Development of a molecular marker for specific detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02 amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters, the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Discrimination of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> races among strains from northwestern Spain by <Emphasis Type="Italic">Brassica</Emphasis> spp. genotypes and rep-PCR

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a severe seedborne disease of Brassica crops around the world. Nine races are recognized, being races 1 and 4 the most aggressive and widespread. The identification of Xcc races affecting Brassica crops in a target area is necessary to establish adequate control measures and breeding strategies. The objectives of this study were to isolate and identify Xcc strains from northwestern Spain by using semi-selective medium and pathogenicity tests, determine the existing races of Xcc in this area by differential series of Brassica spp., and evaluate the use of repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) to differentiate among the nine existing Xcc races. Seventy five isolates recovered from infected fields were identified as Xcc. Race-typing tests determined the presence of the following seven pathogen races: 1, 4, 5, 6, 7, 8 and 9. Race 4 was the most frequent in Brassica oleracea and race 6 in Brassica rapa crops, therefore breeding should be focussed in obtaining resistant varieties to both races. Cluster analysis derived from the combined fingerprints showed four groups, but no clear relationship to race, crop or geographical origin was found. Rep-PCR analysis was found not to be a reliable method to discriminate among Xcc races, therefore race typing of Xcc isolates should be done by using the differential series of Brassica spp. genotypes or another alternative approach.     

Detection of <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> by PCR using novel <Emphasis Type="Italic">virD2</Emphasis> gene-specific primers that discriminate two subgroups

Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.     

Multigene phylogenetic analysis of inter- and intraspecific relationships in <Emphasis Type="Italic">Venturia nashicola</Emphasis> and <Emphasis Type="Italic">V. pirina</Emphasis>

Isolates of Venturia species isolated from pear in Japan, China, Taiwan and Israel were used in this study to analyze their molecular phylogenetic relationship. The nucleotides of rDNA-ITS, partial β-tubulin and elongation factor 1α genes were sequenced directly after PCR. Based on these sequence data two phylogenetic groups could be distinguished. Isolates collected from Asian pears such as Japanese and Chinese pears formed a distinct evolutionary lineage from those derived from European and Syrian pears. This result corroborated the early taxonomic separation of V. nashicola from V. pirina. In addition, trees from single-locus data sets and the combined data set showed that all isolates of V. nashicola were included in a monophyletic group and representative isolates of five pathological races originating from different locations and cultivars formed a single lineage. In contrast, two distinct evolutionary lineages were revealed in V. pirina and isolates of five races were scattered in two lineages. Israeli isolates of race 2 as well as two Japanese isolates of V. pirina formed a distinct lineage from other isolates of this species, while other Israeli isolates belonging to races 1, 3, 4, and 5 were closely related to each other and formed another lineage. It was indicated that the evolution of pathological races in V. nashicola might have occurred relatively recently as compared with V. pirina.     

PCR-based specific detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 4 strains

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2 × 10²cfu.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB118756 and AB118757     

Species-specific PCRs differentiate <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from <Emphasis Type="Italic">R. compacta</Emphasis> as the prevalent cause of white root rot in Japan

Rosellinia compacta, described recently, resembles R. necatrix and also causes white root rot. Here a species-specific PCR was developed for R. compacta, and the two R. necatrix-specific primer sets already available were validated in terms of species specificity. PCRs using the primer sets for R. necatrix amplified specific products exclusively from R. necatrix isolates. The R. compacta-specific primer set exclusively detected R. compacta, which appears to be a rare but widely distributed species. We conclude that R. necatrix is the major cause of the disease in Japan but that the involvement of R. compacta should be studied further.     

Validation of molecular markers for resistance among Pakistani chickpea germplasm to races of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis>

DNA markers in chickpea, targetting resistance genes for different races of Fusarium oxysporum f.sp. ciceris (Foc), have been identified in chickpea, but validation of these markers is essential for effective use in resistance breeding. In view of this, different simple sequence repeats (SSR) markers were analysed in Pakistani germplasm including induced mutants and some local lines. Most of the SSR markers showed good correlation with phenotypic evaluation of genotypes to different races of Foc and may be used effectively in resistance breeding, except those markers for race 3. Markers for race 3 showed deviations from phenotypic data and the reason might be that race 3 is actually Fusarium proliferatum as reported recently and resistance to this race might involve some other major resistance genes. Poor correlation of markers with foc-3 on LG2 in our study and a recent report of independent segregation of foc-2 and foc-3 in near isogenic lines suggested that linkage distances among different resistance genes need further investigation. Moreover three Pakistani mutant lines (97477, CM444/92 and CM368/93) depicted high levels of resistance to Foc races and can be deployed as a valuable source in resistance breeding programmes.     

Characterization of <Emphasis Type="Italic">Phytophthora clandestina</Emphasis> races on <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> in Western Australia

Phytophthora clandestina is a causal agent of root rot disease of subterranean clover in Western Australia (W.A). As a significant number of isolates of P. clandestina from W.A. could not previously be designated using existing differentials, a comprehensive set of subterranean clover (Trifolium subterraneum) cultivars was used as differentials to delineate a broader range of races of the pathogen. One hundred and one isolates of the pathogen collected from W.A. were screened on nine subterranean clover cultivars, of which seven were found to be useful as host differentials. A total of 10 races (in contrast to the five recognized previously) were defined and differentiated using octal nomenclature, presenting a clearer picture of the racial distribution of P. clandestina among W.A. isolates. Differences were found in the race populations between Australian states and are therefore important to the selection/breeding of cultivars for specific regions of Australia to counter the predominant race populations and for enforcing quarantine measures in relation to seed movements within and outside Australia. The octal nomenclature used provides a sound basis for follow-up studies and future race designations. Races 173 and 177 in this study were widely distributed and were the most common races in W.A., and together constitute 80% of the isolates characterized. While six of the seven host differentials were resistant to isolates belonging to race 001 and all were resistant to 000, it is of concern that only one differential was resistant to 157 and 173 and that none of the host differentials were resistant to 177. Our approach to P. clandestina race delineation is clearly conservative and is different from previous studies. The octal nomenclature we applied in this study is not only scientifically sound but also will facilitate rapid recognition and characterization of the races.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

Notes on the Occurrence of Pathogenic Races of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>Found in Hiroshima Prefecture in 1999

The pathogenic race of 59 cultures of Xanthomonas oryzae pv. oryzae, a pathogen of bacterial leaf blight of rice, isolated from six locations in the inland mountainous area of Hiroshima Prefecture in 1999, were determined by a set of traditional differentials. Four races—I, II, V and VII—were found across the area; however, we noticed the composition of the races as well as the dominant race in each location different. All races were avirulent on differential cultivar Te-tep. Races V and VII were new to Hiroshima. The rice cultivars infected with bacterial leaf blight in Hiroshima are thought to be grouped into the Kinmaze group, which does not have any resistance genes. Apparently, a variety of races occurred unexpectedly on the cultivars contrary to stabilizing selection theory. Received 25 February 2000/ Accepted in revised form 13 July 2000     

Gene-based analysis of <Emphasis Type="Italic">Puccinia</Emphasis> species and development of PCR-based marker to detect <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> causing yellow rust of wheat

Stripe rust is considered as the current major rust disease affecting winter cereal production across the world. A quick, reliable PCR-based marker was developed here to detect, identify and rapidly monitor Puccinia striiformis f. sp. tritici (Pst) in wheat-growing areas. Three respective sets of primers, designed from β-tubulin, squalene monooxygenase and ketopantoate reductase genes selected from the full genome of Puccinia striiformis f. sp. tritici, amplified sequences of 239, 358 and 1518 bp, respectively, in Pst pathotypes. A fragment of 1518 bp unique to Pst pathotypes was amplified using primer set PstKeto F1_30/Pst KetoR1_1547 and distinguished the pathogen clearly from different Puccinia spp. and other fungal pathogens. The detection limit of the marker (KetoPstRA₁₅₀₀, accession no. KU240073) by conventional PCR assay was 10 pg. This marker could detect the pathogen in the host before symptom expression. The sensitivity and utility of the marker were further enhanced in a qPCR-based assay that was developed with a newly designed primer set PstKeto F1_1246/Pst KetoR1_1547, which amplified a product of 302 bp and detected as little as 10 fg of DNA. This PCR/qPCR based marker is suitable for studying cultivar resistance, which requires accurate quantification of the pathogen in diseased host tissue.     

Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

Isolation of Molecular Markers Linked to the <Emphasis Type="Italic">Cry</Emphasis> Locus Conferring Resistance to <Emphasis Type="Italic">Cucumber mosaic cucumovirus </Emphasis>Infection in Cowpea

We previously demonstrated that cowpea [Vigna unguiculata (L.) Walp.] cultivar Kurodane-Sanjaku contains the Cry gene, which confers resistance against Cucumber mosaic cucumovirus infection. In this paper, randomly amplified polymorphic DNA (RAPD) analysis was carried out to tag the Cry locus. Bulked segregant analysis for RAPD resulted in many polymorphisms in amplified DNA patterns. Candidates were further screened using parental and/or F₂ cowpea DNAs. As a result, we obtained three RAPD markers, D13/E14-350, WA3-850 and OPE3-500, flanking the Cry locus. In addition, we amplified cowpea sequences coding for the putative nucleotide-binding site (NBS). Degenerate primers based on NBS sequences of tobacco N and Arabidopsis RPS2 disease resistance genes were used for polymerase chain reaction, and resultant products were cloned and sequenced. Among eight independent clones, cowpea resistance gene analog (CRGA) 5 showed a distinct polymorphism when used as a probe for restriction fragment length polymorphism analysis against the susceptible cowpea cultivar PI 189375 and a near-isogenic line for the Cry. Linkage analyses of these molecular markers showed that genetic distances of CRGA5, D13/E14-350, WA3-850 and OPE3-500 to the Cry locus were 0.7, 5.2, 11.5 and 24.5 cM, respectively. Received 16 December 1999/ Accepted in revised form 22 March 2000     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

<Emphasis Type="Italic">Colletotrichum destructivum from</Emphasis> cowpea infecting <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and its identity to <Emphasis Type="Italic">C. higginsianum</Emphasis>

Colletotrichum isolates isolated from cowpea in the Hangzhou area of China were identified as C. destructivum based on morphological characteristics, pathogenicity tests, sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal DNA and the infection process. The ability of the C. destructivum isolates to infect Arabidopsis thaliana was investigated under laboratory conditions and showed a two-phase hemibiotrophic infection process. In addition, the sequences of the rDNA ITS region of C. destructivum isolates from cowpea were identical with 100% similarity to that of isolates of C. higginsianum originating from cruciferous plants. This article presents new evidence in support of C. higginsianum as a synonym of C. destructivum.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.