稻瘟病菌株的DNA指纹分析及小种鉴别

基于对稻瘟病菌(Pyricularia oryzae)基因文库的分析,我们找到了一套含重复顺序的克隆。其中POR6和POR7被证实具有高度的多态性并随机散布于稻瘟病菌生理小种的致病性时,可以获得可分辨的基因组特异的杂交带型。我们还分析了致病性与8个稻瘟病菌株DNA指纹图谱之间的关系,结果表明各个小种组合间的百分相似率S_xy,值与该小种组合间共同侵染的鉴别品种数目有正相关性。     

中国稻瘟病菌的致病性和其DNA指纹图谱间关系的研究

通过在稻瘟病菌Pyricularia oryza。基因组文库中的筛选,找到一个散布的并具有基因组特异性的层重复顺序POR6。本文报道用这一株针对6个日本菌株和26个中国北方菌株进行DNA指纹作图的结果。其中22个中国北方菌株按其杂交带型百分相似率被分成8个株系。一些在我们实验室保存的菌株用传统方法鉴定发现在转管过程中会发生致病性变异。当用POR6作探针与这些菌株NNA的EcoRV酶切片段杂交时,检测出它们的无性世代中出现数条EcoRV多态性片段。     

Karyotypic Variation within Clonal Lineages of the Rice Blast Fungus, Magnaporthe grisea

We have analyzed the karyotype of the rice blast fungus, Magnaporthe grisea, by using pulsed-filed gel electrophoresis. We tested whether the electrophoretic karyotype of an isolate was related to its pathotype, as determined by infection assays, or its genetic lineage, as determined by DNA fingerprinting. Highly reproducible electrophoretic karyotypes were obtained for a collection of U.S. and Chinese isolates representing a diverse collection of pathotypes and genetic lineages. Chromosomes ranged in size from 3 to 10 Mb. Although chromosome number was largely invariant, chromosome length polymorphisms were frequent. Minichromosomes were also found, although their presence was not ubiquitous. They ranged in number from 1 to 3 and in size from 470 kb to 2.2 Mb. Karyotypes were sufficiently variable as to obscure the obvious relatedness of isolates on the basis of pathogenicity assays or genetic lineage analysis by DNA fingerprinting. We documented that the electrophoretic karyotype of an isolate can change after prolonged serial transfer in culture and that this change did not alter the isolate's pathotype. The mechanisms bringing about karyotype variability involve deletions, translocations, and more complex rearrangements. We conclude that karyotypic variability in the rice blast fungus is a reflection of the lack of sexuality in wild populations which leads to the maintenance of neutral genomic rearrangements in clones of the fungus.     

Genotyping with RAPD and microsatellite markers resolves pathotype diversity in the ascochyta blight pathogen of chickpea

 The poor definition of variation in the ascochyta blight fungus (Ascochyta rabiei) has historically hindered breeding for resistance to the chickpea (Cicer arietinum L.) blight disease in West Asia and North Africa. We have employed 14 RAPD markers and an oligonucleotide probe complementary to the microsatellite sequence (GATA)₄ to construct a genotype-specific DNA fragment profile from periodically sampled Syrian field isolates of this fungus. By using conventional pathogenicity tests and genome analysis with RAPD and microsatellite markers, we demonstrated that the DNA markers distinguish variability within and among the major pathotypes of A. rabiei and resolved each pathotypes into several genotypes. The genetic diversity estimate based on DNA marker analysis within pathotypes was highest for the least-aggressive pathotype (pathotype I), followed by the aggressive (pathotype II) and the most-aggressive pathotype (pathotype III). The pair-wise genetic distance estimated for all the isolates varied from 0.00 to 0.39, indicating a range from a clonal to a diverse relationship. On the basis of genome analysis, and information on the spatial and temporal distribution of the pathogen, a general picture of A. rabiei evolution in Syria is proposed. Received: 10 January 1998 / Accepted: 23 January 1998     

Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay.

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.     

Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay.

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.     

Identification of Pathotypes in the Daylily Rust Pathogen Puccinia hemerocallidis

Daylily rust, caused by Puccinia hemerocallidis, has been present in the United States since 2000. In 2003, inoculations with a single isolate of P. hemerocallidis identified daylily cultivars with high levels of resistance to the fungus. The present study was carried out to determine if pathotypes of P. hemerocallidis are present in the south eastern United States. Sixteen isolates of P. hemerocallidis were each inoculated onto leaf segments from 19 daylily cultivars and the resulting disease phenotype assessed. A significant effect of rust isolate on host reaction phenotype was observed for nine of the 19 daylily cultivars. Five of the nine cultivars displayed reaction phenotypes with different isolates of P. hemerocallidis that included at least one susceptible or moderately susceptible and also resistant phenotypes. These results indicate that different pathotypes of the fungus are present in the south east United States. Daylily hybridizers interested in screening for host resistance to P. hemerocallidis will need to include multiple isolates of the fungus to allow for this host specialization.     

Genetic Analysis of Resistance to Rice Blast: A Study on the Inheritance of Resistance to the Blast Disease Pathogen in an F3 Population of Rice

Blast caused by the fungus Magnaporthae grisea (Herbert) Borr. (anamorphe Pyricularia oryza Cav.) is a serious disease of rice (Oryza sativa L.). One method to overcome this disease is to develop disease resistant cultivars. Due to the genetic plasticity in the pathogen genome, there is a continuous threat to the effectiveness of the developed cultivars. Additional studies of the genetics of resistance, virulence stability and functional genomics are required to accelerate research into understanding the molecular basis of blast disease resistance. In this study, individual plants of the F₃ population derived from Pongsu Seribu 2 and Mahsuri were used for pathogenesis assays and inheritance studies of blast resistance. The study was performed with two of the most virulent Malaysian M. grisea pathotypes: P7.2 and P5.0. For blast screening, plants were scored based on the IRRI Standard Evaluation System (SES). F₃ populations showed a segregation ratio of 3R:1S for pathotype P7.2, indicating that resistance to this pathotype is likely controlled by a single nuclear gene. Chi‐square analysis showed that the F₃ families segregated in a 15R:1S ratio for pathotype P5.0. Therefore, locus interactions or epitasis of blast resistance occur against pathotype P5.0 in the F₃ population derived from Pongsu Seribu 2 and Mahsuri. This can be explained by the presence of two independent dominant genes that when present simultaneously, provide resistance to the M. gresia pathotype P5.0. These results indicated that blast resistance in rice is due to the combined effects of multiple loci with major and minor effects. The genetic data generated here will be useful in the breeding of local cultivars for resistance to field blast. The methodology reported here will facilitate the mapping of genes and quantitative trait loci (QTLs) underlying the blast resistance trait.     

Genetic Diversity of Magnaporthe grisea in China as Revealed by DNA Fingerprint Haplotypes and Pathotypes

We determined DNA fingerprint haplotypes and pathotypes of the rice‐blast fungus Magnaporthe grisea collected from 13 areas in China. This DNA fingerprinting analysis, using rep‐PCR, of 381 haplotypes (482 isolates) from China indicated that the M. grisea populations cannot be delineated into region‐specific groups. Analyses of the number of alleles (na), Nei's gene diversity, unbiased genetic distance, and Shannon's Information index among 13 populations showed that clusters were not related to the geographic distance between populations with the exception of the Ningxia (NX) and Jilin (JL) cluster. Among northern populations, NX and JL were more similar to one another than to other populations. Pathogen populations consisting of 121 isolates from China were grouped into 53 pathotypes on the basis of disease reaction in differential rice lines. Isolates assayed for pathotypes were detected based on disease reactions. No correlation was observed between fingerprint groups and pathotypes of the pathogen. High frequency of virulence was found on the rice line Shin2 (Pi‐k^s and Pi‐sh) followed by PiNo.4 (Pi‐ta² and Pi‐sh) and K1 (Pi‐ta), while it was low on Kanto 51 (Pi‐k + ?), K3 (Pi‐k^h), and Fujisaka (Pi‐i and Pi‐sh). Virulence was rare on Toride 1 (Pi‐z^t and Pi‐sh). Tetep (Pi‐k^h + ?) was predicted to be a highly effective, as none of the isolates infected this line. These blast‐resistant rice lines can be used in resistance breeding for the effective management of rice blast in the respective regions of China.     

Clonality and host selection in the wheat pathogenic fungus Puccinia triticina

Clonal reproduction in Puccinia triticina, the cause of wheat leaf rust, has long been hypothesized but has never been demonstrated. Using a population genetics approach and microsatellite markers, we analysed genetic diversity of this fungus at each level of genome organisation. Sampling included isolates from two field populations growing on two cultivars carrying specific resistance genes, completed with isolates representing the main pathotypes identified from a national survey. For the two cultivars, populations differentiated according to the distribution of their genotypes and pathotypes. There was a high proportion of repeated genotypes, combined with a significant linkage disequilibrium and a strong negative value for FIS. These three factors, especially heterozygote excess, strongly support the hypothesis of a high rate of clonal reproduction. Each pathotype matched a unique multilocus genotype, except for a few isolates, which were taken to be mutants of the dominant genotype. We discussed the strong relationship between pathotypes and genotypes as the consequence of clonal reproduction combined with a strong selection exerted by host cultivars.     

Outbreak of Rice Blast Disease at Yeoju of Korea in 2020

Rice blast is the most destructive disease threatening stable rice production in rice-growing areas. Cultivation of disease-resistant rice cultivars is the most effective way to control rice blast disease. However, the rice blast resistance is easy to breakdown within years by blast fungus that continually changes to adapt to new cultivars. Therefore, it is important to continuously monitor the incidence of rice blast disease and race differentiation of rice blast fungus in fields. In 2020, a severe rice blast disease occurred nationwide in Korea. We evaluated the incidence of rice blast disease in Yeoju and compared the weather conditions at the periods of rice blast disease in 2019 and 2020. We investigated the races and avirulence genes of rice blast isolates in Yeoju to identify race diversity and genetic characteristics of the isolates. This study will provide empirical support for rice blast control and the breeding of blastresistant rice cultivars.     

Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes.

The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplification of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, originally from an Australian grasshopper (Praxibulus sp.), through a North America grasshopper resulted in no differences in the resultant RAPD fragment patterns. In the second method, polymorphic RAPD fragments were used to probe the genomic DNA from the three pathotypes, and pathotype-specific fragments were found. In the third method, restriction fragments from genomic DNA of the three pathotypes were cloned and screened for pathotype specificity. A genomic probe specific for each pathotype was isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers. To facilitate the use of RAPD analysis and other molecular tools to identify pathotypes, a method for extracting DNA from resting spores from infected grasshoppers was developed. The DNA from the fractured resting spores was of sufficient integrity to be blotted and probed with the pathotype-specific DNA probes, thus validating the use of these probes for pathotype identification in field-collected grasshoppers.     

Pathotype and Genetic Diversity amongst Indian Isolates of Xanthomonas oryzae pv. oryzae

A number of rice resistance genes, called Xa genes, have been identified that confer resistance against various strains of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. An understanding of pathotype diversity within the target pathogen population is required for identifying the Xa genes that are to be deployed for development of resistant rice cultivars. Among 1024 isolates of Xoo collected from 20 different states of India, 11 major pathotypes were distinguished based on their reaction towards ten Xa genes (Xa1, Xa3, Xa4, xa5, Xa7, xa8, Xa10, Xa11, xa13, Xa21). Isolates belonging to pathotype III showing incompatible interaction towards xa8, xa13 and Xa21 and compatible interaction towards the rest of Xa genes formed the most frequent (41%) and widely distributed pathotype. The vast majority of the assayed Xoo isolates were incompatible with one or more Xa genes. Exceptionally, the isolates of pathotype XI were virulent on all Xa genes, but have restricted distribution. Considering the individual R-genes, Xa21 appeared as the most broadly effective, conferring resistance against 88 % of the isolates, followed in decreasing order by xa13 (84 %), xa8 (64 %), xa5 (30 %), Xa7 (17 %) and Xa4 (14 %). Fifty isolates representing all the eleven pathotypes were analyzed by southern hybridization to determine their genetic relatedness using the IS1112 repeat element of Xoo. Isolates belonging to pathotype XI were the most divergent. The results suggest that one RFLP haplotype that is widely distributed all over India and is represented in strains from five different pathotypes might be an ancestral haplotype. A rice line with xa5, xa13 and Xa21 resistance genes is resistant to all strains, including those belonging to pathotype XI. This three gene combination appears to be the most suitable Xa gene combination to be deployed in Indian rice cultivars.     

Genetic variation in eastern North American and putatively introduced populations of Ceratocystis fimbriata f. platani

The plant pathogenic fungus Ceratocystis fimbriata f. platani attacks Platanus species (London plane, oriental plane and American sycamore) and has killed tens of thousands of plantation trees and street trees in the eastern United States, southern Europe and Modesto, California. Nuclear and mitochondrial DNA fingerprints and alleles of eight polymorphic microsatellite markers of isolates of C. fimbriata from these regions delineated major differences in gene diversities. The 33 isolates from the eastern United States had a moderate degree of gene diversity, and unique genotypes were found at each of seven collection sites. Fingerprints of 27 isolates from 21 collection sites in southern Europe were identical with each other; microsatellite markers were monomorphic within the European population, except that three isolates differed at one locus each, due perhaps to recent mutations. The genetic variability of C. fimbriata f. platani in the eastern United States suggests that the fungus is indigenous to this region. The genetic homogeneity of the fungus in Europe suggests that this population has gone through a recent genetic bottleneck, perhaps from the introduction of a single genotype. This supports the hypothesis that the pathogen was introduced to Europe through Naples, Italy during World War II on infected crating material from the eastern United States. The Californian population may also have resulted from introduction of one or a few related genotypes because it, too, had a single nuclear and mitochondrial genotype and limited variation in microsatellite alleles.     

Population Structure of Puccinia recondita in Western Europe During 1995, as Assessed by Variability in Pathogenicity and Molecular Markers

The population structure of Puccinia recondita f. sp. tritici (Prt) in western Europe was examined by assessing variability in pathogenicity and in randomly amplified polymorphic DNA (RAPD) among 61 single uredinial isolates. The isolates were chosen to represent pathotypes detected in a previous survey of pathogenic variability in the fungus in western Europe in 1995. Thirty‐five pathotypes were identified by assessing infection types produced by the 61 isolates on 24 differential lines, each with a single gene for resistance to Prt. In contrast, only 18 RAPD phenotypes were identified by scoring 19 polymorphic RAPD bands generated with eight RAPD primers. When analysed by cluster and bootstrap analyses, the pathogenicity and RAPD results revealed little evidence for robust distinct clusters among the isolates. Multiple isolates of several pathotypes collected from widely separated locations such as Belgium, Germany, France, Italy and Switzerland had the same RAPD phenotype, providing evidence of clonal migration over considerable distances in western Europe. Some variability (one or two band differences) was observed in RAPD phenotype within several pathotypes, indicating the possible occurrence of genetic changes independent of pathogenicity, and/or the independent development of pathotypes with different genetic backgrounds. Two groups of isolates identified in the 1995 survey, differentiated by pathogenicity for genes Lr3a, Lr3bg, Lr3ka and Lr30, were not distinguished by RAPD phenotype, indicating that the groups probably do not constitute separate lineages within the pathogen population. Little correlation was apparent between the polymorphisms observed in pathogenicity and RAPD phenotypes. The similarity in the genetic backgrounds of the isolates, as assessed by RAPD markers, suggest that the observed differences in pathogenicity may have arisen by selection for specific virulences corresponding to genes for resistance in wheat cultivars grown in the region. Three isolates of pathotype 3, restricted in its distribution to southern France during 1995, were distinct from all other isolates in RAPD phenotype. Circumstantial evidence suggests that this pathotype originated from northern Africa, and that it belongs to a group of leaf rust pathogens specialized to durum wheats.     

Analysis of Pathotypic and Genotypic Diversity of Xanthomonas oryzae pv. oryzae in China

Virulence analysis and restriction fragment length polymorphism (RFLP) were used to evaluated the population structure of Xanthomonas oryzae pv . oryzae ( Xoo ) from the main rice-growing region in China. The pathotype of Xoo was determined for 103 strains by inoculating 13 near-isogenic rice lines using IR24 as the recurrent parent. Sixty-one pathotypes was shared by these strains, on the basis of the consensus of three clustering statistics, and four clusters for pathotype were formed. Cluster 2 consists of strains with high molecular polymorphorism and many pathotypes that are either virulent to a majority of the 13 major resistance ( R ) genes or avirulent only to Xa21 , and is geographically dispersed. The resistance gene Xa21 has broader resistance than others to the strains tested. A probe from a member of the avrBs3/pthA type III effector family, 1376 bp Sph I-digested fragment, was used to screen the genomes of 52 strains tested. Four common bands were found in the DNA fingerprint pattern of Xoo , suggesting basic patterns of evolutionary relationship for members of avrBs3/pthA family and/or the pathogen. Each distinct RFLP banding pattern of each strain was considered as a haplotype; 42 haplotypes were revealed by the probe and divided into four lineages by the same statistics method. It was observed that some isolates with different pathotypes shared the same haplotype and others with different haplotypes harboured identical pathotype. There was a weak correlation between virulent pathotypes and molecular haplotypes.     

Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus, Magnaporthe oryzae

The interaction between rice, Oryza sativa, and rice blast fungus, Magnaporthe oryzae, is triggered by an interaction between the protein products of the host resistant gene, and the pathogen avirulence gene. This interaction follows the ‘gene-for-gene' concept. The resistant gene has effectively protected rice plants from rice blast infection. However, the resistant genes usually break down several years after the release of the resistant rice varieties because the fungus has evolved to new races. The objective of this study is to investigate the nucleotide sequence variation of the AVR-Pita1 gene that influences the adaption of rice blast fungus to overcome the resistant gene, Pi-ta. Thirty rice blast fungus isolates were collected in 2005 and 2010 from infected rice plants in northern and northeastern Thailand. The nucleotide sequences of AVR-Pita1 were amplified and analyzed. Phylogenetic analysis was conducted using the MEGA 5.0 program. The results showed a high level of nucleotide sequence polymorphisms and the positive genetic selection pressure in Thai rice blast isolates. The details of sequence variation analysis were described in this article. The information from this study can be used for rice blast resistant breeding program in the future.     

Strains of Zucchini Yellow Mosaic Virus in Muskmelon (Cucumis melo L.)

According to the reaction of muskmelon line PI 414723, 22 natural isolates of Zucchini Yellow Mosaic Virus (ZYMV) were grouped into two pathotypes. When inoculated by isolates belonging to pathotype 0, most of the PI 414723 plants (over 70%) remained symptomless while few plants developed systemic chloronecrotic spotting and more rarely yellowing, stunting, mosaic and leaf deformations. When contaminated by pathotype 1 all PI 414723 plants developed systemic chloronecrotic spotting. Two variants were obtained from representative strains of these two pathotypes, able to induce yellowing, stunting, mosaic and leaf deformation on all inoculated PI 414723 plants. These variants could not be differentiated from their originating strains either by host range, serology or aphid transmission properties. Therefore they are regarded as belonging to a third group called pathotype 2. No relation was observed between the type of symptom developed on PI 414723 and the ability to induce a rapid wilting reaction of melon cv. Doublon (pathotype F).     

Characterization of Rice Blast Isolates by the Differential System and their Application for Mapping a Resistance Gene,Pi19(t)

To facilitate resistance gene characterization in the present study, the pathogenicities of newly collected blast isolates from rice fields in the Philippines were characterized using international blast differential varieties consisting of 31 monogenic lines that target 24 resistance genes. To classify and designate the blast isolates, we used a new international blast designation system, which has been proposed as a suitable naming system for comparing blast races among different studies. A total of 23 rice blast isolates collected from the Philippines were classified into 16 pathotypes, which showed reaction patterns different from those seen in the standard isolates. Among the blast pathotypes, 11 had differentiating ability for four Pik alleles (Pik, Pik‐m, Pik‐h, and Pik‐p) and Pi1, whereas the standard blast isolates from the Philippines were not able to differentiate these genes. In addition, several blast isolates were avirulent to IRBLt‐K59, IRBL19‐A, and Lijiangxintuanheigu, although the standard differential blast isolates were virulent to these lines. Moreover, two blast isolates were virulent to a monogenic line, IRBL9‐W, which harbours Pi9 and was resistant to all standard differential blast isolates. By using the isolates avirulent to IRBL19‐A, Pi19(t) was successfully mapped in the centromeric region on chromosome 12 with simple sequence repeat markers RM27937 and RM1337. These markers are useful for marker‐assisted Pi19(t) introgression worldwide.     

Biomechanical model for appressorial design in Magnaporthe grisea

The fungus Magnaporthe grisea, commonly referred to as the rice blast fungus, is responsible for destroying from 10% to 30% of the world's rice crop each year. The fungus attaches to the rice leaf and forms a dome-shaped structure, the appressorium, in which enormous pressures are generated that are used to blast a penetration peg through the rice cell walls and infect the plant. We develop a model of the appressorial design in terms of a bioelastic shell that can explain the shape of the appressorium, and its ability to maintain that shape under the enormous increases in turgor pressure that can occur during the penetration phase.