Phylogenetic analysis of Ukrainian BYMV isolates from soybeans and beans

Two isolates of Bean yellow mosaic virus originating from soybean (Glycine max L.) and bean (Phaseolus vulgaris L.) plants that are distributed in Ukraine were examined according to their molecular characteristics. PCR amplification products were sequenced and compared with the corresponding sequences of different BYMV isolates obtained from GeneBank. The dates obtained in phylogenetic analysis showed 98% sequence identities of isolates under study, and, in accordance with the species demarcation criteria and identification guidelines for potyviruses, it was suggested that the Ukrainian isolates are one strain of BYMV. The sequence showed a high degree of sequence identities with other BYMV isolates/strains and shared maximum identity with BYMV strains reported from Russia, Australia, and Argentina. The sequence data have been submitted to NCBI, accession numbers KT923790.1 for soybean isolate and KT923791.1 for bean isolate of BYMV.     

Split Personality of a Potyvirus: To Specialize or Not to Specialize?

Bean yellow mosaic virus (BYMV), genus Potyvirus, has an extensive natural host range encompassing both dicots and monocots. Its phylogenetic groups were considered to consist of an ancestral generalist group and six specialist groups derived from this generalist group during plant domestication. Recombination was suggested to be playing a role in BYMV''s evolution towards host specialization. However, in subsequent phylogenetic analysis of whole genomes, group names based on the original hosts of isolates within each of them were no longer supported. Also, nine groups were found and designated I-IX. Recombination analysis was conducted on the complete coding regions of 33 BYMV genomes and two genomes of the related Clover yellow vein virus (CYVV). This analysis found evidence for 12 firm recombination events within BYMV phylogenetic groups I–VI, but none within groups VII–IX or CYVV. The greatest numbers of recombination events within a sequence (two or three each) occurred in four groups, three which formerly constituted the single ancestral generalist group (I, II and IV), and group VI. The individual sequences in groups III and V had one event each. These findings with whole genomes are consistent with recombination being associated with expanding host ranges, and call into question the proposed role of recombination in the evolution of BYMV, where it was previously suggested to play a role in host specialization. Instead, they (i) indicate that recombination explains the very broad natural host ranges of the three BYMV groups which infect both monocots and dicots (I, II, IV), and (ii) suggest that the three groups with narrow natural host ranges (III, V, VI) which also showed recombination now have the potential to reduce host specificity and broaden their natural host ranges.     

Plasmid distribution in European <Emphasis Type="Italic">Diaporthe helianthi</Emphasis> isolates

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe, which has been sporadically recorded in Italy. A collection of 26 Diaporthe helianthi isolates deriving from different geographic origins was analysed in order to determine the presence of extra-chromosomal genetic determinants and their molecular diversity. Extra-chromosomal bands in total genomic DNAs were identified in every French and the Yugoslavian isolate and in only one Italian isolate, while no Romanian and Argentinean isolates resulted to host any plasmids. When tested for their chemicophysical nature, they were recognised as linear plasmids sized about 2.3 Kb. A more detailed analysis was performed on a plasmid purified from a French isolate (plasmid F). Its intracellular localisation resulted as mitochondrial. Plasmid F was also exploited as a probe in Southern hybridisation experiments, in which it recognised only plasmids present in the genomes of French and Yugoslavian isolates (countries were the disease has a heavy incidence) indicating a strong correlation to geographic origin. An RFLP hybridisation analysis performed on genomic DNAs revealed a homogeneous restriction pattern in all French and Yugoslavian isolates, suggesting molecular homology among plasmids present in those isolates.     

Alfalfa mosaic virus isolates from lucerne in South Australia: biological variability and antigenic similarity

Alfalfa mosaic virus (AMV) was isolated from lucerne (Medicago sativa) plants with a variety of disease symptoms in eight of 13 sites in South Australia indicating that the virus is widespread in the state. The host ranges and symptomatology of the virus isolates varied considerably. Twelve selected local lesion isolates were shown to be distinct when mechanically inoculated to a range of plant species and cultivars. However, agar-gel diffusion and enzyme-linked immunoassay tests with polyclonal antisera prepared against glutaraldehyde-fixed virus preparations of the five most readily distinguishable AMV isolates, failed to reveal significant antigenic differences between the 12 virus isolates. This indicates that serological tests with polyclonal antisera can detect a wide range of AMV variants but would be unlikely to differentiate between strains. The wide host range and variability of AMV precluded the grouping of isolates into strains of the virus.     

基于线粒体细胞色素氧化酶I（COI）序列的猪和兔四个疥螨分离株的系统发育关系分析

为了探讨兔疥螨分离株和猪疥螨分离株的分类地位, 采用PCR技术首次扩增了分离自中国猪和兔的4个疥螨分离株的线粒体细胞色素氧化酶I（COI）基因, 并与GenBank中注册的14个国外疥螨分离株的同源基因进行了比较。序列分析结果显示: 扩增的4个疥螨株COI基因长度均为1 427 bp, 序列间无插入、缺失, A+T含量（73%）明显高于G+C含量（27%）, 碱基组成存在明显偏移。猪和兔的4个疥螨分离株间的COI基因同源性较高（99.1%～100.0%）, 它们与澳大利亚人疥螨株、国外动物疥螨株的同源性范围为98.4%～99.6%。在构建的NJ树中, 分离自中国猪和兔的4个疥螨分离株同澳大利亚人疥螨分离株、国外动物疥螨分离株亲缘关系较近。根据疥螨COI基因同源性分析和系统树构建结果, 我们认为分离自中国猪和兔的4个疥螨分离株与澳大利亚人疥螨分离株以及国外的动物疥螨分离株均应属于同一个种。     

Interaction of Cucumber mosaic virus and Bean yellow mosaic virus in co-infected plants of bean and broad bean

After evaluation of the responses of bean and broad bean common cultivars against an isolate of Cucumber mosaic virus (CMV-K) and Bean yellow mosaic virus (BYMV-K), interaction of isolates was statistically studied on co-infected plants of bean cv. Bountiful and broad bean cv. Lahijan at two trials. Based on viral relative concentration determined by quantitative enzyme-linked immunosorbent assay, BYMV interacts synergistically with CMV in bean at 14 days post inoculation, while in co-infection with BYMV, CMV interacts antagonistically in both host plants at least in one of the two trials. This suggests that CMV/BYMV interaction is dependent on host species and developmental stage of plant. Co-infection like single infection with CMV in bean plants led to significantly decrease in plants’ height and fresh weight than BYMV singly infected and healthy plants, while viral infection of broad bean plants did not significantly affect growth parameters. Decline effect of viral infection (especially co-infection) on chlorophyll and carotenoids value of bean plants was greater than those of broad bean. Viral infection (singly or doubly) caused irregular changes in nutrient elements values of both hosts compared with healthy ones.     

Plant Virology and Next Generation Sequencing: Experiences with a Potyvirus

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I–IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.     

Patterns of spread of two non-persistently aphid-borne viruses in lupin stands under four different infection scenarios

Patterns of spread of Bean yellow mosaic virus (necrotic type, BYMV‐N) and Cucumber mosaic virus (CMV) were examined in stands of narrow‐leafed lupin (Lupinus angustifolius) where naturally occurring aphid vectors moved them from external or internal primary virus sources. The lupin stands were: commercial crops near BYMV‐infected clover pasture with or without an intervening non‐host barrier crop; a large rectangular block with BYMV‐N and CMV sources on opposite sides and a narrow, non‐host barrier crop facing the BYMV‐N source; and a plot within which seed‐infected lupin plants acted as internal CMV sources. When BYMV‐N spread into commercial crops in the absence of a non‐host barrier, there was a steep decline in its incidence with distance from the crop edge. However, when a 20 m‐wide perimeter barrier of oats intervened between the two, there was only a shallow decline. When CMV and BYMV‐N spread from opposite directions into a block with a 0.25 m‐wide oat barrier between it and the BYMV‐N source, the BYMV‐N incidence gradient was shallow but in the opposite direction the CMV gradient was steep. When CMV spread from primary sources within a plot, infection was concentrated in large internal patches. Spread of BYMV‐N was more diffuse with more isolated symptomatic plants and small clusters than occurred with CMV, spread of which was more comprehensive, reacting the near monocyclic and polycyclic patterns of spread with BYMV‐N with CMV respectively. Spread of both viruses was greater along than across rows, especially with CMV. With BYMV‐N, three different phased cycles of secondary spread were evident in the individual symptomatic plants within the small clusters that formed away from the edges of lupin stands. These findings help validate inclusion of perimeter non‐host barriers within an integrated disease management strategy for BYMV‐N in lupin.     

Genetic relationships among clinical and environmental isolates ofVibrio cholerae from Australia

DNA-DNA homology among twenty-nine isolates having the phenotypic properties ofVibrio cholerae was studied using the S1 endonuclease method. Ten strains ofV. cholerae O1 isolated from patients and from the environment in Australia showed greater than 88% homology with the neotype strain ofV. cholerae NCTC 8021. Strains of the non-O1 serotype isolated from a variety of clinical and environmental sources also showed a high level of relatedness, including four luminescent strains and a reference strain of the biotypealbensis. A group of sucrose-negative strains showed a low level of homology (40 to 43%) withV. cholerae, but 75% and 82% homology within the group.     

Adherence and motility characteristics of clinical Acinetobacter baumannii isolates

Acinetobacter baumannii continues to be a major health problem especially in hospital settings. Herein, features that may play a role in persistence and disease potential were investigated in a collection of clinical A. baumannii strains from Australia. Twitching motility was found to be a common trait in A. baumannii international clone I strains and in abundant biofilm formers, whereas swarming motility was only observed in isolates not classified within the international clone lineages. Bioinformatic analysis of the type IV fimbriae revealed a correlation between PilA sequence homology and motility. A high level of variability in adherence to both abiotic surfaces and epithelial cells was found. We report for the first time the motility characteristics of a large number of A. baumannii isolates and present a direct comparison of A. baumannii binding to nasopharyngeal and lung epithelial cells.     

广西不同时期IBV分离株S1基因高变区Ⅰ的遗传变异分析

对广西1985-2007年间分离到的22株传染性支气管炎病毒（IBV）的S1基因高变区I（HVRI）进行序列测定,并与发表的其他IBV参考株及鸽子分离的冠状病毒株的基因序列进行比较和分析。系统进化关系显示毒株可分为5个基因群,其中有16个广西分离株属第1群,它们与鸽子冠状病毒分离株的氨基酸序列同源性较高,与Massachusetts（Mass）型疫苗株的同源性较低。有15个分离株在33-34位和34～35之间分别有4个和3个氨基酸残基的插入,GX-NN6在33～34位和34～35位之间则均有4个氨基酸残基的插入;GX-YL1、GX-NN2与常用的Mass型疫苗株的亲缘关系最近,同属于第Ⅱ群;GX-G、GX-XD与日本同一时期分离的毒株JP Miyazaki 89亲缘关系最近,属于第Ⅲ群;GX-YL6、GX-NN7与欧洲毒株4／91亲缘关系较近,属于第V群。结果表明广西存在着多种类型IBV毒株的流行,毒株S1基因HVRI碱基的突变或插入比较普遍,可导致其氨基酸序列的变化,绝大部分毒株与目前常用的Mass型疫苗株的亲缘关系较低。同一时期的分离株同源性较高,但无明显的地域性差异。     

Recent horizontal transmission of plasmids between natural populations of Escherichia coli and Salmonella enterica.

Seventy-one natural isolates obtained from a Salmonella reference collection were examined for the presence of plasmids closely related to the Escherichia coli F plasmid. The collection consists of several serovars of the S. enterica Typhimurium complex, subspecies I, to which 99% of pathogenic salmonellae belong. Molecular genetic techniques of DNA hybridization, along with PCR and DNA sequencing, were used to examine the occurrence, distribution, and genetic diversity of F-like plasmids among Salmonella strains. The F plasmid genes examined were finO, traD, traY, and repA, which map at dispersed positions on the F plasmid of E. coli. Comparative sequence analysis of each of the four genes in Salmonella plasmids showed them to be homologous (in some cases, virtually identical) to those found in F plasmids of E. coli natural isolates. Furthermore, the frequency of F-like plasmids in Salmonella strains was approximately the same as that observed in the E. coli Reference Collection. However, in Salmonella, the distribution was confined predominately to the serovars Typhimurium and Muenchen. The unexpected finding of a shared pool of F-like plasmids between S. enterica and E. coli demonstrates the significant role of conjugation in the histories of these important bacterial species.     

Re-evaluation of Japanese Phytophthora isolates based on molecular phylogenetic analyses

Over the past 40 years in Japan, Phytophthora isolates have been collected from various diseased host tissues and infested soils and identified using morphological characters. In order to develop a molecular method for the characterization of Japanese Phytophthora species, we obtained nuclear ribosomal ITS and LSU and mitochondrial coxI DNA sequences from 151 isolates representing 21 known species and 10 unidentified isolates. These were compared with similar sequences from representative isolates of known species. Of these, 124 isolates were found to have been correctly identified. Among the remaining 37 isolates, 19 showed high homology with other described species. The remaining 18 isolates showed only low levels of homology with any known species, and generated monophyletic sub-clades in a phylogenetic tree based on the ITS and nLSU regions and the coxI gene. Therefore, these isolates are candidates for new species, falling into six groups. Together, the Japanese isolates were found to represent phylogenetically diverse groups of species. In a sequence variation analysis, the ITS regions and the coxI genes were found to be more variable than the nLSU sequences, suggesting that they will be more useful for Phytophthora identification.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.     

禽源致病性大肠埃希菌安徽分离株I型菌毛pilA基因的序列测定与分析

目的探明大肠埃希菌I型菌毛pilA基因在不同宿主来源菌株间的同源性,为利用Ⅰ型菌毛基因诊断和防治大肠埃希菌病提供理论基础。方法以禽源致病性大肠埃希菌安徽分离株基因组DNA为模板,采用PCR方法扩增Ⅰ型菌毛pilA基因,并进行序列测定与分析。结果 13株不同血清型禽源致病性大肠埃希菌安徽分离株均携带pilA基因,彼此间该基因的核苷酸序列和氨基酸序列同源性分别介于84.9%～99.7%和86.2%～99.2%。pilA基因核苷酸序列在安徽分离株与鸡源参考株、猪源参考株以及人源参考株之间的同源性分别为85.9%～99.7%、85.9%～93.9%和87.5%～100%,氨基酸序列同源性分别为83.1%～99.2%、88.5%～93.8%和90%～100%。系统发育进化树分析显示JD16、JD34、JD11、JD24、YD2、JD8和YD1禽源安徽分离株与人源参考株sPH2的亲缘关系较近,在进化树的同一分支上;GD3、YD5、GD2、YD3、YD5和YD7禽源安徽分离株与鸡源参考株PDI-386和猪源参考株107/86的亲缘关系较近,在进化树中属于同一分支;GD1禽源安徽分离株与鸡源参考株HY1-2和MS2-1的亲缘关系较近,在进化树中属于另一分支。结论大肠埃希菌I型菌毛pilA基因在不同宿主来源菌株及不同血清型菌株之间高度保守,可作为大肠埃希菌病的诊断基因和疫苗候选基因。     

Biological and Serological Variability of Zucchini Yellow Mosaic Virus in Sudan

Zucchini yellow mosaic potyvirus (ZYMV) is prevalent in different cucurbit growing agro-ecosystems in Sudan. A study of the biological and serological variability of isolates originating from different regions was conducted to better understand ZYMV epidemiology and to develop adapted and durable control strategies. Variability was detected among isolates regarding symptomatology, host range and virulence towards the Zym resistance gene in melon ( Cucumis melo L.) PI 414723. Serological variability was also revealed using a set of seven differential monoclonal antibodies (mAbs) raised against a French isolate (ZYMV-E9). Six serotypes were differentiated, but a majority of isolates (88%) reacted with all the mAbs as did the reference strains from Italy and France. All isolates from Sudan were equally well controlled by the resistance genes described in squash ( Cucurhita moschata (Duchesne) Duchesne ex Poir. cvs. Menina and Nigeria) and in cucumber ( Cucumis sativus L. cv. TMG), or by cross protection with the mild ZYMV-WK strain. All isolates were transmitted in a nonpersistent manner by Aphis gossypii Glover and Myzus persicae Sulzer.