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.     

Assessment of biological and molecular variability between and within field isolates of Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate biotroph that causes clubroot, one of the most damaging diseases of crucifers. Differential cultivars and random amplified polymorphic DNA markers were used to assess the extent of genetic diversity among nine single-gall populations of P. brassicae and 37 single-spore isolates (SSI) derived from four of those field samples. Isolates were classified into eight pathotypes, and each isolate was associated with a unique molecular genotype. Virulence and DNA polymorphisms were detected within and between field isolates, and among SSIs from different pathotypes, hosts and geographical origins. The relatively high level of genetic diversity among field isolates was similar to that among SSIs derived from a single-club field isolate. Molecular and pathogenicity-based classifications were not clearly correlated, but isolates belonging to pathotype P1 were clustered. Two RAPD markers were specific to pathotype P1. The finding that genetic differences can occur in P. brassicae field isolates will be an important consideration in resistance genetic studies and in choosing breeding strategies to develop durable clubroot resistance.     

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     

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.     

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.     

Potential loss of clubroot resistance genes from donor parent Brassica rapa subsp. rapifera (ECD 04) during doubled haploid production

Clubroot resistance derived from the oilseed rape/canola Brassica napus ‘Mendel’ has been overcome in some fields in Alberta, Canada, by the emergence of ‘new’ strains of the protist Plasmodiophora brassicae. Resistance to the pathogen was assessed in 112 doubled haploid (DH) lines, derived from B. rapa subsp. rapifera (European clubroot differential (ECD) 04). The lines were evaluated against five single‐spore isolates representing the ‘old’ pathotypes 2, 3, 5, 6 and 8, and 15 field populations representing new strains of P. brassicae. The disease severity index (ID%) data revealed that none of the DH lines were resistant or moderately resistant to the new pathotype 5X (field populations L‐G1, L‐G2, L‐G3) and D‐G3, while 3–42% were resistant or moderately resistant to the other 11 new strains. Using the mean ID induced by the old pathotype 3 (approx. 13.5%) as the baseline, clubroot severity increased by 300–600% when inoculated with the new pathotypes. A significant finding of this study was the fact that ECD 04 showed absolute resistance to all of the old and new P. brassicae strains while the B. napus ‘Mendel’, although resistant to all of the old pathotypes, was resistant to only about 50% of the new strains. Similarly, all of the selected clubroot‐resistant commercial canola cultivars evaluated in this study were susceptible to 87% of the new P. brassicae strains. The molecular data revealed that the breakdown of clubroot resistance in Mendel and the canola cultivars was in part due to the non‐inheritance of the Crr1 gene on the A08 chromosome from ECD 04.     

感染“中四”小麦条锈菌T4菌系SCAR检测标记的开发

正条锈病是由小麦条锈菌(Puccinia striiformis f.sp.tritici)引起的世界范围内小麦上最重要的病害之一。利用抗病品种是防治该病最经济、有效的措施。但是由于小麦条锈菌的高度变异性,品种抗病性很容易被条锈菌新小种所克服。因此,持续监测条锈菌生理小种的动态变化,及时发现新小种,对     

Development of a co-dominant SCAR marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato (Solanum lycopersicum)

Random amplified polymorphism DNA (RAPD) and bulk segregant analysis (BSA) approaches were used to characterize the molecular marker linked to the Phytophthora infestans resistance gene Ph-3 in tomato. A total of 800 RAPD primers were screened. One RAPD marker UBC#602 was identified to be tightly linked to the Ph-3 gene. The marker was successfully converted into a co-dominant sequence characterized amplified region (SCAR) marker. The SCAR marker SCU602 was used to analyze 96 F₂ progenies and fitted the expected 1:2:1 Mendelian segregation ratio. Forty one tomato inbred lines were screened using the SCAR marker in comparison with a reference marker linked to the Ph-3 gene and both markers gave the same results. SCU602 was further validated for association to resistance and its potential in MAS in 72 tomato lines and cultivars. The marker identified three genotypes harbouring the resistance allele. This SCAR marker can be used in breeding programs for the selection of the Ph-3 gene for Phytophthora infestans resistance.     

Electrophoretic karyotyping and mapping of pathotype-specific DNA sequences in Japanese isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG)₅, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate’s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.     

Pathotypic variation in turnip mosaic virus with special reference to European isolates

A collection of 124 isolates of turnip mosaic virus was gathered from around the world, principally from European countries, and characterized by inoculation to four differential lines of Brassica napus (oilseed rape and swede). Three symptom phenotypes were induced—apparent immunity, local infection only, or systemic infection. Twelve distinct patterns, i.e. pathotypes, were observed. Three pathotypes were predominant in the collection; pathotype 1 isolates, which were the most common, did not overcome any of the most extreme sources of resistance in the differential lines. Of the other two, pathotype 3 isolates overcame one of the major sources of resistance and pathotype 4 isolates overcame all sources of resistance. The distribution of pathotypes within Europe was examined. No pathotype was confined to any geographical area, although pathotype 4 isolates were not found in southern Europe or Asia. Most isolates (90) originated from Brassica hosts, while others were from other cruciferae genera (19) or non-crucifers (5). The species of plant that the isolates originated from was not clearly related to the pathotype of the isolates. Resistance to pathotype 1 isolates is controlled by a dominant allele in one of the differential lines, and resistance sources are being examined in the other lines. Isolates belonging to pathotype 1 appeared to be able to mutate readily to overcome the resistance in one of the rape differential lines, but no isolates appeared to mutate to overcome the other major source of resistance in the differentials. The implications of the results for disease control strategies are discussed.     

香蕉枯萎病菌生理小种鉴定及其SCAR标记

 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。     

Identification of <Emphasis Type="Italic">Pratylenchus thornei</Emphasis>, the cereal and legume root-lesion nematode,based on SCAR-PCR and satellite DNA

Two different molecular tools for the diagnosis of the cereal and legume root-lesion nematode Pratylenchus thornei were developed. A randomly amplified DNA (RAPD) fragment specific to P. thornei was identified. After sequencing the fragment, longer primers were designed that complement the terminal sequences of the RAPD fragment, and this pair of specific primers was used to amplify the sequence-characterized amplified region (SCAR). Using the developed pair of SCAR primers, the SCAR fragment specific to P. thornei was easily amplified with DNA extracts obtained from different life stages of the nematode. The described SCAR-PCR-based assay has the potential to be optimized for routine practical diagnostic tests. In addition, the use of a species-specific satellite DNA sequence to distinguish P. thornei from other Pratylenchus spp. is discussed.     

Pathotypic diversity of Hyaloperonospora brassicae collected from Brassica oleracea

Downy mildew caused by Hyaloperonospora brassicae is an economically destructive disease of brassica crops in many growing regions throughout the world. Specialised pathogenicity of downy mildews from different Brassica species and closely related ornamental or wild relatives has been described from host range studies. Pathotypic variation amongst Hyaloperonospora brassicae isolates from Brassica oleracea has also been described; however, a standard set of B. oleracea lines that could enable reproducible classification of H. brassicae pathotypes was poorly developed. For this purpose, we examined the use of eight genetically refined host lines derived from our previous collaborative work on downy mildew resistance as a differential set to characterise pathotypes in the European population of H. brassicae. Interaction phenotypes for each combination of isolate and host line were assessed following drop inoculation of cotyledons and a spectrum of seven phenotypes was observed based on the level of sporulation on cotyledons and visible host responses. Two host lines were resistant or moderately resistant to the entire collection of isolates, and another was universally susceptible. Five lines showed differential responses to the H. brassicae isolates. A minimum of six pathotypes and five major effect resistance genes are proposed to explain all of the observed interaction phenotypes. The B. oleracea lines from this study can be useful for monitoring pathotype frequencies in H. brassicae populations in the same or other vegetable growing regions, and to assess the potential durability of disease control from different combinations of the predicted downy mildew resistance genes.     

Inheritance of mefenoxam resistance in Phytophthora nicotianae populations from a plant nursery

Three sexual crosses involving isolates of P. nicotianae with differing sensitivity to mefenoxam were established to study the inheritance of mefenoxam resistance. Mefenoxam sensitivity was determined by measuring the mycelial growth on both mefenoxam-amended clarified V8 agar and non-amended agar and then calculating the relative growth. When both parents had the same phenotype (both were resistant or both were sensitive), all F ₁ progeny had the parental phenotype and no segregation for a major effect gene was observed in sensitivity to mefenoxam. However, variations in the mycelial growth of progeny indicated the segregation of minor-effect genes. When the cross involving the mefenoxam-resistant isolate 3A4 and a sensitive parent, the F ₁ progeny segregated for mefenoxam resistance in a ratio of 1:1 (resistant: sensitive), indicating that the mefenoxam resistance is controled by a single dominant gene. Mating type was not linked to the mefenoxam-resistance gene locus. One RAPD marker linked in trans to the MEX locus was obtained by bulked segregant analysis using RAPD markers and was converted to a sequence characterized amplified region marker (SCAR). The SCAR maker identified in this study is a limited but useful tool for differentiating homozygous resistant isolates from sensitive isolates of P. nicotianae.     

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.     

Genetic diversity of the root‐knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava‐damaging species

The objectives of this work were to evaluate the genetic variability of Meloidogyne enterolobii by molecular markers, and develop species‐specific molecular markers for application in detection. Sixteen M. enterolobii isolates from different geographical regions (Brazil and other countries) and hosts were used in this study. The identification and purification of the populations were carried out based on isoenzyme phenotype. The DNA amplification of the intergenic region (IGS) of the rDNA and of the region between the cytochrome oxidase subunit II (COII) and 16S rRNA genes (mtDNA) produced specific fragments of the expected size for this nematode, i.e. 780 and 705 bp, respectively. Intraspecific variability among the isolates was evaluated with three different neutral molecular markers: AFLP, ISSR and RAPD. The results showed a low level of diversity among the isolates tested, indicating that M. enterolobii is a genetically homogeneous root‐knot nematode species. The RAPD method allowed the identification of a species‐specific RAPD fragment for M. enterolobii. This fragment was cloned and sequenced, and from the sequence obtained, a set of primers was designed and tested. The amplification of a 520‐bp‐long fragment occurred only for the 16 isolates of M. enterolobii and not for the 10 other Meloidogyne species tested. In addition, positive detection was achieved in a single individual female, egg‐mass and second stage juvenile of this nematode. This SCAR species‐specific marker for M. enterolobii represents a new molecular tool to be used in the detection of this nematode from field samples and as a routine diagnostic test for quarantine devices .     

Vegetative compatibility of cotton-defoliating <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in Israel and its pathogenicity to various crop plants

Verticillium dahliae isolates recovered from a new focus of severe Verticillium wilt of cotton in the northeast of Israel were tested for vegetative compatibility using nitrate non-utilizing (nit) mutants and identified as VCG1, which is a new record in Israel. Other cotton isolates of V. dahliae from the northern and southern parts of the country were assigned to VCG2B and VCG4B, respectively. VCG1 isolates induced severe leaf symptoms, stunting and defoliation of cotton cv. Acala SJ-2, and thus were characterized as the cotton-defoliating (D) pathotype, whereas isolates of VCG2B and VCG4B were confirmed as the earlier described defoliating-like (DL) and non-defoliating (ND) pathotypes, respectively. This is the first record of the D-pathotype in Israel. The host range of representative isolates of each VCG-associated pathotype was investigated using a number of cultivated plants. Overall, the D isolates were more virulent than DL isolates on all tested host plants, but the order of hosts (from highly susceptible to resistant) was the same: okra (Hibiscus esculentus local cultivar), cotton (Gossypium hirsutum cv. Acala SJ2), watermelon (Citrullus lanatus cv. Crimson Sweet), safflower (Carthamus tinctorius cv. PI 251264), sunflower (Helianthus annuum cv. 2053), eggplant (Solanum melongena cv. Black Beauty), and tomato (Lycopersicon esculentum cv. Rehovot 13). The pattern of virulence of ND isolates differed from that of D and DL isolates, so that the former were highly virulent on eggplant but mildly virulent on cotton. Tomato was resistant to all cotton V. dahliae isolates tested. RAPD and specific PCR assays confirmed that the D isolates from Israel were similar to those originating from other countries.     

Genotypic diversity in barley powdery mildew populations in northern France

Diversity in populations of Erysiphe graminis ( Blumeria graminis ) f.sp. hordei was studied with virulence and molecular markers. Isolates were sampled in two locations in northern France from a winter barley cultivar (Plaisant) and a spring cultivar (Caruso). Only a few pathotypes (determined by virulence markers) were common. The rest of the population was diverse. Diversity within common pathotypes, estimated by five RAPD and two SCAR markers, was generally high, except for one pathotype, which was frequent on Plaisant. This pathotype carried only one virulence, Va22, out of the 11 virulences tested. It appeared as a clonal lineage, which had occurred previously, at least in 1992, in northern France, demonstrating survival of asexual lineages in populations that often reproduce sexually.     

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