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1.
Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g–1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.  相似文献   

2.
The sensitivity of a bioassay in detecting soil inoculum of Colletotrichum coccodes and Helminthosporium solani was examined using potato minitubers and microplants. Tests were conducted on soils which were collected from fields in which the interval after a previous potato crop differed, and which were also artificially infested with conidia or microsclerotia. For C. coccodes , determining plant infection based on the occurrence of infected roots after 9–12 weeks was a sensitive method for detecting and quantifying the amount of inoculum in soil. Infestations of less than 0·4 microsclerotia per g soil were detected in artificially infested soils. A semiselective medium, developed for isolating C. gloeosporioides from pepper, detected soil infestations by C. coccodes as low as nine conidia or one microsclerotium per g soil in artificially infested soil. For H. solani , infection on minitubers was a sensitive measure, with soil inoculum of fewer than 10 conidia per g soil being detected. Soil infestation could be quantified by assessing the percentage surface area of minitubers covered by sporulating lesions, which was strongly related to the amount of soil infestation. The results of these bioassay tests were compared with published results for real-time quantitative PCR assays on the same soils. The two methods were in good agreement in artificially infested soils, but the bioassay appeared to be more sensitive with naturally infested soils.  相似文献   

3.
A specific and sensitive PCR assay for the detection of Phytophthora infestans , the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans -specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of rDNA internal transcribed spacer regions of most of the known Phytophthora species. PCR amplification of P. infestans DNA with primers INF FW2 and INF REV generated a 613 bp product, and species specificity was demonstrated against DNA from nine other Phytophthora species and seven potato-blemish pathogens. In a single-round PCR assay, 0·5 pg pure P. infestans DNA was detectable. Sensitivity was increased to 5 fg DNA in a nested PCR assay using Peronsporales-specific-primers in the first round. As few as two sporangia or four zoospores of P. infestans could be detected using the nested assay. Procedures are described for detection of P. infestans in leaves, stem and seed potato tubers before expression of symptoms. A soil assay in which 10 oospores per 0·5 g soil were detectable was developed and validated using samples of field soil. The PCR assay was used to examine the long-term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans in leaf material buried in a replicated experiment under natural field conditions. Oospores were consistently detected using the PCR assay up to 24 months (total length of the study) after burial in soil, whereas the sporangial inoculum was detected for only 12 months after burial. Sporangial inoculum was shown to be nonviable using a baiting assay, whereas leaf material containing oospores remained viable up to 24 months after burial.  相似文献   

4.
In an attempt to better understand the importance of tuber-borne inoculum in black dot development, several potato cultivars were inoculated with various Colletotrichum coccodes isolates. Symptoms developed first on underground organs (starting 2 weeks after inoculation on roots, and later on stolons and tubers) of inoculated plants; stem infections developed only after 7–10 weeks, depending on the cultivar. Infection with C. coccodes resulted in a reduction in numbers of stolons and tubers in cv. Bintje, but not in the later maturing cv. Roseval. Significant isolate by cultivar interactions were detected from the analysis of root symptoms after inoculation of three potato cultivars (Bintje, Spunta and Desiree) with five C. coccodes isolates. Such an interaction was also detected for stolon/tuber symptoms at the latest scoring date (98 days after inoculation), but not at earlier dates (58, 70 and 84 days after inoculation). These results suggest that protocols based on root colonization might be used for investigating cultivar response to black dot and pathogenicity of C. coccodes isolates, and that some specificity exists in the reaction of potato genotypes to this pathogenic fungus.  相似文献   

5.
Black dot, caused by Colletotrichum coccodes , is of potential concern to potato production in France as part of the tuber-blemishing disease complex. The lack of information about the actual distribution of the pathogen in potato-producing areas led to a survey of the occurrence of the disease. Black dot symptoms were observed on roots, stems and/or tubers of the 82 potato cultivars examined in 1994. A baiting bioassay, using cuttings of potato cultivars Bintje and Urgenta, revealed the presence of the pathogen in all 37 soil samples tested, which had been collected throughout the main French potato growing areas. In vitro , growth of five C. coccodes isolates recovered from diseased potatoes grown in western and southern France was severely affected by imazalil, tolchlofos-methyl and, to a lesser extent, mancozeb and thiabendazole. Conversely, iprodione, flutolanil and pencycuron were ineffective in reducing the growth of these isolates. These data indicate that C. coccodes is widespread in French potato cropping areas, that currently popular cultivars are susceptible to the disease, and that at least some of the fungicides commonly applied to seed tubers are ineffective against the pathogen. A better diagnosis of the disease, but also the insensitivity of the pathogen to several chemicals frequently used on seed tubers for controlling black or silver scurfs, might thus provide explanations for the apparent increase in black dot occurrence in recent years.  相似文献   

6.
 马铃薯粉痂菌(Spongospora subterranea f. sp. subterranea)是引起马铃薯粉痂病的病原。本研究根据粉痂菌内部转录间隔区和线粒体DNA的保守区域,分别设计了2对适用于普通PCR的引物A5/A9、C3/C8和1对适用于荧光定量PCR的引物QF/QR,用于检测块茎和土壤样品中的粉痂菌。特异性检测结果表明:引物对A5/A9和C3/C8,以马铃薯粉痂菌DNA为模板,能分别扩增出264和367 bp大小的单一条带,而对其他非靶标DNA无扩增;引物对QF/QR对马铃薯粉痂菌有单一的熔解峰,说明三对引物特异性良好。灵敏性检测结果表明:荧光定量PCR灵敏度为13.8 fg·μL-1,是普通PCR灵敏度的1 000倍。进一步建立循环域值(Ct)与质粒DNA含量的曲线关系,获得标准曲线y=-3.893 9 x+35.228,R2 = 0.9966,呈良好线性关系。通过对不同地区采集的18份带菌种薯和18份带菌土壤进行普通PCR和荧光定量PCR检测,引物A5/A9、C3/C8和QF/QR对带菌种薯检测率均为100%,对带菌土壤的检测率分别为44.44%、66.67%和100%。本研究建立的马铃薯粉痂病菌快速检测方法,能及时、准确地检测带菌种薯和土壤,为马铃薯粉痂病的早期诊断和防治提供依据。  相似文献   

7.
建兰胶孢炭疽菌ITS序列分析及其PCR快速检测   总被引：3，自引：3，他引：0  
由胶孢炭疽菌Colletotrichum gloeosporioides引起的炭疽病是建兰的重要病害.为建立快速检测该病原菌的方法,以ITSl/ITS4为引物,对15个建兰胶孢炭疽菌的ITS进行PCR扩增及测序,将测定的序列与炭疽菌属其它种的ITS序列进行比对分析,设计特异性引物CFl/CR1,并通过常规和巢式PCR对建兰胶孢炭疽菌进行检测.结果显示,15个菌株中有13个菌株ITS序列与该菌的模式种序列相似性高达99％以上,而另外2个菌株相似性则为86％;供试菌株在系统发育树上聚为2个不同的分支;引物CFl/CR1通过常规PCR可从1 ng的建兰胶孢炭疽菌基因组DNA中扩增到目的条带,而利用引物ITSl/ITS4和CF1/CR1通过巢式PCR可从1 pg的基因组DNA中扩增到目的条带,即巢式PCR反应检测灵敏度较常规PCR至少高1 000倍.表明建立的巢式PCR法可从自然感病的建兰叶片组织中检测到胶孢炭疽菌.  相似文献   

8.
Real-time PCR assays for Colletotrichum acutatum , one of the most important pathogens of strawberry worldwide, were developed using primers designed to the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) and the β-tubulin 2 gene. Using TaqMan technology, the ITS-based assay could reliably detect as little as 50 fg genomic DNA, 100 copies of target DNA, or 25 conidia. The β-tubulin-based assay was c . 66 times less sensitive, and therefore less suitable for detection purposes. The TaqMan-ITS assay recognized all C. acutatum isolates tested from various intraspecific molecular groups, while no amplification was observed with several other Colletotrichum species or other strawberry pathogens, indicating the specificity of this assay. Detection and quantification of C. acutatum was demonstrated in artificially and naturally infected strawberry leaves. First, C. acutatum was detected in plant mixes of which only 0·001% of the tissue was infected by C. acutatum . Secondly, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allowed monitoring of growth progression of C. acutatum . This real-time PCR-mediated monitoring of the pathogen was well-correlated with microscopic data, and confirmed that leaf age may play a role in the extent of C. acutatum infection. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material.  相似文献   

9.
甘肃省马铃薯炭疽病的鉴定及室内药剂筛选   总被引：3，自引：0，他引：3  
在甘肃省马铃薯主要种植区的田间和贮藏库中分别采集不同时期发病植株和块茎,进行症状描述,并进行病原菌分离鉴定和致病性测定,以及室内药剂筛选。结果表明,该病害在茎秆上形成褐色长条斑;叶片上症状多不明显;薯块上病斑近圆形或不规则形,略下陷,逐渐褐色腐烂;在发病部位形成分生孢子盘;马铃薯炭疽病菌菌丝无色至浅褐色,有隔膜;分生孢子盘黑褐色,其上着生有分隔和顶部渐尖的刚毛;分生孢子棒状,无色;分生孢子梗无色,具分隔,紧密排列在分生孢子盘上;通过ITS序列同源性分析,与已报道的Colletotrichum coccodes相似性达100%。基于病原形态特征和ITS序列分析结果,鉴定该病原菌为球炭疽菌(Colletotrichum coccodes),其引起的病害为马铃薯炭疽病。经室内毒力测定,甲基硫菌灵、苯醚甲环唑、丙环唑.苯醚甲环唑、苯醚甲环唑.嘧菌酯、肟菌.戊唑醇、咪鲜胺锰盐和噻霉酮对马铃薯炭疽病菌的EC50小于10,理论上对该病害有较好的控制作用。  相似文献   

10.
The incidence and severity of root infection and root galling caused by Spongospora subterranea were assessed in potato plants (cv. Estima) grown under controlled environmental conditions. The effects of temperature, soil type, soil moisture regime and soil inoculum level on infection and root gall development were determined by molecular and visual methods at two plant growth stages. Root gall severity was scored at harvest, after which DNA was extracted from the roots and quantified in a real-time polymerase chain reaction (PCR) assay specific for S. subterranea . Root galling was severe at 17°C, with a disease score of 3·1 on a 0–4 scale, low (0·6) at 12°C, and did not occur at 9°C. The level of inoculum in soil, in the form of artificially added sporosori, had no effect on the incidence and severity of visual symptoms, with 21%, 41% and 33% incidence observed at 5, 15 and 50 sporosori g−1 soil, respectively. Incidence of infection, as detected by the real-time PCR assay, was greater with increasing soil inoculum concentrations, ranging from 48% at 5 sporosori g−1 to 59% (15 sporosori g−1) and 73% (50 sporosori g−1) of plants infected at maturity, but this effect was not statistically significant. No correlation was found between the occurrence of galls on roots and powdery scab on tubers of the same plants.  相似文献   

11.
The incidence of potato pathogens on healthy roots of micropropagated (MP) and seed tuber (ST) plants was examined on successive dates during the growing season in two field experiments. Microplants were grown in a glasshouse for 4–5 weeks in perlite or peal-based substrates, and exposed or not to natural inoculum before planting in the field. The seed tubers originated from stocks of visually clean or moderately blemished tubers and were surface-sterilized or not before planting. Polyscytalum pustulans and Helminthosporium solani only infected roots of ST plants and inoculated MP plants. The incidence of P. pustulans was affected by seed tuber-borne inoculum and, in I year, by the substrate. H. solani was detected infrequently on roots. Rhizoctonia solani was present at low frequencies in most root samples, and more ST than MP plant roots were colonized; there were no substrate effects. In 1 year, increased inoculum levels increased root infection, but only in MP roots. Colletotrichum coccodes occurred at high frequencies and was most common in roots of ST plants. Progeny tubers showed some treatment effects when tested in September and after storage for 6 months, but there were no consistent relationships between root and progeny tuber infection.  相似文献   

12.
Controlled‐environment and field experiments were done to quantify the individual contribution of seed‐tuber and soilborne inoculum of Colletotrichum coccodes in causing black dot disease of potato tubers. Seed‐tuber and soilborne inocula of C. coccodes were quantified using an existing real‐time PCR assay and related to subsequent incidence and severity of disease. In four field trials, a controlled‐environment experiment and through the monitoring of 122 commercial crops, seed‐tuber inoculum was found to be relatively less important than soilborne inoculum in causing black dot, and the level of seed‐tuber inoculum did not significantly affect either the incidence or severity of disease or the percentage of progeny tubers deemed unmarketable. By contrast, soilborne inoculum had the potential to result in high levels of disease and the level of C. coccodes soil infestation (pg DNA g?1 soil) was found to have a significant effect. At soil infestation levels below 100 pg DNA C. coccodes g?1 soil, 7% of commercial crops had an incidence of black dot greater than 20%, increasing to 40% and 57% of crops at levels of 100–1000 pg g?1 and >1000 pg g?1 soil, respectively. These arbitrary threshold levels for soilborne inoculum related to disease risk are discussed. Interpretation of disease risk based on inoculum levels must, in the future, be informed by agronomic variables and potential control strategies.  相似文献   

13.
甘肃省马铃薯炭疽病病原分离与鉴定   总被引：3，自引：1，他引：2  
对甘肃省定西市马铃薯田早死病株进行了病原真菌的分离与鉴定,形态学观察和ITS序列分析表明,该病原菌为球炭疽菌(Colletotrichum coccodes).甘肃省定西市马铃薯田早死病株由C.coccodes引起,该病原菌危害马铃薯在甘肃省首次报道.  相似文献   

14.
Rhizoctonia solani is an important pathogen of potatoes causing stem canker and black scurf. The fungus is a species complex comprised of 13 known anastomosis groups (AGs). AG3-PT is the anastomosis group frequently associated with disease in potatoes. A real-time PCR assay was designed to the rDNA ITS region of AG3-PT isolates to enable the pathogen to be detected directly in tuber and soil samples. The resulting assay was highly specific for AG3-PT, and did not amplify DNA from isolates from other AGs or subgroups of AG3. Using a bulk DNA extraction method capable of extracting from up to 250 g of soil, the assay could detect one individual sclerotium of AG3-PT (weighing 200 μg) in 250 g of soil. The AG3-PT assay was used, with assays for AG2-1, AG5 and AG8 to determine the prevalence of those AGs in UK potato soils and tubers. AG2-1 and AG3-PT were the predominant groups in tubers and soils, although AG3-PT was more frequently isolated from tubers, highlighting its importance as a potato pathogen. AG3-PT was also detected in more than half of the tuber samples tested suggesting the importance of seed borne inoculum.  相似文献   

15.
双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用   总被引：3，自引：0，他引：3  
 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。  相似文献   

16.
North American isolates of Colletotrichum coccodes, representing six vegetative compatibility groups (NA-VCG), were used to study morphological and pathogenic variability. The objective was to determine if variability in conidial and microsclerotial size was related to pathogenicity. Significant differences were detected in length, width, and length/width ratios of conidia as well as in the length and width of microsclerotia among the NA-VCGs. The longest and widest conidia were produced by isolates belonging to NA-VCG1 and the largest microsclerotia were produced by isolates of NA-VCG2. Conidial and microsclerotial lengths and widths also were affected significantly by type of growth medium. There was no relationship between the size of conidia and the size of microsclerotia among the NA-VCGs studied. Conidial and microsclerotial size may affect inoculum potential and survival as isolates of NA-VCG2 have been demonstrated to occur more frequently than other NA-VCGs. Aggressiveness of 17 isolates of C. coccodes representing six NA-VCG's was studied on three potato cultivars using foliar and root inoculation methods. C. coccodes infection reduced tuber weight in all cultivars with both inoculation methods although tuber weight reductions were significantly higher following root inoculations than foliar inoculations. Pathogenic aggressiveness varied among NA-VCGs. Isolates belonging to NA-VCG2 and 3 were the least aggressive on potato foliage and isolates of NA-VCG1, 2, 3, 4, and 5 produced higher microsclerotial density on all three cultivars compared with isolates of NA-VCG6. Across inoculation methods, isolates of C. coccodes belonging to NA-VCG2 and 6 were the most aggressive based on reductions in tuber weight. Umatilla Russet was the most susceptible cultivar to C. coccodes compared to other cultivars regardless of inoculation method. These results demonstrate variability in morphology and pathogenic aggressiveness among the NA-VCGs of C. coccodes but these traits are not related.  相似文献   

17.
To investigate the ability of black dot symptoms to develop on infected potato tubers during storage, the growth of Colletotrichum coccodes was followed in vitro on malt agar at temperatures ranging from 5–27°C, and in vivo on artificially infected potato tubers kept at 5, 10 and 15°C. In vitro , 13 isolates from different geographical origins grew at all temperatures tested; growth started with a delay of 10 days at 5°C and of 4 days at 10°C, and was fastest at 27°C. All isolates had similar growth patterns and produced conidia and sclerotia at all temperatures. Minitubers were successfully infected at 5, 10 and 15°C by depositing either a mycelial plug or a drop of conidial suspension on the tuber surface. Sclerotia were observed after 7 days at the point of inoculation. Symptoms extended in all cases, although more slowly at 5 and 10 than at 15°C. Latent infections were detected in up to 21% of tubers without black dot symptoms at harvest. These results show that latent infections by C. coccodes are probably quite frequent, and that the pathogen is able to develop at low temperatures in controlled conditions. This suggests that black dot symptoms can increase during storage if stores are not adequately managed.  相似文献   

18.
Stem canker and black scurf are diseases of potato caused by the fungus Rhizoctonia solani . Spatiotemporal experimentation and empirical modelling were applied for the first time to investigate the effect of antagonistic Trichoderma harzianum on the dynamics of soilborne R. solani on individual potato plants. Trichoderma harzianum reduced the severity of symptoms, expressed as 'rhizoctonia stem lesion index' (RSI), during the first 7 days post-inoculation when the inoculum of R. solani was placed at certain distances (30–60 mm) from the host. For example, with inoculum at 40 mm from the host, RSI was 6 and 40 with and without T. harzianum , respectively. At later observation times, the antagonistic effect was overcome. Trichoderma harzianum reduced the severity of black scurf on progeny tubers. Furthermore, the mean number of progeny tubers per potato plant was reduced by the biocontrol treatment (means of 6·5 ± 1·1 and 9·9 ± 2·7 tubers per plant with and without T. harzianum , respectively), as was the proportion of small (0·1–20·0 g) tubers (48% and 66% with and without T. harzianum , respectively). Additionally, there were fewer malformed and green-coloured tubers in pots treated with T. harzianum than in those without T. harzianum .  相似文献   

19.
The effects of post‐harvest curing and storage temperature on severity of black dot, caused by Colletotrichum coccodes, were investigated for potato crops grown for different crop durations (days from 50% emergence to harvest) in soils that posed a low, medium and high risk of disease. In field trials over four growing seasons (2005–8), black dot severity at harvest increased with increasing crop duration, within the range 103–146 days from 50% emergence to harvest (P < 0.05). In field trials over three growing seasons (2006–8), black dot severity on tubers at harvest increased significantly with increasing soil inoculum in each year, within the range 43–4787 pg C. coccodes DNA/g soil (P < 0.05). Storage trials were conducted to measure the influence of accumulated post‐harvest temperature on black dot. In 2005, no difference in black dot severity was observed on tubers stored for 20 weeks at 2.5 and 3.5 °C. In 2006 (but not 2007), increasing the duration of curing after harvest from 4 to 14 days increased black dot severity on tubers from 8.9 to 11.2% (P < 0.01) in long duration crops (>131 days after 50% emergence) grown under high (>1000 pg C. coccodes DNA/g soil) soil inoculum. The number of days of curing did not affect disease severity for shorter duration crops grown at high soil inoculum, or on crops grown at medium or low (100–1000 and <100 pg C. coccodes DNA/g soil, respectively) soil inoculum concentrations. Soil inoculum and crop duration together provided a reasonable prediction of black dot severity at harvest and after a 20‐week storage period.  相似文献   

20.
A highly virulent and polyvalent Streptomyces phage was isolated from a potato field near Albany, Western Australia. The efficacy of the isolated phage to disinfest seed potato tubers artificially inoculated with a common scab-causing streptomycete was evaluated. The phage suspension was prepared in a mini-bioreactor. Diseased potatoes were bathed in a phage suspension (1 × 109 plaque-forming units per mL) for 24 h. The suspension was constantly circulated within a novel 25 L phage bath by means of an air-sparging pipe driven from an air compressor. Phage-treated scab-affected seed potatoes planted into free-draining polystyrene boxes containing steam-pasteurized field soil produced tuber progeny with significantly ( P  < 0·05) reduced levels of surface lesions of scab (1·2%) compared with tubers harvested from nonphage-treated tubers (23%). The number of scab lesions was also significantly reduced ( P  < 0·05) by phage treatment of mother tubers. No significant differences were recorded in weight, size or number of harvested tubers from phage-treated or nontreated mother tubers. This is the first in vivo study that has used Streptomyces phage to significantly disinfest seed potatoes of Streptomyces scabies and thereby reduce contamination of soil from seed-tuber-borne inoculum and reduce infection of daughter tubers.  相似文献   

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