The control of bubble diseases and cobweb disease of mushrooms with prochloraz

Fungicides were compared for the control of the mushroom pathogens Verticillium fungicola (dry bubble disease), Mycogone perniciosa (wet bubble disease) and Hypomyces rosellus (cobweb disease). Prochloraz, applied as a manganese complex, consistently gave significantly greater control of a benomyl-resistant strain of V. fungicola. Captafol gave some reduction in disease. Benomyl, thiabendazole and prochloraz all gave significant control of M. perniciosa and H. rosellus. Prochloraz residues in mushrooms were measured by gas chromatography after extraction in acetone and purification by means of solvent partition, When pure prochloraz was added to homogenized mushroom tissue, 77–98% was recovered by this procedure. The residue levels in sporophores from crops treated with prochloraz were low, Prochloraz appears to be an effective fungicide for the control of the major fungal pathogens of the mushroom crop, particularly where benzimidazole-resistant strains of V. fungicola occur.     

检疫性轮枝菌及其近似种的鉴定

 大丽轮枝菌(Verticillium dahliae)和黑白轮枝菌（V. albo-atrum）在世界范围内引起多种作物的黄萎病,属于我国重要进境植物检疫对象。本研究对采自我国部分地区和CBS保存的多种植物病原性轮枝菌,包括黑白轮枝菌、大丽轮枝菌及其变种大丽轮枝菌长孢变种(V. dahliae var. longisporum)、三体轮枝菌(V. tricorpus)、变黑轮枝菌(V. nigrescens)和云状轮枝菌(V. nubilum),采用生物学特性观察,结合rDNA-ITS序列分析的方法,进行了比较和分析。结果表明：不同种类轮枝菌在休眠结构形态上具有一定差异,部分菌株不产生任何休眠结构。各供试菌株在15～25℃范围内均可生长,但黑白轮枝菌在30℃下生长受到强烈抑制,而其他菌株受影响较小。对供试菌株rDNA-ITS序列分析结果表明植物病原性轮枝菌可聚为9个分支,包括三体轮枝菌、变黑轮枝菌、云状轮枝菌、V. theobromae、大丽轮枝菌、大丽轮枝菌长孢变种和3个不同的黑白轮枝菌分支,黑白轮枝菌、大丽轮枝菌及其长孢变种亲缘关系较近。采用生物学性状结合rDNA-ITS序列分析能够更加有效地将两种检疫性轮枝菌从其他植物病原性轮枝菌中区分出来。     

Sources of Verticillium fungicola on a commercial mushroom farm in England

From 1979 to 1981 Verticillium fungicola, a pathogen to Agaricus bisporus, was detected in 47 out of 485 samples of baled peat and in one out of 126 samples of bagged limestone arriving at an isolated mushroom farm in the UK. The pathogen was not detected in 875 samples of water, soil, mushroom spawn or spawned compost. Casing (a mixture of peat and limestone) prepared and used on the farm contained detectable V. fungicola in 25 out of 108 samples. The primary sources of V fungicola were thus the two constituents of casing, especially peat.
Secondary sources of V. fungicola were numerous and included symptomless and diseased mushrooms, pickers and other staff, floors and doors and the principal pests (sciarid flies and mites).
Viable airborne spores of V. fungicola were detected with an Andersen sampler in farm buildings at all stages in crop production. The spore load increased with the age of the crop. Few airborne spores were detected immediately outside affected buildings, and very few at the farm entrance 30 m away. There was no evidence that V. fungicola reached the farm from the surrounding air. despite substantial soil disturbance nearby.
Selective media for V. fungicola are described.     

Comparison of Puccinia graminis f.sp. tritici from South America and Europe

Twenty isolates of Puccinia graminis f.sp. tritici from South America were compared with 19 from Europe using virulence, isozymes and random amplified polymorphic DNA (RAPD) markers. The isozyme and virulence patterns for these isolates were also compared with those of 11 isolates representative of the common race clusters in North America. All three types of marker showed a level of similarity between the South American and European isolates comparable with that between isolates from the same continent. The average similarity coefficients between the South American and European isolates were 0.65 for virulence, 0.67 for isozymes, and 0.70 for RAPD markers. Among South American isolates the values were 0.63 for virulence, 0.64 for isozymes and 0.72 for RAPDs. For the South American and European isolates, correlation between the similarity matrices based on RAPDs and on isozyme markers, respectively ( r  = 0.52), was higher than that between the RAPD and virulence matrices ( r  = 0.32) or between isozyme and virulence matrices ( r  = 0.16). The North American isolates had a comparable level of similarity for virulence and isozymes to both the South American and European populations. There was no clear distinction between the South American, North American and European isolates, which is consistent with the hypothesis that these populations may have had a common origin.     

Phylogenetic Analyses of Phytopathogenic Isolates of Verticillium spp

ABSTRACT To better understand the genetic relationships between Verticillium dahliae isolates from lettuce and other phytopathogenic Verticillium spp. isolates from various hosts and geographic locations, the complete intergenic spacer (IGS) region of the nuclear ribosomal RNA gene (rDNA) and the beta-tubulin gene were amplified and sequenced. The sequences of the complete IGS region and the beta-tubulin gene were used alone and in combination to infer genetic relationships among different isolates of Verticillium with the maximum-likelihood distance method. Phylogenetic analyses set sequences into four distinct groups comprising isolates of V. albo-atrum, V. tricorpus, and V. dahliae from cruciferous and noncruciferous hosts. Within the four Verticillium groups, isolates of V. dahliae from cruciferous hosts displayed the closest affinity to V. dahliae from noncruciferous hosts. Isolates of V. dahliae from noncruciferous hosts could be further divided into four subgroups based on sequence similarities within the IGS region. Cross-pathogenicity tests demonstrated that most Verticillium isolates were as virulent on other hosts as on their hosts of origin. A phenogram based on the cross pathogenicity of individual isolates resembled those derived from the IGS and beta-tubulin sequence comparisons. On the basis of the data presented, the potential origin of some isolates of V. dahliae pathogenic on lettuce is proposed.     

<Emphasis Type="Italic">Verticillium tricorpus</Emphasis> causing lettuce wilt in Japan differs genetically from California lettuce isolates

In January 2002, Verticillium wilt of lettuce (Lactuca sativa L.) caused by Verticillium tricorpus occurred in upland paddy fields in Hyogo Prefecture for the first time in Japan. This fungal species was first isolated from lettuce in California, USA. In the present study, the genetic relationships between the American and Japanese isolates of V. tricorpus from lettuce were analyzed to determine whether the pathogen could have migrated to Japan from the USA, the major lettuce-seed supplier for Japan. Nucleotide sequences of the rDNA internal transcribed spacer regions, as well as the genes coding for translation elongation factor 1-alpha and RNA polymerase II were compared among American and Japanese V. tricorpus isolates from lettuce. The Japanese isolates of V. tricorpus were distinct from the American. Random amplified polymorphic DNA analyses also supported this conclusion. These results demonstrated that Verticillium wilt on lettuce caused by V. tricorpus in Japan was not related to the isolates causing the disease in California.     

Comparative Study of Genetic Diversity and Pathogenicity Among Populations of Verticillium Dahliae from Cotton in Spain and Israel

Genetic diversity and phenotypic diversity in Verticillium dahliae populations on cotton were studied among 62 isolates from Spain and 49 isolates from Israel, using vegetative compatibility grouping (VCG), virulence and molecular assays. In Spain, defoliating V. dahliae isolates (D pathotype) belong to VCG1, and non-defoliating isolates (ND) belong to VCG2A (often associated with tomato) and VCG4B (often associated with potato). The D pathotype was not identified in Israel. The ND pathotype in Israel is comprised of VCG2B and VCG4B. Isolates in VCG2B and VCG4B ranged in virulence from weakly virulent to highly virulent. The highly virulent isolates induced either partial defoliation or no defoliation. Virulence characteristics varied with inoculation method and cotton cultivar. Highly virulent isolates from Israel were as virulent as D isolates from Spain under conditions conducive to severe disease. The D pathotype is pathologically and genetically homogeneous, whereas the ND pathotype is heterogeneous with respect to virulence, VCG, and molecular markers based on single-primer RAPD and on PCR primer pairs.     

Diversity among isolates ofVerticillium lecanii as expressed by DNA polymorphism and virulence towardsBemisia tabaci

Thirty-six isolates ofVerticillium lecanii andVerticillium sp. were taken from different hosts (both insects and rusts) and geographical locations. The isolates were analyzed for genomic variability, as expressed by random amplified polymorphic DNA (RAPD), in relation to virulence onBemisia tabaci. Virulence on larvae ofB. tabaci within these isolates ranged from 0% to 83%. RAPD analysis was performed employing two different arbitrary decamer primers and the calculated similarity coefficients were subjected to cluster analysis using the unweighted average linkage (UPGMA) algorithm. The dendrograms obtained with each of the two primers were identical. Eight cluster groups and three unclustered isolates were obtained by selecting a similarity level of 80%. The amplification pattern of DNA obtained by RAPD for the various isolates suggested thatV. lecanii is a highly diverse species. No correlation could be established between virulence and either RAPD polymorphism of the fungal isolates or the insect host from which they were isolated. Generally, no correlation could be established between the clustering ofV lecanii strain and geographical location although a limited number of strains obtained from Russia and Georgia were assembled in the same cluster and those from Kazakhstan were clonal.     

小麦纹枯病菌核糖体基因内转录区序列比较

 对7个从江苏省小麦纹枯病样本分离到的丝核菌菌株,进行形态学鉴定、融合群分类和致病性测定,提取病菌的DNA,采用通用引物ITS1(TCC GTA GGT GAA CCT GCG G)和ITS4(TCC TCC GCT TAT TGA TAT GC),扩增病菌的rDNA内转录区(ITS),并对扩增产物进行了测序.用这些序列在NCBI中进行BLAST分析,得到与这些菌株亲缘关系最近的菌株序列,并明确了这些菌株的分类地位.对以上的菌株序列进行Alignment分析,结果表明,病菌的5.8S rDNA序列高度保守,而ITS区的可变性则相对较高,在双核和多核丝核菌、双核丝核菌CAG1融合群和非CAG1融合群菌株间存在差异,可用于反映菌株间的进化关系和双核丝核菌种下分类.     

Molecular Characterisation of Alternaria linicola and its Detection in Linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.     

Intraspecific diversity within avocado field isolates of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from south-east Spain

Fifty-five isolates of Rosellinia necatrix, the cause of common avocado white root rot disease, were collected from south-east Spain and characterised according to their virulence behaviour and their molecular patterns to assess broader levels of genetic diversity. Virulence properties were revealed by in vitro inoculation on avocado plants. Differences in reaction types showed variability among these isolates. No sequence differences were observed when the internal transcribed spacer 1 (ITS1) and ITS2 regions and DNA fragments of the β-tubulin, adenosine triphosphatase and translation elongation factor 1 genes were explored in representive isolates from five virulence groups. Random amplified polymorphic DNA (RAPD) amplifications were also performed for each isolate using 19 random primers. Four of these primers revealed polymorphism among isolates and repetitive and discriminative bands were used to build an unweighted pair group with arithmetic mean tree. However, RAPD clustering showed low stability, and no correlation between RAPD and virulence groups was observed, possibly indicating high levels of sexual recombination.     

棉花黄萎病菌致病力测定及评价方法研究

 棉花黄萎病菌致病力测定及评价是抗病育种及病害综合防治的基础。本文以我国三大棉区的167个黄萎菌菌株为对象,研究致病力测定及评价中不同批次之间病情指数的校准及致病力类型划分标准等关键技术环节。结果表明,以中等致病力类型菌株Vd076为校正菌株,以感病性稳定的冀棉11为校正鉴别寄主,以校正菌株在校正鉴别寄主上病情指数达到50.0±5.0时的调查结果进行各菌株的致病力评价,可获得较好的校正效果,使不同批次试验数据具有可比性;依据聚类分析结果,制定了致病力类型划分标准,强、中、弱3种致病力类型的平均校正病指分别为﹥40.0、20.1~40.0和20.0;通过对不同鉴别寄主组合的分析,推荐陆地棉中棉所41号、豫棉21、鲁棉研28、中棉所35号、中棉所8号、冀棉11作为鉴别寄主。该研究为棉花黄萎病菌致病力变异等相关研究工作提供了技术支撑。     

Evidence of Two Formae Speciales in Venturia inaequalis, Responsible for Apple and Pyracantha Scab

ABSTRACT Genetic relationships, mating crosses, and host specificity of Venturia inaequalis isolates from Malus spp. and of Spilocaea pyracanthae isolates from Pyracantha spp. were evaluated. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS1-5.8S to ITS2) revealed a total similarity between these two putative species. ITS restriction fragment length polymorphism carried out with five restriction enzymes on a collection of 28 isolates confirmed a lack of diversity in this region between and within these two populations. Additional isolates from three related species (V. pirina, V. nashicola, and S. eriobotryae) were divided into two distinct monophyletic groups in a phylogenetic tree using ITS sequence comparison. These groups were related to their anamorph (i.e., Spilocaea or Fusicladium). When inoculated on their host of origin, fields isolates caused typical symptoms of scab disease, and a host specificity was demonstrated by cross pathogenicity of isolates from Malus x domestica and Pyracantha spp. Mating on dried leaves in vitro between one isolate of each putative species led to production of numerous perithecia. Ninety-six sporulating monoascosporic progenies were isolated from this cross. Based on these genetic and pathogenic data, we proposed that pathogens responsible for scab on Malus spp. and Pyracantha spp. are considered as two formae speciales belonging to V. inaequalis.     

Genetic variability and virulence among Verticillium albo-atrum isolates from hop

Verticillium wilt, caused by Verticillium albo-atrum or V. dahliae, is an important disease of many worldwide crop species. In Europe, V. albo-atrum isolates infecting hop express different levels of virulence, inducing mild or lethal disease syndromes, and it is therefore an attractive model for studying the virulence of this pathogen. In this work, eleven amplified fragment length polymorphism (AFLP) primer combinations were used to analyze genetic variability among 55 V. albo-atrum hop isolates from four European hop growing regions, as well as isolates from other hosts and V. dahliae isolates. Cluster analysis divided V. albo-atrum and V. dahliae isolates into two well-separated groups. Within the V. dahliae cluster, isolates were separated without host specific grouping, although no host adapted isolates were included. In V. albo-atrum, the alfalfa isolates were distinct from isolates of other hosts, where a high association with virulence was observed in hop and tomato isolates. All lethal hop isolates were genetically different from mild hop isolates. The lethal hop isolates from England and Slovenia expressed the same virulence phenotype, although they showed a different AFLP pattern. The mild hop isolates formed two subgroups, to which isolates clustered irrespective of geographical location. These data suggest multiple origins of V. albo-atrum hop isolates, and the possible appearance of new virulent isolates in the future in other hop growing regions.     

Sequences of the internal transcribed spacers of the ribosomal RNA genes and relationships between isolates of Verticillium alboatrum and V. dahliae

The sequences of the internal transcribed spacers (ITS) 1 and 2 of the rRNA genes of 38 Verticillium alboatrum and V. dahliae isolates have been determined. The isolates represented RFLP groups of both species, V. dahliae vegetative compatibility groups and pathotypes, and V. alboatrum 'group 2'(the majority of the V. alboatrum isolates studied were in'group 1'). The ITS sequences of a single V. tricorpus isolate were also determined. RFLP groups L and NL of V. atboatrum were distinct, with a maximum of three nucleotide differences between any isolate. Most haploid V. dahliae isolates were identical and separated from most L/NL V. alboatrum isolates by differences at five or six positions. A few haploid V. dahtiae isolates, not forming any obvious grouping, differed from the majority, each at a single position. Diploid isolates of V. dahtiae were identical but only one nucleotide difference separated them from some RFLP group L V. alboatrum isolates. At least six differences separated the diploid isolates of V. dahliae from the haploid ones. The'group 2' V. atboatrum isolates were more distantly related to'group 1'isolates (at least 17 positions different) than was V. tricorpus. The possibility of defining specific primers for use in PCR to discriminate species and subspecific groups is discussed.     

Genetic variability of Colletotrichum lindemuthianum in wild populations of common bean

The genetic structure of wild populations of Colletotrichum lindemuthianum was evaluated for virulence and molecular markers. Forty-five isolates were collected from five wild common bean populations located in their South-Andean centre of origin. The five pathogen populations were monomorphic in their ITS regions, but 45 polymorphic markers were identified using RAPDs. Polymorphism for virulence was also observed; 15 pathotypes were characterized on an international set of 12 differentials. A molecular variance analysis ( AMOVA ) showed that a very large part of the total genetic variation was within populations. Statistical analysis showed that there was a weak though significant differentiation among the five populations for the RAPD and virulence markers. A positive and significant correlation was found between geographic distance and the distances from RAPD and virulence data, suggesting migration between adjacent populations along the Argentinian transect. Our results suggest that the Andean wild isolates of C. lindemuthianum do not reflect all the putative diversity found in the isolates from cultivated common bean.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Host Range Specificity in Verticillium dahliae

ABSTRACT Verticillium dahliae isolates from artichoke, bell pepper, cabbage, cauliflower, chili pepper, cotton, eggplant, lettuce, mint, potato, strawberry, tomato, and watermelon and V. albo-atrum from alfalfa were evaluated for their pathogenicity on all 14 hosts. One-month-old seedlings were inoculated with a spore suspension of about 10(7) conidia per ml using a root-dip technique and incubated in the greenhouse. Disease incidence and severity, plant height, and root and shoot dry weights were recorded 6 weeks after inoculation. Bell pepper, cabbage, cauliflower, cotton, eggplant, and mint isolates exhibited host specificity and differential pathogenicity on other hosts, whereas isolates from artichoke, lettuce, potato, strawberry, tomato, and watermelon did not. Bell pepper was resistant to all Verticillium isolates except isolates from bell pepper and eggplant. Thus, host specificity exists in some isolates of V. dahliae. The same isolates were characterized for vegetative compatibility groups (VCGs) through complementation of nitrate nonutilizing (nit) mutants. Cabbage and cauliflower isolates did not produce nit mutants. The isolate from cotton belonged to VCG 1; isolates from bell pepper, eggplant, potato, and tomato, to VCG 4; and the remaining isolates, to VCG 2. These isolates were also analyzed using the random amplified polymorphic DNA (RAPD) method. Forty random primers were screened, and eighteen of them amplified DNA from Verticillium. Based on RAPD banding patterns, cabbage and cauliflower isolates formed a unique group, distinct from other V. dahliae and V. albo-atrum groups. Minor genetic variations were observed among V. dahliae isolates from other hosts, regardless of whether they were host specific or not. There was no correlation among pathogenicity, VCGs, and RAPD banding patterns. Even though the isolates belonged to different VCGs, they shared similar RAPD profiles. These results suggest that management of Verticillium wilt in some crops through crop rotation is a distinct possibility.     

Molecular characterization of Endothia gyrosa isolates from Eucalyptus in South Africa and Australia

Endothia gyrosa is a canker pathogen best known as the causal agent of pin oak blight in North America, and causes cankers on other woody hosts such as Castanea spp. and Liquidambar spp. In South Africa, Australia and Tasmania, a fungus identified as E. gyrosa has been recorded on Eucalyptus spp. Some morphological differences exist between the North American fungus and the isolates from Eucalyptus . Phylogenetic relationships between E. gyrosa from North America and E. gyrosa from South Africa and Australia, as well as that of the related fungi Cryphonectria parasitica and C. cubensis , were studied using PCR-based restriction fragment length polymorphism (RFLP) and sequences of the internal transcribed spacer (ITS) region of the rRNA operon. Endothia gyrosa isolates from South Africa produced the same RFLP banding patterns as those from Australia, which differed markedly from North American isolates of E. gyrosa . In a phylogram based on the DNA sequences, the Australian and South African isolates of E. gyrosa resided in a single, well resolved clade, distinct from North American isolates. Isolates of C. parasitica grouped in the same clade as the South African and Australian isolates of E. gyrosa , but C. cubensis was distantly related to them. The molecular data suggest that the E. gyrosa isolates from South Africa and Australia represent a distinct taxon, and probably belong to the genus Cryphonectria .