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1.
The Oomycete Plasmopara viticola is the causal organism of downy mildew on grapevine (Vitis spp.). In order to set up the techniques for investigating downy mildew disease dynamics and genetic structure, co-dominant, neutral, highly reproducible and polymorphic microsatellite markers for P. viticola were developed. Five markers, two with a (TC)n repeat (loci BER and ISA), two with a (TC)n(AC)n repeat (loci CES and REX) and one with a (CT)n(CTAT)n repeat (locus GOB), were selected. Simple sequence repeat (SSR) markers revealed different degrees of polymorphism within 190 oil spots (disease symptoms) collected from an infected Italian vineyard. The most polymorphic SSR marker GOB showed 43 alleles (Nei's expected gene diversity He = 0.89) while CES, ISA, BER and REX showed 14 (He = 0.71), 4 (He = 0.57), 3 (He = 0.24) and 1 allele (He = 0), respectively. A high throughput DNA extraction method, that allowed molecular analysis of this obligate pathogen directly in the host without any isolation procedure, was developed. The quality and quantity of oil spots did not influence the SSR analysis. Amplified SSR loci were separated by electrophoresis on a Beckman–Coulter 2000XL sequencer and automatically analysed. The objective of this study was to develop molecular biological tools and methods that allow high throughput analysis of the downy mildew populations.  相似文献   

2.
Members of the Fusarium graminearum species complex (FGSC) cause Fusarium head blight in small cereal grains all over the world. To determine the species and trichothecene chemotype composition and population structure of FGSC in Jiangsu and Anhui provinces, an area where epidemics occur regularly, 891 isolates were collected in two consecutive years (2011 and 2012) and characterized with species- and chemotype-specific polymerase chain reaction. Of the 891 isolates typed, 83 were F. graminearum sensu stricto (s. str.) and 808 were F. asiaticum. All 83 F. graminearum s. str. isolates were of a 3ADON (26.51 %) or 15ADON (73.49 %) type, while F. asiaticum isolates included 696 3ADON producers, 46 15ADON producers, and 66 NIV producers. Eight variable number tandem repeat (VNTR) markers were tested on a representative 384 F. asiaticum isolates from 55 sampling sites. VNTR analysis showed high gene diversity and genotypic diversity but low linkage disequilibrium in both populations Fg2011 and Fg2012 grouped based on the year of collection. Low genetic differentiation (F ST ? =?0.026) and high gene flow (N m ?=?15.13) was observed between the two populations and among subpopulations within the same population (N m ?=?3.53 to 48.37), indicating that few influence of temporal and spatial variations on population differentiation in this area. Similar result was obtained from 3ADON, 15ADON and NIV populations or carbendazim resistant and sensitive populations, indicating that chemotype of Fusarium isolates and carbendazim application had minor influence on population subdivision.  相似文献   

3.
Downy mildew (Plasmopara viticola) is one of the most important diseases in grape-growing areas worldwide, including Brazil. To examine pathogen population biology and structure, P. viticola was sampled during the 2015/16 growing season from 516 lesions on nine grape cultivars in 11 locations in subtropical areas of São Paulo State, Brazil. For identification of cryptic species, a subsample of 130 isolates was subjected to cleaved amplified polymorphic sequence (CAPS) analysis, and for 91 of these isolates the ITS1 region was sequenced. These analyses suggest that the population of P. viticola in São Paulo State consists of a single cryptic species, P. viticola clade aestivalis. Seven microsatellite markers were used to determine the genetic structure of all 516 P. viticola isolates, identifying 23 alleles and 55 multilocus genotypes (MLGs). Among these MLGs, 34.5% were clonal and represented 93% of the isolates sampled. Four dominant genotypes were present in at least five different locations, corresponding to 65.7% of the isolates sampled. Genotypic diversity (Ĝ = 0.21–0.89) and clonal fraction (0.58–0.96) varied among locations (populations). Most populations showed significant deviation from Hardy–Weinberg expectations; in addition, excess of heterozygosity was verified for many loci. However, principal coordinate analysis revealed no clusters among locations and no significant isolation by distance was found, suggesting high levels of migration. The results indicate that downy mildew epidemics result from multiple clonal infections caused by a few genotypes of P. viticola, and reproduction of P. viticola in São Paulo State is predominantly asexual.  相似文献   

4.
Cymoxanil has been used for over 30 years to control grape downy mildew (Plasmopara viticola) in European vineyards, prevalently in mixture with other fungicides active on this disease. In the 1990’s cases of P. viticola resistant to cymoxanil were detected using a leaf disc assay. In this study, we establish that the presence of only 1 % of resistant isolates in a P. viticola population will allow the detection of cymoxanil resistance in the leaf disc assay. A poor correlation (R?=?0.194) was observed between the leaf disc assay and a whole- plant test for 38 P. viticola field populations collected in 2004. Over 60 % of these populations were characterized as fully sensitive in a whole-plant assay compared to 10 % in the leaf disc assay. Five P. viticola field isolates resistant to cymoxanil reverted to full sensitivity after six to nine transfers to untreated vines, indicating that cymoxanil resistance in P. viticola is unstable. Two European P. viticola populations sensitive to cymoxanil became resistant when transferred 12–14 times on vines treated with cymoxanil. In contrast, two populations originating from the USA and three monozoospore isolates from France retained full sensitivity to the fungicide after 13 cycles on cymoxanil-treated plants. Whole-plant experiments were conducted in the laboratory to compare the efficiency of spray programs to delay the development of cymoxanil resistance. Whereas the continuous use of cymoxanil alone quickly selected for resistance, the mixture of cymoxanil and folpet applied either continuously or in strict or block alternation effectively prevented the development of resistance over 10 generations of the fungus. These results demonstrate that resistance to cymoxanil in P. viticola can be managed with appropriate spray programs.  相似文献   

5.
为了解小麦全蚀病病菌的群体组成和遗传多样性,采用8对多态性较好的微卫星标记,对我国黄淮麦区的116个小麦全蚀病菌株进行了分析。8个SSR标记的平均等位基因数为4.25个,多态性信息含量的平均值为0.66。供试菌株的平均有效等位基因数和基因遗传多样性指数分别为1.46和0.27,漯河群体的遗传多样性水平最高,周口群体最低。不同群体间遗传距离均较小,为0.0199~0.1153,其中徐州和周口群体间的遗传距离相对较大,而周口和驻马店群体间的遗传距离相对较小。分子方差分析结果显示,小麦全蚀病菌群体间和群体内均存在着一定的遗传分化,群体间的遗传变异占总变异的10%,群体内遗传变异占90%。6个群体间的基因流为3.5。根据SSR多态性,对来源不同菌株的UPGMA聚类分析结果显示,小麦全蚀病菌群体结构与地理来源有一定的关系。  相似文献   

6.
The antifungal properties of two phosphonates (fosetyl-Al and a fertilizer) and acibenzolar-S-methyl (ASM) were evaluated to assess their potential for protecting grapevine leaves against grapevine mildews (Plasmopara viticola and Erysiphe necator), and to determine their effects on the development of various mildew isolates, taking into account the inter- and intra-species variability of the pathogens. The phosphonates directly and significantly inhibited the growth of these pathogens. By contrast, ASM had no direct effect on spore production and growth of P. viticola and of E. necator at 1.9 mM. Applied before inoculation, the mean EC50 of ASM was 0.50?±?0.04 mM and 1.00?±?0.07 mM for downy and powdery mildew isolates, respectively. The EC50 of the fosetyl-aluminium (FOS) was 0.50?±?0.02 mM for downy mildew and the EC50 for powdery mildew varied depending on the genetic group under consideration (0.89?±?0.32 mM for group B 3.30?±?0.46 mM for group A, respectively). The EC50 of the potassium phosphonate fertilizer (PK2) was 0.96?±?0.45 mM for downy and 6.9?±?0.76 mM for powdery mildew isolates. These compounds showed differences in their efficacy depending on the variability of mildews and could be an alternative or additional method to traditional pest management in the grapevine.  相似文献   

7.
陕西省苹果树腐烂病菌基因多态性的ISSR分析   总被引:3,自引:1,他引:2  
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。  相似文献   

8.
The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes and is one of the largest gene families in plant genomes. In this study, resistance gene analogs (RGAs) were isolated from peanuts based on the NBS domain. A full-length cDNA, PnAG3, was obtained by rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the length of PnAG3 was 1882 bp, which included a complete open reading frame of 1335 bp that encoded for the PnAG3 protein composed of 444 amino acids. Multiple analyses showed that this protein had homology with known resistance proteins, the highest being 48.01% with a resistance protein from Arachis cardenasii. The polypeptide has a typical non-TIR-NBS-LRR gene structure. Real-time fluorescence quantitative PCR analysis showed that after Aspergillus flavus infection, expression of the PnAG3 gene in J11 (A. flavus-resistant species) increased by 16.68, 11.16 and 25.96 in the seed coat, kernel and pericarp, respectively. However, it only increased 2–3 times in JH1012 (A. flavus-sensitive species). Cloning of the putative resistance gene from peanut provides a basis for studying the structure and function of peanut disease resistance-related genes and disease resistance genetic breeding in peanuts.  相似文献   

9.
为明确不同栽培模式下葡萄霜霉病菌Plasmopara viticola的遗传结构、遗传多样性及遗传分化水平,于2014-2015年定期采集露地和避雨2种栽培模式下的葡萄霜霉病菌菌株,利用6对SSR引物对该病菌基因型、遗传多样性及遗传分化进行对比分析。结果表明,露地和避雨栽培模式下葡萄霜霉病菌群体的Nei’s基因多样性指数大于0.14,香农多样性指数大于0.31,2种栽培模式下群体具有丰富的遗传多样性,但避雨栽培模式可显著降低群体等位基因数和等位基因频率。露地栽培模式下该病菌群体的流行模式呈现中等水平无性繁殖,2年初侵染和再侵染对病害流行的贡献率分别约占26.1%和73.9%;避雨栽培模式下葡萄霜霉病菌群体的流行模式则呈现高等水平无性繁殖,初侵染和再侵染对病害流行的贡献率分别约占4.3%和95.7%。卵孢子的形成对于葡萄霜霉病菌种群遗传变异和有效越冬起着关键的作用。2014-2015年露地栽培模式下葡萄霜霉病菌群体的主效流行基因型对病害流行的贡献率分别为44.5%和51.8%;而其在避雨栽培模式下葡萄霜霉病菌群体的贡献率分别可达84.2%和87.1%。同一年份的露地和避雨栽培模式下葡萄霜霉病菌群体的主效基因型种类相同,2个群体间的等位基因频率呈现显著正相关性,且二者之间存在频繁的基因交流,推测避雨栽培模式下葡萄霜霉病的初侵染源自于避雨设施附近的露地栽培病株上再侵染形成的飞散传播孢子囊。  相似文献   

10.
Fusarium culmorum is a haploid, worldwide occurring phytopathogenic fungus causing seedling blight, foot rot, and head blight of cereals and producing the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) associated with health hazards in human and animals. The fungus reproduces asexually by conidiospores, a teleomorph is not known. We analyzed for the first time naturally occurring F. culmorum populations collected randomly in the field from infected wheat heads. A total of 186 isolates, from three populations from Germany (GER), Russia (RUS), Syria (SYR), as well as an international collection (INT) for comparison, were genotyped by 10 microsatellite (SSR, single sequence repeat) markers. A high genetic diversity within the three natural populations and the INT population as well was detected. About 90 % of multi-locus haplotypes (MLH) were unique across populations. The largest part of variance (81 %) was found within populations. Accordingly, population subdivision was low, fixation indices were significant only in one out of six comparisons, while estimates of gene flow (N m ) ranged from 0.8–4.8. Linkage equilibrium was revealed by the index of multi-locus association and the quotient of observed and expected variance when two linked markers were deleted. DON and NIV chemotypes grouped closely together in a principle coordinate analysis. SYR isolates were partly separated from GER and RUS populations. All population-genetic parameters were in a similar range compared to those for the sexually propagating species F. graminearum. In conclusion, results support the hypothesis of a recombining structure in F. culmorum as revealed by the high genetic variation within populations, a low fixation index and low gametic phase disequilibrium within populations.  相似文献   

11.
为研究苹果小吉丁虫Agrilus mali Matsumura的种群遗传结构,采用磁珠富集法以生物素标记探针(AC)_(12)和(AG)_(12)构建苹果小吉丁虫微卫星文库,根据阳性克隆测序获得的微卫星侧翼序列设计引物,筛选和开发苹果小吉丁虫多态性微卫星标记,并利用开发的微卫星标记对其不同地理种群的遗传结构进行分析。结果显示,从微卫星文库中的300个阳性克隆中筛选得到248个含有微卫星片段的克隆子,阳性克隆率为82.7%,其中完美型微卫星131个,占52.8%;不完美型90个,占36.3%;混合型27个,占10.9%,表明磁珠富集法开发新微卫星标记的效率较高。从获得的248个苹果小吉丁虫微卫星中筛选获得7个多态性微卫星位点,这7个位点在苹果小吉丁虫8个地理种群样本中的等位基因数为10~23个,有效等位基因数为1.674~12.218个,多态信息含量为0.304~0.796。8个苹果小吉丁虫种群的观测杂合度为0.095~0.676,期望杂合度为0.469~0.755,Shannon指数为0.791~1.621,遗传距离为0.071~0.788。采自新疆维吾尔自治区的7个苹果小吉丁虫种群之间遗传相似度较高,但其与辽宁省种群的遗传相似度较低。  相似文献   

12.
Plasmopara viticola is an economically important pathogen of grapevine. Early detection of P. viticola infection can lead to improved fungicide treatment. Our study aimed to determine whether chlorophyll fluorescence (Chl-F) imaging can be used to reveal early stages of P. viticola infection under conditions similar to those occurring in commercial vineyards. Maximum (FV/FM) and effective quantum yield of photosystem II (ΦPSII) were identified as the most sensitive reporters of the infection. Heterogeneous distribution of FV/FM and ΦPSII in artificially inoculated leaves was associated with the presence of the developing mycelium 3 days before the occurrence of visible symptoms and 5 days before the release of spores. Significant changes of FV/FM and ΦPSII were spatially coincident with localised spots of inoculation across the leaf lamina. Reduction of FV/FM was restricted to the leaf area that later yielded sporulation, while the area with significantly lower ΦPSII was larger and probably reflected the leaf parts in which photosynthesis was impaired. Our results indicate that Chl-F can be used for the early detection of P. viticola infection. Because P. viticola does not expand systemically in the host tissues and the effects of infection are localised, Chl-F imaging at high resolution is necessary to reveal the disease in the field.  相似文献   

13.
During Plasmopara viticola epidemics only few genotypes produce most of the secondary lesions and dominate in the population. Selection of dominant genotypes is hypothesized to be linked to environmental conditions and can occur rapidly, particularly if there is also difference between genotypes in terms of fitness and aggressiveness. Measurements of aggressiveness components can largely determine the rate of epidemic development, although the components of aggressiveness do not take into account potential direct competition between genotypes. Differences in aggressiveness have been also reported to be greater under non-optimal conditions suggesting for genotype adaptation to different conditions. To evaluate differences in latency at non-optimal conditions, we characterized genotypes deriving from different climatic regions at three different temperatures (15, 25 and 35 °C) and we found no differences. To investigate whether other factors may impact on competition between P. viticola genotypes, we evaluated polycyclic infections of P. viticola by co-inoculating three genotypes with similar aggressiveness components in two different co-inoculation experiments and an increasing prevalence of one of the two genotypes was observed. Competition was not related to the origin of the genotype and we hypothesize that competitive selection is modulated by differences in the secretion of effector molecules which can contribute to the establishment of dominant genotypes over an epidemic season.  相似文献   

14.
A repetitive sequence-based (rep)-polymerase chain reaction (PCR) and inter-simple sequence repeat (ISSR)-PCR were used to molecular type Pseudomonas syringae pv. syringae (PSS) strains isolated from barley and wheat plants with bacterial black node symptoms grown in 22 different locations and six different seed-production districts in Japan. Eighteen genomic fingerprinting (GF) genotypes were obtained from the combined results of BOX-, REP-, and GTG5-PCR, indicating that the PSS population in Japan has high genetic diversity. The result based on logistic regression indicated that the population of GF genotype A was significantly related to a seed-producing district and that the epidemic of PSS strains in fields originated mainly from seed infection. This study will be applicable to future studies of the molecular epidemiology of bacterial plant diseases that have multiple infection routes.  相似文献   

15.
The oomycete Phytophthora colocasiae that causes taro leaf blight is the most devastating disease of taro and is widely distributed worldwide. Molecular and phenotypic techniques were employed for assessing and exploiting the genetic variability among four populations of P. colocasiae obtained from a fine spatial scale (multiple leaf blight lesions on single taro leaf). Phenotypic characters such as virulence, morphology and mating type showed no variation. ITS characterization revealed detectable polymorphism among isolates of P. colocasiae. The mean number of haplotypes (H), haplotype diversity (HD), nucleotide diversity (π), and nucleotide substitution rate (θ) among analyzed sequences were 6.75, 1.00, 0.069, and 0.088 respectively. High levels of inter and intra specific variation were detected by random amplified polymorphic DNA (RAPD) assays. Moderate genetic diversity (H?=?0.2651) was observed among populations of P. colocasiae. Analysis of molecular variance (AMOVA) confirmed that most of the genetic variability was confined to within a population (63.54 %). The coefficient of genetic differentiation among populations (G ST ) was 0.2007 and estimates of gene flow (Nm) among populations was 1.991 migrants per generation. Cluster analysis using UPGMA revealed that individuals from the same population failed to cluster in one distinct group. The results of the study reveal considerable genetic diversity among and within populations of P. colocasiae obtained from fine spatial scale. The possible mechanisms and implications of this genetic variation are discussed.  相似文献   

16.
The effectiveness of Quinone outside Inhibitor (QoI) fungicides against grape downy mildew in European vineyards has significantly decreased in the last decade. One nucleotide polymorphism, G143A in the cytochrome b gene of Plasmopara viticola, is involved in resistance to QoIs. Previous genetic examination on the mitochondrial genomes showed four major haplotypes (IR, IS, IIR, IIS) coexisting in European vineyards. A resistant allele (G143A) was present in IR and IIR haplotypes. The purpose of the present study was to estimate the diversity of the different mitochondrial haplotypes and their distribution in QoI-resistant populations before evaluating the potential cost of the resistant mutation G143A in P. viticola population. From 2000 to 2004, the frequencies of resistant isolates ranged from 0% to 23.25% with an average of 4.64 % among the populations examined. To evaluate the fitness of sensitive and resistant isolates, a comparison of different biological parameters including latent period, spore production and infection frequency was performed, enabling a fitness index (FI) to be determined. Resistant isolates exhibited greater infection frequency than sensitive isolates, whereas no significant difference was found in sporulation ability and latent period between sensitive and resistant isolates. To further investigate competitiveness among isolates, an assay including two resistant isolates in different proportion with a sensitive isolate was conducted on eight asexual growing cycles in the absence of a QoI fungicide. The competitiveness of resistant isolates varied according to their fitness parameters, suggesting that there is no noticeable cost of QoI resistance in controlled conditions in Plasmopara viticola.  相似文献   

17.
Fusarium oxysporum f. sp. melongenae (Fomg), causal agent of Fusarium wilt of eggplant, is a serious pathogen in open fields and greenhouses. Inter-simple sequence repeat (ISSR) banding profiles, sequence analyses of inter-transcribed-spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and actin (actA) DNA regions were employed in this study to determine genetic diversity and population structure of Fomg isolates obtained from Turkey. For ISSR study, (ACTG)5, (GACAC)3, (GACA)4, (GATA)4, HVH(TG)7 and (CA)8RG primers were selected from a set of 16. Discriminative ability of the primers revealed with various indices including polymorphic information content (PIC), and mean PIC value was calculated as 0.26. The ISSR data revealed 31 loci belonging to 202 Fomg isolates and 14 of them were found to be polymorphic. The isolates on neighbor joining ISSR tree were grouped into two major clusters which separated Fomg and outgroup isolates. Population structure was investigated based on bayesian modeling and results indicated five subpopulations (K = 5, ?K = 205.42). Mean genetic and geographical distances among sampling locations revealed only a weak and insignificant correlation (r = 0.583, P = 0.06). Phylogenetic analyses were carried out with ITS, TEF-1α and actA DNA regions with a selected subset of 30 Fomg, along with one non-host and one outgroup isolates. Since ITS region were not able to provide a meaningful separation, TEF-1α and actA sequences of each organism were concatenated individually to build a dendrogram. The clustering tree successfully separated the Fomg, non-host and outgroup isolates in which all Fomg were located on the same branch, forming a monophyletic group in the dendrogram.  相似文献   

18.
A method for evaluating the potential threat of selection for resistance to organically-based fungicides in populations of P. viticola is needed to screen a large panel of products alternative to copper in organic viticulture. Populations from an unexposed plot were compared throughout one season with a population sprayed with azoxystrobin (Quadris), reported as engendering selection pressure and resistance, and a population sprayed with an organically-based fungicide (Mycosan). The evolution of the three populations was followed with neutral specific SSR markers and with the specific marker for strobilurin resistance, as control of selection for resistant mutants. A reduction in genetic diversity of the P. viticola population was observed in the population sprayed with azoxystrobin, consistent with directional selection toward higher resistance, confirmed by an enhanced frequency of resistant mutants with respect to the unexposed population. In contrast, a higher diversity and a reduced frequency of resistant mutants were observed in the population sprayed with the organically-based fungicide. Assessing a reduction of genotypic diversity allows the detection of selection for resistance and constitutes a valid instrument for screening a large panel of products with non-specific, different and possibly indirect modes of action.  相似文献   

19.
棉铃虫对菊酯类药剂的抗性狭义遗传力   总被引:1,自引:0,他引:1  
通过单对交配设计不同的交配家系,采用半同胞法分析了棉铃虫对三氟氯氰菊酯和氰戊菊酯的抗性狭义遗传力,并对2种菊酯类药剂的抗性发展速度进行评价。结果表明,棉铃虫对三氟氯氰菊酯和氰戊菊酯的抗性狭义遗传力分别为0.2476±0.0248和0.4625±0.1578,说明棉铃虫对氰戊菊酯的抗性发展速度要快于三氟氯氰菊酯。另外,通过方差分析发现,在棉铃虫对拟除虫菊酯类药剂的抗性遗传中,母体效应对其没有影响。  相似文献   

20.
中4是中国小麦条锈菌生理小种的重要鉴别寄主之一。采用常规杂交遗传分析法和花粉母细胞染色体镜检,明确中4抗条锈病基因遗传组成,并探讨利用中国春ph1b突变体分析小麦近缘属(种)抗条锈病基因。将中4分别与中国春ph1b突变体和感病品种铭贤169杂交,对亲本及其杂交后代进行苗期抗条锈性鉴定和遗传分析,发现中4对条锈菌小种CY31和CY32的抗病性由1对显性基因控制;通过等位性分析和抗谱比较,发现中4对小种CY32的抗病基因与T. spelta album、Moro及K733中的抗条锈病基因不同,对中国春ph1b突变体×中4组合的F2代植株染色体数目及其核型变化的研究表明,F2代单株的抗条锈性与来自中间偃麦草X组染色体增加有关,并导致F2单株染色体数目发生变化,且X组染色体在F2代群体对小种CY31表现为抗病和感病植株中随机分布。  相似文献   

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