Characterization of Verticillium dahliae Isolates from Potato on Hokkaido by Random Amplified Polymorphic DNA (RAPD) and REP-PCR Analyses

Verticillium dahliae isolates from potato on the island of Hokkaido (potato isolates) and those belonging to pathotypes A (eggplant pathotype), B (tomato pathotype) and C (sweet pepper pathotype) were divided into three distinct groups by RAPD and REP-PCR. The three DNA groups I, II, III consisted of pathotypes A and C, pathotype B and potato isolates, respectively. The potato isolates were assigned to pathotype A on the basis of pathogenicity. Another set of potato isolates was further collected from eight potato cropping regions on Hokkaido to further examine the relationships among them in detail. Only one of these isolates was identified as DNA group II, but all the others were classified as DNA group III. Isolates from daikon, eggplant, and melon on Hokkaido also belonged to DNA group III. These results suggest that V. dahliae isolates from Hokkaido are unique at the DNA level and different from other pathotype A isolates in Japan. Received 28 February 2000/ Accepted in revised form 6 November 2000     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

Differentiation of Cotton-defoliating and Nondefoliating Pathotypes of Verticillium dahliae by RAPD and Specific PCR Analyses

Severe Verticillium wilt of cotton in southern Spain is associated with the spread of a highly virulent, defoliating (D) pathotype of Verticillium dahliae. Eleven of the D and 15 of a mildly virulent, nondefoliating (ND) pathotype were analyzed by random amplified polymorphic DNA (RAPD) using the polymerase chain reaction (PCR). Six of 21 primers tested generated pathotype-associated RAPD bands. Another 21 V. dahliae isolates were compared in blind trials both by RAPD-PCR using the six selected primers and pathogenicity tests on cotton cultivars. There was a 100% correlation between pathotype characterization by each method. Unweighted paired group method with arithmetic averages cluster analysis was used to divide the 47 V. dahliae isolates into two clusters that correlated with the D or ND pathotypes. There was more diversity among ND isolates than among D isolates, these latter isolates being almost identical. ND- and D-associated RAPD bands of 2.0 and 1.0kb, respectively, were cloned, sequenced, and used to design specific primers for the D and ND pathotypes. These pathotype-associated RAPD bands were present only in the genome of the pathotype from which they were amplified, as shown by Southern hybridization. The specific primers amplified only one DNA band of the expected size, and in the correct pathotype, when used for PCR with high annealing temperature. These specific primers successfully characterized V. dahliae cotton isolates from China and California as to D or ND pathotypes, thus demonstrating the validity and wide applicability of the results.     

Specific Detection of Tomato Pathotype of Verticillium dahliae by PCR Assays

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     

Pathogenicity and RAPD analysis of Phytophthora nicotianae pathogenic to pepper in Tunisia

Nine isolates of Phtophthora nicotianae were isolated from infected pepper plants. Their pathogenicity was studied in Capsicum annuum in comparison with P. nicotianae isolates from tomato and tobacco. The pathogenicity test showed that pepper isolates of P. nicotianae are adapted to their host. Banding patterns obtained by RAPD analysis with six oligonucleotide primers revealed polymorphism that grouped the isolates independently of the plant host. The polygenic dendrogram showed that pepper isolates were more similar to tomato isolates than to tobacco isolates. The RAPD bands of 1300 and 1500 bp, detected with primers OPD-01 and OPD-10, respectively, appeared specific to the most pathogenic pepper isolates. The OPK-08-1950 seems specific to the isolates of P. nicotianae from tomato. These results suggest that host specified might occur in P. nicotianae and that may be due to interspecific hybridization events resulting in novel pathogenic behavior.     

Pepper resistance-breaking tobamoviruses: Can they co-exist in single pepper plants?

We previously reported a procedure for identifying pathotypes of the tobamoviruses infecting the L-resistant genotypes of pepper based on the polymerase chain reaction (PCR) (Tenllado et al., 1994). We have now used this method to assess pathotype incidence in virus isolates from field pepper samples representing three successive epidemic episodes in southeastern Spain, and to analyse the interaction of pathotypes during experimental infections in pepper plants. The majority of virus isolates corresponded to the P_1,2 pathotype, and only three out of twenty behaved as P_1,2,3 pathotypes. Interestingly, restriction enzyme analysis of the PCR-amplified products distinguished two restriction subgroups on each P_1,2 and P_1,2,3 pathotype, referred to as restrictopatterns I₁ and I₂ or II₁ and II₂, respectively. Experimental coinoculations of pepper plants with mixtures of two different pathotypes showed coexistence between them when inoculations proceeded simultaneously. However, reciprocal cross-protection was observed between P_1,2 and P_1,2,3 pathotypes when they were successively inoculated, while no cross-protection was observed between P₁ and either P_1,2 or P_1,2,3 pathotypes. The potential of the PCR-based method for detecting heterogeneity within P_1,2 and P_1,2,3 pathotypes, and the possibility of genetically engineered resistance to those viruses by genetic transformation with viral coat protein genes are briefly discussed.     

Pathogenic and genetic variation among isolates of Corynespora cassiicola in Japan

In order to develop a method for discrimination of Corynespora cassiicola isolates pathogenic to sweet pepper among Japanese isolates, this study analysed pathogenic variations of 64 Japanese isolates of C. cassiicola on perilla, cucumber, tomato, aubergine and sweet pepper, and their multigene phylogeny. Japanese isolates were divided into seven pathogenicity groups (PG1–PG7). The virulence of isolates in PG1–PG5 was restricted to perilla, cucumber, tomato, aubergine and sweet pepper, respectively. Isolates in PG6 were virulent to sweet pepper, tomato and aubergine. Isolates in PG7 were avirulent to all tested plants. Multigene phylogenetic analysis of the isolates based on β‐tubulin, translation elongation factor 1‐α, calmodulin and actin genes showed three divergent clusters, MP‐A, MP‐B and MP‐C. These clusters included all isolates in PG1, PG2, PG8 and PG9 (MP‐A), PG3 and PG5 (MP‐B) and PG4 and PG6 (MP‐C). Isolates in PG7 were distributed amongst all clusters. Furthermore, random amplified polymorphic DNA (RAPD) analysis using universal primers, Q17 (5′‐GAAGCCCTTG‐3′) and Q13 (5′‐GGAGTGGACA‐3′), facilitated discrimination of isolates virulent on sweet pepper amongst isolates in MP‐B and MP‐C, respectively. Together, a combination of the multigene analysis and the RAPD technique allowed the discrimination of the isolates virulent to sweet pepper.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.     

Development of a Pathotype Specific SCAR Marker in Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate biotroph that causes clubroot, one of the most damaging diseases of crucifers. Breeding of clubroot-resistant plants has been hampered by the large variation of pathogenicity in P. brassicae and by the lack of an efficient means for detecting specific isolates. To improve the practicality of P. brassicae pathotype-identification, a molecular approach was developed. RAPD profiles of 37 single-spore-derived isolates belonging to seven different pathotypes were compared. A RAPD marker, OPL14₁₂₀₀, was found in the molecular pattern of all the isolates belonging to one particular pathotype (P1), pathogenic on all differential hosts tested. The DNA band corresponding to this marker was cloned and sequenced. No significant homology to previously characterised nucleotide sequences was found. Primers were designed to specifically amplify the OPL14₁₂₀₀ band. The SCAR marker was observed in all isolates belonging to pathotype P1 and was absent in isolates belonging to other pathotypes and in the different plant hosts analysed. The SCAR marker was also generated from direct amplification of DNA from clubs (mixture of host and pathogen DNA) developed after infection by P1 isolates. This molecular marker may be a valuable tool for rapid and reliable identification of P. brassicae P1 isolates in areas where resistant varieties are cultivated.     

Serological Analysis and Coat Protein Sequence Determination of Potato Virus Y (PVY) Pepper Pathotypes and Differentiation from Other PVY Strains

The serological relationships of Potato Virus Y (PVY) isolates belonging to the pepper pathotypes 0, 1 and 1-2 were established by enzyme-linked immunosorbent assay (ELISA). PVY pepper pathotypes did not react with monoclonal antibodies which typically recognize non-pepper strains within the PVY group, leading to discrimination between these two groups of strains. No serological differences were found between the three PVY pepper pathotypes. The coat protein (CP) nucleotide and predicted amino acid sequences of the three different PVY pepper pathotypes were determined. The highest sequence similarity was found between pathotypes 0 and 1 (99.2%), while the lowest occurred between these two and pathotype 1–2 (98.1%). PVY strains from potato and tobacco appeared more distantly related. Phenetic analysis of the CP amino acid sequences showed that the PVY pepper pathotypes formed a tightly clustered group separate from other PVY strains.     

华南水稻白叶枯病菌致病性分化检测与分析

为了解华南稻区水稻白叶枯病菌的致病性分化和变异动态,采集华南地区水稻白叶枯病病叶标样分离病原菌,应用中国鉴别寄主IR26、南粳15、爪哇14、特特普、金刚30和国际水稻已知抗病基因的近等基因系IRBB5、IRBB13、IRBB3、IRBB14、IRBB2、IR24两套鉴别寄主,在水稻孕穗期采用剪叶法接种,依据寄主和菌株的互作反应检测病菌的致病性分化。结果显示,参试菌株可划分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅸ六个致病型和R1、R2、R3、R4、R5、R8、R10七个致病小种。Ⅴ、Ⅳ致病型和R8、R5小种出现频率分别为27.40%、19.30%和44.67%、15.34%,为华南稻区优势种群。Ⅸ、Ⅴ、Ⅳ致病型和R8、R5小种对500份华南稻区品种资源的致病率依次为96.40%、95.00%、50.40%、62.00%和42.60%;Ⅸ致病型毒性最强且发展很快;强致病菌系Ⅴ型已替代Ⅳ型发展为华南优势致病菌系。     

Development of simple sequence repeat markers for the classification of the clubroot pathogen <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious diseases in cruciferous crops. To classify isolates, we developed simple sequence repeat (SSR) markers for P. brassicae. Twenty-four Japanese isolates were used in this study: 12 isolates of an unknown pathotype from the Kyoto Prefecture, as well as 12 isolates of known pathotypes, including three single-spore lines. From the 12 isolates from Kyoto Prefecture, 11 were classified into either pathotype 2 (three isolates) or 4 (eight isolates). We designed 23 SSR markers based on the P. brassicae genome, of which 11 markers from intergenic regions showed polymorphisms in the 24 isolates. Many haploid isolates belonging to pathotypes 2 and 4 were monomorphic, and typical alleles were detected in some isolates not belonging to pathotype 4. Two bands were detected for eight SSR loci in five isolates, indicating that different genotypes were mixed in these isolates. We constructed a phylogram based on the 11 polymorphic SSRs. Pathotypes 2 and 4 formed a cluster, from which pathotypes 3 and 1 were successively placed. These results strongly suggest a close genetic relationship between isolates in pathotypes 2 and 4, consistent with our finding that isolates in these two pathotypes were found at one collection site. In combination with pathotype classification and other marker systems, the SSR markers can be used for more detailed analyses to improve the control of clubroot disease.     

Genetic and pathogenic diversity within Ascochyta rabiei(Pass.) Lab. populations in Pakistan causing blight of chickpea (Cicer arietinum L.)

Pathogenic and genetic diversity in Ascochyta rabiei populations in Pakistan were evaluated. Biological pathotyping of 130 A. rabiei isolates (obtained from hierarchically collected samples) was conducted on a set of three chickpea differentials, i.e. ILC 1929 (susceptible), ILC 482 (tolerant) and ILC 3279 (resistant), under controlled conditions. Disease severity data were recorded 12 days after inoculation. Statistical analysis grouped the isolates into three pathotype classes. Four isolates belonged to pathotype I (least aggressive), 79 isolates to pathotype II (medium aggressive) and 47 isolates to pathotype-III (highly aggressive).Genetic analysis was performed using RAPDs and oligonucleotide fingerprinting, where Hinf I-digested DNA was hybridized to the³²P-endlabeled oligonucleotide probes (CAA)₅, (GAA)₅, (GA)₈, (CA)₈and (GATA)₄. Dendrograms produced by cluster analysis discriminated 46 genotypes in the A. rabiei population of Pakistan. Genetic distances and relatedness between isolates were calculated. At a genetic distance of 0.3, genotypes were divided into six distinct genotype groups A, B, C, D, E and F containing 16, 11, 2, 5, 5 and 7 isolates, respectively. Most of the genotypes were area specific or predominated in certain areas but did not belong to a distinct pathotype, while most of the aggressive isolates (pathotype III) occurred in Northern Punjab and in the North Western Frontier Province.     

Electrophoretic karyotype variation among pathotypes of Fusarium oxysporum f.sp. dianthi

Karyotype analysis by pulsed-field gel electrophoresis was applied to characterize isolates of Fusarium oxysporum f.sp. dianthi , the causal agent of Fusarium wilt on carnation. Eleven distinct chromosomal DNA patterns were detected among 38 pathogenic isolates, and the total genome size was estimated to range from 23·7 to 36·4 Mb. Except for isolates belonging to pathotypes 2 and 4 , all members of the same pathotype shared overlapping electrophoretic karyotypes. Karyotypes of isolates assigned to pathotypes 1 and 8 showed a high degree of similarity, in accordance with VCG and RFLP analysis. The same electrophoretic karyotype was also shared by members of pathotypes 2 and 5, thus confirming results obtained by both VCG and RFLP grouping, A single representative of pathotype 6, previously confined to the same VCG and RFLP group as pathotypes 2 and 5, had a slightly different chromosomal pattern. Isolates assigned to pathotype 4 showed four related karyotypes which partially differed in both the number and size of chromosomal bands. However, all strains assigned to this pathotype shared a basic profile of nine chromosomal bands, while two low-molecular-weight bands were present or absent. The findings are discussed with regard both to the suitability of race distinction in the case of the special form dianthi of F. oxysporum and to the use of karyotype analysis by PFGE as a tool for the study of the population genetics of this fungus.     

Differential reaction of sweet pepper to infection with the crinivirus tomato chlorosis virus probably depends on the viral variant

The tomato chlorosis virus (ToCV), transmitted by whitefly species of the genera Bemisia and Trialeurodes in a semipersistent manner, causes significant losses in solanaceous crops including tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum). Worldwide reports of natural and experimental infection of sweet pepper plants with ToCV are contradictory, raising the question of whether the critical factor determining infection is related to the susceptibility of sweet pepper cultivars or the genetics of virus isolates. In this work, ToCV isolates obtained from different hosts and geographical origins were biologically and molecularly analysed, transmitted by B. tabaci MEAM1 and MED, and the reaction of different sweet pepper cultivars was evaluated under different environmental conditions. Brazilian ToCV isolates from tomato, potato (S. tuberosum), S. americanum, and Physalis angulata did not infect plants of five sweet pepper cultivars when transmitted by B. tabaci MEAM1. Temperatures did not affect the sweet pepper susceptibility to tomato-ToCV isolates from São Paulo, Brazil, and Florida, USA. However, sweet pepper-ToCV isolates from Spain and São Paulo, Brazil, were transmitted efficiently to sweet pepper plants by B. tabaci MEAM1 and MED. Although the results indicated that ToCV isolates from naturally infected sweet pepper plants seem to be better adapted to plants of C. annuum, phylogenetic analyses based on the complete nucleotide sequences of RNA1 and RNA2 as well as the p22 gene did not reveal significant nucleotide differences among them. Additional studies are needed to identify intrinsic characteristics of ToCV isolates that favour infection of sweet pepper plants.     

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers AB469331to AB469354.     

Pathogenicity of plant and soil isolates of <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> on tomato and pepper

Crown and root rot of tomato and sweet pepper can be caused by Phytophthora parasitica. In this work, 23 P. parasitica isolates from diseased pepper or tomato plants as well as 54 isolates from 23 monocrop tomato soils (from Spain and Chile) and one from a pepper soil were studied for their host–pathogen response. Results show significant host specificity for the isolates from tomato plants and tomato soils (63 of 64 isolates were unable to cause disease in pepper). None of the pepper plant/soil isolates showed pathogenicity on tomato, and only four of 14 reproduced their pathogenicity on pepper. Only one tomato isolate was pathogenic to both Solanaceae species. Two different inoculation protocols were evaluated (substrate irrigation and stem cutting). All isolates which expressed pathogenicity when stem inoculated also did it when root inoculated, but not vice-versa. Therefore, the recommended test protocol for tomato and pepper breeding programmes is that based on root inoculation by irrigation.     

A classification of <Emphasis Type="Italic">Pepper yellow mosaic virus</Emphasis> isolates into pathotypes

Pepper yellow mosaic virus (PepYMV) is the most important potyvirus infecting sweet pepper in Brazil. In this study, twenty isolates of PepYMV were obtained from commercial sweet pepper crops. To confirm virus identity, the coat protein gene was completely sequenced for eleven of these isolates, and partially sequenced for the other nine isolates. The amino acid identities obtained were above 93% when compared with the sequence of a characterized PepYMV isolate (AF348610). Extracts of Nicotiana tabacum cv. TNN plants infected with the different isolates were used to inoculate the differential series of Capsicum spp cultivars containing the genes pvr2 ¹ , pvr2 ² , pvr2 ³ , pvr2 ⁴ , and Pvr4. Using the same criteria established for Potato virus Y (PVY), fourteen isolates of PepYMV could be classified as known pathotypes described for PVY, that is: 1.2 (2 isolates), 1.3 (6) and 1.2.3 (6). The remaining six isolates, 1.3 (2) and 1.2.3 (4) could not be classified into the typical pathotypes of PVY because they were also virulent on Serrano Criollo de Morellos—334 (C.M 334) which carries the pvr2 ³ and Pvr4 genes. To classify the PepYMV into pathotypes and counter the biological diversity found in this species we propose the utilization of 2^x for the ability to overcome the correspondent allele of the pvr2 locus and 4 for the capacity to break down the Pvr4 gene. Using this criterion we could classify the PepYMV into five pathotypes: 2¹.2²; 2¹.2³; 2¹.2².2³; 2¹.2³. 4 and 2¹.2².2³. 4.