Phylogenetic Analyses of Phytopathogenic Isolates of Verticillium spp

ABSTRACT To better understand the genetic relationships between Verticillium dahliae isolates from lettuce and other phytopathogenic Verticillium spp. isolates from various hosts and geographic locations, the complete intergenic spacer (IGS) region of the nuclear ribosomal RNA gene (rDNA) and the beta-tubulin gene were amplified and sequenced. The sequences of the complete IGS region and the beta-tubulin gene were used alone and in combination to infer genetic relationships among different isolates of Verticillium with the maximum-likelihood distance method. Phylogenetic analyses set sequences into four distinct groups comprising isolates of V. albo-atrum, V. tricorpus, and V. dahliae from cruciferous and noncruciferous hosts. Within the four Verticillium groups, isolates of V. dahliae from cruciferous hosts displayed the closest affinity to V. dahliae from noncruciferous hosts. Isolates of V. dahliae from noncruciferous hosts could be further divided into four subgroups based on sequence similarities within the IGS region. Cross-pathogenicity tests demonstrated that most Verticillium isolates were as virulent on other hosts as on their hosts of origin. A phenogram based on the cross pathogenicity of individual isolates resembled those derived from the IGS and beta-tubulin sequence comparisons. On the basis of the data presented, the potential origin of some isolates of V. dahliae pathogenic on lettuce is proposed.     

Isolates of Verticillium dahliae Pathogenic to Crucifers Are of at Least Three Distinct Molecular Types

ABSTRACT Diverse isolates of the soilborne wilt fungi Verticillium dahliae and V. albo-atrum were studied to understand the nature and origins of those infecting cruciferous hosts. All isolates from cruciferous crops produced microsclerotia, and the majority produced long conidia with a high nuclear DNA content; these isolates were divided into two groups by amplified fragment length polymorphism (AFLP) analysis. One group could be subdivided by other criteria such as rRNA sequences and mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis. Two crucifer isolates were short spored and had a low nuclear DNA content. The results are consistent with the crucifer isolates being interspecific hybrids. The long-spored isolates are best regarded as amphihaploids (or allodiploids) with the AFLP groups probably each representing separate interspecific hybridization events. The short-spored crucifer isolates appear to be derived from interspecific hybrids and are here called 'secondary haploids'. Molecular evidence suggests that one parent in the crosses was similar to V. dahliae. The other parent of the amphihaploids seems to have been more similar to V. albo-atrum than to V. dahliae, but was distinct from all isolates of either species so far studied. The implications for the taxonomy of crucifer isolates are discussed and the use of the name V. longisporum, proposed elsewhere for just some of these isolates, is discouraged.     

Restriction fragment length polymorphisms (RFLPs) and the relationships of some host-adapted isolates of Verticillium dahliae

The relationships of two host-adapted pathotypes of Verticillium dahliae have been examined at the molecular level using restriction fragment length polymorphisms. Isolates obtained from and adapted to Mentha × piperita (peppermint), which were presumed to be haploid, formed a distinct subspecific group (referred to as M) related to the previously described non-host-adapted subspecific group A of V. dahliae. The limited molecular variation found among the four group M isolates was not related to geographic origin. Isolates from several cruciferous hosts (and one from Beta vulgaris (sugar beet)), which are thought to be natural, stable diploids, formed another distinct group (referred to as D) that was markedly different from all previously described subspecific groupings in both V. dahliae and V. alboatrum. This group of isolates might better be regarded as a separate species. Again, only limited variation was found within the D group. Polymorphisms revealed by two probes distinguished two isolates derived from Brassica rapa (Chinese cabbage) from the six other isolates (four from Brassica napus (oilseed rape) and one each from Raphanus raphanistrum (wild radish) and Beta vulgaris).     

Molecular variation within some Japanese isolates of Verticillium dahliae

Eight isolates of Verticillium dahliae from Japan, classified into four groups based on pathogenicity to differential hosts, were compared with isolates in previously defined RFLP groups. Within each of two of the pathogenicity groups (JB and JC) the pairs of haploid isolates were closely related but those in a third group (JA; isolates not pathogenic to sweet pepper or tomato) were not. Only one of the six haploid isolates (one of the two in the JA group) could be placed in an existing RFLP group. The two diploid isolates (the JD pathogenicity group) were similar to RFLP group D and only distantly related to the six haploid isolates. Of the Japanese pathogenicity groups, only JD corresponded to an existing RFLP group.     

检疫性轮枝菌及其近似种的鉴定

 大丽轮枝菌(Verticillium dahliae)和黑白轮枝菌（V. albo-atrum）在世界范围内引起多种作物的黄萎病,属于我国重要进境植物检疫对象。本研究对采自我国部分地区和CBS保存的多种植物病原性轮枝菌,包括黑白轮枝菌、大丽轮枝菌及其变种大丽轮枝菌长孢变种(V. dahliae var. longisporum)、三体轮枝菌(V. tricorpus)、变黑轮枝菌(V. nigrescens)和云状轮枝菌(V. nubilum),采用生物学特性观察,结合rDNA-ITS序列分析的方法,进行了比较和分析。结果表明：不同种类轮枝菌在休眠结构形态上具有一定差异,部分菌株不产生任何休眠结构。各供试菌株在15～25℃范围内均可生长,但黑白轮枝菌在30℃下生长受到强烈抑制,而其他菌株受影响较小。对供试菌株rDNA-ITS序列分析结果表明植物病原性轮枝菌可聚为9个分支,包括三体轮枝菌、变黑轮枝菌、云状轮枝菌、V. theobromae、大丽轮枝菌、大丽轮枝菌长孢变种和3个不同的黑白轮枝菌分支,黑白轮枝菌、大丽轮枝菌及其长孢变种亲缘关系较近。采用生物学性状结合rDNA-ITS序列分析能够更加有效地将两种检疫性轮枝菌从其他植物病原性轮枝菌中区分出来。     

Correlation of molecular markers and biological properties in Verticillium dahliae and the possible origins of some isolates

Haploid and amphihaploid Verticillium dahliae isolates were studied using PCR-based molecular markers which: (i) discriminate the defoliating and nondefoliating pathotypes (two primer pairs INTD2f/r and INTND2f/r), and (ii) are species-specific (primer pair 19/22). The results were compared with some known biological and other molecular properties of the isolates. Five discrete sequences of the 19/22 amplicon were found. Sequence 4 was associated with both defoliating isolates from Spain and nondefoliating isolates from Spain and USA; these pathotypes were separated by the primer pairs INTD2f/r and INTND2f/r, but the data showed that the primer espdef01 (derived from the 19/22 amplicon) cannot be used for this purpose. Amplicon sizes and sequences with primers 19/22 divided amphihaploid isolates from crucifers (thought to be interspecific hybrids) into those corresponding to the previously reported α and β groups. The β-group isolates had either sequence 4 or 5 (these two differing by a single base). The distinct amplicon sequence 3 given by the α-group isolates demonstrated that the V. dahliae -like 'parent' of this group was molecularly unlike any haploid isolate yet studied. The overall results are discussed in relation to phytosanitary considerations and the probability of defoliating or crucifer pathotypes arising de novo within Europe, either by selection or by interspecific hybridizations.     

Development of an assay for rapid detection and quantification of Verticillium dahliae in soil

ABSTRACT Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R(2) = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.     

Mitochondrial haplotype analysis as a tool for differentiating isolates of Verticillium dahliae

The ability to monitor mitochondrial background in Verticillium dahliae may provide an additional tool for population studies and monitoring clonal populations. Published mitochondrial genome sequences of V. dahliae (DQ351941) were used to design primers for amplification of spacer regions for assessment of mitochondrial haplotype differences among isolates. Five regions were examined (5,229 bp, or 19% of the total genome size) for 30 isolates representing a range in vegetative compatibility group (VCG), host, and geographic origin. Observed differences among isolates were due to single nucleotide polymorphisms, different numbers of bases in specific homopolymeric regions, and copies of subrepeated sequences. When the differences observed for each locus were totaled there were 28 total groupings; when the results of each locus for individual isolates were combined there were 15 mitochondrial haplotypes. Some of the observed groupings correlated with VCG. For example, five VCG-1A and VCG-1B isolates from California, Spain, and Greece had identical haplotypes; however, this was not observed for VCG-2 or VCG-4 isolates. While some VCG-2 isolates also were identical and fell into a single haplotype, five haplotypes were found for this group (five other haplotypes were observed for other isolates that had not been characterized to VCG but grouped with VCG-2 isolates in the phylogenetic analysis). Likewise, five VCG-4 isolates fell into four mitochondrial haplotypes, one of which was identical to the largest VCG-2 grouping. A heterokaryon self-incompatible isolate that was reported in the literature to cluster with VCG-2 isolates by amplified fragment length polymorphism analysis was identical with VCG-1 isolates for four of the five loci, but was intermediate between VCG-1 and VCG-2 in the haplotype analysis. Phylogenetic analysis with these regions revealed the mitochondrial background of VCG-1 and VCG2-B to be monophyletic but VCG-2A and VCG-4 could not be separated. The results obtained indicate that there is variation in mitochondrial haplotypes and this type of analysis may be a useful for characterization of isolates. While data from all five regions was used for the haplotype separation in this study, depending on the VCG or the level of variability observed within a population it is possible to use fewer loci.     

Effects of crop rotation and removal of crop debris on the soil population of two isolates of Verticillium dahliae

Microsclerotia of Verticillium dahliae are produced in large numbers on senescing parts of host plants and remain viable in the soil for many years. Changes in the population density, i.e. density of microsclerotia, in the soil were measured in micro-plots using two isolates of V. dahtiae , specific to either field bean or potato, several crop sequences comprising potato, field beans and barley, and either the removal of aerial debris of the crops or incorporation into soil.
Potato was more susceptible to the potato isolate and field bean more susceptible to the field bean isolate. Removal of debris of potato and field bean reduced numbers of microsclerotia in the soil in the subsequent years, but removal of barley straw had no effect. Initially non-infested control micro-plots became infested, probably by the growth of potato roots into the naturally infested subsoil. The rate of increase of the microsclerotial population in the non-infested control micro-plots was larger than in the initially infested treatments, because more colonized debris was produced. It is concluded that removal of aerial debris of host crops is important to reduce the soil population of V. dahtiae.     

Genetic and Morphological Diversity of Temperate and Tropical Isolates of Phytophthora capsici

ABSTRACT Phytophthora capsici is a diverse species causing disease on a broad range of both temperate and tropical plants. In this study, we used cultural characteristics, amplified fragment length polymorphism (AFLP), and DNA sequence analyses of the ribosomal internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase II (cox II) genes to characterize temperate and tropical isolates from a wide range of host species. All but one temperate isolate grew at 35 degrees C, while all tropical isolates did not. All but two tropical isolates formed chlamydospores, while temperate isolates did not. There was strong bootstrap support for separation of temperate and tropical isolates using AFLP analysis; however, the temperate isolates appeared as a subgroup within the observed variation of the tropical isolates. The majority of temperate isolates clustered within a single clade with low variation regardless of host or geographical origin, while the tropical isolates were more variable and grouped into three distinct clades. Two clades of tropical isolates grouped together and were affiliated closely with the temperate isolates, while the third tropical clade was more distantly related. Phylogenetic analysis of the ITS regions resulted in similar groupings and variation within and between the temperate and tropical isolates as with the AFLP results. Sequence divergence among isolates and clades was low, with more variation within the tropical isolates than within the temperate isolates. Analysis of other species revealed shorter branch lengths separating temperate and tropical isolates than were observed in comparisons among other phylogenetically closely related species in the genus. Analysis of cox II sequence data was less clear. Although the temperate and tropical isolates grouped together apart from other species, there was no bootstrap support for separating these isolates. Restriction fragment length polymorphism (RFLP) analysis of the ITS regions separated the temperate and tropical isolates, as in the AFLP and ITS phylogenetic analyses. However, RFLP analysis of the cox I and II gene cluster did not distinguish between temperate and tropical isolates. The differences in grouping of isolates in these two RFLP studies should be helpful in identifying isolate subgroups. Our data do not fully clarify whether or not temperate and tropical isolates should be separated into different species. The available worldwide data are incomplete and the full range of variation in the species is not yet known. We suggest refraining from using the epithet P. tropicalis until more data are available.     

Detection of Fusarium oxysporum f.sp. vasinfectum in cotton tissue by polymerase chain reaction

A polymerase chain reaction assay was developed for the detection of Fusarium oxysporum f.sp. vasinfectum (FOV), a serious wilt pathogen of cotton in many parts of the world. Based on small nucleotide differences in internal transcribed spacer sequences between 18S, 5.8S and 28S ribosomal DNAs, primers Fov1 (5'-CCCCTGTGAACATACCTTACT-3') and Fov 2 (5'-ACCAGTAACGAGGGTTTTACT-3') were selected. These primers unambiguously amplified a 400-bp DNA fragment of all the FOV isolates tested (from Angola, Brazil, China and the USA) but did not amplify any other isolates of mycoflora associated with cotton, such as F. moniliforme , Verticillium albo-atrum , V. dahliae , Aspergillus sp., F. oxysporum , F. sambucinum or F. solani . A control PCR assay was developed employing the universal primer pair ITS1 and ITS2 which amplified a fragment of approximately 220 bp from all isolates tested. This control assay demonstrated that all fungal DNAs were readily amplifiable, thus confirming that the lack of amplification with Fov1 and Fov2 primers was a result of primer specificity and not of other possible causes, such as DNA degradation or the presence of PCR inhibitors. The assay was effective on samples from the stems, leaves, roots and calli, and from plant tissues both with and without symptoms. This detection system proved to be accurate and sensitive and could aid not only diagnosis but also disease monitoring and forecasting.     

红花黄萎病病原菌鉴定

2014年在石河子大学试验站和实验农场种植的红花出现了一种植株矮化,叶片发黄,枯死的病害,切开茎秆,维管束变成褐色。采用常规组织分离法对病组织茎秆进行分离、纯化获得单孢纯培养菌株;通过常规纸钵撕底沾根法对其进行致病性测定;用形态学和 rDNA-ITS 序列分析对病原菌进行鉴定。结果表明,分离菌株的菌落形态、分生孢子梗和分生孢子的形态都与大丽轮枝菌Verticillium dahliae 一致;经分子生物学鉴定,该菌的 rDNA-ITS 序列与中国棉花黄萎病菌V．dahliae 的 ITS 序列（登录号 KT803074）同源性为99％以上。故将引起新疆红花黄萎病的病原菌鉴定为大丽轮枝菌V．dahliae。     

新疆榆树黄萎病病原菌鉴定

2013年以来,在石河子大学农学院试验站榆树苗上,出现了一种病害,可导致叶片褪绿,变黄,萎蔫,甚至枯死,剖茎检查可见维管束变成褐色。采用常规组织分离法对病茎组织进行分离、纯化获得单孢纯培养菌株;通过常规纸钵撕底蘸根法对其进行致病性测定;用形态学和rDNA-ITS序列分析方法对病原菌进行鉴定。结果表明,分离菌株的菌落形态、分生孢子梗和分生孢子的形态都与大丽轮枝菌(Verticillium dahliae)一致;经分子生物学鉴定,该菌的rDNA-ITS序列与中国棉花黄萎病菌(V.dahliae)的ITS序列(登录号EU 835817.1)相似性达99.0%,故将引起新疆榆树黄萎病的病原菌鉴定为大丽轮枝菌(V.dahliae)。     

Molecular, physiological and pathological characterization of Corynespora leaf spot fungi from rubber plantations in Sri Lanka

The plant pathogenic fungus Corynespora cassiicola causes a severe leaf spot disease on more than 70 host plant species including Hevea brasiliensis . Genetic variability in 32 isolates of C. cassiicola collected from diverse hosts and locations in Sri Lanka and Australia was assessed using restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region of ribosomal DNA and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis of total fungal DNA. Amplified ITS fragments from all 32 C. cassiicola isolates exhibited an identical size, and restriction analysis with seven different restriction endonucleases revealed identity in all of the detected DNA fragments. This finding of high genetic relatedness was further supported by the cloning and DNA sequencing of the ITS2 region from one Sri Lankan and one Australian isolate. However, RAPD-PCR profiles generated by 15 oligonucleotide decamer primers revealed significant polymorphism between groups of organisms. Genetic relationships among the isolates were determined by cluster analysis of the RAPD-PCR data and seven different RAPD groups were identified. Isolates showed strong correlations between the assigned RAPD group and the location and host plant genotype from which the isolate was collected. Correlations were also observed between the RAPD group, growth of the isolate and pathogenicity on different plant hosts.     

Variation for pathogenicity on tomato among parasexual recombinants of Verticillium dahliae

Ten haploid prototrophic recombinant isolates (HPRs) were obtained from each of 15 parasexual crosses between complementary autotrophs derived from nine tomato isolates and one eggplant isolate of V. dahliae , including those identified as race 1 and race 2. These HPRs were tested for pathogenicity to the tomato cultivar Roma which is susceptible to both races. HPRs from a 'selfed' race 2 × race 2 cross were as pathogenic as the wild-type parent. The pathogenicity of HPRs derived from all other crosses was variable, and generally lower than the parental mean. A particularly marked reduction in pathogenicity, compared with the parental mean, was observed for HPRs recovered from two crosses between isolates belonging to different heterokaryon compatibility groups. The results suggest that pathogenicity to cv. Roma is controlled by complex interactions between genes at numerous loci.     

烟草靶斑病菌菌丝融合群及ITS序列分析

 烟草靶斑病是2006年我国新报道发生的一种叶部病害^［1］,其病原的无性世代为立枯丝核菌(Rhizoctonia solani Kühn),有性世代为瓜亡革菌(Thanatephorus cucumeris (Frank)Donk)。该病菌主要危害叶部形成病斑,对烟草的产量和品质影响显著,目前该病害主要分布在辽宁省丹东和铁岭地区,并呈现出迅速蔓延趋势。烟草靶斑病最早由巴西报道,此后,哥斯达黎加、美国、南非和津巴布韦也相继发生^［2,3］。     

Influences of Cropping Practices on Verticillium dahliae Populations in Commercial Processing Tomato Fields in Ontario

ABSTRACT The abundance of Verticillium dahliae in the soil and the incidence of V. dahliae-infected plants were determined for 12 commercial processing tomato fields in Kent County, Ontario. Comparison of the data with those from a previous survey of fields in adjacent Essex County showed that soil inoculum levels and incidence of infection were generally lower in Kent County fields and that race 2 V. dahliae was not common in Kent County. From the two counties, 128 isolates were characterized by restriction fragment length polymorphism (RFLP) analysis, using the subspecies-specific repetitive DNA sequence E18. A subset of these isolates was also characterized by vegetative compatibility and DNA hybridization analysis with a second subspecies-specific DNA sequence. Isolates with E18 RFLP profiles highly similar to those of isolates previously collected from potato fields in North America were prevalent in Essex County tomato fields but not common in Kent County fields. The data are consistent with the hypothesis that the group I isolates were introduced into southwestern Ontario with potato and that the different cultural practices in Essex County and Kent County have contributed to the differences in the accumulation of these isolates in the two regions.     

Sibling species of cercospora associated with gray leaf spot of maize

ABSTRACT Monoconidial isolates of the fungus causing gray leaf spot of maize were obtained from diseased leaves collected throughout the United States and analyzed for genetic variability at 111 amplified fragment length polymorphism (AFLP) loci. Cluster analysis revealed two very distinct groups of Cercospora zeae-maydis isolates. Both groups were found to be relatively uniform internally with an average genetic similarity among isolates of approximately 93 and 94%, respectively. The groups were separated from each other by a genetic distance of approximately 80%, a distance greater than that separating each group from the sorghum pathogen, C. sorghi (67 to 70%). Characteristics and dimensions of conidia and conid-iophores produced on infected plants or nutrient media were unreliable criteria for taxonomic differentiation of isolates composing the two groups of C. zeae-maydis. Nucleotide sequences of 5.8S ribosomal DNA (rDNA) and the internal transcribed spacer (ITS) regions were identical within each group but different between the two groups and different from C. sorghi. Restriction fragment length polymorphisms generated by digestion of the 5.8S rDNA and ITS regions with TaqI readily distinguished each group and C. sorghi. Isolates in one group were generally distributed throughout maize-producing regions of the United States; isolates in the other group were localized in the eastern third of the country. Both types were present in the same fields at some locations. The genetic distance based on AFLP profiles and different ITS nucleotide sequences between the two morphologically indistinguishable groups indicate that they are sibling species. Although it is unlikely that breeding for resistance to gray leaf spot will be confounded by local or regional variation in the pathogen, a vigilant approach is warranted, because two pathogenic species exist with unknown abilities to evolve new pathotypes.     

河南商丘地区棉花黄萎病菌分离鉴定和致病力分析

为探讨河南商丘地区棉花黄萎病菌的致病型群体变异,对该地区棉花上分离的8株单孢菌株的菌落形态、显微结构、致病力、ITS序列、系统进化及菌体蛋白等方面进行了研究。结果表明:这些菌株均属于棉花黄萎病菌Verticilliumdahliae;系统进化树显示8株菌株并没有聚在同一进化枝上;8株黄萎菌菌株存在致病力差异,SQ4菌株致病力最强,属于落叶型,而其它致病力较弱的7个菌株属于非落叶型;不同致病力的菌株间蛋白谱带存在差异。