Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

西藏珠穆朗玛峰国家级自然保护区鸟类群落结构与多样性

通过样线法调查并综合有关文献,录得珠峰保护区鸟类342种,并对其鸟类群落结构与多样性进行了分析。其中留鸟218种,夏候鸟67种,冬候鸟43种,旅鸟及迷鸟14种;国家一级保护鸟类8种,二级保护鸟类31种;东洋界132种,古北界156种,广布种54种,特有种19种。多样性指数2.4340,均匀性指数0.4371。研究表明,喜马拉雅山脉将保护区明显阻隔为南坡和北坡两种不同的生态景观。南坡鸟类群落以森林鸟类为主,东洋界成分占67%,垂直分带明显,多样性系数3.3983,均匀度指数0.6396;北坡鸟类群落以湿地和荒漠鸟类为主,古北界成分占77%。呈斑块状分布,多样性指数1.8751,均匀度指数0.4199,南、北坡鸟类群落的相似百分率为15.70,差异显著。     

Application of rep-PCR fingerprinting for genotyping of Escherichia coli strains in Wojnowskie Wschodnie and Wojnowskie Zachodnie lake

The paper presents usefulness of application of the PCR-based fingerprinting method, which uses enterobacterial repetitive intergenic consensus primers ERIC (rep-PCR (ERIC)) in analysing and characterising Escherichia coli population in the water environment. 46 E. coli isolates of homogenous biochemical properties were analysed. The received results prove considerable genomic diversity among the analysed isolates. The used technique has turned to be a reproducible and rapid method with a considerable differentiation power. The introductory research has revealed that the technique may be successfully used in qualitative research, for intra-species differentiation of microorganisms occurring in water environment.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Responses of white spruce (Picea glauca) to experimental warming at a subarctic alpine treeline

From 2001 to 2004 we experimentally warmed 40 large, naturally established, white spruce [Picea glauca (Moench) Voss] seedlings at alpine treeline in southwest Yukon, Canada, using passive open‐top chambers (OTCs) distributed equally between opposing north and south‐facing slopes. Our goal was to test the hypothesis that an increase in temperature consistent with global climate warming would elicit a positive growth response. OTCs increased growing season air temperatures by 1.8°C and annual growing degree‐days by one‐third. In response, warmed seedlings grew significantly taller and had higher photosynthetic rates compared with control seedlings. On the south aspect, soil temperatures averaged 1.0°C warmer and the snow‐free period was nearly 1 month longer. These seedlings grew longer branches and wider annual rings than seedlings on the north aspect, but had reduced Photosystem‐II efficiency and experienced higher winter needle mortality. The presence of OTCs tended to reduce winter dieback over the course of the experiment. These results indicate that climate warming will enhance vertical growth rates of young conifers, with implications for future changes to the structure and elevation of treeline contingent upon exposure‐related differences. Our results suggest that the growth of seedlings on north‐facing slopes is limited by low soil temperature in the presence of permafrost, while growth on south‐facing slopes appears limited by winter desiccation and cold‐induced photoinhibition.     

Microclimate variability in alpine ecosystems as stepping stones for non‐native plant establishment above their current elevational limit

Alpine environments are currently relatively free from non‐native plant species, although their presence and abundance have recently been on the rise. It is however still unclear whether the observed low invasion levels in these areas are due to an inherent resistance of the alpine zone to invasions or whether an exponential increase in invasion is just a matter of time. Using a seed‐addition experiment on north‐ and south‐facing slopes (cf. microclimatic gradient) on two mountains in subarctic Sweden, we tested the establishment of six non‐native species at an elevation above their current distribution limits and under experimentally enhanced anthropogenic pressures (disturbance, added nutrients and increased propagule pressure). We found a large microclimatic variability in cumulative growing degree days (GDD) (range = 500.77°C, SD = 120.70°C) due to both physiographic (e.g. aspect) and biophysical (e.g. vegetation cover) features, the latter being altered by the experimental disturbance. Non‐native species establishment and biomass production were positively correlated with GDD along the studied microclimatic gradient. However, even though establishment on the north‐facing slopes caught up with that on the south‐facing slopes throughout the growing season, biomass production was limited on the north‐facing slopes due to a shorter growing season. On top of this microclimatic effect, all experimentally imposed anthropogenic factors enhanced non‐native species success. The observed microclimatic effect indicates a potential for non‐native species to use warm microsites as stepping stones for their establishment towards the cold end of the gradient. Combined with anthropogenic pressures this result suggests an increasing risk for plant invasion in cold ecosystems, as such stepping stones in alpine ecosystems are likely to be more common in a future that will combine a warming climate with persistent anthropogenic pressures.     

Phenotypic and genotypic diversity among strains of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> in comparison with related species

Intra-specific diversity of 200 Aureobasidium pullulans strains isolated from different sources and their relatives Kabatiella lini CBS 125.21 T and Hormonema prunorum CBS 933.72 T were studied by assessment of macromorphological, and physiological tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (SDS–PAGE) of whole-cell proteins as well as enterobacterial repetitive intergenic consensus (ERIC)-, repetitive extragenic palindromic (REP)- and BOX-PCR techniques (collectively known as rep-PCR). Rep-PCR is an efficient procedure for discrimination of A. pullulans in terms of simplicity and rapidity. RFLP-PCR technique was applied for the identification of A. pullulans isolates and distinction from related species. This technique was insufficient for investigation of intra-specific diversity. The tested strains of A. pullulans could be divided into two groups based on their macromorphological, protein patterns obtained after SDS-PAGE as well as rep-PCR patterns. The first group of strains shared similar characteristics and was very different from the second one, designated as “complex group”, consisting of strains with very little similarities within the group. Phenetic analysis of ERIC banding patterns failed to group the isolates on the basis of their substrate or geographical origin. Using 18S rDNA gene sequence analysis of selected isolates, three strains: HoHe3 km, A. pullulans DSM 62074 and H. prunorum CBS 933.72 T were distinguished from all other analysed members of genera Aureobasidium and Kabatiella. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

23株不同地理来源嗜酸硫杆菌的系统发育及其遗传差异

【目的】研究不同地理来源嗜酸硫杆菌的系统发育及其遗传差异,以及基因指纹图谱技术聚类与嗜酸硫杆菌地理来源的相关性。【方法】采用16S-23S r RNA间隔区(ITS)序列建立系统发育树,并结合ERIC和BOXAIR两种引物进行rep-PCR,以及rus基因扩增,对不同地理来源嗜酸硫杆菌进行分析。【结果】分离自不同样点的23株嗜酸硫杆菌遗传差异显著,依据ITS序列系统发育树被划分为5个大类群,与rep-PCR指纹图谱的分类结果较为接近,其中Acidithiobacillus ferrooxidans在ITS系统发育和BOXAIR-PCR指纹聚类分析中被划分为2个类群,但在ERIC-PCR中归为1个类群,rus基因分组中,在系统发育和聚类分析中处于同一类群的菌株拥有不同类型的rus基因,说明嗜酸硫杆菌的亚铁氧化途径与系统发育类群无明显相关性;ITS基因拥有区分近缘种或亚种的能力,且BOXAIR-PCR的分辨能力较强,非常适于嗜酸硫杆菌的遗传差异分析。     

rep-PCR fingerptinting as a tool for the analysis of genomic diversity in Escherichia coli strains isolated from an aqueous/freshwater environment

The rep-PCR fingerprinting method, with the support of ERIC and REP primers, was used to analyse the genomic diversity of 93 E. coli strains isolated from lake water samples drawn at two different depths. The applied UPGMA for DNA analysis did not reveale any genomic similarities between the 48 E. coli strains derived from the subsurface-zone water and the 43 of the bottom-zone water. The considerable genomic diversity of the E. coli of the surface zone was expressed as a dendrogram in the form of 8 similarity groups comprising strains isolated from samples drawn over one month. The bottom-zone strains, which display a lesser degree of genomic diversity (5 similarity groups), showed distinct common features in their DNA fingerprints. In the similarity dendrogram for the bottom-zone, strains derived in different months of sampling were segregated into the same similarity groups. Applying REP primers in rep-PCR generates more complex fingerprints increasing the discriminatory power of the analysis, whereas the ERIC primer generates less complex fingerprint patterns, and is thus clearer to interpret.     

Genetic Diversity of <Emphasis Type="Italic">Arcobacter</Emphasis> Species of Animal and Human Origin in Andhra Pradesh,India

Arcobacter is an emerging foodborne pathogen having zoonotic significance. Enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive sequence-based PCR (rep-PCR) analysis of a total of 41 Arcobacter isolates revealed a greater degree of genetic diversity. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power for ERIC and rep-PCR was found to be 0.997 and 0.996, respectively. Close clustering between isolates of animal and human origin are indicative of probable zoonotic significance.     

A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads

Five typing methods were evaluated, utilising 63 strains of fluorescent pseudomonads, to assess their usefulness as tools to study the bacterial diversity within this complex group. The methods used were Biolog metabolic profiling, restriction fragment length polymorphism ribotyping, PCR ribotyping, and repetitive element sequence-based PCR (rep-PCR) utilising BOX and enterobacterial repetitive intergenic consensus (ERIC) primers. Cluster analysis of the results clearly demonstrated the considerable homogeneity of Pseudomonas aeruginosa isolates and, conversely, the heterogeneity within the other species, in particular P. putida and P. fluorescens, which need further taxonomic investigation. Biolog metabolic profiling enabled the best differentiation among the species. Rep-PCR proved to be highly discriminatory, more so than the other DNA fingerprinting techniques, demonstrating its suitability for the analysis of highly clonal isolates. RFLP ribotyping, PCR ribotyping, and rep-PCR produced specific clusters of P. aeruginosa isolates, which corresponded to their origins of isolation, hence we recommend these methods for intraspecific typing of bacteria.     

Genotyping Assessment of Phosphate Solubilizing Bacteria Isolated from Rhizospheric Soil from Central Regions of Lucknow

Abstract

Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous (P) to an available form which is a major concern in Indian agriculture. In this study, 21 isolates having phosphate solubilizing capability were isolated from different regions of Lucknow, India. Among all, six efficient PSB were confirmed by using in vitro P estimation and 16S rRNA universal primers. The similarity detection was done using random amplified polymorphic DNA (RAPD) finger printing for genotyping the PSB isolates and to determine genetic relatedness between them. Twenty different OPA primers were tested among which four primers produced prominent, highly reproducible, and polymorphic bands. An average of 10.5 polymorphic bands per primer with the amplified DNA fragments ranging from 200 to 2000?bp in size. A dendrogram constructed from these data indicated 25–76% homology. Highest similarity was found in between Bacillus anthracis and Bacillus cereus with 33.8% similarity while least dissimilarity was found in B. anthracis and Pseudomonas fragi with 12% of similarity. These findings provide that there is a great genetic diversity between bacterial isolates from different geographical regions and RAPD can be used as a specific, time consuming and also proves as a reliable molecular tool which helps in strain level discrimination.     

Latitudinal patterns and regionalization of plant diversity along a 4270‐km gradient in continental Chile

According to the global latitudinal diversity gradient, a decrease in animal and plant species richness exists from the tropics towards higher latitudes. The aim of this study was to describe the latitudinal distribution patterns of Chilean continental flora and delineate biogeographic regions along a 4270‐km north–south gradient. We reviewed plant lists for each of the 39 parallels of continental Chile to build a database of the geographical distribution of vascular plant species comprising 184 families, 957 genera and 3787 species, which corresponded to 100%, 94.9% and 74.2% of the richness previously defined for Chile, respectively. Using this latitudinal presence–absence species matrix, we identified areas with high plant richness and endemism and performed a Cluster analysis using Jaccard index to delineate biogeographic regions. This study found that richness at family, genus and species levels follow a unimodal 4270‐km latitudinal distribution curve, with a concentration of richness in central Chile (31–42°S). The 37th parallel south (central Chile) presented the highest richness for all taxonomic levels and in specific zones the endemism (22–37°S) was especially high. This unimodal pattern contrasts the global latitudinal diversity gradient shown by other studies in the Northern hemisphere. Seven floristic regions were identified in this latitudinal gradient: tropical (18–22°S), north Mediterranean (23–28°S), central Mediterranean (29–32°S), south Mediterranean (33–37°S), north temperate (38–42°S), south temperate (43–52°S) and Austral (53–56°S). This regionalization coincides with previous bioclimatic classifications and illustrates the high heterogeneity of the biodiversity in Chile and the need for a reconsideration of governmental conservation strategies to protect this diversity throughout Chile.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

Rep-PCR Identifies Both Inter- and Intra-Specific Mitochondrial Genome Differences in Carthamus

Mitochondria are derived from ancient prokaryotic endosymbionts, and their genomes exhibit similarities to prokaryote genomes. Therefore, it was hypothesized that the molecular techniques suitable for distinguishing prokaryotic genomes could also be used to assess mitochondrial diversity. The rep-PCR (repetitive element palindromic-PCR) technique, based on the repetitive sequences found in bacterial genomes, has been used extensively for identifying and distinguishing bacterial strains. This study was undertaken to evaluate the utility of rep-PCR for identifying mitochondrial (mt) genome diversity in safflower (Carthamus tinctorius L.) and its wild relatives. Using three sets of commonly used primers, BOX, ERIC and REP, both inter-specific and intra-specific mt genome diversities in Carthamus were identified. To confirm that the amplicons obtained with rep-PCR were derived from mitochondrial genomes, we cloned and sequenced six randomly chosen bands from rep-PCR gels and demonstrated that the amplified products were mitochondrial-genome-specific. The advantages of rep-PCR in assessing chondriome variability are discussed.     

Change in the temperature preferences of <Emphasis Type="Italic">Beauveria bassiana sensu lato</Emphasis> isolates in the latitude gradient of Siberia and Kazakhstan

The radial growth of twenty isolates of the entomopathogenic fungus Beauveria bassiana sensu lato from different natural zones of Western Siberia and Kazakhstan (from 65 to 43°N) was tested under different temperatures (5–35°C). It was shown that the thermotolerance of the fungal isolates increased significantly from the north to south. The cold activity of the cultures did not significantly correlate with the latitude of origin and the sum positive temperatures of the regions. A distinct group of the steppe thermotolerance isolates was shown by the analysis of genomic polymorphism using seven intermicrosatellite DNA markers (ISSR). The steppe isolates had high levels of virulence to the wax moth Galleria mellonella and the Colorado potato beetle Leptinotarsa decemlineata at high temperatures (>30°C) compared to that of the forest-steppe isolates. The obtained data indicate that the use of isolates from the steppe zone will be most promising for the insect pest control under the conditions of continental and arid climate.     

Patterns of β‐diversity in a Mexican tropical dry forest

Abstract. Patterns of β‐diversity in a highly diverse tropical dry forest tree community are described; the contribution of environmental heterogeneity and distance to β‐diversity was assessed. Significant differences in elevation, insolation, slope and soil water holding capacity (p < 0.01), variables related to water availability, were found among 830 m × 100 m transects laid along contrasting slopes of a system of three parallel microbasins. A gradient in elevation and insolation was found within north‐facing transects, among 10 m × 10 m sites; south‐facing transects showed an elevation gradient while crest transects showed a gradient in water holding capacity. In total 119 species were registered, with 27 to 64 species per transect, and 4 to 16 species per site. A large β‐diversity was found among and within transects; two indices of β‐diversity consistently showed a higher β‐diversity within transects than among them. Among transects, 64% of the variance in species composition could be attributed to the environmental variables; an additional 22% to the spatial distribution of sites. Within transects, 42% of the deviance in β‐diversity values was explained by insolation, and 19% by distance. β‐diversity increased with distance and with difference in insolation among sites; north‐facing transects, those with most contrasting insolation conditions, had the steepest increase in β‐diversity with distance. Such increase was clearly associated with changes in species composition, not with changes in species richness.     

Molecular Typing of Paenibacillus larvae Strains Isolated from Bulgarian Apiaries Based on Repetitive Element Polymerase Chain Reaction (Rep-PCR)

The aim of the present study was to perform molecular typing of Paenibacillus larvae (P. larvae) isolates from Bulgarian apiaries with repetitive element polymerase chain reaction (rep-PCR) using BOX A1R, MBO REP1, and ERIC primers. A total of 96 isolates collected from brood combs with clinical symptoms of American foulbrood originating from apiaries located in different geographical regions of Bulgaria, a reference strain P. larvae NBIMCC 8478 and 30 commercial honey samples with Bulgarian origin were included in the study. Rep-PCR fingerprinting analysis revealed two genotypes ab and AB of P. larvae isolates from brood combs and honey samples. A combination of genotypes ab/AB was detected in one apiary and honey sample. The prevailing genotype ab was found in 78.1 % of brood combs isolates as well as in the reference strain whereas genotype AB was determined in 21.9 % of isolates. The examination of honey samples confirmed the preponderance of ab genotype which was demonstrated in 20 of 30 samples analyzed. In conclusion, the genetic epidemiology of P. larvae revealed two genotypes—ab and AB for Bulgarian strains. Developed protocols for molecular typing of P. larvae are reliable and may be used to trace the source of infection.     

Nitrogen fixation and nodule occupancy by native strains of Rhizobium on different cultivars of common bean (Phaseolus vulgaris L.)

A field experiment under rainfed conditions was conducted in Durango, México, to assess N2-fixation of three cultivars of common bean (Phaseolus vulgaris L.) using ¹⁵N-methodology. In addition, diversity of rhizobial isolates obtained from nodules of the different plant genotypes was evaluated by intrinsic antibiotic resistance (IAR), PCR using enterobacterial repetitive intergenic consensus (ERIC) primers, PCR-RFLP analysis of the 16S rRNA gene and multilocus enzyme electrophoresis (MLEE). Selected isolates were used to determine acetylene reduction and competitive ability under greenhouse conditions. The three cultivars tested did not show high variation in N2-fixation, the %Ndfa values ranged from 19 to 26%. Variability in N2-fixation efficiency among various native rhizobial isolates was very high and our results indicate that differences in competitive abilitiy exist also. PCR-RFLP of the 16S rRNA gene and MLEE revealed that most of the isolates belong to the species Rhizobium etli. Intrinsic antibiotic resistance analysis and ERIC-PCR showed high diversity among isolates. In contrast, our results using MLEE show low genetic diversity (H = 0.105).