我国三地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响

【目的】Wolbachia 是广泛存在于节肢动物和丝状线虫体内的一类共生菌, 能够以多种方式对宿主产生影响。精卵细胞质不亲和（CI）是其引起的最普遍的表型, 即感染Wolbachia的雄性宿主与未感染或感染不同品系的雌性宿主交配后, 不能产生后代或后代极少, 而感染同品系Wolbachia的雌雄宿主交配后则能正常产生后代。我们前期研究发现, 湖北武汉、 云南六库和天津3个地区黑腹果蝇Drosophila melanogaster被Wolbachia感染。本研究旨在明确这3个地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响。【方法】利用Clustal X软件对Wolbachia的wsp基因序列进行比对, 利用MEGA软件构建系统发育树。采用多位点序列分型（MLST）的方法对Wolbachia进行分型。通过区内交配和区之间杂交的方式研究不同地区黑腹果蝇体内Wolbachia 的关系及其对果蝇生殖的影响。【结果】湖北武汉、 云南六库和天津3个地区黑腹果蝇中感染的Wolbachia都是属于A大组的Mel亚群。这3个地区果蝇感染的Wolbachia的序列类型（ST）不同, Wolbachia之间存在一定的差异。湖北武汉和天津果蝇中的Wolbachia能引起强烈的CI表型, 而云南六库果蝇中的Wolbachia引起的CI强度相对较弱。武汉果蝇中Wolbachia不能完全挽救天津果蝇中Wolbachia引起的CI表型, 而天津果蝇中Wolbachia也不能完全挽救武汉果蝇中Wolbachia引起的CI表型。【结论】武汉和天津地区黑腹果蝇中的Wolbachia可能距离较远。Wolbachia的长期共生可能对黑腹果蝇的进化产生了一定的影响, 湖北武汉与云南六库的黑腹果蝇中感染的Wolbachia属于不同的序列类型, 这2个地区的黑腹果蝇已发生了一定的分歧, 产生了一定的生殖隔离。     

Multi-Loci Sequence Typing (MLST) for Two Lacto-Acid Bacteria (LAB) Species: <Emphasis Type="Italic">Pediococcus parvulus</Emphasis> and <Emphasis Type="Italic">P. damnosus</Emphasis>

The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P. parvulus when using either conventional PCR or Real Time PCR.     

Habitat fragmentation as a result of biotic and abiotic factors controls pathogen transmission throughout a host population

1. The hypothesis that habitat fragmentation (biotic or abiotic) alters the transmission of disease within a population is explored using field data from a well-studied amphibian-pathogen system. 2. We used the Ambystoma tigrinum-A. tigrinum virus (ATV) model system to show how habitat fragmentation as a result of emergent vegetation and habitat management affects disease transmission dynamics in ponds across a landscape. 3. We quantified variation in ATV infection over time and across the landscape. ATV infection was significantly higher in ponds modified for livestock use (P = 0.032). Disease incidence decreased with increased amounts of emergent vegetation (P < 0.001). These factors appear to control disease transmission by altering the host contact rate and with it disease transmission. 4. A field experiment to test the effect of emergent vegetation on the distribution of larvae in ponds demonstrated a behavioural change in larvae found in sparsely vegetated ponds. Microhabitat choices resulted in larvae being concentrated at the pond edge resulting in a 'halo effect' in sparsely vegetated ponds, whereas larvae in heavily vegetated ponds were distributed more evenly throughout. Microhabitat choice affects the effective density that larvae experience. This 'halo effect' increases contact rates in the shallows of sparsely vegetated ponds and increases the transmission of a directly transmitted pathogen. 5. Despite recurrent epidemics of a lethal Ranavirus in tiger salamanders on the Kaibab Plateau, Arizona, USA, these populations persist. We discuss the implications of our results in the context of density-dependent transmission and homogeneous mixing, two increases key assumptions of epidemiological theory.     

Development and characterization of microsatellite markers in <Emphasis Type="Italic">Castanea sativa</Emphasis> (Mill.)

Thirty-three simple sequence repeat (SSR) markers were isolated andcharacterized in Castanea sativa (Mill.) from the cultivarGarrone Nero. For the identification of SSR loci, primers were designed on eachside flanking the repeat region and they were initially tested on 5 chestnutsamples using chemiluminescence detection. Twenty four loci where shown to bepolymorphic and the number of alleles detected per locus varied from 2 to 7.Fourteen loci were chosen for the analysis of 20 cultivars grown in North Italyusing the semi-automatic system ABI PRISM 377. These 14 markers showed a highlevel of genetic polymorphism with a total of 90 alleles; the number of allelesranged from 4 to 10 per locus, with an average level of 6.4. The mean expectedand observed heterozygosity were 0.724 (range: 0.649–0.835) and 0.793(range: 0.350–0.950) respectively. The estimated frequency of nullallelesshowed a positive value for 3 loci, but except for 1 locus, the values wereverylow. The total value for the probability of identity was 7.04 ×10^–11. Paternity exclusion probability was very high (0.999),sufficiently high to study pollen flow.     

Frequent occurrence of multidrug‐resistant CC17 Enterococcus faecium among clinical isolates in Sweden

Aims: To screen for the globally spread cluster of Enterococcus faecium, clonal complex 17 (CC17) and characterize the genetic profile of Swedish clinical Ent. faecium isolates. Methods: A total of 203 consecutive isolates collected from 2004 to 2007 from patients with bacteraemia in Sweden. All isolates were genotyped using multiple‐locus variable‐number tandem repeat analysis (MLVA) and 20 isolates representing different MLVA types (MT) were chosen for multilocus sequence typing (MLST). Minimal inhibitory concentrations against clinically relevant antibiotics were determined with agar dilution. Presence of the virulence genes esp and hyl was investigated using PCR. Results: A total of 65% (n = 109) of all isolates belonged to MT‐1, and the second most common MLVA type was MT‐159 (13%, n = 21). MLST analysis confirmed the presence of CC17 during the entire study period. The number of isolates resistant to gentamicin and vancomycin, as well as the presence of hyl, increased significantly during the investigation period. Conclusions: The present study demonstrates that nosocomial infections caused by Ent. faecium CC17 are commonly occurring in Sweden. Significance and Impact of the Study: This is the first report of CC17 Ent. faecium in Sweden. The increase of antibiotic resistance and virulence indicates that these strains are further adapting to the hospital environment.     

Differentiation of probiotic and environmental Saccharomyces cerevisiae strains in animal feed

Aims: To establish an identification system for probiotic Saccharomyces cerevisiae strains based on artificial neural network (ANN)–assisted Fourier‐transform infrared (FTIR) spectroscopy to improve quality control of animal feed. Methods and Results: The ANN‐based system for differentiating environmental from probiotic S. cerevisiae strains comprises five authorized feed additive strains plus environmental strains isolated from different habitats. A total of 108 isolates were used as reference strains to create the ANN. DHPLC analysis and δ‐PCR were used as reference methods to type probiotic yeast isolates. The performance of the FTIR‐ANN was tested in an internal validation using unknown spectra of each reference strain. This validation step yielded a classification rate of 99·1 %. For an external validation, a test data set comprising 965 spectra of 63 probiotic and environmental S. cerevisiae isolates unknown to the ANN was used, resulting in a classification rate of 98·2 %. Conclusions: Our results demonstrate that probiotic S. cerevisiae strains in feed can be differentiated successfully from environmental isolates using both genotypic approaches and ANN‐based FTIR spectroscopy. Significance and Impact of the Study: FTIR‐based artificial neural network analysis provides a rapid and inexpensive technique for yeast identification both at the species and at the strain level in routine diagnostic laboratories, using a single sample preparation.     

桑椹肥大性菌核病病原菌生物学特性及流行性

【目的】对桑椹灾害性真菌病害——桑椹肥大性菌核病病原菌,即桑实杯盘菌(Ciboria shiraiana)的生物学特性进行研究,分析其流行性。【方法】采用人工接种、调查等研究方法,对C.shiraiana在无性生长阶段中菌丝侵染能力,菌核的休眠期,有性生长阶段中子囊孢子的结构、释放、数目以及萌发等进行研究,并对菌核萌发的物候期进行调查。【结果】C.shiraiana菌丝对桑雌花没有侵染能力;C.shiraiana菌核具有休眠期,低温处理6周以上的菌核才能萌发形成子囊盘;1个菌核可萌发1–15个子囊盘,直径为1.5 cm的子囊盘能产生高达(5.6–6.3)×10~7个子囊孢子;C.shiraiana子囊孢子在酸性环境中的萌发率明显高于在中性和碱性环境中的萌发率;C.shiraiana菌核萌发形成子囊盘产生子囊孢子的物候期,从1月下旬开始到4月中旬结束,其中在3月中旬萌发子囊盘的数目达到最高值。【结论】桑椹肥大性菌核病属于典型的流行性侵染病,在果桑栽培上容易造成毁灭性危害,生产上必须高度重视该病的防控。     

Comparative sequence analysis of recA gene among Vibrio cholerae isolates from Iran with globally reported sequences

Aims: To study the genetic relatedness between V. cholerae isolates from Iran and other countries based on housekeeping gene recA sequence analysis. Methods and Results: A 995‐bp region of the recA gene from 24 V. cholerae isolates obtained from human and surface water origins in Iran over a 5‐year period was sequenced and compared with the sequence data from the isolates belonging to other places. Cluster analysis of the constructed dendrogram based on recA sequence divergence for our clinical isolates showed one sequence type (ST), whereas environmental isolates revealed eight STs. Interestingly, one of our environmental isolates was intermixed with clinical isolates in the largest cluster containing the epidemic strains. Our 24 isolates plus 198 global isolates available in the GenBank showed 77 sequence types (STs) with at least one nucleotide difference. Conclusions: Our result suggested that recA sequencing is a reliable analysis method for understanding the relatedness of the local isolates with the isolates obtained elsewhere. Significance and Impact of the Study: Understanding the genetic relatedness between V. cholerae isolates could give insights into the health care system for better control and prevention of the cholera.     

A multilocus sequence analysis of Xanthomonas campestris reveals a complex structure within crucifer-attacking pathovars of this species

Previous classification of Xanthomonas campestris has defined six pathovars (aberrans, armoraciae, barbareae, campestris, incanae, and raphani) that cause diseases on cruciferous plants. However, pathogenicity assays with a range of strains and different hosts identifies only three types of symptom: black rot, leaf spot and bacterial blight. These findings raise the question of the genetic relatedness between strains assigned to different pathovars or symptom phenotypes. Here we have addressed this issue by multilocus sequence analysis of 42 strains. The X. campestris species was polymorphic at the 8 loci analysed and had a high genetic diversity; 23 sequence types were identified of which 16 were unique. All strains that induce black rot (pathovars aberrans and campestris) were genetically close but split in two groups. Only three clonal complexes were found, all within pathovar campestris. The assignment of the genome-sequenced strain 756C to pathovar raphani suggested from disease symptoms was confirmed, although this group of strains was particularly polymorphic. Strains belonging to pathovars barbareae and incanae were closely related, but distinct from pathovar campestris. There is evidence of genetic exchanges of housekeeping genes within this species as deduced from a clear incongruence between individual gene phylogenies and from network structures from SplitsTree analysis. Overall this study showed that the high genetic diversity derived equally from recombination and point mutation accumulation. However, X. campestris remains a species with a clonal evolution driven by a differential adaptation to cruciferous hosts.