首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 78 毫秒
1.
Xylella fastidiosa (Xf) is a gram-negative bacterial plant pathogen that can infect over 500 plant species. While it is endemic in America, X. fastidiosa subsp. pauca was reported for the first time in Europe in 2013 on olive trees in southern Italy. The availability of fast, sensitive, and reliable diagnostic tools is indispensable for managing current and future outbreaks of Xf. In this paper, we use the OXford Nanopore Technologies (ONT) MinION platform for detecting and identifying Xf at species, subspecies, and sequence type (ST) level. Two workflows were developed: the first one provided a “shotgun” strategy, that is, exploring the possibility of detecting Xf within DNA extracted from plant samples. This allowed detection of Xf by direct DNA sequencing and identifying the subspecies only in samples with high bacterial levels. Nanopore amplicon sequencing was pursued as a second workflow. This consists of PCR amplification of a set of seven multilocus sequence typing (MLST) fragments, officially adopted for identifying Xf at type strain level, followed by Nanopore-sequencing of the amplicons and an ad hoc pipeline to generate MLST consensus calls. This combined approach, which takes only a few hours, allowed the detection and identification of Xf at ST level in plant material with low bacterial infection.  相似文献   

2.
Interactions between Verticillium dahliae and Colletotrichum coccodes , major causal agents of potato early dying (PED) syndrome, were studied in four potato cultivars that differ in their susceptibility to these pathogens . Aseptic plantlets of Nicola, Desiree, Alpha and Cara were inoculated with identical concentrations of each pathogen or with a mixture of the pathogens, and grown for 4 weeks in a monitored growth chamber. Coinoculation of Nicola with both pathogens caused more severe foliar disease symptoms and crown rot and greater C. coccodes colonization, than inoculation with each pathogen separately. Significant reductions in weight and height were also observed in plants coinoculated with both pathogens, as compared with plants inoculated with each pathogen separately or noninoculated plants. In Desiree, more roots were covered with C. coccodes sclerotia and disease symptoms were significantly more severe in plants inoculated with both pathogens together. However, plant weight and height were similar to those of plants inoculated with C. coccodes only. In Alpha, disease symptoms and levels of sclerotia in the roots were not affected by simultaneous inoculation with both pathogens. Weight and height of all plants were similar, whether inoculated with each pathogen separately or with both pathogens together. In Cara, plants inoculated with the mixture or either pathogen alone were smaller than the noninoculated control. Disease symptoms and occurrence of sclerotia were similar in plants inoculated with the combination and with a single pathogen. Compared with the effects of inoculation with either pathogen, simultaneous inoculation with both pathogens can, in some cultivars, increase the incidence of PED syndrome and thus severely decrease yields.  相似文献   

3.
Sanogo S 《Phytopathology》2007,97(1):37-43
ABSTRACT Phytophthora capsici and Verticillium dahliae are two mycelial microorganisms associated with wilt symptoms on chile pepper (Capsicum annuum). Both pathogens occur in the same field and can infect a single plant. This study examined the nature of the co-occurrence of P. capsici and V. dahliae. Chile pepper plants were inoculated with each pathogen separately or with both pathogens concomitantly or sequentially. In concomitant inoculations, plants were inoculated with a mixture of zoospores of P. capsici and conidia of V. dahliae. In sequential inoculations, plants were inoculated with zoospores of P. capsici 4 days prior to inoculation with conidia of V. dahliae, or plants were inoculated with conidia of V. dahliae 4 days prior to inoculation with zoospores of P. capsici. Stem necrosis and leaf wilting were visible 3 to 4 days earlier in plants inoculated with both P. capsici and V. dahliae than in plants inoculated with P. capsici alone. Stem necrosis and generalized plant wilting were observed in plants inoculated with P. capsici alone, and stem necrosis, generalized plant wilting, and vascular discoloration were observed in plants inoculated with both P. capsici and V. dahliae by 21 days after inoculation. These symptoms were not observed in control plants or plants inoculated with V. dahliae alone. The frequency of recovery of V. dahliae from stems was approximately 85 to 140% higher across inoculum levels when plants were inoculated with both P. capsici and V. dahliae than when plants were inoculated by V. dahliae alone. Similarly, the frequency of recovery of V. dahliae from roots was approximately 13 to 40% higher across inoculum levels when plants were inoculated with both P. capsici and V. dahliae than when plants were inoculated by V. dahliae alone. There was no apparent antagonism between the two pathogens when they were paired on growth media. In general, when P. capsici and V. dahliae were paired on growth media, mycelial growth of each pathogen grown alone was not significantly different from mycelial growth when the pathogens were paired. Results suggest that wilt development is hastened by the presence of both P. capsici and V. dahliae in the same plants. The presence of P. capsici and V. dahliae in the same inoculum court enhanced infection and colonization of chile pepper by V. dahliae.  相似文献   

4.
Plant parasitic nematodes are generally soilborne pathogens that attack plants and cause economic losses in many crops. The infested plants show nonspecific symptoms or, often, are symptomless; therefore, diagnosis is performed by taking soil and root tissue samples. Here, we show that a combination of different infrared spectra analysis and machine learning algorithms can be used to detect plant parasitic nematode infestations before symptoms become visible, using leaves instead of roots and soil as samples. We found that tomato and guava plants infested with Meloidogyne enterorlobii produced different spectral patterns compared to uninfested plants. Using partial spectra from 1,450 to 900/cm as the "fingerprint region", principal component analyses indicated that after 5 (tomatoes) or 8 weeks (guava), plants with no visible symptoms of infestations were positively diagnosed. To improve the early detection response, we used machine learning modelling. A support vector machine (SVM) was used to obtain more robust, accurate models. The SVM model contained 34 support vectors, 17 for each level. The overall performance of the model was >97% and the total accuracy was significantly higher, demonstrating the absence of chance prediction. The best prediction of infestation was obtained at the second and fourth weeks for tomatoes and guavas, respectively, reducing the diagnostic time by half. The combined application of these techniques reduces the processing time from field to laboratory and shows enormous advantages by avoiding root and soil sampling.  相似文献   

5.
P. Legrand 《EPPO Bulletin》2015,45(2):240-251
Biological assays, often referred to as woody indexing and herbaceous indexing, may be used to detect and identify plant viruses and other graft‐transmissible pathogens to ensure that plants for planting are free from regulated pests. However, many factors can influence the results of biological indexing, which affect the analytical specificity, analytical sensitivity and reliability (repeatability and reproducibility) of these tests. This review focuses on the following: the environmental factors, the role of the inoculum source, the choice of the indicator plant, the expression of symptoms, the number of repetitions, the use of positive controls and healthy controls, the means of transmission of pathogens, the differentiation of strains, the influence of isolates or strains of pathogens, mixed infections, and the bulking of samples for testing. Some indicator plants which are highly susceptible to many viruses and virus‐like diseases may be used to detect a wide range of pathogens. However, comparing the results from bioassays with those from serological or molecular tests has shown the limits for detection and identification of many pathogens. It is concluded therefore that biological assays should only be considered for use in conjunction with serological or molecular tests to detect and identify pathogens. Bio‐amplification by indicator plants to amplify a pathogen to levels that can be detected by another test method remains of interest.  相似文献   

6.
ABSTRACT Although Ustilago hordei infects barley seedlings, symptoms of the disease covered smut are not visible until heading. Natural or artificial inoculation usually results in inconsistent infection, even in highly susceptible lines. Thus, breeding for resistance to covered smut is time consuming and difficult. The ribosomal DNA internal transcribed spacer (ITS) regions of U. hordei were sequenced and a primer pair was developed for polymerase chain reaction (PCR). These primers amplified a 574-bp fragment from DNA of Ustilago spp., but did not amplify DNA from barley or other common barley pathogens. DNA extracted from as few as four U. hordei sporidia was detected by this method. U. hordei DNA was amplified from leaf tissue of inoculated susceptible and resistant plants at different stages of plant development in differential varieties. Growth of the fungus in different leaves of an individual plant was variable. Several highly resistant varieties were shown to contain U. hordei DNA in the first three to four leaves, but not in later leaves. Thus, although the fungus can infect many resistant plants, the plants remain symptomless. Detection of U. hordei prior to heading should assist efforts for breeding for resistance and provide clues concerning the mechanisms of resistance employed by different resistance genes.  相似文献   

7.
The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Real-time polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multi-disciplinary studies.  相似文献   

8.
Molecular detection of phytopathogens is increasingly being applied to identify regulated organisms at the border in many parts of the world. However, even with molecular tests, complete phenotyping and identification of a strain is often time consuming and sometimes inconclusive. In this study, a leaf-based pathogenicity test was used to separate pseudomonads into two groups, Group A containing pathogens, and Group B containing saprotrophs. Comparative genomics of 56 pseudomonad genomes from different plant hosts (including 29 strains from kiwifruit) agreed with kiwifruit pathogenicity test results, placing pathogens into Group A and saprotrophs into Group B. Sixteen loci were found unique to Group A. A PCR assay was developed for amplification of one of these loci, the trehalose phosphatase gene. The generation of this 655 bp amplicon was associated with production of water-soaked lesions on inoculated kiwifruit leaves by pseudomonads in Group A. This test was validated for further strains from all seven pathogenic Pseudomonas phylogroups, non-pathogenic pseudomonads, and other bacterial genera. The sensitivity of the PCR was comparable to the limit of recovery of pseudomonads by culturing. This simple PCR assay could be used as part of a testing pipeline at the border and for general surveillance for screening plants with and without symptoms, offering the potential to detect uncharacterized pseudomonads that may pose a biosecurity risk. The method was shown to be able to rapidly identify pathogens cultured from plant material with symptoms, or, more importantly, to detect pathogens directly from plant tissue.  相似文献   

9.
Spread of Phytophthora root and crown rot in three pot plant species was studied on ebb-and-flow benches where the nutrient solution was recirculated. The plant species and their respective pathogens were: Saintpaulia ionantha – P. nicotianae, Gerbera jamesonii – P. cryptogea, and Spathiphyllum wallissii – Phytophthora spp. Ebb-and-flow benches were infested with the pathogen using different methods: 18–25% of the plants on a bench were inoculated or potted in soil infested with the pathogen or the nutrient solution was infested by either zoospores or mycelium fragments. More than 80% of the inoculated Saintpaulia plants and 22% of plants potted in infested soil developed disease but no spread of the disease was observed. Infestation of the nutrient solution did not result in any diseased Saintpaulia plant. More than 70% of the Gerbera plants developed disease as a result of spread of the pathogen irrespective of the infestation method used. No significant spread of the disease was observed with inoculated Spathiphyllum plants nor from plants potted in infested soil. A few Spathiphyllum plants developed disease symptoms after infestation of the nutrient solution with zoospores. In one experiment, nearly all Spathiphyllum plants were diseased after infestation of the nutrient solution with mycelium fragments. The presence of an irrigation mat significantly reduced the spread of the Phytophthora disease in Gerbera and Spathiphyllum. The possibility of an irrigation mat acting as a filter for zoospores is discussed.  相似文献   

10.
DNA amplification by polymerase chain reaction was used to specifically detect phytoplasmas associated with severe decline diseases of European stone fruits. PCR primers were designed according to the partial sequence of a nonribosomal genomic fragment of European stone fruit yellows phytoplasmas obtained by direct sequencing of a specific PCR product. A PCR assay was developed which resulted in specific amplification of a 237 bp-DNA fragment from total DNA extracts derived from over 300 stone fruit samples. No PCR product was obtained with DNA from healthy controls or plants diseased with various other phytoplasmas, e.g. the closely related apple proliferation and pear decline phytoplasmas. Phytoplasma infection was checked in all samples by PCR amplification with universal ribosomal primers. Detection rate with specific and universal primers was correlated by 97%. European stone fruit yellows phytoplasmas were detected in samples of 114 out of 139 examined orchards which represent the major stone fruit growing regions of France. Typical symptoms like chlorotic leaf roll in summer and off-season growth in winter were correlated by 95% to the presence of phytoplasmas. However, phytoplasmas were also detected in 51% of samples derived from trees showing non-specific symptoms. A comparison study including 201 samples showed that 81% of the PCR-positive samples were also tested positive using fluorescence microscopy with DAPI staining.  相似文献   

11.
转CP基因线辣椒对CMV和CMV-RNA的抗病性比较   总被引：7，自引：0，他引：7  
 本试验以转化CMV-CP和TMV-CP基因线辣椒纯合系作试材,比较了接种CMV粒体和CMV-RNA后的发病特点和叶片中的病毒含量。结果表明:转化线辣椒不仅能抵抗CMV粒体的侵染,而且还能抵抗CMV-RNA的侵染。不论接种CMV粒体或CMV-RNA,CP(+)线辣椒的系统症状都延迟出现,显症株率和病害严重度级别大幅度降低,病毒增殖和运转受到抑制,接种叶片与新生叶片中的病毒含量明显减低。这一结果证实CMV-RNA不能克服线辣椒由CP基因介导的抗病性。  相似文献   

12.
During the spring of 2001, approximately 10 000 yellow passion flower plants, from two orchards in the county of Livramento de Nossa Senhora, Bahia State, Brazil, exhibited intense yellow mosaic symptoms and drastic reduction of the leaf lamina and plant development. A large population of whiteflies ( Bemisia tabaci ) was also found colonizing the plants. All field samples collected tested positive for Passion fruit woodiness virus in DAS-ELISA. Five out of 20 passion flower plants inoculated with adult whiteflies collected from diseased plants in the field developed symptoms 20–30 days after inoculation. Two of these plants gave a positive reaction in TAS-ELISA using antiserum against a begomovirus. Degenerated PCR primers amplified viral DNA fragments from the DNA-A and DNA-B components of a begomovirus infecting these plants. The fragment corresponding to the core region of the coat protein (DNA-A) was cloned and sequenced. A phylogenetic analysis placed this begomovirus isolated from passion flower in the same clade of the New World begomoviruses as several other species from Brazil. Based on the symptoms induced by this virus alone, the disease was tentatively named passion flower little leaf mosaic.  相似文献   

13.
Four of five weeks after planting a group of potato plants ‘Bintje’ was inoculated with potato virus X (PVX). Other groups were inoculated at intervals of 14 days. Tubers produced by plants inoculated 35 days after planting were all infected. The plants inoculated 49 days or later after planting produced few infected tubers. The latter had developed mature plant resistance against PVX infection. The ribosome and RNA contents of leaves were measured by application of adsorption chromatography. A rapid decrease in ribosome and RNA contents occurred in plants at the time of rapid increase in the rate of mature plant resistance. The decrease was most distinct in the fifteenth leaf and therefore the contents in this leaf seem to give a good indication of the rapid increase in resistance.  相似文献   

14.
  相似文献   

15.
A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.  相似文献   

16.
Newly emerging or re‐emerging diseases are a constant and significant threat to agricultural production, so prompt and accurate identification of the causative agents is required for rapid and appropriate disease management. Classical methods of pathogen detection can be successfully supplemented by next‐generation sequencing (NGS), whereby sequence analysis can help in the discovery of new or emerging diseases. In 2007, hop growers in Slovenia reported the appearance of severely stunted hop plants, a phenomenon that spread rapidly within hop gardens and among farms. Classical diagnostic methods were unable to detect a new pathogen; therefore, single step high‐throughput parallel sequencing of total RNA and small RNAs from plants with and without symptoms was employed to identify a novel pathogen. The sequences were assembled de novo and also mapped to reference genomes, resulting in identification of a novel sequence of Citrus bark cracking viroid (CBCVd) in the stunted hop plants. Furthermore, the presence of this novel pathogen on hop was confirmed by RT‐PCR analysis of 59 plants with symptoms from 15 hop gardens, representing the main outbreak locations identified by systematic disease monitoring, and small RNA Illumina sequencing of the bulked RNA sample. The high infectivity of the newly identified CBCVd was also confirmed by biolistic inoculation of two hop cultivars, which developed aggressive symptoms in controlled conditions. This study shows the feasibility of deep sequencing for the identification of causative agents of new diseases in hop and other plants.  相似文献   

17.
The development of a rapid PCR assay for detection of Fusarium moniliforme   总被引：1，自引：0，他引：1  
The fungus Fusarium moniliforme infects a wide range of crops throughout the world. In maize (Zea mays L.) it causes seedling blight and root, stalk, and ear rots. A simple procedure that can be used to detect infection by F. moliliforme from infected plant tissues has been developed. A F. moniliforme genomic library was prepared and used to identify the recombinant clones containing fungal DNA sequences not hybridizing with the DNA of the host plant, maize. Based on the nucleotide sequence information obtained from the F. moniliforme pUCF2 genomic clone, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by the polymerase chain reaction. An amplification product was obtained with F. moniliforme DNA preparations whereas no amplified DNA was detected with DNAs from other fungal pathogens, including various Fusarium species, or from the host plant. This PCR analysis was successfully employed to identify F. moniliforme directly from the mycelia that develop from naturally infected maize seeds, with no need to obtain pure fungal cultures for reliable diagnosis. The protocol can be used for the diagnosis of infected plants and soils in epidemiological studies of Fusarium diseases, for seed health testing, and for evaluation of susceptibility to colonization in commercial maize hybrids.  相似文献   

18.
大麦黄矮病毒-GAV在燕麦植株体内运动规律的初步研究   总被引：2，自引：0，他引：2  
 利用RT-PCR方法研究了大麦黄矮病毒-GAV在燕麦植株内的移动规律。先将介体麦二叉蚜(Schizaphis graminum)在BYDV-GAV新鲜病叶上饲毒,再将获毒蚜虫放置到二叶期的健康燕麦植株接种48h,随后分期提取接种植株的第1~6片叶和根组织的总RNA,利用特异引物扩增BYDV-GAV的外壳蛋白(CP)基因以检测病毒在燕麦植株内的复制和移动。结果表明,在接种5d后,接种叶片(第2片叶)呈现阳性,接种7d后,植株新生的第4片叶被侵染,接种9d后,部分的第3片叶呈现阳性,至接种16d,几乎所有的叶片均呈现阳性。仅在接种的第5、7和9d收集的根组织呈现阳性,而所有的第1片叶均为阴性,可能是由于这些组织内病毒含量太低所致。本研究初步揭示了BYDV-GAV长距离运动的规律并且发现该病毒在燕麦根部从接种到系统发病都没有进行大量增殖,为今后进一步研究病毒运动机制选取适当的植物材料提供了基本信息。  相似文献   

19.
Hélias  rivon  & Jouan 《Plant pathology》2000,49(1):23-32
Infection of seed tubers by pectinolytic Erwinia species can lead to the development of various symptoms during vegetative growth of potato crops, including non-emergence of plants, chlorosis, wilting, haulm desiccation and typical blackleg. The relationships between types of symptoms and yield are poorly documented, and are investigated by following the development of symptoms in potato plants grown under field conditions from seed tubers artificially inoculated with E. carotovora ssp. atroseptica ( Eca ) , and measuring the yield of each plant. Symptoms were classified into five main types (non-emergence, wilting/chlorosis, blackleg, haulm desiccation and plant death). Each plant was scored for types of symptom on four successive dates; plants without visible symptoms were scored as healthy. The method of inoculation and inoculum concentration proved major factors for the subsequent development of symptoms. Disease development was more severe after vacuum infiltration of bacteria into seed tubers than after shaking tubers in contaminated sand. Disease usually progressed from chlorosis and/or wilting to partial or total desiccation on a given plant. Yield losses varied according to symptom type, but the relationship between symptoms recorded and yield also depended on scoring dates. The data suggest that the beginning of tuber growth might be the most suitable stage for predicting yield losses from symptom observations. In both cultivars studied (Bintje, highly susceptible, and Désirée, moderately resistant), the yield of symptomless plants growing from inoculated seed tubers was significantly less than that of control plants, indicating that the presence of bacteria on the seed tuber was detrimental, even in the absence of visible symptoms. Differences in symptom expression in the field between cultivars matched the level of visible infection of tubers at harvest, as Bintje tubers showed a higher incidence of rot than Désirée tubers.  相似文献   

20.
高通量测序技术(next-generation sequencing,NGS)平台提供了一种高效、快速、低成本、深度测序DNA的解决方案,2009年该技术开始被应用于植物病毒学领域,包括新病毒的发现,病害病原的鉴定,病毒基因组多样性及进化的研究,显著加快了植物病毒学的发展进程。迄今,应用高通量测序技术已经成功鉴定了上百种新的植物病毒和类病毒。双生病毒是一类对多种作物造成毁灭性危害的DNA病毒,多发生于草本作物。然而利用高通量测序技术,从柑橘、葡萄、苹果和桑树等多种多年生木本植物中检测到了新的双生病毒,显示出了高通量测序技术所独有的、传统检测技术所不具备的优势。本文围绕高通量测度技术在植物病毒学领域的应用进行概述,重点阐述NGS用于检测木本植物双生病毒的几个实例。  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号