacteria and Actinobacteria), and stable C decomposition genes and C-degrading enzymes. Specifically, among the key functional genes correlated with priming effect, which were identified in orders Rhizobiales and Pseudonocardiales, considerably promoted SOC priming. Overall, our findings indicate that afforestation alters soil microbial community structure and function, particularly with respect to enhancing stable soil C decomposition genes, which may promote SOC priming. The findings of the present study could enhance our understanding of fresh C input-induced changes associated with C mineralization in the context of the revegetation of ecologically fragile areas.

Biotic and abiotic factors causing the collapse of Robinia pseudoacacia L. veteran trees in urban environments.

Robinia pseudoacacia L. is an interesting example of how one plant species can be considered invasive or useful depending on its environment. In the past this tree species was planted for decorative purposes and for wood in Poland. For many years it was recommended in poor and degraded habitats because it facilitated late-successional plant species. The aim of this study was to verify if black locust can still be regarded as a resistant tree species in urban greenery. The health condition of old tree specimens growing along streets and in parks was compared. The occurrence of pests and pathogens on R. pseudoacacia trees was assessed and the content of mineral elements in leaves was examined. The research results showed that the health of black locust trees growing in the urban environment in Polish cities, especially along streets (in comparison to park sites), deteriorated significantly due to the interaction of harmful biotic and abiotic factors. Increased level of toxic metals (Fe, Zn, Pb, Mn and Cd) in plant tissues and the accumulation of pests and pathogens negatively influenced the health of R. pseudoacacia.

Effects of sodium sulfide application on the growth of Robinia pseudoacacia, heavy metal immobilization, and soil microbial activity in Pb-Zn polluted soil.

Sodium sulfide (Na2S) is usually used as an amendment in industrial sewage treatment. To evaluate the effects of Na2S on the growth of Robinia pseudoacacia (black locust), heavy metal immobilization, and soil microbial activity, the R. pseudoacacia biomass and nutrient content and the soil heavy metal bioavailability, enzyme activity, and arbuscular mycorrhizal (AM) fungal community were measured by a single-factor pot experiment. The Pb-Zn-contaminated soil was collected from a Pb-Zn mine that had been remediated by R. pseudoacacia for five years. Three pollution levels (unpolluted, mildly polluted, and severely polluted) were evaluated by the pollution load index. Na2S application increased the shoot biomass under severe and mild contamination. In soil, Na2S application decreased the bioavailable Pb and Zn contents under severe and mild contamination, which resulted in a decrease in the Pb and Zn content in R. pseudoacacia. However, Na2S application did not affect the total Pb content per plant and enhanced the total Zn content per plant because of the higher biomass of the plants under Na2S application. Increased phosphatase activity and increased available phosphorous content may promote the uptake of phosphorus in R. pseudoacacia. Moreover, Na2S application is beneficial to the diversity of AM fungi under mild and severe pollution. Overall, Na2S application has great potential for enhancing soil heavy metal immobilization, enhancing soil microbial activity, and improving the growth of R. pseudoacacia in polluted soils. Therefore, Na2S is suitable for use in Pb-Zn remediation to ameliorate environmental heavy metal pollution.

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.

Soil microbial community structure in the rhizosphere of Robinia pseudoacacia L. seedlings exposed to elevated air temperature and cadmium-contaminated soils for 4 years.

The co-occurrence of heavy metal contamination of soils and increasing air temperature can affect the microbial community in rhizosphere soils by altering the allocation of plant photosynthates to roots. Here, we investigated the community structure of bacteria, fungi, ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in the rhizosphere of Robinia pseudoacacia L. seedlings exposed to elevated air temperature (+1.99 °C) and cadmium (Cd) for 4 years. Elevated temperature increased the richness of bacterial and AOA communities by 15.1% to 43.8% and by 1.4% to 18.6%, respectively, and decreased fungal and AOB richness by 3.7% to 28.7% and by 2.1% to 30.6%, respectively, under Cd exposure. Elevated temperature combined with Cd exposure decreased fungal diversity by 1.5% to 14.0%. However, elevated temperature decreased the diversity of bacteria, AOB and AOA by 1.4%, 17.4% and 10.1%, respectively, under 1.0 mg Cd kg-1 dry soil and increased the diversity of these taxa by 1.5%, 15.3% and 9.2%, respectively, under 5.0 mg Cd kg-1 dry soil relative to Cd exposure alone. Elevated temperature led to increased abundance of genera such as Methylobacterium, Stenotrophomonas, and Archangium and decreased abundance of genera including Ramlibacter, Microascus and Nitrosospira under Cd exposure. Over all, 4 years of exposure to elevated temperature had a greater effect on the community structure of bacteria, fungi, AOB and AOA when combined with Cd pollution.

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).

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).

Changes in arbuscular mycorrhizal fungal attributes along a chronosequence of black locust (Robinia pseudoacacia) plantations can be attributed to the plantation-induced variation in soil properties.

Arbuscular mycorrhizal (AM) fungi form symbioses with the majority of terrestrial plant species, and their community influences many important ecosystem processes, including ecological succession. Understanding the successional changes of AM fungal communities in afforested zones over time is of primary interest in forest ecology. Black locust (Robinia pseudoacacia) has been widely planted on the Loess Plateau of China to prevent soil erosion. We sampled fine roots and rhizosphere soils in black locust plantations consisting of stands of 0, 11, 23, 35 and 46years of age to measure soil properties, AM fungal colonization level, and spore density and to describe the composition of AM fungal communities in roots and soils using 454 sequencing. With increasing stand age, AM fungal spore density and soil NO3-N and available K contents increased, dehydrogenase and alkaline phosphatase activities decreased, and soil catalase activity and the level of root colonization by arbuscules and hyphae first increased and then decreased. Roots and soils hosted different AM fungal diversity and communities. In soils, AM fungal diversity and community composition did not vary with stand age. In roots, the relative abundance of Claroideoglomus, together with Chao1 richness and OTU richness, peaked at the intermediate stage (35years) and then declined, and the relative abundance of Glomus decreased linearly with tree age, whereas the relative abundance of the dominant genus Rhizophagus did not vary with stand age. Soil available K and NO3-N largely explained the shift in the structure of the root-colonizing AM fungal community along the chronosequence. Soil enzyme activities were also associated with changes in AM fungal spore abundance and root colonization level. All the results presented here suggest that the successional changes in AM fungal communities in black locust plantations occurring over time can largely be attributed to plantation-induced changes in soil nutrient levels.

Specialist nectar-yeasts decline with urbanization in Berlin.

Nectar yeasts are common inhabitants of insect-pollinated flowers but factors determining their distribution are not well understood. We studied the influence of host identity, environmental factors related to pollution/urbanization, and the distance to a target beehive on local distribution of nectar yeasts within Robinia pseudoacacia L. and Tilia tomentosa Moench in Berlin, Germany. Nectar samples of six individuals per species were collected at seven sites in a 2 km radius from each target beehive and plated on YM-Agar to visualise the different morphotypes, which were then identified by sequencing a section of the 26S rDNA gene. Multivariate linear models were used to analyze the effects of all investigated factors on yeast occurrence per tree. Yeast distribution was mainly driven by host identity. The influence of the environmental factors (NO2, height of construction, soil sealing) strongly depended on the radius around the tree, similar to the distance of the sampled beehive. Incidence of specialist nectar-borne yeast species decreased with increasing pollution/urbanization index. Given that specialist yeast species gave way to generalist yeasts that have a reduced dependency on pollinators for between-flower dispersal, our results indicate that increased urbanization may restrict the movement of nectar-specialized yeasts, via limitations of pollinator foraging behavior.

Elevated CO2 increases glomalin-related soil protein (GRSP) in the rhizosphere of Robinia pseudoacacia L. seedlings in Pb- and Cd-contaminated soils.

Glomalin-related soil protein (GRSP), which contains glycoproteins produced by arbuscular mycorrhizal fungi (AMF), as well as non-mycorrhizal-related heat-stable proteins, lipids, and humic materials, is generally categorized into two fractions: easily extractable GRSP (EE-GRSP) and total GRSP (T-GRSP). GRSP plays an important role in soil carbon (C) sequestration and can stabilize heavy metals such as lead (Pb), cadmium (Cd), and manganese (Mn). Soil contamination by heavy metals is occurring in conjunction with rising atmospheric CO2 in natural ecosystems due to human activities. However, the response of GRSP to elevated CO2 combined with heavy metal contamination has not been widely reported. Here, we investigated the response of GRSP to elevated CO2 in the rhizosphere of Robinia pseudoacacia L. seedlings in Pb- and Cd-contaminated soils. Elevated CO2 (700 µmol mol-1) significantly increased T- and EE- GRSP concentrations in soils contaminated with Cd, Pb or Cd + Pb. GRSP contributed more carbon to the rhizosphere soil organic carbon pool under elevated CO2 + heavy metals than under ambient CO2. The amount of Cd and Pb bound to GRSP was significantly higher under elevated (compared to ambient) CO2; and elevated CO2 increased the ratio of GRSP-bound Cd and Pb to total Cd and Pb. However, available Cd and Pb in rhizosphere soil under increased elevated CO2 compared to ambient CO2. The combination of both metals and elevated CO2 led to a significant increase in available Pb in rhizosphere soil compared to the Pb treatment alone. In conclusion, increased GRSP produced under elevated CO2 could contribute to sequestration of soil pollutants by adsorption of Cd and Pb.

Responsiveness of soil nitrogen fractions and bacterial communities to afforestation in the Loess Hilly Region (LHR) of China.

In the present paper, we investigated the effects of afforestation on nitrogen fractions and microbial communities. A total of 24 soil samples were collected from farmland (FL) and three afforested lands, namely Robinia pseudoacacia L (RP), Caragana korshinskii Kom (CK), and abandoned land (AL), which have been arable for the past 40 years. Quantitative PCR and Illumina sequencing of 16S rRNA genes were used to analyze soil bacterial abundance, diversity, and composition. Additionally, soil nitrogen (N) stocks and fractions were estimated. The results showed that soil N stock, N fractions, and bacterial abundance and diversity increased following afforestation. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla of soil bacterial compositions. Overall, soil bacterial compositions generally changed from Actinobacteria (Acidobacteria)-dominant to Proteobacteria-dominant following afforestation. Soil N fractions, especially for dissolved organic nitrogen (DON), were significantly correlated with most bacterial groups and bacterial diversity, while potential competitive interactions between Proteobacteria (order Rhizobiales) and Cyanobacteria were suggested. In contrast, nitrate nitrogen (NO3(-)-N) influenced soil bacterial compositions less than other N fractions. Therefore, the present study demonstrated that bacterial diversity and specific species respond to farmland-to-forest conversion and hence have the potential to affect N dynamic processes in the Loess Plateau.

Symbiosis of Arbuscular Mycorrhizal Fungi and Robinia pseudoacacia L. Improves Root Tensile Strength and Soil Aggregate Stability.

Robinia pseudoacacia L. (black locust) is a widely planted tree species on Loess Plateau for revegetation. Due to its symbiosis forming capability with arbuscular mycorrhizal (AM) fungi, we explored the influence of arbuscular mycorrhizal fungi on plant biomass, root morphology, root tensile strength and soil aggregate stability in a pot experiment. We inoculated R. pseudoacacia with/without AM fungus (Rhizophagus irregularis or Glomus versiforme), and measured root colonization, plant growth, root morphological characters, root tensile force and tensile strength, and parameters for soil aggregate stability at twelve weeks after inoculation. AM fungi colonized more than 70% plant root, significantly improved plant growth. Meanwhile, AM fungi elevated root morphological parameters, root tensile force, root tensile strength, Glomalin-related soil protein (GRSP) content in soil, and parameters for soil aggregate stability such as water stable aggregate (WSA), mean weight diameter (MWD) and geometric mean diameter (GMD). Root length was highly correlated with WSA, MWD and GMD, while hyphae length was highly correlated with GRSP content. The improved R. pseudoacacia growth, root tensile strength and soil aggregate stability indicated that AM fungi could accelerate soil fixation and stabilization with R. pseudoacacia, and its function in revegetation on Loess Plateau deserves more attention.

The roles of arbuscular mycorrhizal fungi (AMF) in phytoremediation and tree-herb interactions in Pb contaminated soil.

Understanding the roles of arbuscular mycorrhizal fungi (AMF) in plant interaction is essential for optimizing plant distribution to restore degraded ecosystems. This study investigated the effects of AMF and the presence of legume or grass herbs on phytoremediation with a legume tree, Robinia pseudoacacia, in Pb polluted soil. In monoculture, mycorrhizal dependency of legumes was higher than that of grass, and AMF benefited the plant biomass of legumes but had no effect on grass. Mycorrhizal colonization of plant was enhanced by legume neighbors but inhibited by grass neighbor in co-culture system. N, P, S and Mg concentrations of mycorrhizal legumes were larger than these of non-mycorrhizal legumes. Legume herbs decreased soil pH and thereby increased the Pb concentrations of plants. The neighbor effects of legumes shifted from negative to positive with increasing Pb stress levels, whereas grass provided a negative effect on the growth of legume tree. AMF enhanced the competition but equalized growth of legume-legume under unpolluted and Pb stress conditions, respectively. In conclusion, (1) AMF mediate plant interaction through directly influencing plant biomass, and/or indirectly influencing plant photosynthesis, macronutrient acquisition, (2) legume tree inoculated with AMF and co-planted with legume herbs provides an effective way for Pb phytoremediation.

Aquaporin gene expression and physiological responses of Robinia pseudoacacia L. to the mycorrhizal fungus Rhizophagus irregularis and drought stress.

The influence of arbuscular mycorrhiza (AM) and drought stress on aquaporin (AQP) gene expression, water status, and photosynthesis was investigated in black locust (Robinia pseudoacacia L.). Seedlings were grown in potted soil inoculated without or with the AM fungus Rhizophagus irregularis, under well-watered and drought stress conditions. Six full-length AQP complementary DNAs (cDNAs) were isolated from Robinia pseudoacacia, named RpTIP1;1, RpTIP1;3, RpTIP2;1, RpPIP1;1, RpPIP1;3, and RpPIP2;1. A phylogenetic analysis of deduced amino acid sequences demonstrated that putative proteins coded by these RpAQP genes belong to the water channel protein family. Expression analysis revealed higher RpPIP expression in roots while RpTIP expression was higher in leaves, except for RpTIP1;3. AM symbiosis regulated host plant AQPs, and the expression of RpAQP genes in mycorrhizal plants depended on soil water condition and plant tissue. Positive effects were observed for plant physiological parameters in AM plants, which had higher dry mass and lower water saturation deficit and electrolyte leakage than non-AM plants. Rhizophagus irregularis inoculation also slightly increased leaf net photosynthetic rate and stomatal conductance under well-watered and drought stress conditions. These findings suggest that AM symbiosis can enhance the drought tolerance in Robinia pseudoacacia plants by regulating the expression of RpAQP genes, and by improving plant biomass, tissue water status, and leaf photosynthesis in host seedlings.

A translationally controlled tumor protein gene Rpf41 is required for the nodulation of Robinia pseudoacacia.

Translationally controlled tumor protein (TCTP) is fundamental for the regulation of development and general growth in eukaryotes. Its multiple functions have been deduced from its involvement in several cell pathways, but its potential involvement in symbiotic nodulation of legumes cannot be suggested a priori. In the present work, we identified and characterized from the woody leguminous tree Robinia pseudoacacia a homolog of TCTP, Rpf41, which was up-regulated in the infected roots at 15 days post-inoculation but decreased in the matured nodules. Subcellular location assay showed that Rpf41 protein was located in the plasma membrane, cytoplasm, nucleus, and also maybe in cytoskeleton. Knockdown of Rpf41 via RNA interference (RNAi) resulted in the impaired development of both nodule and root hair. Compared with wild plants, the root and stem length, fresh weight and nodule number per plant was decreased dramatically in Rpf41 RNAi plants. The number of ITs or nodule primordia was also significantly reduced in the Rpf41 RNAi roots. The analyses of nodule ultrastructure showed that the infected cell development in Rpf41 RNAi nodules remained in zone II, which had fewer infected cells. Furthermore, the symbiosomes displayed noticeable shrinkage of bacteroid and peribacteroid space enlargement in the infected cells of Rpf41 RNAi nodules. In the deeper cell layers, a more remarkable aberration of the infected cell ultrastructure was observed, and electron-transparent lesions in the bacteroid cytoplasm were detected. These results identify TCTP as an important regulator of symbiotic nodulation in legume for the first time, and it may be involved in symbiotic cell differentiation and preventing premature aging of the young nodules in R. pseudoacacia.

The Combined Effects of Arbuscular Mycorrhizal Fungi (AMF) and Lead (Pb) Stress on Pb Accumulation, Plant Growth Parameters, Photosynthesis, and Antioxidant Enzymes in Robinia pseudoacacia L.

Arbuscular mycorrhizal fungi (AMF) are considered as a potential biotechnological tool for improving phytostabilization efficiency and plant tolerance to heavy metal-contaminated soils. However, the mechanisms through which AMF help to alleviate metal toxicity in plants are still poorly understood. A greenhouse experiment was conducted to evaluate the effects of two AMF species (Funneliformis mosseae and Rhizophagus intraradices) on the growth, Pb accumulation, photosynthesis and antioxidant enzyme activities of a leguminous tree (Robinia pseudoacacia L.) at Pb addition levels of 0, 500, 1000 and 2000 mg kg(-1) soil. AMF symbiosis decreased Pb concentrations in the leaves and promoted the accumulation of biomass as well as photosynthetic pigment contents. Mycorrhizal plants had higher gas exchange capacity, non-photochemistry efficiency, and photochemistry efficiency compared with non-mycorrhizal plants. The enzymatic activities of superoxide dismutase (SOD), ascorbate peroxidases (APX) and glutathione peroxidase (GPX) were enhanced, and hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were reduced in mycorrhizal plants. These findings suggested that AMF symbiosis could protect plants by alleviating cellular oxidative damage in response to Pb stress. Furthermore, mycorrhizal dependency on plants increased with increasing Pb stress levels, indicating that AMF inoculation likely played a more important role in plant Pb tolerance in heavily contaminated soils. Overall, both F. mosseae and R. intraradices were able to maintain efficient symbiosis with R. pseudoacacia in Pb polluted soils. AMF symbiosis can improve photosynthesis and reactive oxygen species (ROS) scavenging capabilities and decrease Pb concentrations in leaves to alleviate Pb toxicity in R. pseudoacacia. Our results suggest that the application of the two AMF species associated with R. pseudoacacia could be a promising strategy for enhancing the phytostabilization efficiency of Pb contaminated soils.

Paenibacillus enshidis sp. nov., Isolated from the Nodules of Robinia pseudoacacia L.

A Gram-positive, motile, endospore-forming, rod-shaped bacterium, designated RP-207(T), was isolated from the nodules of Robinia pseudoacacia L. plants planted in Enshi District, Hubei, PR China. Phylogenetic analyses based on the 16S rRNA gene sequence showed that the novel strain was affiliated to the genus Paenibacillus, with its closest relatives being Paenibacillus xylanilyticus XIL14(T) (95.6%), Paenibacillus peoriae DSM8320(T) (95.3%) and Paenibacillus polymyxa DSM 36(T) (95.3%). The DNA G+C content was 47.0 mol%. DNA-DNA hybridization value between strain RP-207(T) and P. xylanilyticus XIL14(T) was 40.1%. The diamino acid found in the cell wall peptidoglycan was meso-diaminopimelic. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unidentified amino-phospholipid and an unknown phospholipid. The predominant menaquinone was menaquinone-7 (MK-7), and the major fatty acid was anteiso-C15:0 and C16:0. On the basis of its physiological and biochemical characteristics and the level of DNA-DNA hybridization, strain RP-207(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus enshidis sp. nov. is proposed. The type strain is RP-207(T) (=CCTCC AB 2013275(T) = KCTC 33519(T)).

Copper tolerance mechanisms of Mesorhizobium amorphae and its role in aiding phytostabilization by Robinia pseudoacacia in copper contaminated soil.

The legume-rhizobium symbiosis has been proposed as an important system for phytoremediation of heavy metal contaminated soils due to its beneficial activity of symbiotic nitrogen fixation. However, little is known about metal resistant mechanism of rhizobia and the role of metal resistance determinants in phytoremediation. In this study, copper resistance mechanisms were investigated for a multiple metal resistant plant growth promoting rhizobium, Mesorhizobium amorphae 186. Three categories of determinants involved in copper resistance were identified through transposon mutagenesis, including genes encoding a P-type ATPase (CopA), hypothetical proteins, and other proteins (a GTP-binding protein and a ribosomal protein). Among these determinants, copA played the dominant role in copper homeostasis of M. amorphae 186. Mutagenesis of a hypothetical gene lipA in mutant MlipA exhibited pleiotropic phenotypes including sensitivity to copper, blocked symbiotic capacity and inhibited growth. In addition, the expression of cusB encoding part of an RND-type efflux system was induced by copper. To explore the possible role of copper resistance mechanism in phytoremediation of copper contaminated soil, the symbiotic nodulation and nitrogen fixation abilities were compared using a wild-type strain, a copA-defective mutant, and a lipA-defective mutant. Results showed that a copA deletion did not affect the symbiotic capacity of rhizobia under uncontaminated condition, but the protective role of copA in symbiotic processes at high copper concentration is likely concentration-dependent. In contrast, inoculation of a lipA-defective strain led to significant decreases in the functional nodule numbers, total N content, plant biomass and leghemoglobin expression level of Robinia pseudoacacia even under conditions of uncontaminated soil. Moreover, plants inoculated with lipA-defective strain accumulated much less copper than both the wild-type strain and the copA-defective strain, suggesting an important role of a healthy symbiotic relationship between legume and rhizobia in phytostabilization.

Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China.

To better understand the diversity of metal resistance genetic determinant from microbes that survived at metal tailings in northwest of China, a highly elevated level of heavy metal containing region, genomic analyses was conducted using genome sequence of three native metal-resistant plant growth promoting bacteria (PGPB). It shows that: Mesorhizobium amorphae CCNWGS0123 contains metal transporters from P-type ATPase, CDF (Cation Diffusion Facilitator), HupE/UreJ and CHR (chromate ion transporter) family involved in copper, zinc, nickel as well as chromate resistance and homeostasis. Meanwhile, the putative CopA/CueO system is expected to mediate copper resistance in Sinorhizobium meliloti CCNWSX0020 while ZntA transporter, assisted with putative CzcD, determines zinc tolerance in Agrobacterium tumefaciens CCNWGS0286. The greenhouse experiment provides the consistent evidence of the plant growth promoting effects of these microbes on their hosts by nitrogen fixation and/or indoleacetic acid (IAA) secretion, indicating a potential in-site phytoremediation usage in the mining tailing regions of China.