Phenotypic and genetic diversity of Paenibacillus azotofixans strains isolated from the rhizoplane or rhizosphere soil of different grasses

Fifty-three strains identified as Paenibacillus azotofixans were isolated from the rhizoplane and rhizosphere of different grasses and from soil. To study the diversity within this species, four approaches were used: assessment of homology with a nifKDH probe in hybridization experiments; use of a selected 20-mer primer to produce RAPD profiles and of BOX-PCR to generate genomic fingerprintings; and phenotypic tests using the API50CH system. The API tests performed with the 53 P. azotofixans strains showed that all strains produced acid from 15 carbohydrates; using six other carbohydrates (sorbitol, dulcitol, tagatose, starch, glycogen and D -arabitol), the strains could be divided in five groups of related strains. All strains tested showed homology to Klebsiella pneumoniae nifKDH genes, resulting in 14 different hybridization patterns with this probe. Using RAPD-fingerprinting with one appropriate primer, 23 different amplification patterns were observed. The BOX-PCR approach confirmed the grouping suggested by the RAPD fingerprinting. A comparison of the 53 strains by similarity matrix analysis using the data obtained in all approaches resulted in a phenogram, grouping them into five broad groups at 74% similarity and into 27 subgroups at 94% similarity. At 100% similarity, 31 groups of strains could be formed, indicating a high degree of diversity among the strains tested. Overall, the diversity was independent from the origin of strains, since a variety of different groups was isolated from each plant studied. However, some clusters were dominant in wheat and sugarcane samples. The results indicated that the methods used here are sensitive indicators of diversity among the strains studied and can be applied as efficient and reliable means for further ecological and biogeographical studies.     

Diversity of pseudomonads isolated from three different plant rhizospheres

AIMS: To study the diversity of the Pseudomonas populations isolated from three different plant rhizospheres, namely pearl millet, cotton and paddy, grown in saline soils along the coastline of Southern India. METHODS AND RESULTS: The Pseudomonas populations were analysed for their biochemical characters and genetic diversity using molecular tools including RAPD and PCR-RFLP. The biochemical characterization, antibiotic resistance assay and RAPD profiles revealed a largely homogeneous population. Even in PCR-RFLP restriction studies, only two groups of isolates were seen. One group was predominant in all three rhizospheres, while the other minor group consisted of salt-sensitive isolates restricted to the paddy rhizosphere alone. CONCLUSIONS: It was observed that increasing salinity caused a predominant selection of salt-tolerant species, in particular Ps. pseudoalcaligenes and Ps. alcaligenes, irrespective of the host rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has reinstated the importance of the soil over the host plant with regard to rhizosphere populations. It has also resulted in the isolation of several salt-tolerant Pseudomonas strains, which are being screened for their biological control activity against common plant pathogens of the coastal agri-ecosystem.     

Technological and molecular diversity of Lactobacillus plantarum strains isolated from naturally fermented sourdoughs

Thirty Lactobacillus (L.) plantarum strains, isolated from sourdough, were identified by biochemical tests as well as 16S rDNA sequencing and differentiated on the basis of technological properties, such as amylase, protease, phytase and antirope activities. These properties were shown to be widely differing among the strains, indicating a significant technological diversity. Genetic differentiation was achieved by restriction endonuclease analysis-pulsed field gel electrophoresis (REA-PFGE) that allowed the L. plantarum strains to be divided into 10 different genomic groups. Moreover, 32 different starters were employed in dough making experiments; each starter consisted of a single strain of L. plantarum associated with a maltose positive or a maltose negative yeast. The technological properties of the doughs were greatly influenced by the type of strain included in the starter. The time of leavening and the acidification activities detected in the dough were enhanced by the presence of L. plantarum strains. The bacterial and yeast contents and fermentation properties were statistically treated by principal component analysis (PCA), which allowed the discrimination of different typologies of dough. The study of the peculiar characteristics of different strains of L. plantarum is fundamental for a better understanding of their potential in affecting the nutritional value, quality and stability of the baked goods. L. plantarum strains are able to differentially influence the dough quality when employed as starters.     

不同寄主来源寄生疫霉菌株的遗传变异分析

从300条RAPD随机引物中筛选出扩增多态性丰富的13条引物,对寄主来源不同的29个寄生疫霉（Phytophthora parasitica）菌株进行基因组DNA遗传变异分析。用筛选出的13条引物对供试菌株进行RAPD-PCR扩增,共产生139条RAPD条带,其中133条为多态性条带,多态检测率为95.7%。利用PopGene Version 1.31软件对供试菌株间的遗传距离进行聚类分析并构建系统树状图,供试29个菌株被划分为5个遗传聚类组,不同菌株间具有丰富的遗传变异。其中寄主为烟草(Nicotiana spp.)、腊梅(Chimonanthus praecox)、凤尾兰(Yucca gloriosa)和西番莲(Passiflora edulis)的菌株的遗传结构与其寄主来源具有明显的相关性,而寄主为刺槐(Sophora chinensis)和番茄(Lycopersicum esculentum)的菌株与其寄主来源的相关性较小,来源于不同寄主植物的菌株之间遗传距离较远。结果提示在寄生疫霉与其不同寄主植物的长期协同进化过程中,寄主对病原菌的遗传结构具有一定的影响。     

Identification of nitrogen-fixing Paenibacillus from different plant rhizospheres and a novel nifH gene detected in the P. stellifer

A total of 534 isolates were selectively obtained from different plant rhizospheres based on their growth on nitrogen-free medium and their resistance to 80 degrees C for 15 min. Of the 534 isolates, 23 isolates had nifH gene and exhibited nitrogenase activities. Based on 16S rDNA sequence, G + C content assay and DNA-DNA hybridization, by the 23 isolates, which were divided into four monophyletic clusters, all belonged to the Paenibacillus genus. NifH gene deduced amino acid alignment analysis revealed that cluster I, including 15 isolates, showed the highest NifH identity with Paenibacillus genus; while cluster II identified as P stellifer by DNA-DNA hybridization was consistent with four uncultured bacterial clones. This study suggested that the nitrogen-fixing Paenibacillus were distributed in various ecosystems and prevalent in different plant rhizospheres. It was the first demonstration that nitrogen fixation existed in P. jamilae and P. stellifer. In eight isolates identified as P. stellfer species, a novel nifH gene was detected in Paenibacillus.     

Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Biotechnological potential of plant growth-promoting bacteria from the roots and rhizospheres of endemic plants in ironstone vegetation in southeastern Brazil

Microorganisms associated with plants have a great biotechnological potential, but investigations of these microorganisms associated with native plants in peculiar environments has been incipient. The objective of this study was to analyze the plant growth-promoting bacteria potential of cultivable bacteria associated with rare plants from the ferruginous rocky fields of the Brazilian Iron Quadrangle. The roots and rhizospheres of nine endemic plants species and samples of a root found in a lateritiric duricrust (canga) cave were collected, the culturable bacteria isolated and prospected for distinct biotechnological and ecological potentials. Out of the 148 isolates obtained, 8 (5.4%) showed potential to promote plant growth, whereas 4 (2.7%) isolates acted as biocontrol agents against Xanthomonas citri pathotype A (Xac306), reducing the cancrotic lesions by more than 60% when co-inoculated with this phytopathogen in Citrus sinensis plants. Moreover, other 4 (2.7%) isolates were classified as potential bioremediation agents, being able to withstand high concentrations of arsenite (5 mM As³⁺) and arsenate (800 mM As⁵⁺), by removing up to 35% and 15% of this metalloid in solution, respectively. These same four isolates had a positive influence on the growth of both the roots and the aerial parts when inoculated with tomato seeds in the soil contaminated with arsenic. This is the first time that an investigation highlights the potentialities of bacteria associated with rare plants of ferruginous rocky fields as a reservoir of microbiota of biotechnological and ecological interest, highlighting the importance of conservation of this area that is undergoing intense anthropic activity.

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Taxonomic diversity of Nitrosovibrio strains isolated from building sandstones

Abstract Five strains of Nitrosovibrio , isolated from building sandstones of historic monuments in Germany, were compared. They were characterized by the presence or absence of carboxysomes, generation time, tolerance to ammonium chloride and sodium chloride, and temperature optima. Three strains were distinguishable by DNA homologies, ultrastructure, and growth mode. Only two strains were closely related, indicated e.g. by a DNA homology of 72%. Taxonomical diversity of the endolithic Nitrosovibrio strains may be related to the existence of microniches in building sandstones. Cell aggregation was observed for endolithic cells and for cells of isolated Nitrosovibrio strains. This could be a response to acidic environments.     

Taxonomic and functional diversity of pseudomonads isolated from the roots of field-grown canola

Among the most important rhizosphere bacteria are the pseudomonads, which are aggressive colonizers and utilize a wide range of substrates as carbon sources. The objective of this study was to determine if the taxonomic or metabolic diversity of pseudomonads differed among field-grown canola cultivars. Bacteria (n=2257) were isolated from the rhizosphere and root interior of six cultivars of field-grown canola, including three transgenic varieties. The bacteria were identified by fatty acid methyl ester (FAME) analysis, and about 35% were identified as Pseudomonas species. The most abundant species were Pseudomonas putida and Pseudomonas chlororaphis. Dendrograms based on FAME analysis revealed that many pseudomonad strains were found in all of the canola cultivars. Pseudomonads of the same strain were found in both the rhizosphere and the root interior of canola plants, suggesting that endophytic bacteria were a subset of the rhizosphere community. Because metabolic profiling provides more useful information than taxonomy, P. putida and P. chlororaphis isolates were characterized for their ability to utilize carbon substrates and produce several enzymes. Bacteria isolated from different plant cultivars had different carbon utilization profiles, but when only carbon substrates found in root exudates were analyzed, the cultivar effect was less pronounced. These characterizations also demonstrated that bacteria that were determined by FAME to be the same strain were metabolically different, suggesting functional redundancy among Pseudomonas isolates. The results of this study suggest that pseudomonads were functionally diverse. They differed in their metabolic potential among the canola cultivars from which they were isolated. Because bacteria capable of using many substrates can effectively adapt to new environments, these results have implications for the use of pseudomonads as biofertilizers, biological control agents and plant growth-promoting bacteria in canola.     

Evaluation of the diversity of Paenibacillus polymyxa strains by using the DNA of bacteriophage IPy1 as a probe in hybridization experiments

AIMS: To evaluate the genetic diversity within the species Paenibacillus polymyxa. METHODS: Southern hybridization was performed on 102 strains of P. polymyxa using DNA from the phage IPy1 as a probe. Results: All 102 strains hybridized to phage IPy1 DNA. Data from different hybridization patterns obtained were used to construct a dendrogram in which 53 genotypic groups were split into two main clusters. One cluster contained strains from the rhizospheres of sorghum and maize planted in Cerrado soil, Brazil, and the majority of strains received from two culture collections. The other cluster contained strains isolated from different Brazilian soils and rhizospheres and strains deposited in a third culture collection. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study appears to be a new and a very useful tool to study the diversity within this species.     

药用植物内生芽孢杆菌的多样性和系统发育研究

[目的]了解药用植物内生芽孢杆菌的生物多样性.[方法]采用数值分类、16S rDNA PCR RFLP、BOX-PCR指纹图谱和16S rDNA序列分析技术对分离于几种药用植物的内生芽孢杆菌和已知参比菌株进行表型、遗传多样性及系统发育研究.[结果]供试菌株在数值分类聚类分析中在84%的相似水平上产生13个表观群.16S rDNAPCR-RFLP分析表明供试菌株表现出丰富的遗传多样性.BOX-PCR指纹图谱分析进一步证明药用植物的内生芽孢杆菌的基因组也具有多样性,聚群的结果与数值分类有较好一致性.用软件在Genbank中进行所得序列的同源性检索,并构建系统发育树.由16S rDNA序列分析可知,供试的代表菌株SCAU11与球形芽孢杆菌(Bacillus sphaericus)亲缘关系最近,SCAU78和SCAU25为枯草芽孢杆菌(Bacillus subtilis)的两个亚种,代表菌株SCAU39与巨大芽孢杆菌(Bacillus megaterium)的亲缘关系最近.[结论]研究结果表明药用植物内生芽孢杆菌具有明显的表型和遗传多样性.     

Genetic diversity of Bradyrhizobium strains isolated from Arachis hypogaea

Rhizobia are used exclusively in agricultural systems for enhancing the ability of legumes to fix atmospheric nitrogen. Knowledge about the indigenous population is necessary for the selection and application of inoculant strains. In this study, we have assessed the genetic diversity of Bradyrhizobium strains isolated from the host plant, Arachis hypogaea along the coastline of Tamil Nadu. Different populations collected from varying environmental conditions were analysed for salt and pH tolerance. Genetic diversity among the strains was studied using RAPD markers and PCR-RFLP of 16S rDNA and nifD genes. The approaches used in this study yielded consistent results, which revealed a high degree of heterogeneity among strains and detection of two distinct genetic groups.     

Genetic diversity and population structure of Saccharomyces cerevisiae strains isolated from different grape varieties and winemaking regions

We herein evaluate intraspecific genetic diversity of fermentative vineyard-associated S. cerevisiae strains and evaluate relationships between grape varieties and geographical location on populational structures. From the musts obtained from 288 grape samples, collected from two wine regions (16 vineyards, nine grape varieties), 94 spontaneous fermentations were concluded and 2820 yeast isolates were obtained that belonged mainly (92%) to the species S. cerevisiae. Isolates were classified in 321 strains by the use of ten microsatellite markers. A high strain diversity (8-43 strains per fermentation) was associated with high percentage (60-100%) of fermenting samples per vineyard, whereas a lower percentage of spontaneous fermentations (0-40%) corresponded to a rather low strain diversity (1-10 strains per fermentation).For the majority of the populations, observed heterozygosity (Ho) was about two to five times lower than the expected heterozygosity (He). The inferred ancestry showed a very high degree of admixture and divergence was observed between both grape variety and geographical region. Analysis of molecular variance showed that 81-93% of the total genetic variation existed within populations, while significant differentiation within the groups could be detected. Results from AMOVA analysis and clustering of allelic frequencies agree in the distinction of genetically more dispersed populations from the larger wine region compared to the less extended region. Our data show that grape variety is a driver of populational structures, because vineyards with distinct varieties harbor genetically more differentiated S. cerevisiae populations. Conversely, S. cerevisiae strains from vineyards in close proximity (5-10 km) that contain the same grape variety tend to be less divergent. Populational similarities did not correlate with the distance between vineyards of the two wine regions. Globally, our results show that populations of S. cerevisiae in vineyards may occur locally due to multi-factorial influences, one of them being the grape variety.     

Comparison of phenotypical and molecular methods for the identification of bacterial strains isolated from a deep subsurface environment

Vol. 61, no. 9, p. 3406: the Acknowledgments should read as follows. "This work was supported by the CNRS and by grants of the European Community and ANDRA as part of the project Archimede-Argiles and of the Direction de la Recherche et des Etudes Doctorales. V.B. was a recipient of a CIFRE fellowship." [This corrects the article on p. 3400 in vol. 61.].     

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.     

蒙古沙冬青及其伴生植物AM真菌物种多样性

于2015年7月在内蒙古乌海、磴口2个样地选取蒙古沙冬青(Ammopiptanthus mongolicus)及其伴生植物为研究对象,分别采集0-20 cm和20-40 cm土层根围土样,利用高通量测序方法分析了不同植物AM真菌群落结构和物种多样性,发掘新物种,为补充和完善AM真菌分类体系提供依据。试验共分离鉴定3纲5目6科9属89个AM真菌OTU,属包括Glomus,Funneliformis,Diversispora,Claroideoglomus,Rhizophagus,Septoglomus,Scutellospora,Ambispora和Paraglomus。与形态学研究结果相比,高通量鉴定结果在属水平上更丰富。同一土层不同样地,蒙古沙冬青AM真菌丰度和多样性指数高于伴生植物;同一土层不同植物,AM真菌丰度和多样性指数表现为磴口高于乌海;同一植物不同样地,AM真菌ACE指数和Chao1指数20-40 cm土层高于0-20 cm土层,Simpson指数和Shannon指数0-20 cm土层高于20-40 cm土层。RDA分析表明,AM真菌ACE指数和Chao1指数与土壤碱解N显著正相关,与酸性磷酸酶显著负相关;Simpson指数和Shannon指数与碱性磷酸酶显著正相关,与pH显著负相关。结果表明,蒙古沙冬青AM真菌物种多样性高于伴生植物,同一样地,寄主植物与土壤深度共同作用对AM真菌群落组成有显著影响。     

Genotypic and phenotypic diversity of Lactobacillus rossiae strains isolated from sourdough

AIM: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae. METHODS AND RESULTS: Genotypic identification was carried out by partial 16S rRNA gene sequence analysis. Genetic diversity was evaluated by RAPD-PCR analysis. Phenotypic diversity was evaluated through fermentative profile by Biolog system, proteinase and peptidase activities using synthetic substrates, and acidification capacity and amino acid profile during sourdough fermentation. The genetic analyses excluded clonal relatedness among the strains used. A large phenotypic diversity was found. It mainly concerned the capacity to use carbon sources available in sourdough during fermentation, the quotient of fermentation and the peptidase activities, especially towards proline containing synthetic substrates. The free amino acid profiles differed either for the total concentration or for the type of amino acids. With a few exceptions, proteinase activity towards wheat albumins and globulins was weak. CONCLUSIONS: Overall, no relationships between genetic and physiological analyses were found, and the strains examined showed a marked genetic and phenotypic heterogeneity. L. rossiae strains had interesting properties for application in sourdough fermentation. Although some strains combined several technological traits, the association of more strains seemed to be a requisite to get optimal sourdough characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: It represents the first characterization of the diversity within the L. rossiae species. Besides, it may represent an example of computerized analysis of genotypic and phenotypic information to select strains for improving sourdough characteristics.