Sensitive real-time PCR detection of Xanthomonas fragariae in strawberry plants

Xanthomonas fragariae , the causal agent of angular leaf spot on strawberry, is a quarantine organism in strawberry propagation material in the European Union. For the reliable screening of planting material for latent infections, a real-time PCR assay based on Taqman® chemistry for the detection of X. fragariae was developed. Primers and probe sequences were based on a DNA fragment amplified by a previously reported X. fragariae -specific technique. The sequence of this genomic fragment had no significant similarity with any published GenBank sequence. Specificity of the designed assay was tested with an extended range of X. fragariae collection strains and isolates, with other Xanthomonas spp. and with unidentified bacterial isolates from strawberry plants. A nested PCR, which until now was the reference method for sensitive detection in planta , cross-reacted with the reference strain of Xanthomonas campestris pv. campestris . In combination with an elaborated DNA extraction procedure, the Taqman® PCR enabled reliable detection down to 300 colony forming units in a 100 mg strawberry leaf sample. The assay offers a new tool for epidemiological research and for sanitary control of plant material with low level or latent infections of X. fragariae .     

Nested Pcr (polymerase chain reaction) for detection of Xanthomonas fragariae in symptomless strawberry plants

Journal of Plant Diseases and Protection - For rapid and sensitive detection of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, a nested polymerase chain reaction (Pcr)...     

Development of Xanthomonas fragariae populations and disease progression in strawberry plants after spray‐inoculation of leaves

Xanthomonas fragariae is the causative agent of angular leaf spot disease of strawberry. Greenhouse experiments were conducted using a X. fragariae isolate tagged with a green fluorescent protein (GFP) for detailed population dynamic studies in and on leaves after spray‐inoculation. The GFP‐tagged bacteria were monitored with dilution plating of leaf washings and leaf extracts, and analysis of intact leaves using a non‐invasive monitoring system called PathoScreen, based on laser radiation of fluorescent cells in plant tissues and signal recording with a sensitive camera. PathoScreen was also used to monitor bacteria grown on an agar medium after leaf printing. During the first 3 days after inoculation, bacterial populations washed off leaves rapidly decreased by at least a factor of 1000, after which populations remained stable until 14 days post‐inoculation (dpi), when symptoms first started to appear. Thereafter, populations increased to a level of 10¹² colony‐forming units (CFU) g^?1 of leaf material or higher. Similarly, densities in leaf extracts were low during the first 3 days after inoculation, at a level of 100–1000 CFU g^?1 of leaf tissue. Gradually populations increased to a level of 10⁹–10¹² CFU g^?1 at 28 dpi. Higher densities of epiphytic populations were found on the abaxial side than on the adaxial leaf side during the first 2 weeks after inoculation. After spray‐inoculation of leaves, bacterial populations released from infected plants remained low until symptoms appeared, after which plants became highly infectious, in particular under high humidity.     

Resistance and systemic dispersal of Xanthomonas fragariae in strawberry germplasm (Fragaria L.)

The angular leaf spot disease caused by Xanthomonas fragariae is an important plant disease with major impact for the strawberry nursery industry. Currently there is no plant protection product available for controlling the disease effectively. Planting of resistant cultivars seems to be promising, but all commercially used cultivars are susceptible and no donor with a high level of resistance has yet been found. Therefore, a total of 145 genotypes from the Fruit Genebank Dresden (Germany) were evaluated for resistance to X. fragariae by artificial inoculation. Six genotypes were classified as partly resistant, out of which only two (US4808 and US4809) are octoploid. Fragaria vesca f. alba, Fragaria nilgerrensis ‘Yunnan’, F. vesca ‘Illa Martin’ and F. moschata ‘Bauwens’ were also classified as partially resistant, but they are only of limited use for breeding because of their variable ploidy level. Fully resistant genotypes could not be detected. The systemic dispersal of the bacteria in strawberry plants was investigated after inoculation of leaves with X. fragariae strain XF3.9.C and the GFP‐tagged strain XF3.9.C_(pKAN). The systemic spread was evaluated after 3, 7, 14 and 28 days post‐inoculation (dpi) by nested PCR and fluorescence microscopy. After 3 dpi, X. fragariae could be found in all tissues tested including the inoculated leaf, its petiole, the rhizome, the heart bud up to the youngest fully expanded leaf and its petiole. The systemic spread was also detectable in partially resistant genotypes.     

Effect of temperature and host genotype on the production of inoculum by Phytophthora fragariae var. fragariae from the roots of infected strawberry plants

A bioassay was used to monitor the release of inoculum in drainage water from strawberry plants inoculated with zoospores of Phytophthora fragariae var. fragariae. The fungus was detected in drainage water from plants that had been held at temperatures between 2 and 20 C. but not from plants held at 26°C. The lag phase before secondary inoculum was first released, the maximum and total amounts of inoculum released, and the length of time over which inoculum was released were all greater at the lower temperature regimes, especially those below 10 C. The results were consistent with observations on the effect of temperature on zoospore production from agar discs and on zoospore motility: more zoospores were produced at lower temperatures and they remained motile for longer. From this it is concluded that the inoculum detected consists mainly of motile zoospores. In most experiments with standardized suspensions c. 10-15 were sufficient to initiate infection of the plants in the bioassay. In general, more inoculum was produced by host genotype/fungal isolate combinations in which there were marked root rot symptoms than in combinations in which the host was resistant.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.     

利用实时荧光PCR技术检测风信子黄腐病菌

风信子黄腐病菌是我国禁止入境的病原细菌之一,我国目前尚无该病的发病报道.本研究根据风信子黄腐病菌基因组16S-23S ribosomal RNA intergenic spacer保守序列,设计并合成了1对特异性引物和1条具有稳定点突变特异性探针进行实时荧光PCR检测,风信子黄腐病菌有很强的荧光信号,供试的其它7种病原细菌菌株均没有荧光产生.与常规PCR相比,实时荧光PCR检测特异性强,灵敏度高,适合病害的快速诊断和口岸检验检疫应用.经优化反应条件,建立了稳定的风信子黄腐病菌实时荧光PCR检测方法.     

草莓角斑病菌检测鉴定技术研究进展

本文综述了近年来草莓角斑病菌(Xanthomonas fragariae)检测鉴定技术的研究进展.传统的鉴定方法主要有依赖田间症状识别、寄主接种鉴定、培养性状、生理生化鉴定、脂肪酸分析法(FAP)等;应用血清学鉴定检测方法主要有酶联免疫吸附法(ELISA)、菌落免疫荧光染色法(IFAS)等,分子生物学检测方法主要有限制性片段长度多态性(RFLP)、聚合酶链反应(PCR)、巢式PCR、实时荧光PCR等.对于XF的检测鉴定不能单单依靠一种方法,应将多种方法结合起来进行最终判定,因此建立一套完整的检测鉴定技术体系十分必要.     

草莓角斑病菌传入中国的风险分析

草莓角斑病菌是一种严重为害草莓生产的细菌。本文从其分布状况、寄主、经济重要性、传入与定殖可能性和风险管理难度等方面进行了综合分析评估,结果表明该病菌属于特别危险的有害生物。并根据风险分析的结果提出了防止其传入我国的管理措施。     

Development and optimization of a real-time detection assay for Xanthomonas fragariae in strawberry crown tissue with receiver operating characteristic curve analysis

Angular leaf spot of strawberry is caused by the bacterium Xanthomonas fragariae. The disease is transmitted primarily through systemically infected nursery stock. This creates problems for nurseries wishing to export plants to Europe because of quarantine restrictions. Currently, field inspections for symptoms are used to certify plants free of X. fragariae, but visual inspection is not useful for detecting plants infected systemically. To detect systemic infections, polymerase chain reaction (PCR) is the desired tool because of its sensitivity, specificity, and ease of use. In this study, we developed three sets of real-time PCR primers and probes and determined optimal reaction conditions for use of these primers for the detection of the bacterium X. fragariae in strawberry crown tissue. Real time detection proved to be both more sensitive and specific than standard PCR. Moreover, the detection of X. fragariae in crown tissue extract was possible with real-time PCR but not with standard PCR which is a significant improvement over standard PCR. The information on sensitivity and specificity of the primer sets was used to evaluate the performance of these primers with receiver operating characteristic (ROC) curve analysis under different tolerances. The results of this analysis can be used to provide guidance on threshold selection to manage disease below unacceptable levels. The results of this research may be useful to regulators and inspectors who must certify that plants meet European and Mediterranean Plant Protection Organization standards.     

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum- specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.     

Occurrence of strawberry marginal chlorosis caused by “Candidatus Phlomobacter fragariae” in Japan

In February 2004, a disease of strawberry (Fragaria × ananassa Duch.), causing little-leaf, proliferation, malformation of fruits, and marginal chlorosis of leaves, occurred in Ehime Prefecture, Japan. The causal pathogen was identified as a phloem-restricted bacterium-like organism, “Candidatus Phlomobacter fragariae,” based on polymerase chain reaction (PCR) detection, electron microscopy, and sequence analysis of PCR products. This is the first report of strawberry marginal chlorosis in Asia. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB246669.     

Bacterial leaf blight of strawberry (Fragaria (x) ananassa) caused by a pathovar of Xanthomonas arboricola, not similar to Xanthomonas fragariae Kennedy & King. Description of the causal organism as Xanthomonas arboricola pv. fragariae (pv. nov., comb. nov.)

A new bacterial disease of strawberry is described. This disease, called bacterial leaf blight of strawberry, is characterized by dry, brown necrotic leaf spots and large brown V-shaped lesions along the leaf margin, midrib and major veins. Symptoms are different from angular leaf spot of strawberry caused by the bacterium Xanthomonas fragariae . Strains of the bacterial leaf blight pathogen were characterized in a polyphasic approach by biochemical tests, fatty acid analysis, protein electrophoresis, serology, PCR, pigment analysis, ice-nucleation activity, AFLP analysis, DNA:DNA hybridization, pathogenicity and host range tests, and compared with a number of reference strains of X. fragariae and other Xanthomonas species. Bacterial leaf blight strains formed a homogeneous group in all tests, completely different from X. fragariae . They were the only strains causing leaf blight of strawberry upon artificial inoculation into strawberry. Fatty acid and protein electrophoretic analysis showed that the strains belong to the phenon X. campestris ( sensu latu , including pathovars now classified as belonging to X. arboricola ). AFLP analysis and DNA:DNA hybridization further clarified their taxonomic position as belonging to X. arboricola. The name X. arboricola pv. fragariae is proposed for the bacterium causing leaf blight of strawberry with strain PD2780 (LMG 19145) as pathovar type strain. Criteria for routine identification are given and the taxonomic status is discussed.     

进境苹果果实中梨火疫病菌的套式PCR检测

 针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。     

Eradication of the first outbreak of Xanthomonas fragariae in the United Kingdom

Xanthomonas fragariae was identified in the UK in strawberry fruiting crops in October 2004. As this pathogen had not been confirmed in the UK before and is listed as a quarantine organism by the EU and EPPO, emergency official action was taken to contain and eventually eradicate this pest. In order to eradicate this disease the affected growers were given the option of either destroying the crop to eradicate the disease immediately or maintaining the infected plants for the life of the crop for fruit production with hygiene measures to prevent the spread of the disease to uninfected crops. The affected growers chose to maintain the crops with hygiene measures to contain the disease. The crops continued to be monitored and no further symptoms were identified in either the infected crop or other crops on the farms.     

Detection of Erwinia herbicola pv. gypsophilae in gypsophila plants by PCR

Three PCR primer pairs, based on the cytokinins (etz) or IAA biosynthetic genes, were used for detecting Erwinia herbicola pv. gypsophilae in Gypsophila paniculata plants. The primers were specific to all gall-forming E. herbicola strains and distinguished them from saprophytic strains associated with gypsophila plants or from other gall-forming bacteria. In pure culture of the pathogen, less than one bacterial cell was detected with nested PCR using the etz primers - an increase of 100-fold in sensitivity as compared with single-round PCR. In the presence of plant extract a reduction of tenfold in sensitivity was observed by nested PCR. When cells were grown on a semi-selective medium prior to PCR (Bio-PCR), five cells from pure culture of the pathogen were detected. The bacteria could be detected by nested-PCR or Bio-PCR in symptomless gypsophila cuttings after 7 days. The Bio-PCR procedure described in this study can be used to establish disease-free nuclear stock of mother plants of gypsophila.     

Survival,distribution and genetic variability of inoculum of the strawberry red core pathogen,Phytophthora fragariae var. fragariae,in soil

An experimental field infested with Phytophthora fragariae var. fragariae (Pff) and used for strawberry red core fungicide and cultivar resistance trials until 1981 was surveyed for the presence of inoculum of the pathogen 11 and 12 years later. Alpine strawberries, highly susceptible to all races of Pff, were grown from true seed and planted as a bait crop on a 0·5 m‐spaced grid. Rapid and widespread red core infection was observed, which provided good evidence that oospores had survived in soil for this extended period. Site elevation and the distribution of red core infected plants showed a strong correlation, with a higher frequency of infected and dead plants in the lowest areas of the field. The race designation of 18 recovered isolates were determined and AFLP fingerprint patterns of some of these and their single‐spore derivatives were analysed. The isolates differed little in race type, and the majority were genetically identical at 433 AFLP loci. Races used to inoculate the site in the 1970s were recovered. The fingerprints of the single variant isolate matched that of an isolation made by Hickman in the 1950s, originally used to inoculate the site. Clearly Pff is a very stable and long‐lived pathogen able to retain its genetic integrity and lie dormant in soil for many years, ensuring its survival between epidemiologically favourable conditions which occur erratically.     

Simple diagnosis using ethanol immersion of strawberry plants with latent infection by Colletotrichum acutatum, Dendrophoma obscurans, and Fusarium oxysporum f. sp. fragariae

Simple diagnosis by ethanol immersion (SDEI) to detect Glomerella cingulata was used to detect three other fungi that also cause latent infection of strawberry plants. Signs on strawberry leaves with asymptomatic latent infection by Colletotrichum acutatum became visible using SDEI. Salmon-pink conidial masses were produced in the acervuli on the treated leaves 5 days after incubation at 28°C. In the case of Dendrophoma obscurans, pycnidia with amber conidial masses formed 5 days after incubation at 28°C. The pycnidia were observed mainly on the ribs, and conidial masses exuded from the ostiole. These macroscopic conidial masses were similar to those of G. cingulata and C. acutatum. When water was dripped onto a lesion caused by D. obscurans, the pycnidia exuded white filamentous conidial masses, making the distinction of D. obscurans from G. cingulata or C. acutatum. On petioles with latent infection by Fusarium oxysporum f. sp. fragariae, white aerial hyphae grew out from the vascular tissues on the cut surface 3 days after incubation at 28°C and were easily observed by eye or with a loupe. Thus, SDEI was also useful for diagnosing latent infection of strawberry plants by C. acutatum, D. obscurans, and F. oxysporum f. sp. fragariae.     

Detection of Xanthomonas campestris pv. pelargonii in geranium and greenhouse nutrient solution by serological and PCR techniques

Specificity of a new monoclonal antibody, 2H5, to Xanthomonas campestris pv. pelargonii, causal agent of geranium bacterial blight, was determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence tests on 14 strains of X. c. pelargonii, 12 strains of other X. campestris pathovars, 3 strains of other Xanthomonas spp., 3 strains of other plant pathogens, and 43 saprophytic bacteria isolated from geranium. X. c. pelargonii was detected in tissue from symptomatic and asymptomatic geraniums sampled from commercial growers, and artificially inoculated plants, by monoclonal antibody-based tests. The intensity of response in ELISA was only moderately correlated (r = 0.56) with symptom severity, while symptom severity was not correlated (r = 0.16) with the number of fluorescing cells in immunofluorescence. The bimodal frequency distribution of ELISA and immunofluorescence results served to validate arbitrarily chosen positive/negative threshold values. Most positive ELISA and immunofluorescence test results were confirmed by the polymerase chain reaction (PCR) using published primers (Manulis et al., 1994. Appl. Environ. Microbiol 60, 4094-4099). In contrast to plant tissue, the bacterium was detected in greenhouse nutrient solution with greater sensitivity by immunofluorescence and PCR than by ELISA. Sensitivity of detection was enhanced 100-fold by concentration of the bacteria by centrifugation.