Occurrence of Fusarium oxysporum f. sp. melonis Race 1 in Japan

In 1994, Fusarium wilt of melon cultivars which are resistant to races 0 and 2 of Fusarium oxysporum f. sp. melonis was observed in southern area of the Lake Biwa region, Shiga prefecture. In commercial fields, mature plants of cv. Amus which were grafted onto cv. Enken Daigi 2, and of cv. FR Amus showed yellowing, wilting and finally death before harvesting of fruits. Diseased plants had vascular and root discolorations, and their stem sections yielded typical colonies of F. oxysporum. When the Shiga strains were tested for their pathogenicity to 12 species of cucurbits, they caused wilts only on melon. Using race differential cultivars of melon, the Shiga strains were classified as race 1 of F. oxysporum f. sp. melonis, which has not been reported in Japan. To further characterize their pathogenicity, the strains were used to inoculate 46 additional cultivars of melon, oriental melon and oriental pickling melon. All the race 1 strains were pathogenic to the cultivars tested, and their host range was apparently different from those of strains belonging to other races (races 0, 2 and 1,2y). DNA fingerprinting with a repetitive DNA sequence, FOLR3, differentiated race 1 strains from strains of races 0 and 2, but not from race 1,2y strains. Received 2 July 1999/ Accepted in revised form 30 September 1999     

Diversity of Phytophthora Clandestina Isolated From Subterranean Clover in Southern Australia: Analysis of Virulence and RAPD Profiles

The virulence spectrum of 112 isolates of Phytophthora clandestina collected from 56 sites in four subterranean clover-growing states in southern Australia was determined using differential cultivars of subterranean clover. Five races were detected, with race 0 in all states except New South Wales, race 1 in all states, race 2 only in Victoria, race 3 only in New South Wales, and race 4 in Victoria and Western Australia. The level of genotypic diversity among the different P. clandestina populations was investigated using five RAPD primers. Among 30 bands amplified, only two were polymorphic. This enabled identification of four multilocus RAPD genotypes. Three of the four genotypes occurred in all four states. Races 2 and 3 occurred with RAPD genotypes 1 and 2 only whereas races 0 and 1 occurred in all four multilocus RAPD genotypes. These results indicate that the pathogenicity spectrum of P. clandestina can change rapidly.     

Production of Beauvericin by Different Races of Fusarium Oxysporum F. sp. Melonis, The Fusarium Wilt Agent of Muskmelon

Fourty-four strains of Fusarium oxysporum were isolated from plants of melon with Fusarium wilt symptoms. Among these strains, thirty-nine were characterized for their pathogenicity on melon. Thirty-seven strains belonged to known races of F. oxysporum f. sp. melonis, while two strains were non-pathogenic. Four strains belonged to race 0, seven to race 1, four to race 2, and twenty-two to race 1,2. Beauvericin was produced by thirty-six strains in a range from 1 to 310gg^–1. Eight isolates of race 1,2 did not produce the toxin. In addition, of the two non-pathogenic strains, only one strain produced the toxin (290gg^–1). The production of enniatin A₁, enniatin B₁, and enniatin B was also investigated. Enniatin B was the only enniatin detected, being produced by eleven strains belonging to all the races, with a range of production from traces to 60gg^–1. Finally, melon fruits belonging to two different cultivars (Cantalupo and Amarillo) were artificially inoculated with one strain of F. oxysporum f. sp. melonis (ITEM 3464). Beauvericin was detected in the fruit tissues of both cultivars at a level of 11.2 and 73.8gg^–1, respectively. These data suggest that the production of both the toxins is not related to the pathogenicity of F. oxysporum f. sp. melonis, nor to the differential specificity of the races. The results confirm that beauvericin is a common metabolite of phytopathogenic Fusarium species.     

Development of sequence tagged site markers to identify races of Fusarium oxysporum f. sp. lactucae

By random amplified polymorphic DNA (RAPD) analysis of the representative isolates of each race of Fusarium oxysporum f. sp. lactucae, RAPD fragments of 0.6, 1.6, and 2.9kb were obtained. The 0.6-kb RAPD fragment was common to the representative isolates of all three races. Amplification of the 1.6- and 2.9-kb fragments were unique to the isolates of races 1 and 2, respectively. Sequence tagged site (STS) marker FLA0001, FLA0101, and FLA0201 were generated from the 0.6-, 1.6-, and 2.9-kb RAPD fragments, respectively. Polymerase chain reaction (PCR) analysis showed that FLA0001 was common to all 49 isolates of F. oxysporum f. sp. lactucae. FLA0101 was specifically generated from all 23 isolates of race 1 but not from races 2 or 3. FLA0201 was specifically amplified from all 12 isolates of race 2 but not from races 1 or 3. In two isolates of F. oxysporum f. sp. lactucum, PCR amplified FLA0001 and FLA0101 but not FLA0201. On the other hand, these STS markers were not detected from isolates of five other formae speciales. Because these STS markers were not generated from isolates of other plant pathogenic fungi, bacteria, or plant materials examined in this study, PCR analysis combined with the three STS markers should be a useful means for rapid identification of races of F. oxysporum f. sp. lactucae.     

Occurrence ofFusarium oxysporum F.SP.Melonis race 1,2 on muskmelon in Israel

Fusarium oxysporum f.sp.melonis race 1,2 was isolated from wilted muskmelon plants from two sites in Israel. The isolates caused disease in all the Israeli and French differential cultivars tested, regardless of whether they were susceptible or carrying resistance genesFom 1 orFom 2. This is the first record ofF.o. f.sp.melonis race 1,2 in Israel.     

Development of a Specific Polymerase Chain Reaction-Based Assay for the Identification of Fusarium oxysporum f. sp. ciceris and Its Pathogenic Races 0, 1A, 5, and 6

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.     

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.     

Molecular analysis of Spanish populations of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">dianthi</Emphasis> demonstrates a high genetic diversity and identifies virulence groups in races 1 and 2 of the pathogen

Fusarium wilt, caused by Fusarium oxysporum f. sp. dianthi (Fod), is the most important carnation disease worldwide. The knowledge of the diversity of the soil population of the pathogen is essential for the choice of suitable resistant cultivars. We examined the genetic diversity of Fod isolates collected during the period 1998–2008, originating from soils and carnation plants in the most important growing areas in Spain. Additionally, we have included some Fod isolates from Italy as a reference. Random amplified polymorphic DNA (RAPD) fragments generated by single-primer PCR were used to compare the relationship between isolates. UPGMA analysis of the RAPD data separated Fod isolates into three clusters (A, B, and C), and this distribution was more related to aggressiveness than to the race of the isolates. The results obtained in PCR amplifications using specific primers for race 1 and race 2, and SCAR primers developed in this work, correlated with the molecular groups previously determined from the RAPD analysis, and provided new molecular markers for the precise identification of the isolates. Results from successive pathogenicity tests showed that molecular differences between isolates of the same race corresponded with differences in aggressiveness. Isolates of races 1 and 2 in cluster A (R1I and R2I isolates) and cluster C (R1-type isolates) were all highly aggressive, whereas isolates of races 1 and 2 in cluster B (R1II and R2II isolates) showed a low aggressiveness profile. The usefulness of the molecular markers described in this study has been proved in double-blind tests with Fod isolates collected in 2008. Results from this work indicate a change in the composition of the Spanish Fod population over time, and this temporal variation could be related to the continuous change in the commercial carnation cultivars used by growers. This is the first report of genetic diversity among Fod isolates in the same race.     

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。     

Characterization,genetic diversity and distribution of Xanthomonas campestris pv. campestris races causing black rot disease in cruciferous crops of India

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a disease of crucifer crops. The objective of this study was to characterize races of Xcc, their distribution and genetic diversity in India. Two hundred and seventeen isolates of bacteria were obtained from 12 different black rot‐infected crucifer crops from 19 states of India; these were identified as Xcc based on morphology, hrpF gene and 16S rRNA gene based molecular markers and pathogenicity tests. Characterization of races was performed by using a set of seven differential crucifer hosts, comprising two cultivars of turnip (Brassica rapa var. rapa) and cultivars of Indian mustard (B. juncea), Ethiopian mustard (B. carinata), rapeseed mustard (B. napus), cauliflower (B. oleracea) and Savoy cabbage (B. oleracea var. sabauda). Races 1, 4 and 6 of Xcc were identified and, among these races, race 1 followed by race 4 dominated most of the states of India. Genetic diversity of the Indian isolates of Xcc was analysed using repetitive sequence‐based PCR (rep‐PCR) including primers for REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (amplifying with BOX A1 R primer) repetitive elements. This method of fingerprinting grouped the isolates into 56 different DNA types (clusters) with a 75% similarity coefficient. Among these clusters, DNA types 22 and 53 contained two different races 1 and 4, whereas DNA type 12 contained races 1, 4 and 6. However, no clear relationship was observed between fingerprints and races, hosts or geographical origin.     

小麦叶锈菌生理小种MFR的分子鉴定研究

 用AFLP方法对来自中国和墨西哥的23个小麦叶锈菌生理小种进行分析,共筛选了64对引物,获得一对引物(M₀₅/E₀₃)可在MFR小种中扩增出一条特异性DNA片段,进行回收、克隆、测序,结果表明该片段具有325个碱基。根据特异性片段序列设计出SCAR标记引物,对60个叶锈菌生理小种分离物进行回检结果表明,研制的SCAR标记能够准确区分MFR生理小种。本实验结果为小麦锈菌生理小种分子检测体系的建立奠定了基础     

The wheat stem rust pathogen in the central region of the Russian Federation

A total of 387 isolates of Puccinia graminis f. sp. tritici (Pgt) collected in the central region of the Russian Federation from 2000 to 2009 was analysed with North American differential sets comprising 16 genotypes for samples of 2000–2006 and 20 genotypes for samples from 2007–2009. Forty‐five races were identified. The race composition of the local population underwent changes during this period. Race MKBT was the predominant race in the earlier years, but TKNT and TKNTF were in the majority later. During 2000–2009 there were no stem rust epidemics in the region. It was assumed that the local pathogen population cycled on wild grasses (including Elytrigia, Agropyron, Festuca, Dactylis, Phleum and Lolium spp.) and not only on wheat. The existence of host communities of wheat stem rust was supported by random amplified polymorphic DNA (RAPD) markers produced with high‐GC primers. The local population of Pgt was considered to be sexual based on the relatively high diversity of races isolated from various hosts and the absence of correlation between virulence attributes and molecular markers.     

Identification of Pathogenic Races 0, 1B/C, 5, And 6 Of Fusarium Oxysporum F. Sp. Ciceris With Random Amplified Polymorphic DNA (RAPD)

Ninety-nine isolates of Fusarium oxysporum f. sp. ciceris (Foc), representative of the two pathotypes (yellowing and wilt) and the eight races described (races 0, 1A, 1B/C, 2, 3, 4, 5, and 6), were used in this study. Sixty isolates were analyzed by the RAPD technique using DNA bulks for each race and 40 primers. Bands presumably specific for a DNA bulk were identified and this specificity was confirmed by further RAPD analysis of individual isolates in each DNA bulk. Primers OPI-09, OPI-18, OPF-06, OPF-10, and OPF-12 generated RAPD marker bands for races 0, 1B/C, 2, 3, 4, 5, and 6. The reliability and utility of this procedure was validated in blind trials using 39 new Foc isolates. Ten of the 39 isolates had already been typed to race by pathogenicity tests and 29 were typed both by pathogenicity and RAPD testing in this study. In these blind trials, we assigned the 39 new isolates to a race solely on the basis of their RAPD haplotype. Thus, we concluded that Foc races 0, 1B/C, 5, and 6 can be characterized by the RAPD markers. Cluster analysis of the RAPD data set resulted in three clusters of isolates within Foc. The yellowing isolates were grouped in two distinct clusters which correspond to races 0 and 1B/C. The wilt isolates constitute a third cluster that included races 1A, 2, 3, 4, 5, and 6. These results provide a means of studying the distribution of Foc races, to assist in the early detection of introduced race(s) and to facilitate the efficient deployment of available host resistance.     

Detection of Fusarium oxysporum f. sp. dianthi in Carnation Tissue by PCR Amplification of Transposon Insertions

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.     

Cross protection provides evidence for race-specific avirulence factors inFusarium oxysporum

Simultaneous inoculation with races 1 and 2 of the vascular wilt pathogenFusarium oxysporumf.sp.lycopersiciprovided a high level of protection against race 2 in three tomato cultivars carrying resistance geneI, which confers resistance to race 1 but not race 2. However, simultaneous inoculation did not provide any protection in cultivars lacking this gene. Protection resulted in reduction and delay of wilt symptoms. Similarly, avirulent races ofF. oxysporumf.sp.melonisprotected muskmelon plants against virulent races of the sameforma specialis.A ratio 10:1 between spore concentrations of inducer and challenger organism gave the highest cross protection, but ratio 0.1:1 still provided significant disease reduction. Cross protection was also obtained when inoculation with the inducer organism was performed 6 or 12 h before inoculation with the challenger organism. Autoclaved spores of the inducer did not have any protective effect, indicating that living propagules were required to initiate protection. The results suggest the presence of a gene-for-gene interaction betweenF. oxysporumf.sp.lycopersici-tomato andF. oxysporumf.sp.melonis-muskmelon, in which cross protection against a virulent race is mediated by recognition of a specific elicitor from the avirulent race by the plant resistance gene product and by subsequent induction of the plant defense reaction.     

RAPD技术和聚类分析在小麦条锈病菌生理小种研究中的应用

 试用45个随机引物对我国小麦条锈病菌的6个生理小种(CYR17,SU11,CYR23,CYR28,CYR29和CYR30)进行了RAPD分析,其中10个适宜引物具有鉴别作用,共扩增出89条RAPD图带,多态性为73%。自CYR23、CYR28和CYR29三个生理小种中扩增出了特征性RAPD图带。根据RAPD分析结果,将这一结果与6个小种对不同小麦品种苗期和成株期致病性的聚类分析作了比较。可将供试小种分为两组,第一组包括CYR17、SU11和CYR23,其中前两者的相似性最大;CYR28和CYR29也较相近,属于第二组;新小种CYR30与其它小种的相关性尚待进一步研究。     

Genetic,biochemical and pathogenic diversity of Pseudomonas syringae pv. pisi strains

Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep‐PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep‐PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.     

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.