AFLP-based PCR markers that differentiate spot and net forms of Pyrenophora teres

Specific polymerase chain reaction (PCR) primers were developed from amplified fragment length polymorphism (AFLP) fragments of Pyrenophora teres , the causal agent of net blotch on barley leaves. The primers were designed specifically to amplify DNA from P. teres f. teres (net form) and allow its differentiation from P. teres f. maculata (spot form), which is morphologically very similar to P. teres f. teres in culture. The PCR amplification was carried out successfully from DNA extracted from fungal mycelium. The PCR assay was validated with 60 samples of Pyrenophora species. The amplification with four designed PCR primer pairs provided P. teres form-specific products. No cross-reaction was observed with DNA of several other species, such as P. tritici-repentis , P. graminea and Helminthosporium sativum .     

Development of an international standard set of barley differential genotypes for Pyrenophora teres f. teres

International comparison of virulence profiles of Pyrenophora teres f . teres (Ptt), the cause of barley net blotch, is seriously restricted by inconsistencies in differential testers used among researchers. This paper reports an attempt to develop an appropriate set of differentials to standardize characterization of Ptt populations globally. Fourteen barley genotypes (Canadian Lake Shore (CLS), Harbin, c-8755, c-20019, Manchurian, Tifang, CI 9825, CI 5791, CI 9819, Beecher, CI 9214, Skiff, Prior and Corvette) were selected from among genotypes previously used as Ptt differentials. Three cultivars (Pirkka, Haruna Nijo and Harrington) were included to identify a universally susceptible control. Genotypes were inoculated with approximately 1000 Ptt isolates from Russia, Europe, Australia and Canada. The mean reaction frequency of genotypes ranged from highly resistant (CI 9819, CI 5791, c-8755 and CI 9825) to highly susceptible (Harrington, Haruna Nijo and Pirkka). The best differential abilities were demonstrated by Harbin, CLS, c-20019, Manchurian and Prior. Application of cluster analyses identified genotypes with similar reaction patterns, which supported a reduction of genotypes in the set. When combined with an algorithm comparing the ability of individual genotypes to discriminate among Ptt isolates, a further reduction of genotypes was justified. A new, concise set of barley genotypes for differentiating virulences in Ptt was formulated. It is proposed that these genotypes be adopted as the standard, international differential set to characterize and identify the virulence properties of Ptt populations across environments. The new Ptt differential set consists of the genotypes c-8755, c-20019, CI 5791, CI 9825, CLS, Harbin, Prior, Skiff and Harrington.     

Genetic variation of Pyrenophora teres f. teres isolates in Western Australia and emergence of a Cyp51A fungicide resistance mutation

Genome-wide, unlinked, simple sequence repeat markers were used to examine genetic variation and relationships within Pyrenophora teres f. teres, a common pathogen of barley, in Western Australia. Despite the region's geographic isolation, the isolates showed relatively high allelic variation compared to similar studies, averaging 7.11 alleles per locus. Principal component, Bayesian clustering and distance differentiation parameters provided evidence for both regional genotypic subdivision together with juxtaposing of isolates possessing different genetic backgrounds. Genotyping of fungicide resistant Cyp51A isolates indicated a single mutation event occurred followed by recombination and long-distance regional dispersal over hundreds of kilometres. Selection of recently emergent favourable alleles such as the Cyp51A mutation and a cultivar virulence may provide an explanation, at least in part, for juxtaposed genotypes. Factors affecting genotypic composition and the movement of new genotypes are discussed in the context of grower practices and pathogen epidemiology, together with the implications for resistance breeding.     

Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance

Resistance to QoI fungicides in Pyrenophora teres (Dreschsler) and P. tritici-repentis (Died.) Dreschsler was detected in 2003 in France and in Sweden and Denmark respectively. Molecular analysis revealed the presence of the F129L mutation in resistant isolates of both pathogens. In 2004, the frequency of the F129L mutation in populations of both pathogens further increased. The G143A mutation was also detected in a few isolates of P. tritici-repentis from Denmark and Germany. In 2005, the F129L mutation in P. teres increased in frequency and geographical distribution in France and the UK but remained below 2% in Germany, Switzerland, Belgium and Ireland. In P. tritici-repentis, both mutations were found in a significant proportion of the isolates from Sweden, Denmark and Germany. The G143A mutation conferred a significantly higher level of resistance (higher EC50 values) to Qo inhibitors (QoIs) than did the F129L mutation. In greenhouse trials, resistant isolates with G143A were not well controlled on plants sprayed with recommended field rates, whereas satisfactory control of isolates with F129L was achieved. For the F129L mutation, three different single nucleotide polymorphisms (SNPs), TTA, TTG and CTC, can code for L (leucine) in P. teres, whereas only the CTC codon was detected in P. tritici-repentis isolates. In two out of 250 isolates of P. tritici-repentis from 2005, a mutation at position 137 (G137R) was detected at very low frequency. This mutation conferred similar resistance levels to F129L. The structure of the cytochrome b gene of P. tritici-repentis is significantly different from that of P. teres: an intron directly after amino acid position 143 was detected in P. teres which is not present in P. tritici-repentis. This gene structure suggests that resistance based on the G143A mutation may not occur in P. teres because it is lethal. No G143A isolates were found in any P. teres populations. Although different mutations may evolve in P. tritici-repentis, the G143A mutation will have the strongest impact on field performance of QoI fungicides.     

Population genetic structure of four regional populations of the barley pathogen Pyrenophora teres f. maculata in Iran is characterized by high genetic diversity and sexual recombination

The leaf spot form of the barley disease net blotch, caused by the fungus Pyrenophora teres f. maculata (PTM), is an increasingly important foliar disease of barley. Studies of population genetic structure and reproductive mode are necessary to make predictions of the evolutionary potential of the pathogen. Sources of resistance to PTM have been found in Iranian landraces, which may have the potential to improve plant breeding efforts. However, little is known about the population genetic structure of this fungus in Iran. In this study, we analysed the frequency of the mating type genes to assess the potential for sexual mating of PTM collected from four provinces—Khuzestan, Hamadan, Golestan, and East Azerbaijan—and we investigated the population genetic structure using seven simple sequence repeat markers. High genotype diversity, linkage equilibrium, and equal ratios of mating types frequencies in the PTM populations at Khuzestan and Hamadan support the occurrence of sexual reproduction in these populations, while in Golestan and East Azerbaijan populations, significant gametic disequilibrium and relatively low genotype diversity suggest a higher incidence of clonality or different demographic histories. Unequal mating type frequencies in Golestan confirm a predominance of asexual reproduction. Finally, we found significant evidence for strong population structure with most of the genetic variation represented within regional populations (89%). Overall, our study provides evidence for high genetic variation in Iranian PTM populations, which may be the basis for rapid adaptive evolution in this pathosystem. This highlights the need for integrated efforts to control the disease.     

Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres

A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.     

Genetic structure of a Pyrenophora teres f. teres population over time in an Australian barley field as revealed by Diversity Arrays Technology markers

Net form of net blotch caused by Pyrenophora teres f. teres (Ptt) is a major foliar disease of barley (Hordeum vulgare) worldwide. Knowledge of the evolution of Ptt pathogen populations is important for development of durable host-plant resistance. This study was conducted to investigate changes in genetic structure of a Ptt population within a barley field during three cropping years. The susceptible barley cultivar Henley was inoculated with Ptt isolate NB050. Leaf samples were collected during the years 2013–15 and 174 single spore Ptt isolates stored. Genotyping using Diversity Arrays Technology markers identified that 25% of isolates were clones of the inoculated isolate and 75% of isolates were multilocus genotypes (MLGs) differing from the original inoculated genotype. The novel genotypes probably originated from a combination of windborne spores from neighbouring fields, infected seed and sexual recombination in the field. The rapid change in the genotypic composition of the Ptt population in this study suggests adaptive potential of novel genotypes and demonstrates the need for barley breeders to use multiple sources of host-plant resistance to safeguard against resistance being overcome.     

Derivation and testing of a model to predict selection for fungicide resistance

A mathematical model was derived to predict selection for fungicide resistance in foliar pathogens of cereal crops. The model was tested against independent data from four field experiments quantifying selection for the G143A mutation conferring resistance to a quinone outside inhibitor (QoI) fungicide in powdery mildew (Blumeria graminis f.sp. hordei) on spring barley (Hordeum vulgare). Fungicide treatments with azoxystrobin differed in the total applied dose and spray number. For each treatment, we calculated the observed selection ratio as the ratio of the frequency of the resistant strain after the last and before the first spray. The model accurately predicted the variation in observed selection ratios with total applied fungicide dose and number of sprays for three of the four experiments. Underprediction of selection ratios in one experiment was attributed to the particularly late epidemic onset in that experiment. When the equation representing epidemic development was modified to account for the late epidemic, predicted and observed selection ratios at that site were in close agreement. On a scatter plot of observed selection ratios on predicted selection ratios, for all four experiments, the 1:1 line explained 89–92% of the variance in the mean of observed selection ratios. To our knowledge, this is the first fungicide resistance model for plant pathogens to be rigorously tested against field data. The model can be used with some degree of confidence, to identify anti‐resistance treatment strategies which are likely to be effective and would justify the resources required for experimental testing.     

Mining wild barley for powdery mildew resistance

Powdery mildew, caused by Blumeria graminis f. sp. hordei (Bgh), is a worldwide disease problem on barley (Hordeum vulgare) with potentially severe impact on yield. Historically, resistance genes have been identified chiefly from cultivated lines and landraces; however, wild barley (H. vulgare subsp. spontaneum) accessions have proven to be extraordinarily rich sources of powdery mildew resistance. This study describes the characterization of a collection of 316 wild barley accessions, known as the Wild Barley Diversity Collection (WBDC), for resistance to powdery mildew and the genetic location of powdery mildew resistance loci. The WBDC was phenotyped for reaction to 40 different Bgh isolates at the seedling stage and genotyped with 10 508 molecular markers. Accessions resistant to all 40 isolates of Bgh were not found; however, three accessions (WBDC 053, 085 and 089) exhibited resistance to 38 of the isolates. Gene postulation analyses revealed that many accessions, while resistant, contained none of the 12 genes present in the Pallas near‐isogenic lines Mla1, Mla3, Mla6, Mla7, Mla9, Mla12, Mla13, Mlk1, MlLa, Mlg, Mlat and Ml(Ru2), suggesting that the accessions carry novel genes or gene combinations. A genome‐wide association study of powdery mildew resistance in the WBDC identified 21 significant marker‐trait associations that resolved into 15 quantitative trait loci. Seven of these loci have not been previously associated with powdery mildew resistance. Taken together, these results demonstrate that the WBDC is a rich source of powdery mildew resistance, and provide genetic tools for incorporating the resistance into barley breeding programmes.     

香蕉枯萎病菌致病力分化与ISSR遗传多样性分析

香蕉枯萎病是一种破坏香蕉维管束的全株性土传病害。本研究旨在探讨香蕉枯萎病菌致病力分化和遗传多样性。以30株采自我国广西的香蕉枯萎病菌,16株分别来自澳大利亚和我国广东、海南、福建、云南等地的香蕉枯萎病菌为对象,采用伤根灌淋法测定香蕉枯萎病菌的致病力,然后用筛选到的ISSR引物对46个香蕉枯萎病菌菌株和4个对照菌株(3个非致病性尖孢镰刀菌和1个茄腐镰刀菌)进行ISSR遗传多样性分析。结果显示,分离到广西香蕉枯萎病菌1号生理小种(FOC1)8株,致病力强、中、弱类型比例分别为62.5%、12.5%和25%;广西香蕉枯萎病菌4号生理小种(FOC4)22株,致病力强、中、弱类型比例分别为18.18%、63.64%和18.18%。14条ISSR引物扩增出237个条带,多态性条带161个,多态性比例为67.93%,遗传相似系数0.76～0.96。聚类分析显示,以遗传距离0.80为阈值时菌株被分为8个类群,所占比例分别为4%、10%、60%、16%、4%、2%、2%、2%。第三类群全部为香蕉枯萎病菌4号生理小种。第一、二、四和五类群总量的70.59%为香蕉枯萎病菌1号生理小种。第八类群为香蕉枯萎病菌3号生理小种。结果表明,在香蕉枯萎病菌与寄主协同进化中,广西的FOC1和FOC4出现明显致病力分化。1号生理小种的遗传多样性比4号生理小种丰富。广西香蕉枯萎病菌4号生理小种与海南、广东的FOC4遗传相似性较高。香蕉枯萎病菌生理小种类型与遗传多样性相关。致病力变异与遗传多样性无相关性。研究结果对香蕉枯萎病菌种群扩张机制探讨、遗传动态分析以及有效防控措施的制定具有一定的指导意义。     

Genetics of host-pathogen interactions in the <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> (net form) – barley (<Emphasis Type="Italic">Hordeum vulgare)</Emphasis> pathosystem

The genetics of host-pathogen interactions in the Hordeum vulgare – P. teres f. teres pathosystem was studied in twelve resistant barley accessions, i.e. CI 9825, CI 9819, Diamond, CI 4922, CI 5401, Harbin, c-8755, c-21849, c-8721 c-23874, c-19979, c-15811. F₂ analyses of crosses with susceptible genotypes employing various isolates (from Europe, USA, Canada, and Australia) revealed that resistance is mostly isolate-specific and controlled by one or two genes. Segregation in ascospore progeny from two crosses between isolates of different origin revealed that avirulence in P. teres is also determined by one or two genes. An epistatic effect of suppressor genes on avirulence genes is proposed for the genetics of virulence to Diamond, Harbin, CI 5401 and c-8721 in the fungal crosses D (181-6 × A80) and F (H-22 × 92-178/9). Segregation in F₂ of crosses of three new sources of resistance (c-23874, c-19979, c-15811) to the susceptible cv. Pirkka was studied in laboratory and greenhouse tests by using seven P. teres isolates, i.e. 181-6, d8-3, d8-4, d9-1, d9-4, F4 and F74. In addition, virulence to these barley accessions of ascospore progeny from crosses of the same isolates was studied. Based on these studies it was concluded that depending on the isolate used, resistance of c-23874 is determined at least by two genes and in c-19979 and c-15811 by three genes. The results of this parallel analyses of genetics of resistance and genetics of virulence allows the postulation of a gene–for–gene interaction in the P. teres – H. vulgare pathosystem.     

Diversity and differentiation in two populations of Gibberella circinata in South Africa

Gibberella circinata [anamorph Fusarium circinatum (=  F. subglutinans f.sp. pini )] causes pitch canker and is an important pathogen in South African pine nurseries. The initial outbreak of the pitch canker fungus was limited to a single nursery at Ngodwana in Mpumalanga Province. Subsequently, several other pine nurseries in South Africa became infected. Most of these outbreaks were relatively small except for the outbreak in the Klipkraal nursery (Mpumalanga Province). The genetic diversity, population differentiation and relative frequencies of the sexual and asexual cycles among two South African subpopulations were determined to establish whether immigration, mutation and/or recombination contributed towards population structure. The allelic diversity of the initial population (Ngodwana) was observed to be lower (0·16) than that of the more recent Klipkraal population (0·25). Approximately 4% ( G _ST = 0·04) of total gene diversity could be attributed to differences among the subpopulations. Furthermore, six new vegetative compatibility groups (VCGs) have been identified since the initial outbreak of G. circinata in South Africa 10 years ago. The relatively low allelic diversity and low level of genetic differentiation suggest restricted gene flow among subpopulations, and indicate that the pathogen has been introduced recently. However, the amount of allelic and VCG diversity suggests that multiple genotypes have been introduced into South Africa. The increases in effective population number, allelic diversity and new VCGs over the past 10 years suggest that sexual reproduction might be occurring.     

苦瓜枯萎病菌的鉴定及其遗传多样性分析

 对分离获得的32株苦瓜枯萎病菌菌株进行形态学特征和寄主专化型测定, 结果表明, 测试的苦瓜枯萎病菌株均为尖孢镰刀菌苦瓜专化型 (Fusarium oxysporum f. sp. momordicae), 这些菌株可以侵染苦瓜和瓠瓜幼苗, 但不侵染其他葫芦科瓜类作物。对苦瓜枯萎病菌菌株的rDNA-ITS区 (ITS1、5.8S和ITS2)序列进行扩增测序, 结果显示其序列长度均为456 bp;聚类分析表明测序菌株与镰刀菌属中尖孢镰刀菌不同专化型的菌株聚为一群。利用RAPD标记技术分析苦瓜枯萎病菌的遗传多样性, 结果显示苦瓜枯萎病菌株与其他葫芦科瓜类作物枯萎病菌株间的遗传相似系数范围为0.59~0.99, 当遗传相似系数为0.85时, 供试的48个菌株分成10个类群 (G_1~10)。在RAPD聚类树中所有苦瓜枯萎病菌株聚在一个分支上 (G₁群), 菌株间的遗传相似系数范围为0.92~1.00, 具有较高的遗传相似性, 且菌株的聚群与地理来源存在一定的相关性。     

2010年西藏小麦条锈菌生理小种群体结构与分析

西藏地区是中国相对独立的小麦种植区,小麦条锈病是西藏冬小麦上最重要的病害.长期以来,对西藏小麦条锈菌生理小种群体结构缺乏全面系统的了解.为了弄清西藏小麦条锈菌生理小种群体结构,本研究从西藏地区小麦条锈病发生的关键地区采集并鉴定了小麦条锈菌标样261份.西藏地区小麦条锈菌群体结构复杂,小种类型数多,主要优势小种以CYR32和CYR33为主,水源11类群为优势类群,Hybrid46类群结构简单,未发现CYR32以外的类型;CYR32之前的小种数较多、其中CYR17、CYR20、CYR31出现频率较高;西藏小麦条锈菌群体结构与内地有着较大的相似性,同时也有其自身的独特性,表现西藏小麦条锈菌优势小种组成与四川、云南两省相似,与青海省差异较大.推测西藏地区小麦条锈菌与四川和云南省存在较密切的菌源交流,与青海省交流较少.     

Resistance in Australian barley (Hordeum vulgare) germplasm to the exotic pathogen Puccinia striiformis f. sp. hordei,causal agent of stripe rust

Eighty‐eight Australian and 10 international barley cultivars were assessed for resistance to the barley stripe (yellow) rust pathogen, Puccinia striiformis f. sp. hordei (Psh). All cultivars were tested for seedling resistance to two UK‐derived isolates of Psh (11.01 and 83.39) that were shown to differ in virulence based on responses on 16 differential barley genotypes. The 98 barley cultivars differed substantially in stripe rust response; 45% were susceptible to Psh 11.01, 53% to Psh 83.39 and 44% to both isolates. The observed diverse infection types (ITs) suggest the presence of both known and uncharacterized resistance. However, further multipathotype tests are required for accurate gene postulation. The Yerong × Franklin (Y×F) doubled haploid (DH) population was phenotypically assessed as seedlings using both Psh isolates. Yerong and Franklin were immune and highly resistant, respectively, to both isolates used in this study. Marker‐trait and QTL mapping identified a major effect on the long arm of chromosome 7H contributed by Franklin in response to all isolates. The resistance of Yerong was mapped to 113·96 and 169·38 cM on chromosome 5HL in response to Psh 11.01 and 83.39, respectively. The Psh resistance sources identified in this study can be used for further genetic analysis and introgression for varietal improvement.     

西藏小麦条锈菌群体结构与遗传多样性

为查明西藏小麦条锈菌Puccinia striiformis f. sp. tritici群体结构和遗传多样性,采用中国鉴别寄主和近等基因系鉴别寄主,以及竞争性等位基因特异性PCR-单核苷酸多态性（kompetitive al-lele specific PCR-single nucleotide polymorphism,KASP-SNP）分子标记对2017年采自西藏的150个小麦条锈菌菌系分别进行表型分析和基因型分析。表型分析结果显示,中国鉴别寄主将150个菌系区分为 12 个已知小种、6 个已知致病类型和 13 个未知致病类型,所有菌系均不能侵染中四和Triticum spelta album鉴别寄主。近等基因系鉴别寄主将150个菌系区分为88个毒性类型,这些毒性类型均不侵染携带抗性基因Yr5、Yr10或Yr15的品种。基因型分析结果显示,26对引物将150个菌系划分为73个基因型,表明西藏小麦条锈菌群体基因型丰富。基因流分析结果表明,波密县与洛扎县小麦条锈菌亚群体之间的基因流N_m最高,达5.86,米林县西部与波密县、洛扎县、巴宜县、米林县东部条锈菌亚群体之间的N_m较低,分别为0.25、0.34、0.42和0.67,表明西藏不同地区条锈菌群体之间基因交流强度差异较大。说明西藏作为我国小麦条锈病的独立流行区,条锈菌群体毒性结构复杂,遗传多样性高。     

Population structure and mating system of Ascochyta rabiei in Tunisia: evidence for the recent introduction of mating type 2

The population structure of Ascochyta rabiei (teleomorph: Didymella rabiei ) in Tunisia was estimated among five populations sampled from the main chickpea growing regions using simple sequence repeat markers (SSR) and a mating type ( MAT ) marker. Mating type 2 isolates ( MAT1-2 ) had reduced genetic and genotypic diversity relative to mating type 1 isolates ( MAT1-1 ). This result, coupled with previous observations of lower overall frequency and restricted geographical distribution of MAT1-2 in Tunisia, and recent (2001) observation of the sexual stage, support the hypothesis of a recent introduction of MAT1-2 . Despite the presence of both mating types in Nabeul, Kef and Jendouba, the hypothesis of random mating was rejected in these locations with multilocus gametic disequilibrium tests. Highly significant genetic differentiation ( θ  = 0·32, G _ST = 0·28, P  < 0·001) was detected among populations and genetic distance and cluster analyses based on pooled allele frequencies revealed that populations from Nabeul and Kef were distinct from those in Beja, Bizerte and Jendouba. More than 70% of total gene diversity ( H _T = 0·55) detected was attributable to variation within populations compared to 28% among populations. This result, coupled with the occurrence of private alleles in each population, suggests that gene flow is currently limited among populations, even those separated by short geographic distances. The presence of two main genetic clusters was confirmed using Bayesian model-based population structure analyses of multilocus genotypes (MLGs) without regard to geographic origin of samples. The presence of MAT1-2 isolates in both clusters suggests at least two independent introductions of MAT1-2 into Tunisia that are likely to be the result of importation and planting of infected chickpea seeds.     

连作条件下土壤中甘蓝枯萎病菌的致病力变化和种群遗传结构分化

 枯萎病菌致病力的变化可能是连作条件下甘蓝枯萎病严重发生的重要原因之一。本研究在建立适度感染的人工病圃的基础上连续种植甘蓝5茬,每茬收获后随机采集土样。利用驹田氏培养基通过稀释平板法对连作土壤中尖孢镰刀菌种群数量的监测结果表明,连作后尖孢镰刀菌的数量由第二茬后的3.047×10⁴ cfu·g^-1土壤增加到第五茬收获后的1.608×10⁵ cfu·g^-1 土壤。对各茬后所分离30株甘蓝枯萎病菌（Fusarium oxysporum f. sp. conglutinans, Foc）的培养性状的观察结果表明,连作后Foc菌株在菌落形态、菌落扩展速率和产孢量等方面均发生明显的变化。用浸根法进行的致病力测定结果表明,随着连茬次数增加,弱致病力菌株占总供试菌株的比例逐渐变小,到第三茬后由第一茬的6.7%下降为0;而强致病力菌株的比例逐渐上升,由第一茬后的6.7%上升到第四茬后的16.7%。利用11条寡聚核苷酸随机引物对受试菌株进行PCR-ISSR扩增,结果显示从第三茬后Foc群体遗传结构出现分化。UPGMA聚类分析结果表明,第三、四和五茬后的Foc菌株都分为A和B两个类群,每个类群又分为Ⅰ和Ⅱ两个亚类群,但同一类群和亚类群中包含不同致病力的菌株,未发现病菌的致病力变化与遗传结构分化之间的相关。