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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).  相似文献   

3.
Host Range Specificity in Verticillium dahliae   总被引:1,自引:0,他引:1  
Bhat RG  Subbarao KV 《Phytopathology》1999,89(12):1218-1225
ABSTRACT Verticillium dahliae isolates from artichoke, bell pepper, cabbage, cauliflower, chili pepper, cotton, eggplant, lettuce, mint, potato, strawberry, tomato, and watermelon and V. albo-atrum from alfalfa were evaluated for their pathogenicity on all 14 hosts. One-month-old seedlings were inoculated with a spore suspension of about 10(7) conidia per ml using a root-dip technique and incubated in the greenhouse. Disease incidence and severity, plant height, and root and shoot dry weights were recorded 6 weeks after inoculation. Bell pepper, cabbage, cauliflower, cotton, eggplant, and mint isolates exhibited host specificity and differential pathogenicity on other hosts, whereas isolates from artichoke, lettuce, potato, strawberry, tomato, and watermelon did not. Bell pepper was resistant to all Verticillium isolates except isolates from bell pepper and eggplant. Thus, host specificity exists in some isolates of V. dahliae. The same isolates were characterized for vegetative compatibility groups (VCGs) through complementation of nitrate nonutilizing (nit) mutants. Cabbage and cauliflower isolates did not produce nit mutants. The isolate from cotton belonged to VCG 1; isolates from bell pepper, eggplant, potato, and tomato, to VCG 4; and the remaining isolates, to VCG 2. These isolates were also analyzed using the random amplified polymorphic DNA (RAPD) method. Forty random primers were screened, and eighteen of them amplified DNA from Verticillium. Based on RAPD banding patterns, cabbage and cauliflower isolates formed a unique group, distinct from other V. dahliae and V. albo-atrum groups. Minor genetic variations were observed among V. dahliae isolates from other hosts, regardless of whether they were host specific or not. There was no correlation among pathogenicity, VCGs, and RAPD banding patterns. Even though the isolates belonged to different VCGs, they shared similar RAPD profiles. These results suggest that management of Verticillium wilt in some crops through crop rotation is a distinct possibility.  相似文献   

4.
ABSTRACT Since 1995, lettuce in coastal California, where more than half of the crop in North America is grown, has consistently suffered from severe outbreaks of Verticillium wilt. The disease is confined to this region, although the pathogen (Verticillium dahliae) and the host are present in other crop production regions in California. Migration of the pathogen with infested spinach seed was previously documented, but the geographic sources of the pathogen, as well as the impact of lettuce seed sparsely infested with V. dahliae produced outside coastal California on the pathogen population in coastal California remain unclear. Population analyses of V. dahliae were completed using 16 microsatellite markers on isolates from lettuce plants in coastal California, infested lettuce seed produced in the neighboring Santa Clara Valley of California, and spinach seed produced in four major spinach seed production regions: Chile, Denmark, the Netherlands, and the United States (Washington State). California produces 80% of spinach in the United States and all seed planted with the majority infested by V. dahliae comes from the above four sources. Three globally distributed genetic populations were identified, indicating sustained migration among these distinct geographic regions with multiple spinach crops produced each year and repeated every year in coastal California. The population structure of V. dahliae from coastal California lettuce plants was heavily influenced by migration from spinach seed imported from Denmark and Washington. Conversely, the sparsely infested lettuce seed had limited or no contribution to the Verticillium wilt epidemic in coastal California. The global trade in plant and seed material is likely contributing to sustained shifts in the population structure of V. dahliae, affecting the equilibrium of native populations, and likely affecting disease epidemiology.  相似文献   

5.
The evolutionary relationships among Verticillium dahliae vegetative compatibility (VCG) subgroups VCG1A, VCG1B, VCG2A, VCG2B, VCG4A, VCG4B, and VCG6 were investigated by parsimony analysis of amplified fragment length polymorphism (AFLP) fingerprints and sequences of six DNA regions (actin, beta-tubulin, calmodulin, and histone 3 genes, the ITS 1 and 2 regions of the rDNA, and a V. dahliae-specific sequence), using 101 isolates of diverse host and geographic origin. Polymorphisms in gene sequences among isolates of different VCGs were very low and individual gene genealogies provided very little resolution at the VCG level. The combined analysis of all DNA regions differentiated all VCG subgroups except for isolates in VCG1A and VCG1B. VCG clonal lineages in V. dahliae and evolutionary relationships among them were resolved independently by analyses of AFLP fingerprints, multiple gene genealogies, and the combined data set of AFLP fingerprinting and multiple gene genealogies. Two main lineages (I and II) were identified with lineage II comprising two closely related subgroups of VCGs. Lineage I included VCG1A, VCG1B, and VCG2B334; and lineage II included, VCG2A and VCG4B (subclade 1); and VCG2B824, VCG4A, and VCG6 (subclade 2). VCG subgroups were monophyletic except for VCG2B that appeared polyphyletic. Limiting the parsimony analysis either to AFLP fingerprints or DNA sequences would have obscured intra-VCG differentiation. Therefore, the dual approach represented by the independent and combined analyses of AFLP fingerprints and DNA sequences was a highly valuable method for the identification of phylogenetic relationships at the intraspecific level in V. dahliae.  相似文献   

6.
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.  相似文献   

7.
ABSTRACT Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathogen recently reported in Arizona. Sequence analysis of the mitochondrial small subunit (mtSSU), translation elongation factor 1-alpha (EF-1alpha) gene, and the nuclear ribosomal DNA intergenic spacer (IGS) region was conducted to resolve relationships among f. sp. lactucae isolates, F. oxysporum isolates from other hosts, and local non-pathogenic isolates. Analysis of mtSSU sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from 13 other F. oxysporum isolates. Analysis of EF-1alpha sequences resulted in moderate resolution, grouping seven formae speciales with the lactucae isolates. Analysis of the IGS region revealed numerous sequence polymorphisms among F. oxysporum formae speciales consisting of insertions, deletions, and single nucleotide transitions and substitutions. Repeat sequence analysis revealed several duplicated subrepeat units that were distributed across much of the region. Based on analysis of the IGS sequence data, lactucae race 1 isolates resolved as a monophyletic group with three other formae speciales of F. oxysporum. In all analyses, lactucae race 2 isolates composed a separate lineage that was phylo-genetically distinct and distantly related to the lactucae race 1 isolates.  相似文献   

8.
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.  相似文献   

9.
检疫性轮枝菌及其近似种的鉴定   总被引:2,自引:0,他引:2  
 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。  相似文献   

10.
In January 2002, Verticillium wilt of lettuce (Lactuca sativa L.) caused by Verticillium tricorpus occurred in upland paddy fields in Hyogo Prefecture for the first time in Japan. This fungal species was first isolated from lettuce in California, USA. In the present study, the genetic relationships between the American and Japanese isolates of V. tricorpus from lettuce were analyzed to determine whether the pathogen could have migrated to Japan from the USA, the major lettuce-seed supplier for Japan. Nucleotide sequences of the rDNA internal transcribed spacer regions, as well as the genes coding for translation elongation factor 1-alpha and RNA polymerase II were compared among American and Japanese V. tricorpus isolates from lettuce. The Japanese isolates of V. tricorpus were distinct from the American. Random amplified polymorphic DNA analyses also supported this conclusion. These results demonstrated that Verticillium wilt on lettuce caused by V. tricorpus in Japan was not related to the isolates causing the disease in California.  相似文献   

11.
ABSTRACT Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the beta-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.  相似文献   

12.
In a survey of verticillium wilt of cultivated and weed hosts in Crete in 1988–89, Verticillium dahliae was the only species isolated. The pathogen was found in 84 out of 231 vegetable crops, and six out of 14 olive orchards. It was also found in two locations on the weed Solanum nigrum. Hosts belonged to 12 plant species in six different botanical families. Natural infection of Vicia faba by V. dahliae is reported for the first time. The physiological races of 92 isolates of V. dahliae were identified using two differential tomato cultivars. Of these isolates, 89 were race 1, one was race 2, and the remaining two were non-pathogenic. Virulence of the various isolates to tomato varied from slightly to highly pathogenic and was independent of the hosts from which they originated.  相似文献   

13.
To estimate the genetic diversity in 30 isolates ofVerticillium lecanii from aphids, whiteflies, mite and black pine in Japan, including two commercialized strains (Mycotal and Vertalec), DNA polymorphisms in ribosomal DNA of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear ribosomal RNA gene of each isolate were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was ~ 580 bp in 27 of the isolates. A 600 bp ITS product was detected in Mycotal and Vertalec. One Japanese isolate produced both the 580 bp and 600 bp products. Enzymatic digestion of the ITS region with Sau3A I,Msp I,Hae III andRsa I revealed RFLPs that consisted of eight haplotypes. Mycotal and Vertalec were specific haplotypes that differed from other isolates. The Japanese isolates had a complex relationship with the original host, but we identified several specific haplotypes common to an aphid origin. Ten distinct IGS haplotypes were detected in the IGS region, some of which were associated with aphid and whitefly origins. These results suggest that the haplotype of rDNA RFLP analysis can be used for studying genetic diversity inV. lecanii.  相似文献   

14.
Isolates of Verticillium dahliae from cocoa (four Brazilian and one Ugandan) were screened against cocoa, aubergine, tomato, cotton, and pepper. Isolates originating from hosts other than cocoa were also inoculated. In general, all inoculated crops were systemically invaded by isolates from cocoa. Isolates from each host tended to be more aggressive on their original host. All isolates from cocoa were pathogenic to cocoa, but exhibited various degrees of aggressiveness to other crops. They induced severe symptoms on aubergine, but few symptoms on tomato, although colonization occurred up to the stem apex in both cases. Likewise, symptomless invasion of pepper was found with some Brazilian isolates. The Ugandan isolate was significantly more aggressive on cotton and pepper than the Brazilian isolates.
A Brazilian isolate from cocoa from the State of Bahia was also used to inoculate 12 common weed species from the same geographical area. Four species showed wilt symptoms, while V. dahliae was readily recovered from the stems of a further four species. The role of alternative hosts on disease spread in cocoa growing areas is discussed.  相似文献   

15.
The sequences of the internal transcribed spacers (ITS) 1 and 2 of the rRNA genes of 38 Verticillium alboatrum and V. dahliae isolates have been determined. The isolates represented RFLP groups of both species, V. dahliae vegetative compatibility groups and pathotypes, and V. alboatrum 'group 2'(the majority of the V. alboatrum isolates studied were in'group 1'). The ITS sequences of a single V. tricorpus isolate were also determined. RFLP groups L and NL of V. atboatrum were distinct, with a maximum of three nucleotide differences between any isolate. Most haploid V. dahliae isolates were identical and separated from most L/NL V. alboatrum isolates by differences at five or six positions. A few haploid V. dahtiae isolates, not forming any obvious grouping, differed from the majority, each at a single position. Diploid isolates of V. dahtiae were identical but only one nucleotide difference separated them from some RFLP group L V. alboatrum isolates. At least six differences separated the diploid isolates of V. dahliae from the haploid ones. The'group 2' V. atboatrum isolates were more distantly related to'group 1'isolates (at least 17 positions different) than was V. tricorpus. The possibility of defining specific primers for use in PCR to discriminate species and subspecific groups is discussed.  相似文献   

16.
新疆榆树黄萎病病原菌鉴定   总被引:1,自引:0,他引:1  
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)。  相似文献   

17.
Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.  相似文献   

18.
ABSTRACT Severe Verticillium dahliae attacks have occurred in artichoke crops in the Comunidad Valenciana region of eastern-central Spain since the late 1990s. Knowledge of genetic and virulence diversity in the pathogen population is a key factor for the management of the disease through disease risk assessment as well as development and use of resistant cultivars. V. dahliae isolates from artichoke (109 isolates) and cotton (three isolates) in that region were characterized by vegetative compatibility grouping (VCG), and specific polymerase chain reaction assays using three sets of primer pairs that differentiate the cotton-defoliating (D) and -nondefoliating (ND) V. dahliae pathotypes. In all, 35 and 39 V. dahliae isolates representative of the identified VCGs and geographic origins were tested for virulence to artichoke cvs. Nun 6374 and Nun 9444, and cotton cv. Acala SJ-2, respectively. Four VCGs were identified among 107 artichoke isolates, and 2 isolates were heterokaryon self-incompatible: VCG1A (one isolate), VCG2A (31 isolates), VCG2B (72 isolates), and VCG4B (three isolates). The three cotton isolates were VCG1A. Isolates in VCG2B were distributed across the region and were the most prevalent isolates in the northern part. Conversely, 83.9% of isolates in VCG2A were recovered from the southern part of the region. Two subgroups of isolates were identified in VCG2B based on heterokaryon compatibility with either international or local tester isolates, which further showed diversity in the amplification of 334- and 824-bp DNA fragments which are markers of the D and ND pathotypes, respectively. Virulence of isolates to artichoke and cotton correlated with VCG but the pattern of correlation varied with the host. VCG1A isolates from artichoke and cotton induced defoliation in cotton but not in artichoke. Collectively, isolates of VCG2B and VCG4B were the most virulent and isolates of VCG1A or HSI were the least virulent to artichoke; but isolates of VCG1A were more virulent to cotton than those of any other VCG. Also, molecular subgrouping in VCG2B determined by amplification of the 334- and 824-bp markers correlated with virulence of isolates to the two hosts tested.  相似文献   

19.
Verticillium dahliae Klebahn is the causal agent of tomato wilt disease. Isolates of V. dahliae can be classified based on pathogenicity to tomato, but the pathotypes are indistinguishable in morphology. We designed PCR primers for specific detection of isolates pathogenic to tomato (tomato pathotype) from the sequences of a pathotype-specific gene, vdt1. With the primer pair Tg5/Tc3, a PCR product (approximately 3.2 kb) specific to tomato pathotype was amplified from the genomic DNA of isolates. Using the primer pair, a tomato pathotype isolate was specifically detected from hypocotyls of inoculated tomato and eggplant. On the other hand, no amplification was observed from non-tomato pathotype isolates of V. dahliae, some other wilt pathogens of tomato and a healthy host plant. Therefore, the primer pair can be useful for pathotype-specific detection of V. dahliae as well as for diagnosis of wilt disease of tomato plant. Received 7 September 2001/ Accepted in revised form 3 December 2001  相似文献   

20.
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。  相似文献   

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