Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.     

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.     

Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes

Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.     

Invasive Robinia pseudoacacia in China is nodulated by Mesorhizobium and Sinorhizobium species that share similar nodulation genes with native American symbionts

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.     

Rhizobia with different symbiotic efficiencies nodulate Acaciella angustissima in Mexico, including Sinorhizobium chiapanecum sp. nov. which has common symbiotic genes with Sinorhizobium mexicanum

Bacteria from nodules of the legume Acaciella angustissima native to the south of Mexico were characterized genetically and their nodulation and competitiveness were evaluated. Phylogenetic studies derived from rpoB gene sequences indicated that A. angustissima is nodulated by Sinorhizobium mexicanum, Rhizobium tropici, Mesorhizobium plurifarium and Agrobacterium tumefaciens and by bacteria related to Sinorhizobium americanum, Sinorhizobium terangae, Rhizobium etli and Rhizobium gallicum . A new lineage related to S. terangae is recognized based on the sequences of gyrA, nolR, recA, rpoB and rrs genes, DNA–DNA hybridization and phenotypic characteristics. The name for this new species is Sinorhizobium chiapanecum and its type strain is ITTG S70^T. The symbiotic genes nodA and nifH were similar to those from S. mexicanum strains, which are Acaciella symbionts as well, with nodA gene sequences grouped within a cluster of nod genes from strains that nodulate plants from the Mimosoideae subfamily of the Leguminosae. Sinorhizobium isolates were the most frequently obtained from A. angustissima nodules and were among the best strains to promote plant growth in A. angustissima and to compete in interstrain nodule competition assays. Lateral transfer of symbiotic genes is not evident among the genera that nodulate A. angustissima ( Rhizobium, Sinorhizobium and Mesorhizobium ) but may occur among the sympatric and closely related sinorhizobia that nodulate Acaciella .     

Burkholderia and Cupriavidus spp. are the preferred symbionts of Mimosa spp. in southern China

Rhizobia were isolated from invasive Mimosa spp. (M.?diplotricha and M.?pudica) in Dehong district of the province of Yunnan in subtropical southern China. Almost all of the 98 isolates were β-rhizobia in the genera Burkholderia and Cupriavidus. These strains were analysed for their distribution characteristics together with strains from a previous study from Sishuangbanna. The proportion of nodules containing each β-rhizobial genus varied between Mimosa species, with Cupriavidus being predominant in M.?diplotricha nodules (63.3% compared to 36.7% occupation with Burkholderia), but with M.?pudica showing a slight preference for Burkholderia over Cupriavidus, with them occupying 56.5% and 43.5% of nodules, respectively. The symbiosis-essential genes nodA and nifH were present in all the Burkholderia and Cupriavidus strains tested, and their phylogenies indicated that these Mimosa symbionts share symbiotic genes with native South American rhizobia. The evolutionary discrepancies among 16S rRNA genes, nodA and nifH of Mimosa spp. symbionts, suggests that the nod and nif genes of β-rhizobia evolved independently.     

Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta-rhizobia

The genetic diversity of 221 Mimosa pudica bacterial symbionts trapped from eight soils from diverse environments in French Guiana was assessed by 16S rRNA PCR-RFLP, REP-PCR fingerprints, as well as by phylogenies of their 16S rRNA and recA housekeeping genes, and by their nifH, nodA and nodC symbiotic genes. Interestingly, we found a large diversity of beta-rhizobia, with Burkholderia phymatum and Burkholderia tuberum being the most frequent and diverse symbiotic species. Other species were also found, such as Burkholderia mimosarum, an unnamed Burkholderia species and, for the first time in South America, Cupriavidus taiwanensis. The sampling site had a strong influence on the diversity of the symbionts sampled, and the specific distributions of symbiotic populations between the soils were related to soil composition in some cases. Some alpha-rhizobial strains taxonomically close to Rhizobium endophyticum were also trapped in one soil, and these carried two copies of the nodA gene, a feature not previously reported. Phylogenies of nodA, nodC and nifH genes showed a monophyly of symbiotic genes for beta-rhizobia isolated from Mimosa spp., indicative of a long history of interaction between beta-rhizobia and Mimosa species. Based on their symbiotic gene phylogenies and legume hosts, B. tuberum was shown to contain two large biovars: one specific to the mimosoid genus Mimosa and one to South African papilionoid legumes.     

Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China

A total of 115 endophytic bacteria were isolated from root nodules of the wild legume Sphaerophysa salsula grown in two ecological regions of Loess Plateau in China. The genetic diversity and phylogeny of the strains were revealed by restriction fragment length polymorphism and sequencing of 16S rRNA gene and enterobacterial repetitive intergenic consensus-PCR. Their symbiotic capacity was checked by nodulation tests and analysis of nifH gene sequence. This is the first systematic study on endophytic bacteria associated with S. salsula root nodules. Fifty of the strains found were symbiotic bacteria belonging to eight putative species in the genera Mesorhizobium, Rhizobium and Sinorhizobium, harboring similar nifH genes; Mesorhizobium gobiense was the main group and 65 strains were nonsymbiotic bacteria related to 17 species in the genera Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus, which were universally coexistent with symbiotic bacteria in the nodules. Differing from other similar studies, the present study is the first time that symbiotic and nonsymbiotic bacteria have been simultaneously isolated from the same root nodules, offering the possibility to accurately reveal the correlation between these two kinds of bacteria. These results provide valuable information about the interactions among the symbiotic bacteria, nonsymbiotic bacteria and their habitats.     

New betaproteobacterial Rhizobium strains able to efficiently nodulate Parapiptadenia rigida (Benth.) Brenan

Among the leguminous trees native to Uruguay, Parapiptadenia rigida (Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with P. rigida. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their nifH and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera Burkholderia, Cupriavidus, and Rhizobium, among which the Burkholderia genotypes were the predominant group. Phylogenetic studies of nifH, nodA, and nodC sequences from the Burkholderia and the Cupriavidus isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the Cupriavidus isolates were able to effectively nodulate Mimosa pudica, the Burkholderia isolates produced white and ineffective nodules on this host.     

Novel alphaproteobacterial root nodule symbiont associated with Lupinus texensis

Phylogenetic analysis of rRNA gene, recA, nodA, nifD, and nifH sequences suggested that nitrogen-fixing symbionts from two populations of Lupinus texensis acquired the capacity for nodule symbiosis separately from other rhizobia in the alphaproteobacteria. Their closest 16S rRNA relatives were the nonsymbiotic taxa Chelatococcus, Bosea, and Balneomonas.     

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia

Ethiopian Bradyrhizobium strains isolated from root nodules of Crotalaria spp., Indigofera spp., Erythina brucei and soybean (Glycine max) represented genetically diverse phylogenetic groups of the genus Bradyrhizobium. Strains were characterized using the amplified fragment length polymorphism fingerprinting technique (AFLP) and multilocus sequence analysis (MLSA) of core and symbiotic genes. Based on phylogenetic analyses of concatenated recA-glnII-rpoB-16S rRNA genes sequences, Bradyrhizobium strains were distributed into fifteen phylogenetic groups under B. japonicum and B. elkanii super clades. Some of the isolates belonged to the species B. yuanmingense, B. elkanii and B. japonicum type I. However, the majority of the isolates represented unnamed Bradyrhizobium genospecies and of these, two unique lineages that most likely represent novel Bradyrhizobium species were identified among Ethiopian strains. The nodulation nodA gene sequence analysis revealed that all Ethiopian Bradyrhizobium isolates belonged to nodA sub-clade III.3. Strains were further classified into 14 groups together with strains from Africa, as well as some originating from the other tropical and subtropics regions. Strains were also clustered into 14 groups in nodY/K phylogeny similarly to the nodA tree. The nifH phylogenies of the Ethiopian Bradyrhizobium were generally also congruent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium. The phylogenies of nodA and nifH genes were also partially congruent with that inferred from the concatenated core genes sequences, reflecting that the strains obtained their symbiotic genes vertically from their ancestor as well as horizontally from more distantly related Bradyrhizobium species.     

Genetic diversity, community structure and distribution of rhizobia in the root nodules of Caragana spp. from arid and semi-arid alkaline deserts, in the north of China

The genetic diversity of 88 Caragana nodule rhizobial isolates, collected from arid and semi-arid alkaline sandy soils in the north of China, was assessed by PCR-RFLP of the 16S rRNA gene and the 16S-23S IGS, as well as the phylogenies of housekeeping genes (atpD, glnII and recA) and symbiotic genes (nodC and nifH). Of the 88 strains, 69 were placed in the genus Mesorhizobium, 16 in Rhizobium and 3 in Bradyrhizobium. Mesorhizobium amorphae, Mesorhizobium septentrionale, Mesorhizobium temperatum and Rhizobium yanglingense were the four predominant microsymbionts associated with Caragana spp. in the surveyed regions, and M. septentrionale was widely distributed among the sampling sites. Phylogenies of nodC and nifH genes showed that two kinds of symbiotic genes existed, corresponding to Mesorhizobium and Rhizobium, respectively. Available phosphorous (P) and potassium (K) contents were the main soil factors correlated with the distribution of these rhizobia in the sampling regions. Positive correlations between the available higher P content/lower K content and the dominance of Mesorhizobium species (M. temperatum, M. amorphae and M. septentrionale), and between the lower P content/higher K content and the dominance of R. yanglingense were found.     

Diverse rhizobia that nodulate two species of Kummerowia in China

A total of 63 bacterial strains were isolated from root nodules of Kummerowia striata and K. stipulacea grown in different geographic regions of China. These bacteria could be divided into fast-growing (FG) rhizobia and slow-growing (SG) rhizobia according to their growth rate. Genetic diversity and taxonomic relationships among these rhizobia were revealed by PCR-based 16 S rDNA RFLP and sequencing, 16 S-IGS RFLP, SDS-PAGE of whole cell soluble proteins, BOX-PCR and symbiotic gene (nifH/nodC) analyses. The symbiotic FG strains were mainly isolated from temperate regions and they were identified as four genomic species in Rhizobium and Sinorhizobium meliloti based on the consensus of grouping results. The SG strains were classified as five genomic species within Bradyrhizobium and they were mainly isolated fron the subtropic and tropical regions. The phylogenetic analyses of nifH and nodC genes showed relationships similar to that of 16 S rDNA but the symbiotic genes of Bradyrhizobium strains isolated from Kummerowia were distinct from those isolated from Arachis and soybean. These results offered evidence for rhizobial biogeography and demonstrated that the Kummerowia-nodulating ability might have evolved independently in different regions in association with distinctive genomic species of rhizobia.     

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.     

The common nodulation genes of Astragalus sinicus rhizobia are conserved despite chromosomal diversity

The nodulation genes of Mesorhizobium sp. (Astragalus sinicus) strain 7653R were cloned by functional complementation of Sinorhizobium meliloti nod mutants. The common nod genes, nodD, nodA, and nodBC, were identified by heterologous hybridization and sequence analysis. The nodA gene was found to be separated from nodBC by approximately 22 kb and was divergently transcribed. The 2. 0-kb nodDBC region was amplified by PCR from 24 rhizobial strains nodulating A. sinicus, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the PCR-amplified nodA gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical nodA sequences. These data indicate that, whereas microsymbionts of A. sinicus exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of nod genes among diverse recipient bacteria.     

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions.     

Genetic diversity of native bradyrhizobia isolated from soybeans (Glycine max L.) in different agricultural-ecological-climatic regions of India

Fifty isolates from root nodules of soybean plants sampled in five agricultural-ecological-climatic regions of India were analyzed by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene, the intergenic spacer region between the 16S and 23S rRNA genes (IGS), and the nifH and nodC genes. Eight haplotypes assigned to the Bradyrhizobium genus were identified, and the genetic diversity was conserved across regions. Sequence analyses of the IGS and the dnaK, glnII, recA, and nifH genes revealed three groups. One of them (26% of isolates) was assigned to Bradyrhizobium liaoningense. A second group (36% of isolates) was identified as B. yuanmingense but likely forms a new biovar able to nodulate soybean plants. The third lineage (38% of isolates) was different from all described Bradyrhizobium species but showed the same symbiotic genotype as B. liaoningense and B. japonicum bv. glycinearum.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。