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1.
ABSTRACT Specific primers and polymerase chain reaction (PCR) assays that identify Fusarium oxysporum f. sp. ciceris and each of the F. oxysporum f. sp. ciceris pathogenic races 0, 1A, 5, and 6 were developed. F. oxysporum f. sp. ciceris- and race-specific random amplified polymorphic DNA (RAPD) markers identified in a previous study were cloned and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. Each cloned RAPD marker was characterized by Southern hybridization analysis of Eco RI-digested genomic DNA of a subset of F. oxysporum f. sp. ciceris and nonpathogenic F. oxysporum isolates. All except two cloned RAPD markers consisted of DNA sequences that were found highly repetitive in the genome of all F. oxysporum f. sp. ciceris races. F. oxysporum f. sp. ciceris isolates representing eight reported races from a wide geographic range, nonpathogenic F. oxysporum isolates, isolates of F. oxysporum f. spp. lycopersici, melonis, niveum, phaseoli, and pisi, and isolates of 47 different Fusarium spp. were tested using the SCAR markers developed. The specific primer pairs amplified a single 1,503-bp product from all F. oxysporum f. sp. ciceris isolates; and single 900- and 1,000-bp products were selectively amplified from race 0 and race 6 isolates, respectively. The specificity of these amplifications was confirmed by hybridization analysis of the PCR products. A race 5-specific identification assay was developed using a touchdown-PCR procedure. A joint use of race 0- and race 6-specific SCAR primers in a single-PCR reaction together with a PCR assay using the race 6-specific primer pair correctly identified race 1A isolates for which no RAPD marker had been found previously. All the PCR assays described herein detected up to 0.1 ng of fungal genomic DNA. The specific SCAR primers and PCR assays developed in this study clearly identify and differentiate isolates of F. oxysporum f. sp. ciceris and of each of its pathogenic races 0, 1A, 5, and 6.  相似文献   

2.
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   

3.
ABSTRACT Strains of the carnation wilt pathogen, Fusarium oxysporum f. sp. dianthi, can be distinguished by DNA fingerprint patterns, using the fungal transposable elements Fot1 and impala as probes for Southern hybridization. The DNA fingerprints correspond to three groups of F. oxysporum f. sp. dianthi strains: the first group includes isolates of races 1 and 8; the second group includes isolates of races 2, 5 and 6; and the third group includes isolates of race 4. Genomic DNAs flanking race-associated insertion sites of Fot1 (from races 1, 2, and 8) or impala (from race 4) were amplified by the inverse polymerase chain reaction (PCR) technique. These regions were cloned and sequenced, and three sets of primers overlapping the 3' or 5' end of the transposon and its genomic insertion were designed. Using fungal genomic DNA as template in PCR experiments, primer pairs generated amplification products of 295, 564 and 1,315 bp, corresponding to races 1 and 8; races 2, 5, and 6; and race 4, respectively. When multiplex PCR was performed with genomic DNA belonging to races 1 and 8, 2, or 4, single amplimers were generated, allowing clear race determination of the isolate tested. PCR was successfully performed on DNA extracted from susceptible carnation cv. Indios infected with isolates representative of races 1, 2, 4, and 8.  相似文献   

4.
Fusarium oxysporum f.sp. gladioli (FOG) race 1 infects both large- and small-flowered Gladiolus cultivars. Race 2 isolates infect only small-flowered cultivars but can be present as epiphytes on large-flowered plants. When 160 arbitrary 10-mer oligonucleotide primers were tested on FOG by PCR to find RAPD markers specific for race 1, the RAPD primer G12 amplified two discriminating DNA fragments, AB (609 bp) and EF (1196 bp), in race 1 isolates only. Both fragments were cloned and sequenced. Two pairs of race 1-specific primers for multiplex PCR were designed. Tests of 112 F. oxysporum isolates by PCR showed that, in almost all cases, race 1 isolates of vegetative compatibility group 0340 could be distinguished with these primers. Seven putative race 1 isolates did not react in multiplex PCR; hybridization studies with labelled AB and EF DNA fragments showed that these isolates belong to separate groups. A bioassay was developed to detect corms that were latently infected with FOG race 1. Gladiolus corms were homogenized and incubated for 5 days at 28°C in a semiselective medium to induce growth of Fusarium . Cultivated mycelium was isolated and subjected to the developed multiplex PCR after standard DNA isolation or disruption by microwave treatment.  相似文献   

5.
In 1995, Fusarium root rot of crisp head lettuce, caused by Fusarium oxysporum f. sp. lactucae, was simultaneously found in the Shiojiri and Kawakami areas of Nagano Prefecture, Japan. The Shiojiri and Kawakami isolates differed in pathogenicity to lettuce cultivars. Because of this distinct physiological specialization, these Shiojiri and Kawakami isolates should be designated as race 1 and race 2, respectively, using lines VP1010 (highly resistant to race 1), VP1013 (highly resistant to race 2) and variety Patriot (highly susceptible to both races) as differential varieties. This is the first report of races of Fusarium oxysporum f. sp. lactucae, Received 21 September 2000/ Accepted in revised form 21 March 2001  相似文献   

6.
One hundred and sixteen isolates of Fusarium oxysporum f. sp. lactucae obtained from 85 fields in three crisphead lettuce-producing areas in Nagano Prefecture, Japan were typed for races using differential cultivars Patriot, Banchu Red Fire and Costa Rica No. 4. They were also grouped into vegetative compatibility groups (VCGs) using complementation tests with nitrate non-utilizing (nit) mutants. Two California strains reported as F. oxysporum f. sp. lactucum, a type culture of F. oxysporum f. sp. lactucae, and 28 avirulent isolates of F. oxysporum obtained from crisphead lettuce were included for comparison. Among Nagano isolates, 66 isolates were identified as race 1, and 50 as race 2. Race 1 strains derived from Shiojiri and Komoro cities and race 2 from Kawakami village and Komoro city. All isolates of race 2 were biotin auxotrophs, and the race could be distinguished based on its requirement for biotin on minimal nitrate agar medium (MM). Pathogenic isolates were classified into two VCGs and three heterokaryon self-incompatible isolates. Strong correlations were found between race and VCG. All the race 1 strains were assigned to VCG 1 except self-incompatible isolates, and all the race 2 strains to VCG 2. The 28 avirulent isolates of F. oxysporum were incompatible with VCG 1 and VCG 2. California strains was vegetatively compatible with VCG 1, and they were assigned to race 1. Based on vegetative compatibility, these two races of F. oxysporum f. sp. lactucae may be genetically distinct, and F. oxysporum f. sp. lactucae race 1 is identical to F. oxysporum f. sp. lactucum. Received 7 May 2002/ Accepted in revised form 6 September 2002  相似文献   

7.
ABSTRACT Fusarium oxysporum f. sp. canariensis causes Fusarium wilt disease on the Canary Island date palm (Phoenix canariensis). To facilitate disease management, a polymerase chain reaction diagnostic method has been developed to rapidly detect the pathogen. A partial genomic library of F. oxysporum f. sp. canariensis isolate 95-913 was used to identify a DNA sequence diagnostic for a lineage containing all tested isolates of F. oxysporum f. sp. canariensis. Two oligonucleotide primers were designed and used to amplify a 567-bp fragment with F. oxysporum f. sp. canariensis DNAs. DNA from 61 outgroup isolates did not amplify using these primers. Once the primers were shown to amplify a 0.567-kb fragment from DNA of all the F. oxysporum f. sp. canariensis isolates tested, a rapid DNA extraction procedure was developed that led to the correct identification of 98% of the tested F. oxysporum f. sp. canariensis isolates.  相似文献   

8.
ABSTRACT An epidemic of vascular wilt caused by Fusarium oxysporum f. sp. erythroxyli is currently occurring on Erythroxylum coca var. coca in the coca-growing regions of the Huallaga Valley in Peru. Random amplified polymorphic DNA (RAPD) analysis of isolates of the pathogen was undertaken to elucidate its genetic complexity, as well as to identify a specific DNA fingerprint for the pathogen. Two hundred isolates of Fusarium were collected from 10 coca-growing regions in Peru. Of these, 187 were confirmed to be F. oxysporum, and 143 of the F. oxysporum were shown to be pathogens of coca by a root-dip pathogenicity test. The pathogens could be grouped into two subpopulations based on RAPD analysis, and no polymorphism in RAPD pattern was observed among isolates of either subpopulation. Both subpopulations were present in the central Huallaga Valley, where earliest reports of the epidemic occurred. RAPD analysis could easily distinguish the isolates of F. oxysporum f. sp. erythroxyli from the nonpathogenic isolates of F. oxysporum from E. coca var. coca, indicating its utility in DNA fingerprinting.  相似文献   

9.
香蕉枯萎病菌生理小种鉴定及其SCAR标记   总被引：8，自引：0，他引：8  
 通过室内人工接种蕉类鉴别寄主,对采集于广东蕉区的18个蕉类枯萎病菌菌株进行鉴定,KP021、KP022、GZ981和JL021 4个菌株属Racel,其余14个菌株属Race4,说明广东蕉区同时存在尖孢镰刀菌古巴专化型Race1和Race4。用RAPD技术对上述18个菌株进行分析,从200条随机引物中筛选出8条引物可产生生理小种RAPD标记12个,其中标记Racel的8个,标记Race4的4个。对这些RAPD标记带分别进行回收、克隆、测序,根据这些特异片段序列分别设计相应的SCAR引物,通过对18个菌株的PCR扩增检验,有4个RAPD标记成功地转化为SCAR标记,其中Race1-SCAR标记1个、Race4-SCAR标记2个、同时能鉴定出2个小种的SCAR标记1个。应用这4个SCAR标记同时对采自田间的9个病菌分离物进行检测,能够准确地鉴定出广东蕉区的尖孢镰刀菌古巴专化型Racel和Race4,这为下一步开展香蕉枯萎病菌生理小种的分子鉴定及各生理小种田间流行动态监测奠定了基础。  相似文献   

10.
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.  相似文献   

11.
Fusarium oxysporum f. sp. lactucae, the causal agent of fusarium wilt of lettuce (Lactuca sativa), occurs in most countries in which lettuce is grown and causes serious economic losses. Three races (1, 2 and 3) of the pathogen have previously been identified on the basis of their ability to cause disease on differential lettuce cultivars, as well as by means of molecular tools developed to characterize different races of this pathogen. Only race 1 has been detected in Europe so far. In this study, two isolates of F. oxysporum, obtained from lettuce plants grown in the Netherlands showing symptoms of wilt, have been characterized by combining the study of pathogenicity with differential cultivars of lettuce and molecular assays to determine whether the isolates are different from the known races of F. oxysporum f. sp. lactucae. This study reports the presence of F. oxysporum f. sp. lactucae for the first time in the Netherlands. The causal pathogen has been identified, using the IRAP‐SCAR technique, as a new race of F. oxysporum f. sp. lactucae. Specific primers have been designed to identify this new race.  相似文献   

12.
Fusarium oxysporum f. sp. cepae, which causes basal rot of onion, consists of seven vegetative compatibility groups (VCGs 0420 to 0426) and several single-member VCGs (SMVs). F. oxysporum f. sp. cepae populations in South Africa and Colorado each consist of one main VCG (namely, VCG 0425 and 0421, respectively). The aim of this study was to develop sequence-characterized amplified region (SCAR) markers for the identification of VCGs 0425 and 0421, using 79 previously characterized F. oxysporum isolates. A second aim was to investigate the prevalence of VCG 0425 among 88 uncharacterized South African onion F. oxysporum isolates using (i) the developed SCAR markers and (ii) inter-retrotransposon (IR)- and random amplified polymorphic DNA (RAPD) fingerprinting. Only two RAPD primers provided informative fingerprints for VCG 0425 isolates but these could not be developed into SCAR markers, although they provided diagnostic fragments for differentiation of VCG 0425 from VCG 0421. IR fingerprinting data were used to develop a multiplex IR-SCAR polymerase chain reaction method for the identification of VCG 0421, VCG 0425, and SMV 4 isolates as a group. Molecular identification of the uncharacterized collection of 88 F. oxysporum isolates (65 F. oxysporum f. sp. cepae and 23 F. oxysporum isolates nonpathogenic to onion) confirmed that VCG 0425 is the main VCG in South Africa, with all but 3 of the 65 F. oxysporum f. sp. cepae isolates having the molecular characteristics of this VCG. Genotyping and VCG testing showed that two of the three aforementioned isolates were new SMVs (SMV 6 and SMV 7), whereas the third (previously known as SMV 3) now belongs to VGC 0247.  相似文献   

13.
 Pathogenic variation among 26 Japanese isolates of Fusarium oxysporum f. sp. lactucae (FOL) was tested using 21 lettuce cultivars to select commercial lettuce cultivars as race differential indicators. Cultivar Costa Rica No. 4 was resistant to race 1 but susceptible to race 2, consistent with the conventional standard differential line VP1010. Cultivar Banchu Red Fire was susceptible to race 1 but resistant to race 2, which showed an opposite type of reaction as another differential line VP1013. Cultivar Patriot was susceptible to both races. The resistance reactions of the three cultivars under field conditions were identical with that observed in the seedlings. Thus cv. Costa Rica No. 4 and cv. Banchu Red Fire can be used as differential hosts to identify pathogenic races of FOL. This differential system showed that all FOL isolates obtained from diseased butterhead lettuce in Fukuoka, Japan were new races (i.e., pathogenic to three cultivars). We propose that the new race be designated race 3. Isolates of FOL, the pathogen of Fusarium wilt in lettuce, obtained from California showed the same reaction as that of race 1. Furthermore, the Japanese isolate SB1-1 (race 1) and California isolate HL-2 belonged to the same vegetative compatibility group. Our results suggest that both of the fungi are the same forma specialis. Received: March 25, 2002 / Accepted: August 26, 2002  相似文献   

14.
Isolates of Fusarium oxysporum f. sp. lactucae obtained from six localities in Japan were divided into three patho-genicity groups. Group 1 was highly pathogenic to lettuce cultivars of crisphead and red leaf types and was less pathogenic to butterhead and green leaf type cultivars. Group 2 was highly pathogenic to butterhead type and less pathogenic to crisphead and leaf types. Group 3 was less pathogenic to all lettuce types than groups 1 and 2. These results indicated pathogenic differentiation in F. oxysporum f. sp. lactucae, roughly relating to horticultural types of host lettuce cultivars. Received 21 February 2001/ Accepted in revised form 28 May 2001  相似文献   

15.
 利用RAPD技术,以随机引物对我国棉花枯萎菌的3个生理小种(3、7、8号小种)共26个菌株进行PCR扩增,从产生的140个RAPD分子标记中寻找到了不同小种的特征性条带,O PF-10513(3号小种)、OPF-08371(7号小种)及OPF-12703(8号小种)。将其纯化后克隆到pGEM-TEasy质粒载体上,并获得了DNA特异性片段的核酸序列。  相似文献   

16.
ABSTRACT Thirty-nine isolates of Fusarium oxysporum were collected from tomato plants displaying wilt symptoms in a field in California 2 years after F. oxysporum f. sp. lycopersici race 3 was first observed at that location. These and other isolates of F. oxysporum f. sp. lycopersici were characterized by pathogenicity, race, and vegetative compatibility group (VCG). Of the 39 California isolates, 22 were in VCG 0030, 11 in VCG 0031, and six in the newly described VCG 0035. Among the isolates in VCG 0030, 13 were race 3, and nine were race 2. Of the isolates in VCG 0031, seven were race 2, one was race 1, and three were nonpathogenic to tomato. All six isolates in VCG 0035 were race 2. Restriction fragment length polymorphisms (RFLPs) and sequencing of the intergenic spacer (IGS) region of rDNA identified five IGS RFLP haplotypes, which coincided with VCGs, among 60 isolates of F. oxysporum from tomato. Five race 3 isolates from California were of the same genomic DNA RFLP haplotype as a race 2 isolate from the same location, and all 13 race 3 isolates clustered together into a subgroup in the neighbor joining tree. Collective evidence suggests that race 3 in California originated from the local race 2 population.  相似文献   

17.
 菠菜枯萎病是由致病性镰刀菌(F.o.f.sp.spinaciae)引起的,是菠菜生产中的重要病害之一。利用常规方法鉴定菠菜枯萎病病原菌需耗费大量时间,并且很难得到正确的结论。随机扩增多态性DNA序列标签(Randomly amplified polymorphic DNA-sequence tagged sites,RAPD-STS)为病原菌鉴定提供了一种有效方法。本研究通过对供试菌株的RAPD分析,克隆出了1个菠菜枯萎病病原菌的特异片段(GenBank登录号:AY337463)。根据测序结果设计了1对菠菜枯萎病病原菌的特异引物,并利用常规PCR和实时定量PCR(real-time PCR)2种方法对病原菌进行了鉴定,并对2种方法的敏感性进行了比较。结果表明,2种PCR方法都可以鉴定菠菜枯萎病病原菌(F.o.f.sp.spinaciae),但二者对病原菌DNA敏感程度不同,常规PCR检测的最低DNA量100Pg,而实时定量PCR检测的最低DNA量是1pg。同时,实时定量PCR还可以对病原菌DNA进行定量分析,并依此估算病原菌的数量。该方法可用于菠菜枯萎病病原菌的快速鉴定和病因诊断。  相似文献   

18.
A group of 133 isolates of the cotton wilt pathogen Fusarium oxysporum Schlecht f sp vasinfectum (Atk) Sny & Hans, representing five races and 20 vegetative compatibility groups within race 1 were used to determine the identity, biosynthetic regulation and taxonomic distribution of polyketide toxins produced by this pathogen. All isolates of F oxysporum f sp vasinfectum produced and secreted the nonaketide naphthazarin quinones, bikaverin and norbikaverin. Most isolates of race 1 (previously denoted as races 1, 2 and 6; and also called race A) also synthesized the heptaketide naphthoquinones, nectriafurone, anhydrofusarubin lactol and 5-O-methyljavanicin. Nine avirulent isolates of F oxysporum from Upland cotton roots, three isolates of race 3 of F oxysporum f sp vasinfectum, and four isolates of F oxysporum f sp vasinfectum from Australia, all of which previously failed to cause disease of Upland cotton (Gossypium hirsutum L) in stem-puncture assays, also failed to synthesize or secrete more than trace amounts of the heptaketide compounds. These results indicate that the heptaketides may have a unique role in the virulence of race 1 to Upland cotton. The synthesis of all polyketide toxins by ATCC isolate 24908 of F oxysporum f sp vasinfectum was regulated by pH, carbon/nitrogen ratios, and availability of calcium in media. Synthesis was greatest below pH 7.0 and increased progressively as carbon/nitrogen ratios were increased by decreasing the amounts of nitrogen added to media. The nonaketides were the major polyketides accumulated in synthetic media at pH 4.5 and below, whereas the heptaketides were predominant at pH 5.0 and above. The heptaketides were the major polyketides formed when 10 F oxysporum f sp vasinfectum race 1 isolates were grown on sterilized stems of Fusarium wilt-susceptible cotton cultivars, but these compounds were not produced on sorghum grain cultures. Both groups of polyketide toxins were apparently secreted by F oxysporum f sp vasinfectum, since half of the toxin in 2-day-old shake culture was present in the supernatant. Secretion was enhanced by calcium. Glutamine and glutamic acid inhibited both nonaketide and heptaketide syntheses, even at low nitrogen  相似文献   

19.
ABSTRACT The capacity of the arbuscular mycorrhizal fungus Glomus intraradices in reducing the presence of Fusarium solani f. sp. phaseoli in bean plants and the surrounding mycorrhizosphere soil was evaluated in a compartmentalized experimental system. Quantification of the pathogen and the symbiont in plant tissues, the soil regions of the mycorrhizosphere (rhizosphere and mycosphere), and the bulk soil was accomplished using specific polymerase chain reaction (PCR) primers in real-time PCR assays, culture-dependant methods, and microscopic determination techniques. Nonmycorrhizal bean plants infected with the pathogen had distinctive Fusarium root rot symptoms, while infected plants previously colonized by G. intraradices remained healthy. The amount of F. solani f. sp. phaseoli genomic DNA was significantly reduced in mycorrhizal bean plants and in each mycorrhizosphere soil compartment. The presence of G. intraradices in the mycorrhizosphere was not significantly modified, although the mycorrhizal colonization of roots was slightly increased in the presence of the pathogen. The results suggest that the reduced presence of Fusarium as well as root rot symptoms are caused by biotic and/or abiotic modifications of the mycorrhizosphere as a result of colonization with G. intraradices.  相似文献   

20.
ABSTRACT A collection of race 1 and race 2 isolates of Fusarium oxysporum f. sp. lycopersici was screened for vegetative compatibility and characterized by random amplified polymorphic DNA (RAPD) analysis to establish the identity and genetic diversity of the isolates. Comparison of RAPD profiles revealed two main groups that coincide with vegetative compatibility groups (VCGs). In addition, several single-member VCGs were identified that could not be grouped in one of the two main RAPD clusters. This suggests that F. oxysporum f. sp. lycopersici is a polyphyletic taxon. To assign avirulence genotypes to race 1 isolates, they were tested for their virulence on a small set of tomato lines (Lycopersicon esculentum), including line OT364. This line was selected because it shows resistance to race 2 isolates but, unlike most other race 2-resistant lines, susceptibility to race 1 isolates. To exclude the influence of other components than those related to the race-specific resistance response, we tested the virulence of race 1 isolates on a susceptible tomato that has become race 2 resistant by introduction of an I-2 transgene. The results show that both line OT364 and the transgenic line were significantly affected by four race 1 isolates, but not by seven other race 1 isolates nor by any race 2 isolates. This allowed a subdivision of race 1 isolates based on the presence or absence of an avirulence gene corresponding to the I-2 resistance gene. The data presented here support a gene-for-gene relationship for the interaction between F. oxysporum f. sp. lycopersici and its host tomato.  相似文献   

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