基于SRAP标记的黄瓜霜霉菌遗传多样性分析

本研究对来自黑龙江、辽宁、河北、山东、江苏5个省12个黄瓜主产区的77个黄瓜霜霉菌菌株,采用SRAP分子标记进行了遗传多样性分析。从35对SRAP引物中筛选出10对引物,共产生9 554条扩增条带,其中9 132条表现多态性,占95.6%。基于SRAP分子标记,77个黄瓜霜霉菌菌株的遗传距离为0.60~1.00,表明黄瓜霜霉菌具有丰富的遗传多样性。通过聚类分析,77个黄瓜霜霉菌菌株聚类为8个类群,并且来自相同地区的菌株大多数聚集于同一类群中,表明黄瓜霜霉菌群体的遗传多样性与其地理来源密切相关。     

Screening of wildcucumis species against downy mildew (Pseudoperonospora cubensis) isolates from cucumbers

Under controlled inoculation, a set of 56 accessions belonging to 19 wild species of the genusCucumis was studied for resistance to seven isolates of cucurbit downy mildew (Pseudoperonospora cubensis (Berk. et Curt.) Rostow.) from cucumber. No resistance toP. cubensis was detected in theseCucumis accessions. In three host accession/pathogen isolate combinations, limited sporulation was observed. Nine newCucumis species are described as hosts forP. cubensis: C. africanus, C. ficifolius, C. figarei, C. meeusii, C. metuliferus, C. myriocarpus, C. leptodermis, C. sagittatus andC. zeyheri. Results are discussed in relation to the origin and evolution ofCucumis species.     

Occurrence of metalaxyl-resistant isolates of Pseudoperonospora cubensis in Israel: a 5-year survey1

The occurrence of metalaxyl-resistant isolates of Pseudoperonospora cubensis, the causal agent of downy mildew in cucurbits, was surveyed during 1980–1984 subsequent to their first appearance in December 1979. Metalaxyl use was abandoned in mid-1981 and resistant isolates diminished in late 1981. Metalaxyl use was renewed in 1982 and a gradual increase in frequency of resistant (R) strains was detected. In 1983, 31 out of 40, and in 1984, 43 out of 47 isolates tested were highly resistant to the fungicide with 65 and 91% of them, respectively, collected from plastic houses or fields not treated with metalaxyl. The survey indicates a rapid build-up of R strains of the fungus and the need for alternative chemicals for combating downy mildew in cucurbits.     

Genetic and pathogenic relatedness of Pseudoperonospora cubensis and P. humuli

The most economically important plant pathogens in the genus Pseudoperonospora (family Peronosporaceae) are Pseudoperonospora cubensis and P. humuli, causal agents of downy mildew on cucurbits and hop, respectively. Recently, P. humuli was reduced to a taxonomic synonym of P. cubensis based on internal transcribed spacer (ITS) sequence data and morphological characteristics. Nomenclature has many practical implications for pathogen identification and regulatory considerations; therefore, further clarification of the genetic and pathogenic relatedness of these organisms is needed. Phylogenetic analyses were conducted considering two nuclear and three mitochondrial loci for 21 isolates of P. cubensis and 14 isolates of P. humuli, and all published ITS sequences of the pathogens in GenBank. There was a consistent separation of the majority of the P. humuli isolates and the P. cubensis isolates in nuclear, mitochondrial, and ITS phylogenetic analyses, with the exception of isolates of P. humuli from Humulus japonicus from Korea. The P. cubensis isolates appeared to contain the P. humuli cluster, which may indicate that P. humuli descended from P. cubensis. Host-specificity experiments were conducted with two reportedly universally susceptible hosts of P. cubensis and two hop cultivars highly susceptible to P. humuli. P. cubensis consistently infected the hop cultivars at very low rates, and sporangiophores invariably emerged from necrotic or chlorotic hypersensitive-like lesions. Only a single sporangiophore of P. humuli was observed on a cucurbit plant during the course of the studies. Together, molecular data and host specificity indicate that there are biologically relevant characteristics that differentiate P. cubensis and P. humuli that may be obfuscated if P. humuli were reduced to a taxonomic synonym of P. cubensis. Thus, we recommend retaining the two species names P. cubensis and P. humuli until the species boundaries can be resolved unambiguously.     

Molecular comparisons amongst wheat bymovirus isolates from Asia, North America and Europe

To study the variation between wheat bymovirus isolates and to resolve uncertainties about the identity of the virus in some countries, leaves of infected plants were obtained from nine sites in China and from one each in Italy, Germany, USA and Canada. The German isolate was obtained from rye and the Canadian isolate was the type strain of wheat spindle streak mosaic virus (WSSMV). In RT-PCR, using primers designed from a partial sequence of a French isolate (tentatively described as WSSMV), genome fragments were obtained from the Italian and the French isolates but not from the Chinese ones. Conversely, products were consistently obtained from the Chinese isolates, but not from the Italian or French ones, when primers were designed from the sequence of a Japanese isolate of wheat yellow mosaic virus (WYMV). Nucleotide sequences were obtained from regions at or near the 3'-terminus of RNA1 of six Chinese isolates and the four from Europe and North America, usually including the coat protein. Nucleotide and amino acid sequence comparisons demonstrated that the European and North American isolates were extremely similar and were therefore WSSMV, while the Chinese isolates were close to the Japanese isolate and were thus WYMV.     

Structure and temporal shifts in virulence of Pseudoperonospora cubensis populations in the Czech Republic

The structure and temporal dynamics of the virulence of Pseudoperonospora cubensis (causal agent of cucurbit downy mildew) were studied in pathogen populations in the Czech Republic from 2001 to 2010. A total of 398 P. cubensis isolates collected from Cucumis (Cm.) sativus, Cm. melo, Cucurbita (Cr.) maxima, Cr. pepo, Cr. moschata and Citrullus lanatus were analysed for variation in virulence (pathotypes). Virulence was evaluated on a differential set of 12 genotypes of cucurbitaceous plants. All isolates of P. cubensis were characterized by their level of virulence (classified according the number of virulence factors, VF; low VF = 1–4, medium VF = 5–8, high VF = 9–12): high (75%), medium (24%) and low (1%). The structure and dynamics of virulence in the pathogen populations were expressed by pathotypes using tetrad numerical codes and a total of 67 different pathotypes of P. cubensis were determined. The most susceptible group of differentials was Cucumis spp., while the lowest frequency of virulence was recorded on Cr. pepo ssp. pepo, Ci. lanatus and Luffa cylindrica. A high proportion (c. 90%) of isolates was able to infect cucurbit species Benincasa hispida and Lagenaria siceraria, which are not commonly cultivated in the Czech Republic or elsewhere in central Europe. In the recent pathogen populations (2008–2010) there was prevailing frequency (70–100%) of isolates with high numbers (9–12) of virulence factors. ‘Super pathotype’ 15.15.15 was often observed in the study within the pathogen populations and was one of the four most frequently recorded pathotypes. Pseudoperonospora cubensis populations shifted to a higher virulence over time. From 2009 the pathogen population changed dramatically and new pathotypes appeared able to establish natural and serious infection of Cucurbita spp. and Ci. lanatus, which was not observed in 2001–2008. Generally, virulence structure and dynamics of P. cubensis populations are extremely variable in the Czech Republic.     

Molecular polymorphisms between populations of Pseudoperonospora cubensis from Greece and the Czech Republic and the phytopathological and phylogenetic implications

Molecular genetic polymorphisms within Pseudoperonospora cubensis isolates of different geographic origins were investigated to establish their phylogenetic relationships and to assess genetic variability between two distant pathogen populations. Thirty isolates originating from Greece (Crete; 15), the Czech Republic (13), the Netherlands (one) and France (one) were analysed by AFLP fingerprinting and ITS 5·8S rDNA sequence analysis. All isolates were obtained from cucumber ( Cucumis sativus ) plants showing typical downy mildew symptoms. Four AFLP primer combinations produced a total of 288 high-quality bands of which 45% were polymorphic, allowing isolates to be grouped into two separate clusters: one including the Central European (Czech Republic) and Western European (the Netherlands and France) and the other the Cretan isolates. Within each AFLP cluster there was some variation, which could be accounted for by geographic origin or pathogenicity. The two populations (Cretan vs. Central and Western European) exhibited a high degree of genetic isolation. There was no clear AFLP grouping of isolates on the basis of pathotypes. No variability was detected in the ITS1 region; however, ITS2 sequences grouped P. cubensis isolates in two subclusters: one with all investigated European and the other with Asian isolates. The two subclusters formed a larger P. cubensis cluster which was differentiated from the cluster of the neighbouring species Pseudoperonospora humuli . Within P. cubensis , AFLP fingerprints could resolve genetically isolated populations, even on small or medium geographic scales, while ITS2 sequence showed differences on a global scale, being only suitable for phylogenetic analyses.     

Relationship between disease severity and escape of Pseudoperonospora cubensis sporangia from a cucumber canopy during downy mildew epidemics

Fundamental to the development of models to predict the spread of cucurbit downy mildew is the ability to determine the escape of Pseudoperonospora cubensis sporangia from infected fields. Aerial concentrations of sporangia, C (sporangia m^?3), were monitored using Rotorod samplers deployed at 0·5 to 3·0 m above a naturally infected cucumber canopy in two sites in central and eastern North Carolina in 2011, where disease severity ranged from 1 to 40%. Standing crop of sporangia was assessed each morning at 07·00 h EDT and ranged from 320 to 16 170 sporangia m^?2. Disease severity and height above the canopy significantly (P < 0·0001) affected C with mean concentration (C_m) being high at moderate disease. Values of C_m decreased rapidly with canopy height and at a height of 2·0 m, C_m was only 7% of values measured at 0·5 m when disease was moderate. Daily total flux (F_D) was dependent on disease severity and ranged from 5·9 to 2242·3 sporangia m^?2. The fraction of available sporangia that escaped the canopy increased from 0·028 to 0·171 as average wind speed above the canopy for periods of high C increased from 1·7 to 3·6 m s^?1. Variations of C_m and F_D with increasing disease were well described (P < 0·0001) by a log‐normal model with 15% as the threshold above which C_m and F_D decreased as disease severity increased. These results indicate that disease severity should be used to adjust sporangia escape in spore transport simulation models that are used to predict the risk of spread of cucurbit downy mildew.     

四川省不同地域的核盘菌遗传多样性分析

 核盘菌(Sclerotinia sclerotiorum)属于世界性分布的植物病原真菌,可以危害油菜等多种经济作物。研究不同地域核盘菌的遗传多样性对了解核盘菌的遗传演化过程和指导病害防控具有重要意义。实验采用序列相关扩增多态性(sequence-related amplified polymorphism,SRAP)标记对四川省17个不同地理来源的66株核盘菌菌株的遗传多样性进行了分析。10对检测引物共获得129个位点,其中123个为多态位点,占95.35%。UPGMA聚类结果显示,在相似性系数为0.7时,66个核盘菌菌株分为5类(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ),分别包含60、2、2、1和1个菌株。在相似性系数为0.74时,第Ⅰ类又可分为3个亚类(Ⅰ-1、Ⅰ-2、Ⅰ-3),分别包含21、37和2个菌株。聚类及组成分分析结果显示,四川省各地区的核盘菌菌株具有较高的遗传多样性,但其遗传变异与菌株地理来源无明显相关性。     

Characterization of a Fusarium poae world-wide collection by using molecular markers

Fusarium poae has been considered as a minor species among those that cause Fusarium Head Blight (FHB) disease but in recent years several researchers have documented a high frequency of occurrence of this species. In this study, a total of 173 F. poae isolates from Argentina, Belgium, Canada, England, Finland, France, Germany, Hungary, Italy, Luxembourg, Poland, Switzerland and Uruguay were evaluated by using inter simple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP) to evaluate genetic variability within F. poae and to amplify MAT idiomorphs as a possible mechanism that could explain part of the variability found in this species. The molecular analysis obtained from both molecular markers showed a high intraspecific variability. However, a partial clustering between F. poae isolates and their geographic origin was obtained by ISSR markers while AFLP showed isolates from different geographic locations distributed throughout the dendrogram. Moreover, ISSR grouped all the F. poae isolates into a different cluster from the F. langsethiae and F. sporotrichioides isolates used as outgroups compared with the dendrogram obtained using AFLP markers. Analysis of molecular variance (AMOVA) indicated a high genetic variability in the F. poae collection, with most of the genetic variability resulting from differences within, rather than between, American and European populations by using both molecular markers. Regarding MAT idiomorphs, for most F. poae isolates both MAT-1 and MAT-2 were present from each isolate.     

Coincidence of virulence shifts and population genetic changes of Pseudoperonospora cubensis in the Czech Republic

Pseudoperonospora cubensis is an oomycete pathogen causing downy mildew disease on a variety of Cucurbitaceae, and has recently re‐emerged as a destructive disease on crops in this family, mainly on cucumber and squash. Multilocus sequence analysis (MLSA) of four mitochondrial and two nuclear DNA regions was used to detect changes in the genetic structure of P. cubensis populations occurring in the Czech Republic that might be associated with recently reported shifts in virulence. The analysed sample set contains 67 P. cubensis isolates collected from 1995 to 2012 in the Czech Republic and some other European countries. Sequence analyses revealed differences and changes in the genetic backgrounds of P. cubensis isolates. While all isolates sampled before 2009 exhibited the genotype of the subspecies of Clade II and were collected from cucumber, all samples collected from other hosts belonged to Clade I (P. cubensis sensu stricto) or were sampled from 2009 onwards. In addition, 67·16% of all post‐2009 isolates from Clade II had two heterozygous positions in their nrITS sequence, which suggests sexual reproduction and/or a mutational origin. Thus, the results indicate that, apart from the rise in prevalence of Clade I, the change in the genetic structure of P. cubensis populations may be linked with a hybridization or, less likely, a mutation event that rendered strains able to infect a broader spectrum of host species.     

Biological properties of the novel fungicide cyazofamid against Phytophthora infestans on tomato and Pseudoperonospora cubensis on cucumber

Cyazofamid (ISO proposed common name), 4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonamide is a novel fungicide exhibiting specific activity against diseases caused by Oomycetes. In tests, cyazofamid at 0.4-1.6 mg litre-1 exhibited excellent preventative activity against Phytophthora infestans on tomato and Pseudoperonospora cubensis on cucumber. Minimum inhibitory concentrations of cyazofamid against both diseases were over 63 times lower than those of mancozeb and at least 16 times lower than those of metalaxyl. Cyazofamid at 1.6-25 mg litre-1 exhibited not only preventative activity, but also stable residual activity and rainfastness. Cyazofamid at 6.3 mg litre-1 reduced zoosporangia formation of P infestans and P cubensis on host plants by 100 and 94% respectively. Cyazofamid also exhibited translaminar and curative activity. Cyazofamid has a new mode of action for fungicides and exhibits no cross-resistance with other currently registered and commonly used fungicides. These properties lead to a high level control by cyazofamid in field.     

陕西省苹果树腐烂病菌基因多态性的ISSR分析

为了从分子水平上揭示苹果树腐烂病菌的群体遗传多样性,采用正交设计对ISSR-PCR体系进行了4因素3水平的筛选,并从47条ISSR引物中筛选出11条多态性较好的引物。对供试的87个分离株进行扩增的结果显示,11条引物在129个位点扩增出稳定的条带,其中多态性位点119个,多态性位点率为92.25%。POPGENE分析显示,病菌种群的遗传多样性和基因多态性丰富,群体间的遗传分化系数(Gst)为0.109,群体内为0.891,群体内多样性大于群体间多样性。两个地理种群间的居群每代迁移数(Nm)为2.046,两者之间存在广泛的基因交流。在遗传相似系数为0.88时,可将21个自然种群划分为9个不同的类群,表明陕西省苹果树腐烂病菌的各个自然种群之间的遗传亲缘关系与其地理来源之间无明显的相关性。     

Characterization of Glomerella cingulata isolates from almond fruit using VCG,molecular, fungicide sensitivity and pathogenicity tests 1

Almond anthracnose caused by Glomerella cingulata is a major disease of this crop in Israel. The pathogen infects young fruit resulting in fruit rot. Leaf wilting and shoot dieback accompany fruit rot, even though the pathogen cannot be isolated from leaves or twigs. Isolates of G. cingulata from diseased almond fruit were compared using vegetative compatibility grouping (VCG), molecular methods, fungicide sensitivity and pathogenicity assays in order to determine the genetic diversity and host specificity among different populations. Polymerase chain reaction amplification of genomic DNA, using four primers produced uniform banding patterns for all the almond isolates from different geographic locations in Israel. HaeIII digestion patterns of A + T-rich DNA, and Southern hybridization of the repetitive nuclear DNA element (GcpR1) to PstI-digested genomic DNA of almond isolates also revealed no polymorphism. Chlorate-resistant nitrate-nonutilizing (nit) mutants were generated and used in heterokaryon tests. Complementary heterokaryons formed between the mutants of different isolates indicated a single VCG. Isolates of G. cingulata from almond had optimal growth temperatures of 20–22°C as opposed to 26–28°C for avocado isolates. In addition, almond isolates of G. cingulata are insensitive to benzimidazole fungicides in contrast to sensitivity of isolates from avocado. In artificial inoculations, almond isolates infected almond, avocado, apple, mango and nectarine fruit at a slower rate than G. cingulata isolates from avocado, apple and mango. Only the anamorph Colletotrichum gloeosporioides has been detected on almond in Israel, whereas isolates of G. cingulata from other hosts produce ascocarps.     

Characterization of Pectobacterium species from Iran using biochemical and molecular methods

Characteristics of forty strains from macerated potato tubers and water-soaked lesions of some ornamental plants were studied in north parts of Iran. The causal organisms isolated from infected tissues were identified as Pectobacterium spp. based on their physiological and biochemical assays and confirmed by species and subspecies specific PCR and RFLP analysis of 16S–23S intergenic transcribed spacer region. Artificial inoculation of isolates to their related hosts generated the same symptoms on potato and ornamental plants, from which the same bacteria were isolated and identified. We detected two groups of atypical isolates in this study. The first group from potato classified as Pectobacterium carotovorum subsp. carotovorum by phenotypic tests but was unable to elicit HR on tobacco leaves, to grow at 37°C and to amplify the pel gene relevant to this subspecies. The second one from ornamental plants which was again characterized as Pectobacterium carotovorum subsp. carotovorum in biochemical assays, produced a unique ITS-RFLP profile different from all of known Pectobacterium species and subspecies. Our findings based on phylogenetic analysis using concatenated partial sequences of housekeeping genes mdh and gapA, indicated the occurrence of P. wasabiae as a novel species in potato storage in Iran. Furthermore we detected a distinct clade of Pectobacterium spp. from some ornamental plants including Schlumbergera bridgesii, Syngonium podophyllum and Iris spp.     

黄瓜-酸黄瓜渐渗系霜霉病抗性及感病前后几种酶活性的变化

 Using resistant and susceptible cultivars as controls, the resistant evaluation to Pseudoperonospora cubensis was carried out in 12 introgression lines from wide cross between cucumber(Cucumis sativus L.) and sour cucumber(Cucumis hystrix Chakr.). Three enzyme activities including POD, SOD and PAL were analyzed before and 7 d after inoculation, and the POD isozyme was detected by polyacrylamide electrophoresis. The inoculation results showed that, of the 12 accessions, 3 were identified as high resistant, 5 were moderately resistant and 4 were moderately susceptible to downy mildew. The enzyme activities of POD, SOD and PAL were greatly increased in resistant accessions after inoculation. PAL enzyme activities showed close correlation with disease rating before or after inoculation, which implicated that PAL enzyme activity might be used to estimate the resistance to downy mildew. POD isozyme electrophoresis showed that the number and intensity for the bands of resistant lines were significantly increased more than those of susceptible lines after inoculation.     

An assessment of genetic diversity within and between populations of Phalaris minor using ISSR markers

Intra- and inter-sample similarities for four populations of the annual grass weed Phalaris minor from Haryana state, India, were examined using inter-simple sequence repeat (ISSR) DNA markers. Levels of polymorphism within and between populations were low in comparison with values reported from other grassy weed species. Analysis of inter-population similarities allowed a partial differentiation of the four populations and of pairs of populations classified by cropping system. Analysis of the intra-population similarity data showed a weak but consistent and statistically significant negative correlation between the molecular similarity of seedlings and the physical distance between their mother plants over distances up to 40 m (the maximum separation tested) in all four populations. The consistency of the observed relationship between molecular similarity and physical separation, and the differences in cultivation practices at the four sites, suggested that the relationship may be a result of localized out-crossing, rather than an effect of localized seed rain. The results of the analyses are discussed in relation to the potential for evolution of multiple traits in the weed in response to changes in the wheat production system in the region.     

Effectiveness of plant extracts of Paeonia suffruticosa and Hedera helix against diseases caused by Phytophthora infestans in tomato and Pseudoperonospora cubensis in cucumber

Journal of Plant Diseases and Protection - The effects of ethanolic extracts (20 % per fresh weight (Fw) or 6 % per dry weight) of 20 different plant species were tested against late blight...     

Comparative study of morphological,cultural and molecular markers for the characterization ofPseudocercosporella herpotrichoides isolates

Nirenberg's classification system and the polymerase chain reaction (PCR) combined with restriction enzyme digestion of an amplified ribosomal DNA fragment, were compared for the characterization of sixty isolates ofPseudocercosporella herpotrichoides, from various geographical areas and with differing fungicide sensitivity. With Nirenberg's system, it was possible to identify most isolates asP. herpotrichoides var.herpotrichoides orP. herpotrichoides var.acuformis. However, identification was slow and sometimes inconclusive as overlap occurred between the two varieties for all criteria examined. Molecular markers identified two distinct types among the isolates tested and generally good correlation was found between the PCR-based assay and Nirenberg's system, but the molecular assay was more accurate and faster.     

Characterization of Macrophomina phaseolina, the charcoal rot pathogen of cluster bean, using conventional techniques and PCR-based molecular markers

Phenotypic and genetic diversity of 59 Macrophomina phaseolina isolates collected from various host species growing in or near cluster bean ( Cyamopsis tetragonoloba ) fields in four states of north and north-west India were characterized using RAPD and PCR–RFLPs of the ITS region. These isolates, and 11 from various hosts from culture collections, were classified into three mycelial phenotypes: dense, feathery and restricted, based on variable growth patterns on nutrient agar containing 120 m m chlorate. Pathogenicity of isolates was evaluated by measuring the length of stem lesions 21 days post-inoculation on the susceptible cluster bean genotype FS 277. Isolates showed considerable variation in aggressiveness, with the isolates from cluster bean with dense chlorate phenotype producing relatively higher lesion lengths on cluster bean plants. The results of the RAPD assay clearly distinguished the isolates on the basis of chlorate phenotype and host origin. Isolates from a single host were generally similar to each other, but differed distinctly from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. A high degree of polymorphism in restriction patterns of the ITS region, including part of 25S rDNA, has been reported for the first time in the charcoal rot fungus.