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The Loess Plateau is one of the key areas for soil and water erosion control in China. Planting vegetation, such as Robinia pseudoacacia, is one of the mainstream methods to prevent soil and water erosion. However, the combination of abundant calcium ions and phosphate in the soil of the Loess Plateau limits the phosphorus nutrition of plants. In the present study, soil samples were collected under the R. pseudoacacia forest, from which two PSB strains with efficient phosphate solubilization capacities, named PSB2 and PSB7, were isolated and screened. The dissolved phosphate concentrations of their culture media were 9.68-fold and 11.61-fold higher, respectively, than that of the control group. After identification, PSB2 was classified as Pseudomonas and PSB7 as Inquilinus. This is the first time that Inquilinus has been isolated as a PSB from calcareous soil in the Loess Plateau. We then investigated the effects of different growth conditions on their phosphate solubilization capacities. Both strains effectively utilized glucose and ammonium nitrogen while maintaining high phosphate solubilization efficiency. In addition, PSB2 preferred to survive under neutral conditions and PSB7 under acidic conditions. Pot experiments indicated that the inoculation with PSB7 significantly increased the phosphorus content in the roots of R. pseudoacacia. These results imply the potential of this PSB as a phosphorus biofertilizer for R. pseudoacacia, which may be beneficial for soil and water management on the Loess Plateau.

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 13 C-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Sodium sulfide (Na 2 S) is usually used as an amendment in industrial sewage treatment. To evaluate the effects of Na 2 S 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. Na 2 S application increased the shoot biomass under severe and mild contamination. In soil, Na 2 S 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, Na 2 S 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 Na 2 S application. Increased phosphatase activity and increased available phosphorous content may promote the uptake of phosphorus in R. pseudoacacia. Moreover, Na 2 S application is beneficial to the diversity of AM fungi under mild and severe pollution. Overall, Na 2 S 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, Na 2 S is suitable for use in Pb-Zn remediation to ameliorate environmental heavy metal pollution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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., (Copyright © 2018. Published by Elsevier B.V.)

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., (Copyright © 2018. Published by Elsevier GmbH.)

A novel endophytic bacterium, designated strain HZ10 T , 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 HZ10 T 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 HZ10 T is Herbaspirillum chlorophenolicum CPW301 T (98.72 % sequence identity). Genome relatedness of the type strains H. chlorophenolicum CPW301 T , Herbaspirillum seropedicae Z67 T and Herbaspirillum aquaticum IEH 4430 T , 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 HZ10 T belongs to the genus Herbaspirillum as a novel species. Based on the results from phylogenetic, chemotaxonomic and physiological analyses, strain HZ10 T represents a novel Herbaspirillum species, for which the name Herbaspirillum robiniae sp. nov. is proposed. The type strain is HZ10 T (=JCM 31754 T =CCTCC AB 2014352 T ).

A novel endophytic bacterium, designated strain HZ7 T , 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 HZ7 T 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 3001 T (99.05 % similarity). Genome relatedness was computed using average nucleotide identity and genome-to-genome distance analysis, both of which strongly supported strain HZ7 T as belonging to the genus Mitsuaria as a representative of a novel species. On the basis of phylogenetic analysis, chemotaxonomic data and physiological characteristics, strain HZ7 T represents a novel species of the genus Mitsuaria, for which the name Mitsuaria noduli sp. nov. is proposed. The type strain is HZ7 T (=JCM 31671 T =CCTCC AB 2014353 T ).

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 NO 3 -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 NO 3 -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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 (NO 2 , 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.

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 CO 2 in natural ecosystems due to human activities. However, the response of GRSP to elevated CO 2 combined with heavy metal contamination has not been widely reported. Here, we investigated the response of GRSP to elevated CO 2 in the rhizosphere of Robinia pseudoacacia L. seedlings in Pb- and Cd-contaminated soils. Elevated CO 2 (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 CO 2  + heavy metals than under ambient CO 2 . The amount of Cd and Pb bound to GRSP was significantly higher under elevated (compared to ambient) CO 2 ; and elevated CO 2 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 CO 2 compared to ambient CO 2 . The combination of both metals and elevated CO 2 led to a significant increase in available Pb in rhizosphere soil compared to the Pb treatment alone. In conclusion, increased GRSP produced under elevated CO 2 could contribute to sequestration of soil pollutants by adsorption of Cd and Pb., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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.

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)).

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.

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., (Copyright © 2014. Published by Elsevier B.V.)

Background: Legume-rhizobium symbiosis is a complex process that is regulated in the host plant cell through gene expression network. Many nodulin genes that are upregulated during different stages of nodulation have been identified in leguminous herbs. However, no nodulin genes in woody legume trees, such as black locust (Robinia pseudoacacia), have yet been reported., Methodology/principal Findings: To identify the nodulin genes involved in R. pseudoacacia-Mesorhizobium amorphae CCNWGS0123 symbiosis, a suppressive subtractive hybridization approach was applied to reveal profiling of differentially expressed genes and two subtracted cDNA libraries each containing 600 clones were constructed. Then, 114 unigenes were identified from forward SSH library by differential screening and the putative functions of these translational products were classified into 13 categories. With a particular interest in regulatory genes, twenty-one upregulated genes encoding potential regulatory proteins were selected based on the result of reverse transcription-polymerase chain reaction (RT-PCR) analysis. They included nine putative transcription genes, eight putative post-translational regulator genes and four membrane protein genes. The expression patterns of these genes were further analyzed by quantitative RT-PCR at different stages of nodule development., Conclusions: The data presented here offer the first insights into the molecular foundation underlying R. pseudoacacia-M. amorphae symbiosis. A number of regulatory genes screened in the present study revealed a high level of regulatory complexity (transcriptional, post-transcriptional, translational and post-translational) that is likely essential to develop symbiosis. In addition, the possible roles of these genes in black locust nodulation are discussed.

This study was carried out to examine the diversity of 34 isolates collected from 11 species of leguminous trees growing in South Korea. Phylogenetic relationships between these 34 isolates and reference strains of the Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Ensifer/Sinorhizobium were analysed by using 16S rRNA gene sequences. Twenty-one isolates were related to Mesorhizobium, four isolates to Rhizobium, and nine isolates to Bradyrhizobium. But none of isolates were related to Sinorhizobium/Ensifer and Azorhizobium. Robinia pseudoacacia and Amorpha fruticosa were nodulated by various genotypes of rhizobia out of them, most of the isolates belonged to the genus Mesorhizobium. The isolates from Lespedeza bicolar belonged to diverse genera of Mesorhizobium, Rhizobium, and Bradyrhizobium. The isolates from Maackia amurensis and Lespedeza maximowiezii var. tomentella were phylogenetically related to the genera of Bradyrhizobium. PCR-based RAPD method and phylogenetic analysis of the 16S rRNA results revealed a high phylogenetic diversity of rhizobial strains nodulating leguminous trees in South Korea. Also, the relationships between host and bacterial phylogenies showed that only Robinia pseudoacacia, and Wisteria floribunda have significantly unique branch length than expected by chance based on phylogenetic tree., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study aimed to identify drought-mediated differences in amino nitrogen (N) composition and content of xylem and phloem in trees having different symbiotic N(2)-fixing bacteria. Under controlled water availability, 1-year-old seedlings of Robinia pseudoacacia (nodules with Rhizobium), Hippophae rhamnoides (symbiosis with Frankia) and Buddleja alternifolia (no such root symbiosis) were exposed to control, medium drought and severe drought, corresponding soil water content of 70-75%, 45-50% and 30-35% of field capacity, respectively. Composition and content of amino compounds in xylem sap and phloem exudates were analysed as a measure of N nutrition. Drought strongly reduced biomass accumulation in all species, but amino N content in xylem and phloem remained unaffected only in R. pseudoacacia. In H. rhamnoides and B. alternifolia, amino N in phloem remained constant, but increased in xylem of both species in response to drought. There were differences in composition of amino compounds in xylem and phloem of the three species in response to drought. Proline concentrations in long-distance transport pathways of all three species were very low, below the limit of detection in phloem of H. rhamnoides and in phloem and xylem of B. alternifolia. Apparently, drought-mediated changes in N composition were much more connected with species-specific changes in C:N ratios. Irrespective of soil water content, the two species with root symbioses did not show similar features for the different types of symbiosis, neither in N composition nor in N content. There was no immediate correlation between symbiotic N fixation and drought-mediated changes in amino N in the transport pathways., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Relatively little information is available regarding the variability of microbial communities inhabiting deeper soil layers. We investigated the distribution of soil microbial communities down to 1.2 m in 5-year-old Robinia pseudoacacia 'Idaho' soil by 454 sequencing of the 16S RNA gene. The average number of sequences per sample was 12,802. The Shannon and Chao 1 indices revealed various relative microbial abundances and even distribution of microbial diversity for all evaluated sample depths. The predicted diversity in the topsoil exceeded that of the corresponding subsoil. The changes in the relative abundance of the major soil bacterial phyla showed decreasing, increasing, or no consistent trends with respect to sampling depth. Despite their novelty, members of the new candidate phyla OD1 and TM7 were widespread. Environmental variables affecting the bacterial community within the environment appeared to differ from those reported previously, especially the lack of detectable effect from pH. Overall, we found that the overall relative abundance fluctuated with the physical and chemical properties of the soil, root system, and sampling depth. Such information may facilitate forest soil management.

The plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286, isolated from the nodules of Robinia pseudoacacia growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of Robinia plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of A. tumefaciens CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies, R. pseudoacacia inoculated with A. tumefaciens CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among A. tumefaciens CCNWGS0286, A. tumefaciens C58, and zinc-sensitive mutants 12-2 (zntA::Tn5) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil.

Gram-negative, rod-shaped bacteria were isolated from Robinia pseudoacacia root nodules. On the basis of the 16S rRNA gene phylogeny, they are closely related to Bradyrhizobium, Rhodopseudomonas and Nitrobacter species (97% sequence similarity), belonging to the class Alphaproteobacteria and family Bradyrhizobiaceae. The results of physiological and biochemical tests together with sequence analysis of housekeeping genes (atpD, dnaK, gyrB, recA and rpoB) allowed differentiation of this group from other validly published Bradyrhizobiaceae genera. NodA, nodC and nifH genes could not be amplified. On the basis of genotypic and phenotypic data, these organisms represent a novel genus and species for which the name Tardiphaga robiniae gen. nov., sp. nov. (LMG 26467(T)=CCUG 61473(T)), is proposed., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Here, we describe the draft genome sequence of Mesorhizobium amorphae strain CCNWGS0123, isolated from nodules of Robinia pseudoacacia growing on zinc-lead mine tailings. A large number of metal(loid) resistance genes, as well as genes reported to promote plant growth, were identified, presenting a great future potential for aiding phytoremediation in metal(loid)-contaminated soil.

Terminal restriction fragment length polymorphism (T-RFLP) was used to examine the community diversity of bacteria and arbuscular mycorrhizal fungi (AMF) and their interrelation in the rhizosphere of 8 plants in the Liudaogou watershed in Shenmu County. The objective was to obtain diversity indices and provide theoretical basis for ecological restoration. Results showed significant variations in the species and abundances of rhizospheric bacteria and AMF associated with 8 plants. Among these, the Shannon diversity index of rhizospheric bacteria was the highest for Robinia pseudoacacia (4.01) and the lowest for Salix babylonica (2.18), whereas the Shannon diversity index of rhizospheric AMF was the highest for Populus simonii (2.07) and the lowest for Hippophae rhamnoides (1.21). Cluster analysis and redundancy analysis indicated a significant difference in associated microbial community structure, while the similarity among community diversity of rhizospheric bacteria and AMF associated with specific plants was also found. There was a significant correlation between diversity indices of bacteria and AMF (P < 0.01). Associated microbial community diversity was influenced primarily by organic matter and total nitrogen content. Our work demonstrated strong impacts of plant species and rhizospheric environment on associated microbial community structure. Due to the high diversity indices of rhizospheric bacteria and AMF, R. pseudoacacia was considered to be a pioneer plant species for vegetation restoration in the Liudaogou watershed.

The endophytic colonisation of Bacillus subtilis strain GXJM08, isolated from roots of Podocarpus imbricatus B1. Enum. P1. Jav., in roots of the leguminous plant Robinia pseudoacacia L. was investigated. Ultrastructure observations showed that B. subtilis caused morphological changes in the root hair and colonised the plant through infected root hairs. The structure of the infection thread was similar to that of rhizobia, but the structure of infected cells was different. B. subtilis is also different from rhizobia and plant pathogens in terms of the formation of a peribacteroid membrane and the mode of penetration through the host cell wall. Our results provide a basis for studying development of the mutualistic symbiotic relationship between B. subtilis and plants, and a basis for studying the mechanism of the B. subtilis-plant interaction., (© 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Previously, five rhizobial strains isolated from root nodules of Robinia pseudoacacia were assigned to the same genospecies on the basis of identical 16S rRNA gene sequences and phylogenetic analyses of the nodA, nodC and nifH genes, in which the five isolates formed a well-supported group that excluded other sequences found in public databases. In this study, the 16S rRNA gene sequence similarities between the isolates and Mesorhizobium mediterraneum UPM-Ca36(T) and Mesorhizobium temperatum SDW018(T) were 99.5 and 99.6 %, respectively. The five isolates were also different from defined Mesorhizobium species using ERIC fingerprint profiles and they formed a novel Mesorhizobium lineage in phylogenetic analyses of recA and atpD gene sequences. DNA-DNA relatedness values between the representative strain, CCNWYC 115(T), and type strains of defined Mesorhizobium species were found to be lower than 47.5 %. These results indicated that the isolates represented a novel genomic species. Therefore, a novel species, Mesorhizobium robiniae sp. nov., is proposed, with type strain CCNWYC 115(T) (=ACCC 14543(T) =HAMBI 3082(T)). Strain CCNWYC 115(T) can form effective nodules only on its original host.

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 μg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere., (Copyright 2010 Elsevier GmbH. All rights reserved.)

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99-100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97-98% for nodC, and 97-100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91-98% and 90-98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51-75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61-64 mol% across M. amorphae strains.

(+/-)-Laetirobin (1) was isolated as a cytostatic lead from Laetiporus sulphureus growing parasitically on the black locust tree, Robinia pseudoacacia, by virtue of a reverse-immunoaffinity system. Using an LC/MS procedure, milligram quantities of (+/-)-laetirobin (1) were obtained, and the structure of 1 was elucidated by X-ray crystallography and confirmed by NMR spectroscopy. Preliminary cellular studies indicated that (+/-)-laetirobin (1) rapidly enters in tumor cells, blocks cell division at a late stage of mitosis, and invokes apoptosis.

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98-99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771-961 kb megaplasmids.

Three lytic phages (PhiRP1, PhiRP2, and PhiRP3) specific for Robinia pseudoacacia rhizobia were isolated from the soil under black locust. They were characterized by their morphology, host range, and some other properties including DNA molecular weights. Studied phages have been found to belong to Siphoviridae family that comprises viruses with long, and noncontractile tails. They had broad host ranges and effectively lysed not only Robinia pseudoacacia microsymbionts but also different Mesorhizobium species. The phages were homogenous in latent periods (300 min) but heterogeneous in burst sizes (100-200 phage particles per one infected cell) and rise periods (90-120 min). They showed a distinct adsorption rate to Robinia pseudoacacia rhizobia (70.4-93.94%). The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from ca. 82 kb to ca. 105 kb.

The aim of this work is to describe the diversity of potentially symbiotic bacteria associated with the invasive introduced legume Robinia pseudoacacia in China. Thirty-three isolates from 33 separate trees and nodules were characterized using restriction length fragment polymorphism and sequencing of 16S rRNA, nodA, nodC and nifH genes. Their 16S rRNA gene patterns and sequences placed them in three clades: 85% of isolates were related to the Mesorhizobium mediterraneum/temperatum group, whereas the remaining were similar either to Mesorhizobium amorphae or to Sinorhizobium meliloti. However, despite their diverse taxonomic positions, the nodA, nodC and nifH genes' phylogenies indicated that these R. pseudoacacia symbionts share similar symbiosis genes, implying gene transfers and a degree of host specificity. Comparison of R. pseudoacacia symbiotic diversity in native and other invaded areas suggests that most Chinese symbionts may not have arrived with the seed but were local bacteria that acquired specific symbiotic genes from native American rhizobia.

The nitrogen-fixing tree black locust (Robinia pseudoacacia L.) seems to affect ectomycorrhizal (ECM) colonization and disease severity of Japanese black pine (Pinus thunbergii Parl.) seedlings. We examined the effect of black locust on the distribution of ECM and pathogenic fungi in soil. DNA was extracted from soil at depths of 0-5 and 5-10 cm, collected from the border between a Japanese black pine- and a black locust-dominated forest, and the distribution of these fungi was investigated by denaturing gradient gel electrophoresis. The effect of soil nutrition and pH on fungal distribution was also examined. Tomentella sp. 1 and Tomentella sp. 2 were not detected from some subplots in the Japanese black pine-dominated forest. Ectomycorrhizas formed by Tomentella spp. were dominant in black locust-dominated subplots and very little in the Japanese black pine-dominated forest. Therefore, the distribution may be influenced by the distribution of inoculum potential, although we could not detect significant relationships between the distribution of Tomentella spp. on pine seedlings and in soils. The other ECM fungi were detected in soils in subplots where the ECM fungi was not detected on pine seedlings, and there was no significant correlation between the distribution of the ECM fungi on pine seedlings and in soils. Therefore, inoculum potential seemed to not always influence the ECM community on roots. The distribution of Lactarius quieticolor and Tomentella sp. 2 in soil at a depth of 0-5 cm positively correlated with soil phosphate (soil P) and that of Tomentella sp. 2 also positively correlated with soil nitrogen (soil N). These results suggest the possibility that the distribution of inoculum potential of the ECM fungi was affected by soil N and soil P. Although the mortality of the pine seedlings was higher in the black locust-dominated area than in the Japanese black pine-dominated area, a pathogenic fungus of pine seedlings, Cylindrocladium pacificum, was detected in soil at depths of 0-5 and 5-10 cm from both these areas. This indicates that the disease severity of pine seedlings in this study was influenced by environmental conditions rather than the distribution of inoculum potential.

The phylogenetic analyses of partial nucLSU rDNA sequence data of three Jobellisia species indicate that J. rhynchostoma is distinct from the core species of Jobellisia. Jobellisia luteola, the type species of the genus, and J. fraterna reside as a strongly supported monophyletic clade in a basal position in a grouping containing the Diaporthales, the Calosphaeriales and the Togniniaceae, while all phylogenies confirm the placement of J. rhynchostoma within the Sordariales. The new family Jobellisiaceae (incertae sedis) is described for Jobellisia. A new perithecial ascomycete genus, Bellojisia (Lasiosphaeriaceae, Sordariales), is introduced for J. rhynchostoma. The fungus produces nonstromatic, long-necked perithecia with a superficial to semi-immersed pyriform venter and carbonaceous three-layered perithecial wall, 1-septate, hyaline, later brown, reniform to navicular ascospores with a polar germ pore formed in unitunicate asci. The fungus was not observed to produce a conidial anamorph in vitro. Both morphological and molecular data suggest Corylomyces selenosporus of the Sordariales is the closest relative of J. rhynchostoma. The other relatives of Bellojisia (viz. Cercophora, Lasiosphaeria and Podospora) recruit from the Lasiosphaeriaceae (Sordariales). Cercophora and Podospora are shown as polyphyletic within the Sordariales, which is in agreement with previous molecular studies.

In this study we examined the role of the nitrogen-fixing tree, Robinia pseudoacacia (black locust), in ectomycorrhizal (ECM) formation and ECM community of Pinus thunbergii (Japanese black pine) seedlings. Two 200 m(2) experimental plots were established at the border between a Japanese black pine- and a black locust-dominated area in a coastal forest. The ECM fungal community of pine seedlings was examined by PCR-RFLP and sequence analysis. We analyzed the relationship between ECM formation, ECM community, growth, and nutrient status of pine seedlings and environmental conditions using the Mantel test and structural equation model. Percentages of ECM root tips, the number of ECM fungal species and ECM diversity on pine seedlings decreased in the black locust-dominated area. Cenococcum geophilum and Russula spp. were dominant in the Japanese black pine-dominated area, whereas Tomentella spp. were dominant in the black locust-dominated area. Nitrogen (N) concentration in soils or pine seedlings strongly influenced the percentage of ECM root tips, the number of ECM fungal species and ECM fungal similarity. These results imply the long-term eutrophication caused by N-fixing trees can change ECM formation and ECM community structure.

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.

The type of the in vitro root interactions of Terfezia terfezioides with the plants Robinia pseudoacacia and Helianthemum ovatum was investigated including detailed anatomical and ultrastructural characterization. No difference in growth was detected at different phosphate concentrations on agar synthetic medium between the inoculated and control plants during a short-time cultivation. The fungal colonization of the roots increased with higher phosphate level in both plant species, but was always lower in R. pseudoacacia roots. Septate hyphae formed frequently intracellular branched coils in dead cortical cells. In H. ovatum intercellular hyphae were observed forming finger-like structures reminiscent of Hartig-net structures in ectomycorrhizae. A loose hyphal envelope covered the root surface of both colonized and noncolonized roots. The features resembled similar structures described earlier during the mycorrhizae of different Terfezia species. Our detailed anatomical and ultrastructural study shows that the in vitro root interactions of the T. terfezioides cannot be considered unambiguously as mycorrhiza.

A study on the microbial ecology in an active slow sand filter, used for disinfecting the circulating plant nutrient solutions, showed that spore-forming plant-associated bacteria belonging to the Bacillus-Paenibacillus complex are well adapted for transmission in the solutions and passage through the filter. Therefore, strains from this bacterial group were suitable candidates for biological control in irrigated and closed plant growth systems. The spore-forming Paenibacillus polymyxa strain PpDGB was selected in in vitro tests as a potent pathogen-antagonist and was tested as a prophylactic protection agent in the plant rhizosphere, especially for cultures stages that are highly susceptible to stress and disease. Plant cuttings, in vitro plants and seeds of different plant types were bacterized and planted in their typical disease-conducive environment where nutrient solutions or water irrigation was applied and further plant development was monitored. Observed plant parameters were plant survival, weight, chlorophyll concentration in the leaf mesophyl, root health and root hair formation. The PpDGB treatment initially induced stress in the plants, which was observed as a transient stop in plant transpiration. This effect caused some necrosis in the most stress-sensitive in vitro plant species. In the other plants this stress period was followed by a significant enhancement in plant growth. In case of seed treatment, more seeds germinated and seedling growth was faster. In the tested formulation, PpDGB enhanced growth but not disease resistance, probably due to simultaneous activation of the residual plant pathogens. Therefore variant formulations have to be tested. The influence of PpDGB on the composition of the bacterial communities in the rhizosphere was assessed by DGGE profiling. In soilless plant cultures, PpDGB-driven profile changes could be observed from the 5th day after the initial treatment. P. polymyxa bacteria were shown to be widely present in association with plants and specific PpDGB detection in plant and rhizosphere was only possible with newly developed strain-specific PCR primers based on Nif H gene sequences. Quantitative PCR based on SYBR Green fluorescence enabled detection of low PpDGB concentrations in the plant rhizosphere.

The paper contains an overview of the results of the studies made on the truffle Terfezia terfezioides, particularly the investigations related to the associations of this fungus with plants. Twelve plant species originated from a natural habitat of the fungus were supposed to be connected with T. terfezioides based on the anatomy of the endogenous fungal structures in their roots. Aseptic experiments were carried out on modified MMN substrates with different phosphate concentrations to study the interaction of T. terfezioides with Robinia pseudoacacia and Helianthemum ovatum. The colonization of the roots of black locust was always weaker than that of Helianthemum. The main characteristics were the intracellular coiled, branched, frequently septated hyphae in dead root cells. The intercellular hyphae formed Hartig-net with finger like structures only in Helianthemum, the interactions could not be considered unambiguously as mycorrhizae. There was no difference between the RFLP profiles of the nr DNA ITS of nineteen fruit bodies collected at the same time from the habitat and the ITS of three randomly chosen specimens were identical on sequence level, too. These invariability makes to design species specific PCR primers possible to check unambiguously the host plants.

From the pathological material of black locust trees showing symptoms of wilting of the foliage or canker of the bark the following Fusarium species were isolated: Fusarium avenaceum (Fr.) Sacc., Fusarium lateritium Nees., Fusarium semitectum Berk. & Rav., Fusarium solani (Mart.) Sacc., Fusarium sulphureum Schlecht. (syn.: Fusarium sambucinum Fuckel f. 6 Wollenw.) The results of the provocation infections of one-year-old black locust seedlings showed that all of the species--except Fusarium solani--are able to cause considerable necrosis in living bark and phloem. Fusarium sulphureum had by far the highest pathogenecity among the tested species. Fusarium semitectum isolated from withered black locust tree also caused necrosis on significant bark area. In the course of the penetration assay Fusarium sulphureum and Fusarium avenaceum were the most successful, and these species can cause cankers on the stem and twigs of black locust without frost effect.