Effects of thiophanate-methyl and azoxystrobin on the composition of Cercospora kikuchii populations with thiophanate-methyl-resistant strains

Azoxystrobin was recently registered in Japan for the control of purple seed stain of soybean caused by Cercospora kikuchii, because the pathogen has developed resistance to thiophanate-methyl. To investigate the effects of these fungicides on the frequency of C. kikuchii strains resistant to thiophanate-methyl and on the genotype structure of the population, we sowed purple-stained seeds, approximately 40% of which were infected with resistant strains, as inocula with asymptomatic seeds and applied thiophanate-methyl and azoxystrobin during the reproductive growth of soybeans. The isolation frequency of resistant strains increased more than 99% by thiophanate-methyl but was not significantly increased by azoxystrobin. In amplified fragment length polymorphism (AFLP) DNA fingerprinting, genotypic diversity was significantly decreased by thiophanate-methyl but was not affected by azoxystrobin. In addition, the similarity of the AFLP genotype structure was increased by thiophanate-methyl but not by azoxystrobin. These results suggest that thiophanate-methyl selectively inhibited the proliferation of sensitive strains, which resulted in a small number of genotypes, most of which were resistant strains. Azoxystrobin was found to nonselectively inhibit proliferation of the pathogen, which retained a large number of genotypes including thiophanate-methyl-sensitive or thiophanate-methyl-resistant strains or both. The nucleotide sequence data for the cytochrome b gene are available in the DDBJ/EMBL/GenBank databases under accession number AB231863.     

Genetic Relationships Between Cercospora kikuchii Populations from South America and Japan

ABSTRACT A collection 160 isolates of Cercospora kikuchii was made from South America and 245 from Japan. DNA fingerprint patterns were analyzed based on amplified fragment length polymorphism among the sample isolates, dividing the isolates into seven lineages (I to VII). Partial nucleotide sequence analyses of the beta-tubulin gene supported this division into seven lineages. Lineages I and III commonly existed in South America and Japan. In all, 136 of the 160 isolates from South America and 223 of the 245 isolates from Japan belonged to lineage I, indicating that lineage I was the major lineage in each area; 5 isolates from South America and 8 isolates from Japan belonged to lineage III. Lineages II (12 isolates) and IV (2 isolates) were specific to Japan and lineages V (3 isolates), VI (1 isolate), and VII (15 isolates) specifically existed in South America. These results suggest that the population genetic structure of C. kikuchii was different between South America and Japan, but the dominance of lineage I was common between the two areas.     

Genetic Analysis of Isolates of Botrytis cinerea Sensitive and Resistant to Benzimidazole and Dicarboximide Fungicides

ABSTRACT A total of 56 isolates of B. cinerea collected from ornamental crops from commercial greenhouses were examined by random amplified polymorphic DNA (RAPD) fingerprint analyses. Isolates were examined as two independent sets of 35 and 36 isolates, with 15 isolates common to both sets. The isolates had four phenotypes: 17 were sensitive to two commonly used fungicides, thiophanate-methyl (a benzimidazole) and vinclozolin (a dicarboximide) (S(T)S(V)); 18 were resistant to both fungicides (R(T)R(V)); 16 were resistant to thiophanate-methyl but sensitive to vinclozolin (R(T)S(V)); and 5 were sensitive to thiophanate-methyl but resistant to vinclozolin (S(T)R(V)). Relationships among the isolates were determined by cluster analyses of mean character differences using the unweighted pair group method using arithmetic average and cladograms were constructed. Isolates were clustered primarily by fungicide-sensitivity phenotype. In one set of greenhouse isolates, 6 of 10 S(T)S(V) isolates clustered together with a bootstrap confidence value of 91%. In the other fingerprint set of greenhouse isolates, 9 of 11 S(T)S(V) isolates clustered together and had a bootstrap confidence value of 98%. Isolates resistant to thiophanate-methyl, vinclozolin, or both fungicides usually were not clustered with other isolates or were clustered with isolates of the same phenotype. To further elucidate these relationships, variant isolates resistant to one or both fungicides were produced on fungicide-amended agar medium from 14 S(T)S(V) greenhouse isolates. These 14 S(T)S(V) parent isolates, 57 resistant variant isolates, and 11 resistant greenhouse isolates were analyzed as three independent RAPD fingerprint sets. Variants selected from eight S(T)S(V) parent isolates were resistant to both thiophanate-methyl and vinclozolin even though parent isolates were exposed to only one of the fungicides. Isolates resistant only to vinclozolin (S(T)R(V)) had fingerprint patterns similar to and clustered with those of parent isolates, while fingerprint patterns of isolates resistant to thiophanate-methyl (i.e., R(T)R(V) or R(T)S(V)), regardless of sensitivity to vinclozolin, clustered differently from both those of S(T)S(V) parent isolates and those of S(T)R(V) isolates derived from the same parent. R(T)R(V) and R(T)S(V) variant isolates derived from the same fungicide-sensitive parents only clustered with other variants having the same phenotype.     

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.     

Molecular characterization of pyraclostrobin resistance and structural diversity of the cytochrome b gene in Botrytis cinerea from apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

DNA fingerprinting of Pyricularia grisea by rep-PCR using a single primer based on the terminal inverted repeat from either of the transposable elements Pot2 and MGR586

We investigated the use of single primers complementary to sequences in the terminal inverted repeat (TIR) of either Pot2 or MGR586, transposable elements found in Pyricularia grisea, for DNA fingerprinting by repetitive-element-based polymerase chain reaction (rep-PCR). Under standard amplification conditions, rep-PCR with each single primer generated distinct fingerprint patterns among rice-infecting P. grisea isolates collected in Japan. With the Pot2-TIR primer, bands ranging in size from 0.2 to 8 kb and in number from 8 to 13 per isolate were amplified. Although fewer bands were amplified with the MGR586-TIR primer, this molecular technique should be more reliable to identify and classify P. grisea isolates by combining the data of fingerprint patterns from each TIR primer. In a cluster analysis based on DNA fingerprints from this rep-PCR with the Pot2-TIR primer, 10 reference isolates and 12 field isolates from Saga Prefecture in 2002 were separated into six clonal lineages. We also demonstrated that the 12 field isolates belonged to one clonal lineage. Thus, this rep-PCR method using the single primer Pot2-TIR will be useful for the analysis of the population structure of rice blast pathogens.     

进境美国大豆中大豆茎褐腐病菌的截获

 从进境的美国大豆豆秆样品中分离到2株疑似大豆茎褐腐病菌Phialophora gregata的分离物247-8和8300-5,2株分离物在PGM培养基上菌落圆形,边缘规则,白色至暗褐色,表面隆起,粗糙,轮纹状。分生孢子卵形至椭圆形,无色,平滑,单胞,平均大小4.3 μm×1.9 μm。分生孢子梗具有瓶梗状结构,无色,无隔膜或有隔膜,大小(5~16)μm×(2~3) μm,呈桶型或长颈瓶型。特异性引物BSRIGS1/2、BSR1/2和Pgl/4分别扩增分离物247-8的DNA得到预期1 022 bp、483 bp和499 bp的产物;分离物8300-5的DNA经PCR扩增分别得到834 bp、483 bp和499 bp的预期条带。2株分离物的ITS区序列完全一致,与GenBank中大豆茎褐腐病菌(登录号AB190396、DQ459387、DQ459386和AF132804)的序列相似性为100%。人工接种大豆幼嫩植株茎基部均产生大豆茎褐腐病菌的典型症状。根据分离物形态特征、PCR检测、序列分析以及致病性测试结果,将进境美国大豆样品中的分离物247-8和8300-5鉴定为大豆茎褐腐病菌P. gregata。     

VCG and AFLP analyses identify the same groups in the causal agents of mango malformation in Brazil

The causal agents of mango malformation disease in Brazil are a new Fusarium lineage in the Gibberella fujikuroi species complex and Fusarium sterilihyphosum; however information on the genetic and geographical diversity of these pathogens in Brazil is missing. Vegetative compatibility group (VCG) and amplified fragment length polymorphism (AFLP) analyses were used to measure the genetic diversity within these populations. Both techniques identified the same genetic groups. Six VCG and AFLP groups were identified amongst isolates of the new lineage from Brazil. FB-VCG 1/AFLP I was the most widespread group, found in seven of the 13 sites sampled. The second most frequent group was recovered from three sites. The remaining four groups were recovered from single-sites. We think that this lineage represents a genetically and geographically diverse indigenous population that reproduces clonally. In F. sterilihyphosum, group FS-VCG 1/AFLP VII was found at three sites in the southeast region of Brazil. FS-VCG 2/AFLP VIII contained isolates from South Africa but not from Brazil. Fusarium mangiferae isolates from India and South Africa formed one group, while isolates from Egypt and the USA formed a second group. F. sterilihyphosum at present is represented by a small population that might have been introduced only once into a restricted area. The clonal nature of the observed populations suggests that these fungi either occur naturally on indigenous hosts and have jumped to the introduced mango host (introduced in Brazil) or that they originated with mango and went through a severe population bottleneck when they were introduced to Brazil from India or Southeast Asia.     

西南地区稻瘟病菌群体遗传多样性分析

为明确西南地区稻瘟病菌Magnaporthe grisea(Hebert)Barr群体遗传结构及其多样性水平,选用13对SSR引物对来自18个县(市)的221个稻瘟病菌单孢菌株进行PCR扩增,利用最长距离法和生物学软件进行聚类分析和群体遗传多样性分析。结果显示,13对SSR引物均能扩增出一条大小相同且清晰的条带,多态位点百分率高达100%。221个菌株在0.16相异水平上可划分为13个遗传宗谱,宗谱SCL01含205个菌株,占总菌株数的92.76%,为优势宗谱;宗谱SCL02~SCL013为劣势宗谱,差异极大。在群体水平上,菌源丰富的8个区域稻瘟病菌群体的Nei’s基因多样性指数为0.2133,Shannon信息指数为0.3588,具有丰富的遗传多样性,且群体间差异较大;这8个种群基于UPGMA法大都聚为一类,种群遗传谱系与地理区域分布呈一定相关性,群体遗传多样性均值为0.2518,存在一定的遗传分化,且群体内多样性大于群体间,总遗传变异的59.37%存在于群体内。总体上,西南地区稻瘟病菌群体结构既有明显的优势宗谱,又存在许多复杂多变的特异性小宗谱,具有丰富的遗传多样性,与地理分布关系较为密切。     

Isozyme and amplified fragment length polymorphisms fromCephalosporium maydis in Egypt

Isoenzyme and amplified fragment length polymorphisms (AFLP) variation within a set of 48 isolates ofCephalosporium maydis was characterized. These isolates included ten cultures that have served as standards in the Egyptian maize resistance breeding program and 38 additional strains collected from 11 governates in Egypt during the 1997 growing season. Eight isozymes also were tested, but only five (acid phosphatase, fumerase, gtucose 4-phosphate isomerase, isocitrate dehydrogenase, and malate dehydrogenase) produced identifiable bands and all five of these enzymes were monomorphic. Sixty-eight AFLP primer-pair combinations were used and 865 bands were scored, of which 288 (33%) were polymorphic and could be used to discriminate four distinct subgroups, or lineages. Representatives from only two of the four lineages are included in the set of ten strains that has been used to challenge new lines in the Egyptian maize breeding program. From among these 68 primer-pair combinations, we identified a set of four AFLP primer-pairs that were strongly correlated (Pearson‘sr > 0.85) with the full data set that can be used as markers to determine the distribution of these lineages and to identify new lineages in field populations.     

大豆枯萎病菌尖孢镰孢遗传多样性及大豆品种抗性

 了解大豆枯萎病菌的群体遗传特征及明确大豆种质对大豆枯萎病的抗性,对抗病育种、抗性品种的合理布局以及制定更有效的病害防治策略具有重要的参考价值。本研究利用随机扩增多态性DNA(random amplified polymorphic DNA,RAPD),对采自我国不同地区的大豆枯萎病菌—尖孢镰孢菌(Fusarium oxysporum)进行遗传多样性分析,筛选到10个多态性随机引物,共扩增出75条RAPD条带,其中55条为多态性条带,占73.3%。利用UPGMA法对DNA扩增图谱进行聚类分析,以相似系数0.68为阈值,55个分离物可分为9个遗传聚类组,表明我国大豆枯萎病菌具有丰富的种内遗传多样性,所划分的群体与分离物来源地不相关。同时,对上述分离物进行致病性分析,发现我国的大豆枯萎病菌具有明显的致病力分化现象。进一步利用3个代表性分离物对来自我国不同大豆产区的180个大豆品种(资源)进行抗大豆枯萎病鉴定,发现皖豆28、中黄13、中黄51、中作X08076和5D034等5个品种对大豆枯萎病具有良好抗性,占供试材料的2.8%,表明不同大豆品种对枯萎病的抗性存在一定的差异。     

Molecular and Pathotype Analysis of the Rice Blast Fungus in North Vietnam

Rice blast caused by Pyricularia oryzae is a devastating disease worldwide. In Vietnam, rice blast is especially severe in the Red River Delta in the North. The genetic diversity of 114 P. oryzae isolates collected from rice in 2001 in the Red River Delta and nine additional Vietnamese P. oryzae isolates was analysed using Amplified Fragment Length Polymorphism (AFLP). DNA similarity and cluster analysis based on 160 polymorphic AFLP markers showed twelve different AFLP genetic groups among the 123 field isolates. Isolates collected from japonica hosts clustered separately from indica host isolates with at least 60% dissimilarity with little evidence for gene flow between the two populations. In the 2001 population originating from indica hosts, three genetic groups were predominant and represented 99% of the isolates sampled. One predominant clonal lineage represented 59% of the 2001 indica host population and was found in eleven provinces in the Red River Delta of North Vietnam. Significant genotype flow could be demonstrated between the indica population south of Red river and the indica population north of Red river. There was significant linkage disequilibrium between the AFLP loci within the indica population, indicating that this is not a random mating population. Pathogenicity tests of 25 isolates selected from the 12 AFLP groups on a set of 29 differential rice lines revealed two avirulent isolates and 23 pathotypes. Different combinations of known resistance genes were found to have potential for blast resistance breeding for North Vietnam. First two authors contributed equally     

Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.     

DNA sequence analysis of ribosomal ITS region 1 of Phytophthora vignae f. sp. adzukicola, the pathogen that causes stem rot of adzuki bean

Sequences of the internal transcribed spacer (ITS) region 1 were used to examine the phylogenetic relationships among races of 19 isolates of Phytophthora vignae f. sp. adzukicola and between this forma specialis and three isolates of the closely related P. vignae f. sp. vignae. The ITS 1 sequences were highly conserved (> 98.7% similarity) among representatives of both formae speciales groups. The results of this study indicate that P. vignae is a monophyletic group. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession nos. AB120062–AB120080 and AB120122     

Genetic structure of Iranian <Emphasis Type="Italic">Pyricularia grisea</Emphasis> populations based on rep-PCR fingerprinting

The population structure of the rice blast fungus Pyricularia grisea was analyzed in two major rice-growing provinces of Iran using rep-PCR DNA fingerprinting. A total of 221 monoconidial isolates of the fungus was collected from 12 cultivars at ten regions during 1997–2000. Long-PCR conditions were used to amplify sequences lying between adjacent Pot2 elements. The frequencies of Pot2 lineages (isolates with 70% amplicon similarity) and haplotypes within lineages were determined. Phenetic analysis differentiated five Pot2 fingerprint lineages, designated A, B, C, D and E. The most common fingerprint group, Lineage E, was recovered from all rice cultivars sampled and was distributed throughout the region. Haplotype E6, the most common haplotype within lineage E, was recovered from almost all regions. Lineage A, the second most common lineage, was found mainly in the western part of the sampled region. Haplotype A1 was found in most sites in the western province. Lineage A occurred at relatively high frequency on the susceptible local cultivar Binam, suggesting that lineage A is specifically adapted to Binam. To test this hypothesis, 193 additional isolates were recovered from four fields at two sites separated by approximately 100 km. This second, field-specific collection of isolates contained lineages A, C, D, and E. Approximately 64% and 29% of the isolates recovered from Binam (the shared cv. at two sites) grouped into lineages A and E, respectively. The other two susceptible cultivars at these sites were infected by lineage E at frequencies of 100% and 71%. Overall, these data indicated a low level of genetic diversity in the Iranian P. grisea population similar to that reported in other countries.     

Field Resistance to Strobilurin (Q(o)I) Fungicides in Pyricularia grisea Caused by Mutations in the Mitochondrial Cytochrome b Gene

ABSTRACT Gray leaf spot caused by Pyricularia grisea is a highly destructive disease of perennial ryegrass turf. Control of gray leaf spot is dependent on the use of preventative fungicide treatments. Strobilurin-based (Q(o)I) fungicides, which inhibit the cytochrome bc(1) respiratory complex, have proven to be very effective against gray leaf spot. However, in August 2000, disease was diagnosed in Q(o)I-treated perennial ryegrass turf on golf courses in Lexington, KY, Champaign, IL, and Bloomington, IL. To determine if resistance was due to a mutation in the fungicide target, the cytochrome b gene (CYTB) was amplified from baseline and resistant isolates. Nucleotide sequence analysis revealed an intronless coding region of 1,179 bp. Isolates that were resistant to Q(o)I fungicides possessed one of two different mutant alleles, each of which carried a single point mutation. The first mutant allele had a guanine-to-cytosine transition at nucleotide position +428, resulting in a replacement of glycine 143 by alanine (G143A). Mutant allele two exhibited a cytosine-to-adenine transversion at position +387, causing a phenylalanine-to-leucine change (F129L). Cleavable amplified polymorphic sequence analysis revealed that neither mutation was present in a collection of baseline isolates collected before Q(o)I fungicide use and indicated that suspected Q(o)I- resistant isolates found in 2001 in Indiana and Maryland possessed the F129L mutation. The Pyricularia grisea isolates possessing the G143A substitution were significantly more resistant to azoxystrobin and trifloxystrobin, in vitro, than those having F129L. DNA fingerprinting of resistant isolates revealed that the mutations occurred in just five genetic backgrounds, suggesting that field resistance to the Q(o)I fungicides in Pyricularia grisea is due to a small number of ancestral mutations.     

Evidence of a new Tomato yellow leaf curl virus in Japan and its detection using PCR

A new isolate of Tomato yellow leaf curl virus (TYLCV) has been identified from tomato plants in Kochi Prefecture in Japan and designated TYLCV-[Tosa]. The complete nucleotide sequence of the isolate was determined and found to consist of 2781 nt. In phylogenetic analyses of entire nucleotide sequences, TYLCV-[Tosa] was delineated as a single branch and was more closely related to TYLCV-[Almeria] than TYLCV isolates Ng, Sz, or Ai reported in Japan, which had spread since 1996. Isolate TYLCV-[Tosa] is suggested to be a newly introduced, novel isolate of TYLCV that dispersed into Kochi Prefecture. In addition, a rapid method using the polymerase chain reaction to separate TYLCV isolates into four genetic groups was established. This method would be useful for reliable diagnosis based on genetic differences among isolates of TYLCV.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB192965 and AB192966     

Amplified Fragment Length Polymorphism Diversity in Cephalosporium maydis from Egypt

ABSTRACT Cephalosporium maydis, the causal agent of late wilt of maize, was first described in Egypt in the 1960s, where it can cause yield losses of up to 40% in susceptible plantings. We characterized 866 isolates of C. maydis collected from 14 governates in Egypt, 7 in the Nile River Delta and 7 in southern (Middle and Upper) Egypt, with amplified fragment length polymorphism (AFLP) markers. The four AFLP primer-pair combinations generated 68 bands, 25 of which were polymorphic, resulting in 52 clonal haplotypes that clustered the 866 isolates into four phylogenetic lineages. Three lineages were found in both the Nile River Delta and southern Egypt. Lineage IV, the most diverse group (20 haplotypes), was recovered only from governates in the Nile River Delta. In some locations, one lineage dominated (up to 98% of the isolates recovered) and, from some fields, only a single haplotype was recovered. Under field conditions in Egypt, there is no evidence that C. maydis reproduces sexually. The nonuniform geographic distribution of the pathogen lineages within the country could be due to differences in climate or in the farming system, because host material differs in susceptibility and C. maydis lineages differ in pathogenicity.     

不同类型Monilinia fructicola菌株的温度适应性及其对苯并咪唑类杀菌剂和乙霉威的交互抗性

褐腐病菌Monilinia fructicola是引起多种果树褐腐病的重要病原菌,前期研究发现该病原菌对甲基硫菌灵的抗性与Tub2蛋白的多个氨基酸变异有关。为明确不同类型菌株的温度适应性及乙霉威是否对所有抗性类型菌株均具有抑菌活性,本研究测定了敏感型菌株S及3种抗性类型包括R_(E198A)、R_(E198Q)及R_(F200Y)型菌株的温度敏感性及其对甲基硫菌灵、多菌灵和乙霉威的敏感性差异,同时分析了不同突变类型菌株对苯并咪唑类药剂和乙霉威的交互抗性。结果表明,4种类型菌株的最适生长温度为21~22 ℃,其中R_(E198Q)型菌株的最适生长温度最低,且其在低温13 ℃和高温28 ℃条件下,生长速率显著低于其他3类菌株,而S、R_(E198A)及R_(F200Y)型菌株在所有温度条件下的生长速率均无显著差异。3种抗性类型菌株对甲基硫菌灵的敏感性有所差异,其中R_(E198Q)型表现较为敏感,R_(F200Y)型在低浓度下与R_(E198A)型无明显差异,但在高浓度下对甲基硫菌灵表现较为敏感;对多菌灵的敏感性差异与甲基硫菌灵相似;只是R_(E198Q) 和R_(F200Y)型菌株对多菌灵比甲基硫菌灵更为敏感。乙霉威的敏感性测定结果表明,R_(E198A)型菌株表现敏感,S类型表现为不敏感性,R_(E198Q)及 R_(F200Y)两种类型均表现为抗性,且抗性水平差别不大。线性相关分析结果表明,仅R_(E198A)型菌株和S型菌株对苯并咪唑类杀菌剂和乙霉威敏感性存在显著负相关性 (甲基硫菌灵和乙霉威:r = ?0.992,p = 0.000;多菌灵和乙霉威:r = ?0.982,p = 0.001)。综合分析结果表明,在3类抗性菌株中,仅R_(E198A)型菌株对苯并咪唑类杀菌剂和乙霉威存在负交互抗性。