Genetic Variability Within and Among Mycelial Compatibility Groups of Sclerotium rolfsii in South Africa

ABSTRACT Isolates of Sclerotium rolfsii, the causal organism of stem rot or southern blight of groundnut, can be placed in mycelial compatibility groups (MCGs) based on hyphal interactions between isolates. The aim of this study was to determine whether amplified fragment length polymorphism (AFLP) analysis was a suitable technique to assess genetic variability between isolates and MCGs of S. rolfsii. For preliminary genetic analysis, 10 isolates were selected from each of two MCGs and compared with each other using the restriction enzymes EcoRI and MseI and 4 primer pairs. The number of polymorphisms ranged from 10 to 36 per primer combination, with an average of 22.5. AFLP analysis clearly showed genotypic differences (22%) among MCGs B and C, with a maximum variation of 6.41% between any two isolates per group using four primer pairs. Certain isolates could not be distinguished from each other. A more in-depth study of 10 isolates from MCG B, using 8 additional primer pairs, showed small genetic differences (maximum of 4.2% and minimum of 0.2%) between isolates. These results suggested that DNA could be pooled for comparison of MCGs. Pooled DNA from isolates within groups using 20 primer pairs confirmed differences between 9 MCGs. This technique effectively differentiated MCGs of S. rolfsii from each other and also detected differences between isolates within a single MCG.     

Genetic variation among asexual progeny of Phytophthora infestans detected with RAPD and AFLP markers

Genotypic variation among 32 single-zoospore isolates (SZI) of Phytophthora infestans , derived asexually from two hyphal-tip parental isolates (PI-105 and PI-1) of the US-8 genotype, was assessed with 80 random amplified polymorphic DNA (RAPD) primers and 18 amplified fragment length polymorphic DNA (AFLP) primer pairs. In previous investigations, the SZIs from parental isolate PI-105 showed high levels of virulence variability and were differentiated into 14 races, whereas the SZIs from PI-1 showed identical virulence to the parent. The purpose of this investigation was to determine if phenotypic variation observed among SZIs of P. infestans could be detected at the DNA level in these isolates. Polymorphism was detected with 51 RAPD primers and with all 18 AFLP primer pairs in PI-105 SZIs. In SZIs from PI-1, polymorphism was also detected with 25 RAPD primers and 17 AFLP primer pairs. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated the SZIs from parent PI-105 into six virulence groups, 11 RAPD groups and three AFLP groups. Cluster analysis of PI-1 SZIs, which all belong to the same virulence group, differentiated them into four RAPD groups and six AFLP groups. No close correlation among RAPD, AFLP and virulence groups could be established within the two progenies of SZIs. Results of this study suggest that there is a considerable level of inherent genetic variability among SZIs derived asexually from the same parental isolate. The possible mechanisms and implications of this genetic variation are discussed.     

Molecular analyses of colletotrichum species from almond and other fruits

ABSTRACT Isolates of Colletotrichum spp. from almond, avocado, and strawberry from Israel and isolates of the pink subpopulation from almond from the United States were characterized by various molecular methods and compared with morphological identification. Taxon-specific primer analysis grouped the avocado isolates within the species C. gloeosporioides and the U.S. almond and Israeli strawberry isolates within the species C. acutatum. However, the Israeli almond isolates, previously identified morphologically as C. gloeosporioides, reacted with C. acutatum-specific primers. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses determined that each population from almond and strawberry was distinct and clonal. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS 1-5.8S-ITS 2) revealed a similarity of between 97.03 and 98.72% among almond isolates from Israel, C. acutatum almond isolates from the United States, and C. acutatum strawberry isolates from Israel. Similarity of the above populations to that of C. gloeosporioides of avocado was between 92.42 and 92.86%. DNA sequence analysis of the entire ITS region supported the phylogeny inferred from the ITS 1 tree of 14 different Colletotrichum species. Although morphological criteria indicated that the Israeli isolates from almond are unique, this population was grouped within the C. acutatum species according to molecular analyses.     

Pathogenic and genetic variability among Colletotrichum lindemuthianum isolates of different geographic origins

The pathogenic and genetic diversity of Colletotrichum lindemuthianum isolates collected from a total of 10 Central and South American, European and African countries were characterized using common bean differential cultivar pathogenicity tests and amplified fragment length polymorphism (AFLP) analysis. On the basis of pathogenicity tests, 74 isolates were attributed to 30 different pathogenic races using the CIAT-defined binary race-classification system. Twenty-one races were restricted geographically, being exclusive to different countries. Race 9 was the most widespread, being detected in four different countries. Cluster analysis of consensus AFLP data generated using three selective AFLP primer combinations grouped 86 isolates (including the 74 subjected to pathogenicity tests) into three clusters (with one isolate as an outlier). This analysis showed that the majority of South and Central American isolates were divided among two clusters, and that the limited number of European and African isolates used in this study were genetically most similar to Central American isolates. Overall, the results of this research showed some associations between the genetic diversity of the isolates and their country of origin, but not between genetic diversity and race classification of isolates.     

Sibling species of cercospora associated with gray leaf spot of maize

ABSTRACT Monoconidial isolates of the fungus causing gray leaf spot of maize were obtained from diseased leaves collected throughout the United States and analyzed for genetic variability at 111 amplified fragment length polymorphism (AFLP) loci. Cluster analysis revealed two very distinct groups of Cercospora zeae-maydis isolates. Both groups were found to be relatively uniform internally with an average genetic similarity among isolates of approximately 93 and 94%, respectively. The groups were separated from each other by a genetic distance of approximately 80%, a distance greater than that separating each group from the sorghum pathogen, C. sorghi (67 to 70%). Characteristics and dimensions of conidia and conid-iophores produced on infected plants or nutrient media were unreliable criteria for taxonomic differentiation of isolates composing the two groups of C. zeae-maydis. Nucleotide sequences of 5.8S ribosomal DNA (rDNA) and the internal transcribed spacer (ITS) regions were identical within each group but different between the two groups and different from C. sorghi. Restriction fragment length polymorphisms generated by digestion of the 5.8S rDNA and ITS regions with TaqI readily distinguished each group and C. sorghi. Isolates in one group were generally distributed throughout maize-producing regions of the United States; isolates in the other group were localized in the eastern third of the country. Both types were present in the same fields at some locations. The genetic distance based on AFLP profiles and different ITS nucleotide sequences between the two morphologically indistinguishable groups indicate that they are sibling species. Although it is unlikely that breeding for resistance to gray leaf spot will be confounded by local or regional variation in the pathogen, a vigilant approach is warranted, because two pathogenic species exist with unknown abilities to evolve new pathotypes.     

Incidence of thiophanate-methyl resistance in Cercospora kikuchii within a single lineage based on amplified fragment length polymorphisms in Japan

We collected 247 isolates of Cercospora kikuchii from soybean seeds with typical purple stain symptoms from 15 prefectures in Japan. Of the 247 isolates, 93 were sensitive to thiophanate-methyl, a benzimidazole used to control this soybean disease; the remaining 154 were highly resistant to the fungicide. To examine genetic variability among the population of 247 isolates, we developed amplified fragment length polymorphism (AFLP) markers. An AFLP primer pair generated DNA fingerprint polymorphisms among the sample isolates, and with the unweighted pair-grouping method to cluster arithmetic means of the similarity coefficients among all pairs of the fingerprint patterns, the isolates were divided into four lineages (I to IV). Of the 247 isolates, 225 belonged to lineage I, including all isolates that were resistant to thiophanate-methyl. To determine whether the resistance of these isolates was related to mutations in the β-tubulin gene, we amplified partial nucleotide sequences of the gene from 29 representative isolates, including 12 that were resistant to thiophanate-methyl, by means of the polymerase chain reaction. The resistant isolates had identical nucleotide sequence with a one-step change at codon 198, in which the amino acid glutamic acid had been replaced by alanine. The evidence thus suggests that thiophanate-methyl resistance might have arisen in lineage I, the largest of the four lineages. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB214511 to AB214515     

Characterization of colletotrichum isolates from tamarillo, passiflora, and mango in Colombia and identification of a unique species from the genus

ABSTRACT This study was conducted to identify the species of Colletotrichum infecting tamarillo, mango, and passiflora in Colombia and to assess whether cross-infection between host species is occurring. Isolates of Colletotrichum spp. from tamarillo (n = 54), passiflora (n = 26), and mango (n = 15) were characterized by various molecular methods and by morphological criteria. Morphological characterization grouped the tamarillo isolates as C. acutatum and the passiflora and mango isolates as C. gloeosporioides. Species-specific primer analysis was reliable and confirmed grouping of the tamarillo isolates (besides Tom-6) as C. acutatum and the mango isolates (besides Man-76) as C. gloeosporioides. However, DNA of the passiflora isolates was not amplified by either C. acutatum- or C. gloeosporioides-specific primers, but reacted with a new primer, Col1, designed according to the internal transcribed spacer (ITS) 1 region of these isolates. Isolates Tom-6 and Man-76 also reacted positively with the Col1 primer. All the isolates reacting with the C. acutatum- and C. gloeosporioides-specific primers failed to react with primer Col1. Isolate Pass-35 from passiflora did not react with any of the taxon-specific primers. Arbitrarily primed polymerase chain reaction (ap-PCR), random amplified polymerase DNA (RAPD)-PCR, and A+T-rich DNA analyses delineated representative isolates into subgroups within the designated species. Molecular analyses indicated that the C. acutatum tamarillo isolates were uniform or clonal, whereas the C. gloeosporioides mango isolates and Colletotrichum passiflora isolates were heterogeneous. Likewise, sequence analysis of the complete ITS (ITS1-5.8S-ITS2) region identified certain isolates to their respective species: tamarillo isolates as C. acutatum; mango isolates as C. gloeosporioides; passiflora, Tom-6, and Man-76 isolates as a Colletotrichum sp. as yet undefined; and the Pass-35 isolate as an additional undefined Colletot-richum sp. Molecular analyses of the population of Colletotrichum isolates from passiflora, Tom-6 from tamarillo, and Man-76 from mango indicate that this population may not be host specific.     

Virulence and Molecular Diversity in Cochliobolus sativus

ABSTRACT Spot blotch, caused by the fungal pathogen Cochliobolus sativus, is an important disease of barley in many production areas of the world. To assess genetic diversity in this pathogen, a worldwide collection of C. sativus isolates was evaluated for virulence on barley and DNA polymorphism. Three pathotypes (0, 1, and 2) were identified among the 22 isolates tested in this study and the 36 isolates characterized previously on three barley differentials (ND5883, Bowman, and NDB112) that differ in their resistance to C. sativus. Pathotype 2, which exhibits high virulence on cv. Bowman, was only found in North Dakota, whereas the other two pathotypes occurred in many other regions of the world. Genetic diversity of the 58 C. sativus isolates, together with isolates of three related pathogenic Cochliobolus spp. (C. heterostrophus, C. carbonum, and C. victoriae) was analyzed using amplified fragment length polymorphism (AFLP) markers. A total of 577 polymorphic AFLP markers were recorded among the 70 isolates of the four Cochliobolus spp. using eight primer combinations. Cluster analysis revealed distinct groups corresponding to the four different species, except in one case where race 0 of C. carbonum was placed in an outgroup that may belong to a different species. In C. sativus, 95 polymorphic AFLP markers were detected with the eight primer pairs used, and each isolate exhibited a unique AFLP pattern. Allelic diversity in the pathotype 2 group was lower (0.10) than in the pathotype 0 (0.23) and pathotype 1 (0.15) groups, indicating that pathotype 2 may have arisen more recently. Cluster analysis did not reveal a close correlation between pathotypes and AFLP groups, although two AFLP markers unique to pathotype 2 isolates were identified. This low correlation suggests that genetic exchange may have occurred through parasexual recombination in the fungal population. Some isolates collected from different regions of the world were clustered into the same AFLP group, suggesting that migration of the fungal pathogen around these regions has occurred.     

Genetic Diversity Within Colletotrichum acutatum sensu Simmonds

ABSTRACT Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1-5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.     

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     

Differentiation and quantification of the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis using a PCR assay

Isolates of Tapesia yallundae and Tapesia acuformis were subjected to Random Amplified Polymorphic DNA (RAPD) assay. Amplification products common to isolates of either species were cloned and primers were generated from each sequence for use in conventional PCR. The primer pair derived from a T. yallundae specific RAPD marker amplified a product only from DNA of T. yallundae isolates and not from DNA of a range of other fungal species associated with the stem base disease complex of cereals. Similarly, the primer pair generated from a T. acuformis -specific RAPD marker amplifed a product only from DNA of T. acuformis isolates. Quantitative assays were developed for both species of Tapesia from these primer pairs, using competitive PCR . Competitive PCR was used to determine the level of colonization of seedlings by each species in glasshouse- and field-inoculated cereal hosts and results compared to those for conventional seedling disease assessment.     

Comparison of Colletotrichum orbiculare and Several Allied Colletotrichum spp. for mtDNA RFLPs, Intron RFLP and Sequence Variation, Vegetative Compatibility, and Host Specificity

ABSTRACT Based on spore morphology, appressorium development, sequence similarities of the rDNA, and similarities in amplified restriction fragment length polymorphism (AFLP), it has been proposed that Colletotrichum orbiculare, C. trifolii, C. lindemuthianum, and C. malvarum represent a single phylogenetic species, C. orbiculare. In the current study, the phylogenetic relationship among isolates in the C. orbiculare species complex was reassessed. In all, 72 isolates of C. orbiculare from cultivated cucurbit or weed hosts, C. trifolii from alfalfa, C. lindemuthianum from green bean, and C. malvarum from prickly sida (Sida spinosa) were examined for mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs), RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase gene, and vegetative compatibility. In addition, host specificity was examined in foliar inoculations on cucurbit, bean, and alfalfa hosts. Inoculations also were conducted on cucumber fruit. Genetically distinct isolates, based on vegetative compatibility, within the species complex (C. orbiculare, C. trifolii, and C. malvarum) had an identical mtDNA haplotype (haplotype A) when examined with each of three different restriction enzymes. Isolates of C. lindemuthianum had a very similar mtDNA haplotype to haplotype A, with a single polymorphism detected with the enzyme HaeIII. The four species represent a phylogenetically closely related group based on a statistical analysis of the 900- and 200-bp intron sequences. However, distinct RFLPs in the 900-bp intron were consistently associated with each species and could be used to qualitatively and quantitatively distinguish each species. Furthermore, each of the species showed distinct host specificity, with isolates of C. orbiculare (from cucurbits), C. lindemuthianum, and C. trifolii being pathogenic only on cucurbits, green bean, and alfalfa, respectively. Consequently, distinct and fixed nucleotide, or genotypic (intron sequences and RFLPs) and phenotypic (host specificity) characteristics can be used to distinguish C. orbiculare, C. lindemuthianum, and C. trifolii from one another; therefore, they should be recognized as distinct species. This species delineation is consistent with the most current species concepts in fungi. More isolates and further characterization is needed to determine whether C. orbiculare from cocklebur and C. malvarum represent distinct species. RFLPs of the 900-bp intron may represent a relatively inexpensive, reliable, and useful diagnostic tool for general species differentiation in the genus Colletotrichum.     

The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers

Disease observations and amplified fragment length polymorphism (AFLP) markers were used to study recent developments in the Puccinia striiformis f.sp. tritici population in Denmark. The fungus appeared spontaneously at 10 locations in Denmark in 1997 after it was not observed under natural conditions in 1996. The pattern of disease development and prevailing winds suggested that the fungus reappeared by airborne spores from the south or west. In 1998, disease incidence was more evenly distributed throughout the country. Forty-eight single lesion isolates were collected from most crops where the disease was observed in these years; all except one from 1997 belonged to two pathotypes that were not previously detected in the country, and both possessed the newly discovered Yr17 virulence. The isolates were characterized with AFLP markers together with 28 isolates representing eight of 13 pathotypes observed prior to 1996. Initial screening of 240 Pst I/ Mse I AFLP primer combinations on four isolates showed that a primer combination, on average, revealed 0·4 polymorphisms between any isolate pair. A selection of 21 primer combinations resulted in 28 AFLP markers, which revealed 16 AFLP phenotypes among all 76 isolates. The two Yr17- virulent pathotypes consisted of three AFLP phenotypes, which were observed in both 1997 and 1998; the two most frequent AFLP phenotypes occurred at most sampling locations and often within the same crop. AFLP diversity was larger among samples collected prior to 1996, and also in this period most AFLP phenotypes were observed at different sampling locations. These results are consistent with the features of an entirely asexually reproducing pathogen dispersed by aerial spores across large areas.     

大豆疫霉多态性SSR标记开发及遗传多样性分析

 用FastPCR软件在大豆疫霉全基因组中搜索到1 234个含2~4个重复基元精确SSRs。选择260个SSRs设计引物,经对大豆疫霉5个分离物的基因组DNA检测,有212对(81.5%)有效扩增出SSR特征条带,112对(52.8%)扩增多态性。用18对多态性SSR引物分析了来自美国、中国黑龙江省和福建省大豆疫霉分离物的遗传多样性,在73个分离物中共扩增出112个等位变异,变异范围为4~9,平均为6.22个,表明选择的引物对具有高的多态性。在3个大豆疫霉群体中,黑龙江省和福建省分离物的遗传距离最近,美国和福建省分离物的遗传距离最远。UPGMA聚类将73个分离物划分为6组,其中8个美国分离物(72.73%)和53个中国分离物(85.48%)被聚类在一起,表明大豆疫霉中国分离物与美国分离物可能具有共同的祖先,中国分离物可能为外来种。     

Genetic Variability in the Potato Pathogen Colletotrichum coccodes as Determined by Amplified Fragment Length Polymorphism and Vegetative Compatibility Group Analyses

ABSTRACT Amplified fragment length polymorphism (AFLP) using three primer sets was used to characterize 211 Colletotrichum coccodes isolates from North America, 112 of which were assigned to six vegetative compatibility groups (VCGs) using nitrate nonutilizing (nit) mutants. These isolates clustered into five corresponding groups by unweighted pairgroup method with arithmetic means-based cluster analysis of AFLP banding patterns. Isolates of C. coccodes belonging to NA-VCG1 and NA-VCG3 were closely related, as were isolates belonging to NA-VCG2 and NA-VCG5. Based on bootstrap analysis of AFLP data, the two isolates originally assigned to NA-VCG4 clustered with isolates belonging to NA-VCG2 and NA-VCG5. C. coccodes isolates that clustered with two isolates belonging to NA-VCG6 were the most diverged from other groups, including seven isolates collected from hosts other than potato. As opposed to the bootstrap analysis, a quadratic discriminant analysis (QDA) of AFLP data correctly categorized the two isolates of NA-VCG4. Furthermore, in isolates where VCG determinations had been made, this model correctly classified isolates of all VCGs. QDA classifications were identical to those made by the bootstrap analysis, with the exception of VCG4. Overall, classifications made by the QDA model were strongly correlated (r = 0.970, P < 0.001) to the VCGs assigned by traditional methods. All 99 C. coccodes isolates evaluated only by AFLP also were subjected to QDA, leading to the assignment of a presumptive VCG for each isolate. No isolates of VCG4 or VCG6 were identified by QDA within this population. Symptoms of black dot developed in plants inoculated with isolates collected from both potato and non-potato hosts. However, total yield was not significantly reduced by infection with non-potato isolates. The lack of any additional groups identified by AFLP analysis may be an indicator of a limited level of genetic variation among North American C. coccodes isolates. AFLP is a much more efficient technique for subspecific characterization in C. coccodes than VCG analysis utilizing nit mutants and will provide an effective means by which the population biology of this pathogen can be further investigated worldwide.     

Assessment of Diversity in Claviceps africana and Other Claviceps Species by RAM and AFLP Analyses

ABSTRACT Genetic diversity among isolates of Claviceps africana, the sorghum ergot pathogen, and isolates of other Claviceps spp. causing ergot on sorghum or other hosts, was analyzed by random amplified microsatellite (RAM) and amplified fragment length polymorphism (AFLP) analyses. Of the RAM primer sets tested, one revealed polymorphism in C. africana isolates, with Australian and Indian isolates possessing a unique fragment. AFLP analysis, in addition to clearly distinguishing Claviceps spp., revealed polymorphisms in C. africana. A group of isolates from the United States, Puerto Rico, and South Africa exhibited 95 to 100% similarity with one another. Several isolates from Isabela, Puerto Rico were 100% similar to an isolate from Texas, and another isolate from Puerto Rico was identical with one from Nebraska. Australian and Indian isolates showed greater than 90% similarity with isolates from the United States., Puerto Rico, and South Africa. A number of polymorphisms existed in the United States group, indicating that the recently introduced population contains multiple genotypes. Isolates of C. sorghicola, a newly described sorghum pathogen from Japan, were very distinct from other species via RAM and AFLP analyses, as were isolates from outgroups C. purpurea and C. fusiformis. Both RAM and AFLP analysis will be useful in determining future patterns of intercontinental migration of the sorghum ergot pathogen, with the AFLP method showing greater ability to characterize levels of intraspecific variation.     

Genetic Diversity and the Presence of Two Distinct Groups in Ophiostoma clavigerum Associated with Dendroctonus ponderosae in British Columbia and the Northern Rocky Mountains

ABSTRACT The sapstaining fungal pathogen Ophiostoma clavigerum is associated with the mountain pine beetle (Dendroctonus ponderosae), which is currently the most destructive forest pest in North America. The genetic diversity of O. clavigerum populations collected from five sites in Canada and two sites in the United States was estimated with amplified fragment length polymorphism (AFLP) analysis. Genomic DNA from 170 O. clavigerum isolates was digested with EcoRI and PstI and amplified with six primer sets. A total of 469 AFLP markers consisting of 243 monomorphic and 226 polymorphic loci were scored. The overall genetic diversity of the O. clavigerum population was low (Hs = 0.0531) and the differentiation of the seven O. clavigerum populations was moderate (Phi = 0.143). Genetic distances among the populations were not significantly correlated with geographic distance (r = 0.3235, P = 0.074). Two genetically distinct groups in the O. clavigerum populations were shown by unique AFLP profiles and the unweighted pair group method with arithmetic averages. Further work to characterize biological differences between the two groups will be needed to confirm whether cryptic species are present in the O. clavigerum population.     

采用AFLP技术分析稻曲病菌的遗传多样性Ⅰ:同一块稻田中稻曲病菌的遗传结构

 采用AFLP (Amplified fragment length polymorphism)技术分析了来自北京昌平同一块稻田中不同水稻品种和育种中间材料上稻曲病菌(Ustilaginoidea virens)的遗传多样性。从256对EcoRⅠ和MseⅠ引物中选择30对扩增40个菌株。结果表明,同一块稻田中稻曲病菌菌株间的相似性系数达0.72以上,来自同一小区的多数菌株能聚成亚类;发现从同一水稻品种分离的菌株没有特异性的AFLP谱带。初步推断稻曲病菌与水稻品种不存在明显的专化性互作。     

Phylogenetic analysis of Verticillium dahliae vegetative compatibility groups

The evolutionary relationships among Verticillium dahliae vegetative compatibility (VCG) subgroups VCG1A, VCG1B, VCG2A, VCG2B, VCG4A, VCG4B, and VCG6 were investigated by parsimony analysis of amplified fragment length polymorphism (AFLP) fingerprints and sequences of six DNA regions (actin, beta-tubulin, calmodulin, and histone 3 genes, the ITS 1 and 2 regions of the rDNA, and a V. dahliae-specific sequence), using 101 isolates of diverse host and geographic origin. Polymorphisms in gene sequences among isolates of different VCGs were very low and individual gene genealogies provided very little resolution at the VCG level. The combined analysis of all DNA regions differentiated all VCG subgroups except for isolates in VCG1A and VCG1B. VCG clonal lineages in V. dahliae and evolutionary relationships among them were resolved independently by analyses of AFLP fingerprints, multiple gene genealogies, and the combined data set of AFLP fingerprinting and multiple gene genealogies. Two main lineages (I and II) were identified with lineage II comprising two closely related subgroups of VCGs. Lineage I included VCG1A, VCG1B, and VCG2B334; and lineage II included, VCG2A and VCG4B (subclade 1); and VCG2B824, VCG4A, and VCG6 (subclade 2). VCG subgroups were monophyletic except for VCG2B that appeared polyphyletic. Limiting the parsimony analysis either to AFLP fingerprints or DNA sequences would have obscured intra-VCG differentiation. Therefore, the dual approach represented by the independent and combined analyses of AFLP fingerprints and DNA sequences was a highly valuable method for the identification of phylogenetic relationships at the intraspecific level in V. dahliae.     

Genetic relationships among isolates of Colletotrichum gloeosporioides from Stylosanthes spp. in Africa and Australia using RAPD and ribosomal DNA markers

Thirty-three isolates of Colletotrichum gloeosporioides from various Stylosanthes species collected in Africa and Australia and associated with restricted (type A), extensive (type B) or nontypical anthracnose lesions (type C) were first compared by random amplified polymorphic DNA (RAPD) analysis. A phylogenetic tree was constructed based on 118 reproducible polymorphic bands generated with 16 random primers, using the upgma method. Twenty-nine isolates were grouped in two main clusters, corresponding to types A and B, within which polymorphic subgroups were partially related to geographical origin. Strong similarities were observed among isolates of distant origin. Four isolates presented profiles completely different from the A and B types and were grouped in two additional clusters. To assess the phylogenetic relationship among isolates of various types and origins at the species level, the lnternal Transcribed Spacer region (ITS 1) of the ribosomal DNA was sequenced. Type A isolates and a restricted number of type B isolates selected in the RAPD clusters showed an homology of 99.4–100%. When compared with published sequence data, the isolates that were clustered separately in the phylogenetic tree, had the exact sequence of a C. gloeosporioides strain associated with the rotting of coffee berries, or of C. kahawae , the causal agent of coffee berry disease.