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1.
A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.  相似文献   

2.
The invasive leafhopper Scaphoideus titanus (Hemiptera, Auchenorrhyncha, Cicadellidae) is an important pest of vineyards in Europe as a vector of ‘Candidatus Phytoplasma vitis’, the causal agent of flavescence dorée, which is on the EPPO A2 List of pests recommended for regulation. Scaphoideus titanus has been recorded from the territory of Ukraine for the first time. Specimens were found in 2017 in the Transcarpathian Region (Zakarpattia Oblast).  相似文献   

3.
In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on‐site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real‐time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real‐time PCR. The whole procedure of sample preparation and testing was designed and optimized for on‐site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.  相似文献   

4.
Three experimental treatments consisting of inoculation with an arbuscular mycorrhizal fungus, application of a synthetic antimicrobial peptide or application of a resistance inducer, were evaluated in Madagascar periwinkle as control methods for rubus stunt and stolbur diseases caused by ‘Candidatus Phytoplasma rubi’ and ‘Candidatus Phytoplasma solani’, respectively. Two experiments were conducted under controlled environment conditions. In the first experiment, 4 months after graft‐inoculating the phytoplasmas, the root colonization achieved by Rhizophagus irregularis significantly reduced both disease symptoms and the frequency of detection of the pathogens by real‐time PCR. In the second experiment, the antimicrobial peptide BP100 totally prevented disease symptoms, despite the molecular detection of the phytoplasmas in 75% and 50% of the plants inoculated with ‘Ca. Phytoplasma rubi’ and ‘Ca. Phytoplasma solani’, respectively, and was more effective than benzothiadiazole (BTH) at increasing resistance against the pathogenic infections. A potential combination of early mycorrhizal inoculation and BP100 antimicrobial peptide application is envisaged as a future control strategy for phytoplasma diseases.  相似文献   

5.
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6.
Phytoplasmas are plant pathogenic bacteria that infect more than 700 plant species. Because phytoplasma-resistant cultivars are not available for the vast majority of crops, the most common practice to prevent phytoplasma diseases is to remove infected plants. Therefore, developing a rapid, accurate diagnostic method to detect a phytoplasma infection is important. Here, we developed a phytoplasma detection assay based on loop-mediated isothermal amplification (LAMP) by targeting the groEL gene and 16S rDNA. We designed 19 primer sets for the LAMP assay and evaluated their amplification efficiency, sensitivity, and spectra to select the most suitable primer sets to detect Candidatus Phytoplasma asteris. As a result, DNA was efficiently amplified by one of the primer sets targeting the groEL gene, and LAMP assay sensitivity with this primer set was 10-fold higher than that of the polymerase chain reaction. Moreover, the groEL gene was successfully amplified from several strains of Ca. Phytoplasma asteris by this primer set, indicating that the groEL gene can be used as a LAMP assay target gene for a broad range of phytoplasma strains. Additionally, a simple DNA extraction method that omits the homogenizing and phenol extraction steps was combined with the LAMP assay to develop a simple, rapid, and convenient diagnostic method for detecting phytoplasma.  相似文献   

7.
In this study, the putative phytoplasma species causing coconut lethal yellowing disease in Mozambique and Tanzania were characterized. The 16S rRNA and secA genes were sequenced. Phylogenetic analysis revealed that Mozambican coconut phytoplasmas belong to three different types: ‘Candidatus Phytoplasma palmicola’ 16SrXXII‐A, a second strain that was previously isolated in Tanzania and Kenya (16SrIV‐C), and a third strain that was different from all known lethal yellowing phytoplasma species. The third strain potentially represents a novel species and is closely related to pine phytoplasma. Co‐infection with ‘Ca. Phytoplasma pini’‐related and ‘Ca. Phytoplasma palmicola’ 16SrXXII‐A strains was observed. Furthermore, sequence variation in ‘Ca. Phytoplasma palmicola’ at the population level was consistent with purifying selection and population expansion.  相似文献   

8.
An outbreak of Spartium witches’ broom (SpaWB) in Sicily prompted us to identify and characterize associated phytoplasmas. Over 80 samples of Spanish broom (Spartium junceum) and around 270 individuals of the potential vector Livilla spectabilis were collected and analysed. Single and mixed infections of 16SrV and ‘Candidatus Phytoplasma spartii’ were detected in Spanish broom samples and for the first time in L. spectabilis. The 16SrV isolates were further characterized by multilocus sequence typing (MLST) to determine their phylogenetic relationship with flavescence dorée phytoplasma (FDp) and to evaluate the risk of host-jumping to grapevine. Phylogenetic analysis of most of the analysed genes using the MLST approach grouped S. junceum 16SrV-C isolates with FDp isolates infecting grapevine and Scaphoideus titanus. Notably, phylogenetic analysis of the vmpA gene clustered the S. junceum isolates with FDp genotypes transmitted by S. titanus. This study extends the knowledge of SpaWB epidemiology, focusing on the possible risk of a 16SrV host jump from Spanish broom to grapevine. Spanish broom was identified as a reservoir and potential inoculum source of phytoplasmas that cause severe disease in cultivated crops. Furthermore, the L. spectabilis psyllid may be involved in the epidemiology of this 16SrV-C phytoplasma, although in the absence of in vivo transmission trials. The study further confirms the strong ability of phytoplasmas to adapt to new hosts and vectors, thus leading to potential phytosanitary emergencies.  相似文献   

9.
Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.  相似文献   

10.
Witches’ broom disease (WBD), caused by ‘Candidatus Phytoplasma aurantifolia’, is a serious disease of acid lime (Citrus aurantifolia) in Oman and the UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ‘Candidatus Phytoplasma aurantifolia’ was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semitropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II‐B. Quantitative PCR analysis showed that the concentration of phytoplasma is significantly higher (8800–801 000 copies) in leaves developing WBD symptoms compared to 2–268 copies in symptomless leaves from the same trees and 8–874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavourable environmental conditions. These findings could provide a basis for managing WBD through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression.  相似文献   

11.
Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.  相似文献   

12.
The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.  相似文献   

13.
《EPPO Bulletin》2017,47(2):146-163

Specific scope

This Standard describes a diagnostic protocol for ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’. This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols

Specific approval and amendment

Approved as PM 7/62 Candidatus Phytoplasma mali and PM 7/63 Ca. P. pyri in 2006. Revised in 2017‐02 as a single Standard as PM 7/62 (2) with the addition of ‘Ca. P. prunorum’.  相似文献   

14.
A new method for pest risk assessment and the identification and evaluation of risk‐reducing options is currently under development by the European Food Safety Authority (EFSA) Plant Health Panel. The draft method has been tested on pests of concern to the European Union (EU). The method is adaptable and can focus either on all the steps and sub‐steps of the assessment process or on specific parts if necessary. It is based on assessing changes in pest population abundance as the major driver of the impact on cultivated plants and on the environment. Like other pest risk assessment systems the method asks questions about the likelihood and magnitude of factors that contribute to risk. Responses can be based on data or expert judgment. Crucially, the approach is quantitative, and it captures uncertainty through the provision by risk assessors of quantile estimates of the probability distributions for the assessed variables and parameters. The assessment is based on comparisons between different scenarios, and the method integrates risk‐reducing options where they apply to a scenario, for example current regulation against a scenario where risk‐reducing options are not applied. A strategy has been developed to communicate the results of the risk assessment in a clear, comparable and transparent way, with the aim of providing the requestor of the risk assessment with a useful answer to the question(s) posed to the EFSA Plant Health Panel. The method has been applied to four case studies, two fungi, Ceratocystis platani and Cryphonectria parasitica, the nematode Ditylenchus destructor and the Grapevine flavescence dorée phytoplasma. Selected results from these case studies illustrate the types of output that the method can deliver.  相似文献   

15.
In this study quantitative real‐time PCR was used to follow the seasonal changes of flavescence dorée phytoplasma (FDp) titre in grapevines of cv. Modra frankinja (syn. Blaufränkisch) and cv. Refo?k (syn. Refosco'd'Istria) from two vineyards located in climatically different vine‐growing regions of Slovenia. Besides its known presence in the leaf veins, FDp was also detected in flowers, berry tissues and tendrils. In plants with high concentrations of FDp in tissues with symptoms, phytoplasma was also detected in symptomless tissues. A trend of decreasing FDp titre in all examined symptomless tissues from June to July and an increasing one throughout the growing season in tissues with symptoms was recorded. Accordingly, FDp was present in detectable amounts in flowers, petioles and veins of almost all infected plants in the late spring, and was detected in all examined tissue types in summer, with the highest titre in berries in August. The study showed that in the absence of plant health measurements an FDp infection may spread exponentially by a factor of 40 per year.  相似文献   

16.
This is the first report of Scaphoideus titanus, the main vector of flavescence dorée, for Madeira (Portugal) and also for Macaronesia. This new record currently represents its southernmost occurrence in Europe. This leafhopper is present in most of the primary wine production regions in the north of Madeira island. There were no symptoms of flavescence dorée disease during the monitoring period from 2010 to 2017. The ability of S. titanus to survive in regions with a subtropical climate suggests that it may also live in the most meridional areas of the Mediterranean region where, until now, it has been absent.  相似文献   

17.
Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.  相似文献   

18.
The phloem‐sucking psyllid Cacopsylla picta plays an important role in transmitting the bacterium ‘Candidatus Phytoplasma mali’, the agent associated with apple proliferation disease. The psyllid can ingest ‘Ca. Phytoplasma mali’ from infected apple trees and spread the bacterium by subsequently feeding on uninfected trees. Until now, this has been the most important method of ‘Ca. Phytoplasma mali’ transmission. The aim of this study was to investigate whether infected C. picta are able to transmit ‘Ca. Phytoplasma mali’ directly to their progeny. This method of transmission would allow the bacteria to bypass a time‐consuming reproductive cycle in the host plant. Furthermore, this would cause a high number of infected F1 individuals in the vector population. To address this question, eggs, nymphs and adults derived from infected overwintering adults of C. picta were reared on non‐infected apple saplings and subsequently tested for the presence of ‘Ca. Phytoplasma mali’. In this study it was shown for the first time that infected C. picta individuals transmit ‘Ca. Phytoplasma mali’ to their eggs, nymphs and F1 adults, thus providing the basis for a more detailed understanding of ‘Ca. Phytoplasma mali’ transmission by C. picta.  相似文献   

19.
A rapid DNA extraction and loop‐mediated isothermal amplification (LAMP) procedure was developed and evaluated for the detection of two specific groups of phytoplasmas from infected plant material. Primers based upon the 16–23S intergenic spacer (IGS) region were evaluated in LAMP assays for amplification of group 16SrI (aster yellows group) and group 16SrXXII (Cape St Paul wilt group) phytoplasma strains. DNA could be extracted from leaf material (16SrI phytoplasmas) or coconut trunk borings (16SrXXII phytoplasmas) onto the membranes of lateral flow devices, and small sections of these membranes were then added directly into the LAMP reaction mixture and incubated for 45 min at 65°C. Positive reactions were detected through the hydroxyl napthol blue colorimetric assay within 1 h of the start of DNA extraction, and were confirmed by subsequent agarose gel electrophoresis of the LAMP products. The level of detection was comparable to that obtained by nested PCR using conventional 16S rDNA phytoplasma‐specific primers. Furthermore, the assays were specific for the phytoplasmas they were designed to detect – the 16SrI assay only detected 16SrI phytoplasmas and not those from any other phylogenetic groups, whilst the 16SrXXII assay only detected 16SrXXII phytoplasmas. The DNA extractions and LAMP assay are easy to perform, requiring minimal equipment, and may therefore form the basis of a rapid and reliable field‐detection system for phytoplasmas.  相似文献   

20.
Chinese scholar tree (Sophora japonica) with witches’ broom symptoms was observed in Shandong Province in China. Phytoplasmas were detected in the diseased plants using 16S rDNA amplification with phytoplasma-specific universal primer pairs. On the basis of the results of 16S rDNA sequencing, virtual restriction fragment length polymorphism patterns and phylogenetic analyses, the phytoplasma found in S. japonica with witches’ broom symptoms was confirmed as a ‘Candidatus Phytoplasma ziziphi’-related strain belonging to the Elm yellows group 16SrV. This is the first report of ‘Ca. P. ziziphi’ infecting S. japonica plant with witches’ broom symptoms.  相似文献   

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