番茄细菌性溃疡病苗期接种新方法的研究

 在剪叶、浸根和针刺等细菌病害传统接种方法基础上设计了打顶接种新方法;以番茄细菌性溃疡病菌中国菌株和美国菌株借助打顶法接种佳粉10、合作908和华南红宝石3个国内主栽番茄品种2~3片真叶期幼苗,在昼夜最低、最高温度15~18℃、32~35℃和相对湿度为30%~60%的温室条件下,打顶法接种番茄幼苗后溃疡病发病率为87.5%~100.0%,病情指数随接种菌悬液浓度提高而增大,达到23.96~82.29,3个供试品种之间表现稳定一致;剪叶、浸根和针刺接种方法的发病率普遍在40%以下,病情指数在20以下,3个供试品种之间未表现明显差异;进一步研究显示,使用选择性培养基mSCM能够从打顶法接种后的发病植株上获得培养性状与原接种体一致的分离物,专化性免疫凝聚试剂盒检测到特定的测试线,PCR扩增到614bp的特异性条带,证实该分离物为番茄细菌性溃疡病菌,发病植株为接种体侵染所致。该研究结果表明,打顶接种方法比剪叶法、针刺法和浸根法更适合用于番茄溃疡病菌的致病性测定和评价不同番茄品种苗期的抗病性,具有应用价值。     

Effects of nutrient solution pH on the survival and transmission of Clavibacter michiganensis ssp. michiganensis in hydroponically grown tomatoes

The effect of pH on the survival of Clavibacter michiganensis ssp. michiganensis and its transmission via roots of tomato in hydroponic culture was studied in laboratory and greenhouse experiments. In a laboratory experiment, C . m. ssp. michiganensis could not survive for 24 h in nutrient solutions with a pH of 4·0 or 4·5, while 1, 14, 51 and 62% of inoculum survived at pH 5·0, 5·5, 6·0 and 6·5, respectively. When tomato plants were inoculated with C . m. ssp. michiganensis through wounds on the stems, the bacteria moved downward from the inoculation site to the roots and infectious bacteria were released from the roots into the nutrient solution. Of two pH regimes tested in greenhouse nutrient-film technique (NFT) culture, the C . m. ssp. michiganensis population was significantly lower in pH 5·0 than in pH 6·5 in most sampling data. In treatments in which C . m. ssp. michiganensis was introduced by transplanting two root-inoculated plants, significantly more plants developed canker at pH 6·5 (34 out of 48 plants) than at pH 5·0 (11 out of 48 plants). When the bacterium was introduced by transplanting two stem-inoculated plants at pH 6·5, seven out of 24 plants developed canker. The potential of pH manipulation in controlling tomato bacterial canker in hydroponic culture is discussed.     

The significance of guttation in the secondary spread of Clavibacter michiganensis subsp. michiganensis in tomato greenhouses

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), can spread in commercial tomato greenhouses causing epidemics. Results of greenhouse experiments with Cmm‐contaminated tools demonstrated disease spread for only a limited distance (<4 plants) from infected plants. However, touching symptomless infected plants bearing guttation droplets prior to touching nearby plants spread the pathogen over longer distances within rows (>22 plants). The pathogen was exuded in large numbers in the guttation fluid of infected plants; its presence in the guttation fluid was not influenced by the inoculation procedures, leaf age or the volume of the guttation droplets. Population size of Cmm and the incidence of leaflets with epiphytic bacteria were significantly higher in plants placed in a guttation‐induction chamber than in those kept in a growth chamber with high humidity, suggesting exudation through guttation contributed to the formation of epiphytic populations on leaflets. This new knowledge may provide a simple and environmentally friendly means for decreasing the spread of the disease by avoiding contact with plants during periods when they bear guttation droplets.     

Detection of Clavibacter michiganensis ssp. michiganensis in tomato seeds by immunofluorescence microscopy and dilution plating

A method for detectingClavibacter michiganensis ssp.michiganensis in tomato seeds was evaluated. The method is based on rapid screening of tomato seed lots using indirect immunofluorescence staining (IF), followed by dilution plating of IF positive seed lots. Different polyclonal antisera, prepared againstC. michiganensis ssp.michiganensis were tested for their specificity using IF. All strains ofC. michiganensis ssp.michiganensis tested reacted with the polyclonal antisera. Two of nine saprophytic isolates from tomato seeds were positive with the antisera as well as with the control normal serum, but cells of these isolates were distinct in shape from cells ofC. michiganensis ssp.michiganensis.For extraction of the pathogen from the seed, seeds were either blended with a stomacher or soaked at 4–6 °C. The stomacher method yielded more fluorescent cells in IF than 24 h soaking of seed samples. However, soaking of seeds for 48 h generally yielded less saprophytes and overall higher numbers ofC. michiganensis ssp.michiganensis colonies in dilution plating when compared to blending by a stomacher. SCM medium was generally more selective than KBT and modified CNS medium. However, the efficacy of the medium was dependent on the seed lot and/or extraction method used. Confirmation of suspected colonies with YDC (yeast-dextrose-carbonate medium), IF and a pathogenicity test on tomato seedlings proved to be highly reliable (P>0.95). For routine testing of seed lots it is recommended to screen tomato seed lost after soaking seeds for 24 h at 4–6 °C with IF, followed by plating of IF-positive seed lots on modified CNS and SCM after soaking seeds for an additional 24 h.     

Survival of Clavibacter michiganensis subsp. michiganensis in tomato debris under greenhouse conditions

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis, is one of the most important diseases of tomato worldwide. Once the pathogen has been introduced into an area, i.e. by contaminated seeds or transplants, it survives mainly on host debris. In different geographic areas the survival time of the pathogen in crop residues under field conditions has been very variable, ranging from 2 months in Morocco to 2 years in Iowa (USA). This study took place in the horticultural belt of Buenos Aires – La Plata, Argentina, where greenhouse production prevails, and monoculture with two production cycles per year is a common practice. The aim was to determine the survival time of this pathogen in plant residues left on the soil surface or buried. During three consecutive years, by the end of both production cycles in July (winter) and December (summer), above‐ (stem, petiole) and belowground (root) tissues were placed into nylon netting bags and left on the soil surface or buried at 10 cm depth. The pathogen population was regularly quantified by dilution plating on semiselective medium. In host debris left on the soil surface, bacteria survived 120–260 days for crop production cycles that ended in winter and 45–75 days for those that ended in summer. In stems or roots buried in winter, this period was 45–75 days. It is concluded that host debris, including roots, might be an important primary inoculum source of the pathogen in greenhouses.     

Clonal populations of Clavibacter michiganensis subsp. michiganensis are responsible for the outbreaks of bacterial canker in greenhouse tomatoes in Italy

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected in greenhouses from 17 farms during tomato bacterial canker outbreaks occurring between 2005 and 2008 in Sicily, were analysed by a multiphasic approach. Population studies were conducted to investigate the possible sources of inocula. Cmm strains were characterized by PCR assays targeting virulence genes, fingerprinting techniques, metabolic profiles and virulence. These strains were comparatively analysed with Cmm strains isolated in other parts of Italy over a period of 15 years. Chromosomal genes encoding virulence determinants tomA, ppaA, chpC, and the plasmid‐encoded genes pat‐1 and celA were detected by PCR in all tested strains, except for four Sicilian Cmm strains where the pat‐1 gene was not amplified. Using BOX‐PCR, Cmm strains were differentiated into 13 haplotypes and clonal populations were identified. Cmm strains isolated from different farms in 2008 showed the same BOX‐PCR haplotype. A distinct BOX‐PCR haplotype was obtained from atypical Cmm strains lacking pat‐1 and isolated in 2006/7 from three farms. Cmm strains with two different haplotypes were detected in one farm, whereas the other farms contained strains with only a single haplotype. A new fAFLP protocol based on the amplification of ApaI/MseI fragments was developed and was able to differentiate C. michiganensis subspecies. Different populations were delineated for the multiple outbreaks occurring in Sicily, whereas similar populations were recorded in other Italian regions over a period of 12 years. The results are consistent with previous studies that demonstrate that Cmm outbreaks are associated with propagation material.     

Comparative study of genetic diversity of Clavibacter michiganensis subsp. michiganensis isolates from the Canary Islands by RAPD-PCR, BOX-PCR and AFLP

Molecular characterization of seedborne pathogens is an important issue when discerning their origin and tracking the spread of a disease. In the Canary Islands (Spain), Clavibacter michiganensis subsp. michiganensis (Cmm) was first detected in 2002, causing severe losses in many tomato-growing areas. Fifty four strains of this bacterium isolated from 2002 to 2007 and 19 strains from different countries were characterized for genetic diversity. RAPD-PCR, BOX-PCR and AFLP provided differentiation among Cmm strains whereas no differences were observed with ERIC-PCR, REP-PCR and 16S-23S ITS PCR-RFLP. RAPD-PCR and BOX-PCR revealed high homogeneity among the Canary Island strains (>80 and >75% of similarity, respectively) which could not be grouped based on tomato cultivar, location or year of isolation. By contrast, strains of Cmm from other countries displayed high diversity, providing several clusters, most of which were composed of a single strain. Similarly, AFLP analysis of 29 selected strains of Cmm gave the same profile for the Canarian ones (>90% of similarity) whereas high polymorphism was obtained with strains from different countries. Moreover, two strains, one from the USA and another from Spain, were related to the Canarian strains, according to RAPD-PCR (>60% of similarity), BOX-PCR (>75%) and AFLP analysis (>90%), suggesting a common origin. The circumstances under which the Cmm outbreaks occurred in the Canary Islands and the high homogeneity observed among the Canarian strains would suggest that the bacterium was introduced into the region from only one origin.     

Effects of plant age on disease development and virulence of Clavibacter michiganensis subsp. michiganensis on tomato

The effect of plant age at the time of inoculation on the severity of bacterial wilt and canker disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) was examined in six greenhouse experiments. The period during which inoculations led to wilt and death of tomato plants was defined. This period, designated ‘window of vulnerability’, ranged from transplanting to the 17‐ to 18‐leaf stage. Plants inoculated after this period expressed disease symptoms but did not wilt or die. No significant changes in disease incidence were observed when leaves of different ages were inoculated. Yield accumulation was significantly reduced in plants inoculated within the window of vulnerability compared with those inoculated after this period. Expression of virulence genes, viz. celA, encoding a secreted cellulase, and the serine protease‐encoding pat‐1, chpC and ppaA, was induced during the early stages after inoculation in plants inoculated within the window of vulnerability. Differences in Cmm population between plants inoculated within and outside of this period were insignificant after the first week post‐inoculation, indicating that differences in disease severity, yield loss and expression of virulence determinants are not correlated with Cmm population level. Results suggest that implementation of precautionary measures during the window of vulnerability to avoid secondary spread of Cmm will have a season‐long effect on plant mortality and may minimize, or even prevent, yield losses.     

Molecular typing and spread of Clavibacter michiganensis subsp. michiganensis in greenhouses in Japan

Clavibacter michiganensis subsp. michiganensis (CMM) strains collected between 2005-2008 from greenhouses in different locations in Okayama Prefecture, Japan, were fingerprinted by repetitive sequence-based polymerase chain reaction (rep-PCR) with ERIC and BOX primers. One hundred and eighty strains from eight different locations in Okayama were differentiated into four haplotypes (A to D) based on rep-PCR. Regardless of the year of isolation, location or cultivar of tomato, the strains in each greenhouse and location belonged to the same haplotype, suggesting the strains originated from the previous greenhouse population. Based on Morisita's index of dispersion ( I _δ ), the distribution of diseased plants in the greenhouses, where disbudding and defoliation using either scissors or by hand were carried out in the same direction to promote the spread of CMM, occurred in an aggregated distribution in a quadrant along a row of plants, but the distribution of diseased plants indicated a random distribution in a quadrant along a furrow of plants (two adjoining rows of plants). These results showed that disbudding and defoliation contribute highly to the secondary spread of bacterial canker in commercial greenhouses.     

番茄溃疡病菌PCR快速检测技术

番茄溃疡病是一种严重危害番茄生产的细菌性病害，许多国家将其列为检疫性病害。利用ITS通用引物扩增了番茄溃疡病菌（Clavibacter michiganensis subsp．michiganensis）的ITS序列，并进行克隆测序。根据序列比较结果设计了引物BT1和BT2，该引物特异性好，能专一扩增出268bp电泳条带，而马铃薯环腐病菌等不同亚种、不同属的细菌及健康的番茄材料均无扩增条带。从接种但未显症番茄苗叶片及人工模拟染菌种子上提取总DNA，以此为模板均能稳定地扩增出特异性目的条带。该方法直接对种子或植株进行检测，不需进行病原菌分离培养，快速简便，适用于出入境检验检疫及种苗健康检测领域。     

Effect of phytotoxic compounds produced by Clavibacter michiganensis subsp. michiganensis on resistant and susceptible tomato plants

A phytotoxic fraction of high molecular weight was isolated from the culture filtrate ofClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker of tomato, and partly purified. This high molecular weight fraction consists of sugars and a minor protein moiety and is therefore probably of similar nature to that of the toxin fromC. michiganensis subsp.michiganensis reported earlier in literature.The high molecular weight fraction was albe to induce wilting, the predominant symptom of the disease, as shown in a bioassay with tomato cuttings. However, this wilting reaction turned out to be non-specific in the bioassay, since (partially) resistant and susceptible genotypes responded similarly. No correlation could be found between the degree of virulence of fiveC. michiganensis subsp.michiganensis strains and the amount of the phytotoxic high molecular weight fraction produced in vitro.As the isolated high molecular weight fraction showed a phytotoxic effect on tomato plants it is worthwhile to test its potential for use as a selective agent in in vitro selection.Samenvatting Een fytotoxische fractie werd geïsoleerd uit cultuurfiltraat vanClavibacter michiganensis subsp.michiganensis, de veroorzaker van de bacterieverwelkingsziekte bij tomaat. Een eerste karakterisering toonde aan dat deze toxische fractie hoog-moleculaire component(en) bevat, bestaande uit polysacchariden en een gering percentage eiwit. Dit is in overeenstemming met toxines vanC. michiganensis subsp.michiganensis die al eerder beschreven zijn.Deze hoogmoleculaire toxische fractie was in staat verwelking te induceren van stengeltoppen van verschillendeLycopersicon esculentum enL. peruvianum genotypen in een bioassay. Gewichtsverandering van de stengeltoppen, uitgedrukt als percentage ten opzichte van het begingewicht, werd gebruikt als parameter voor verwelking. De toxische fractie reageerde niet-specifiek in de bioassay, want er werd geen verschil gevonden in respons van (partieel) resistente en gevoelige genotypen. Er bleek geen correlatie te zijn tussen de mate van virulentie van verschillende isolaten vanC. michiganensis subsp.michiganensis en de hoeveelheid van de toxische fractie geproduceerd in vitro.Het mogelijke gebruik van deze hoogmoleculaire toxische fractie als selectief agens bij in vitro selectie zal nader onderzocht worden.     

Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media‐based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 10¹ to 10⁵ CFU per seed; one contaminated seed was mixed into 1000‐seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).     

Characterization of a Clavibacter michiganensis subsp. michiganensis population in Israel

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected during the last decade from different locations in Israel, were analyzed by macrorestriction pulsed-field gel electrophoresis (PFGE). Fifty-eight strains from Israel and 18 from other sources were differentiated into 11 haplotypes with either VspI or DraI restriction enzymes. The strains from Israel formed four distinct groups among which groups A (16 strains) and B (32 strains) constituted the major clusters. These two groups originated from the Besor region, which is the main area for growing tomatoes under cover. Rep-PCR, with either ERIC or BOX primers, confirmed results obtained by PFGE. PCR with primers based on three genes – ppaA, chpC and tomA – that spanned the pathogenicity island of the reference strain NCPPB382, produced the expected products with the tested pathogenic strains. Plasmid analysis of representative strains revealed different profiles of one or two plasmids. However all the strains, including five non-pathogenic ones, reacted positively in PCR with primers based on celA gene, which resides on the plasmid pCM1 of NCPPB382. Southern hybridization of total DNA with a 3.2-kb BglII-fragment of pCM1 containing the celA gene was positive when carried out with 31 strains, but the size of the reacting band was not always the same as that of pCM1, suggesting that the plasmids carrying celA may differ in size. Comparison between the colonization rates of strain Cmm42 (group A) and of Cmm32 (group B) did not show any significant differences. The high diversity of the Cmm strains, on the one hand, and the presence of two persistent groups in the Besor region, on the other hand, suggests that the primary inoculum originated each year from residual plants in the soil rather than from infested seeds, in spite of extensive control measures taken by the growers in this area.     

Temperature at the early stages of Clavibacter michiganensis subsp. michiganensis infection affects bacterial canker development and virulence gene expression

Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial canker and wilt, causes severe economic losses in tomato net‐houses and greenhouses worldwide. In this study, seedlings which were transplanted and inoculated monthly over 2 years wilted and died earlier in the spring (21–24°C) and autumn (18–23°C) than in the winter (15–18°C) and summer (28–31°C): T₅₀ (the time taken for 50% of the plants to wilt or die) was 2 and 3–4 months after inoculation, respectively. A highly significant correlation was found between the average temperatures during the first month after inoculation and T₅₀; the shortest T₅₀ mortality (70 days) was observed for an average temperature of 26°C. Expression of virulence genes (pat‐1, celA, chpC and ppaA) by Cmm was higher in plants inoculated in the spring than in those inoculated in the summer. In another set of experiments, seedlings were inoculated and maintained in controlled‐environment growth chambers for 2 weeks. Subsequently, they were transplanted and maintained in commercial‐type greenhouses for 4–5 months. The temperatures prevailing in the first 48 h after inoculation were found to affect Cmm population size and virulence gene expression and to have season‐long effects on bacterial canker development.     

Differentiation of Clavibacter michiganensis subsp. michiganensis from seed-borne saprophytes using ELISA, Biolog and 16S rDNA sequencing

Specificity of a monoclonal antibody (MAb), Cmm1, to geographically diverse strains of the seed-borne tomato pathogen, Clavibacter michiganensis subsp. michiganensis (Cmm), was assessed and the MAb was tested for its usefulness as a tool to separate the pathogen from saprophytes in naturally infested tomato seed. Of the 236 international Cmm strains tested, 99% reacted with MAb Cmm1. MAb Cmm1 was also strongly reactive with an additional 32 strains isolated from seed that were later identified as Cmm by the Biolog MicroLog™ microbial identification system (Biolog, Inc., Hayward, CA) and 16S rDNA sequence analysis. It correctly differentiated these strains from 12 MAb Cmm1-negative seed strains that possessed similar colony morphology but were later identified as other Gram-positive genera and species. The specificity of MAb Cmm1 to the pathogen and the near universality of the MAb Cmm1-reactive antigen among diverse Cmm strains make this antibody a useful detection and identification tool. The finding that a large proportion of the Cmm strains associated with naturally infested tomato seed were putatively hypovirulent or non-virulent indicates that such populations cannot be ignored and points to a need for studies to determine their significance in host-pathogen interactions.     

Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds

Plasmopara halstedii , the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii . The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µ L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes.     

Quantification of viable cells of Clavibacter michiganensis subsp. michiganensis using a DNA binding dye and a real-time PCR assay

Viable cells of Clavibacter michiganensis subsp. michiganensis (CMM), the causal agent of bacterial canker of tomato, were discriminated from the dead cells by quantitative real-time polymerase chain reaction (PCR), after the bacterial solution was treated with the DNA binding dye ethidium monoazide (EMA). The primers and TaqMan probe, based on the 16S-23S rDNA spacer sequences, were highly specific for CMM at the subspecies level. The detection limit of the direct real-time PCR was 10³ colony forming units per mL (cfu mL⁻¹) in samples and with an apparent sensitivity of 2 cfu of target cells in PCR reaction solution. Application of this method allows for selective quantification of viable cells of CMM and facilitates monitoring the pathogen in tomato seeds.     

Survival of Clavibacter michiganensis ssp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco

The survival and half-life of Clavibacter michiganensis ssp. michiganensis ( C. michiganensis ), the causal agent of bacterial canker of tomato, were determined in infected plant debris under natural field conditions in California, Ohio and Morocco using a semiselective agar medium. The organism survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered in high amounts from tomato stems left on the soil surface for 314 days in Ohio and California, USA, and for 194 and 132 days in Melk Zhar and Aït Melloul, Morocco, respectively; it was recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Aït Melloul and Melk Zhar. The half-life of the pathogen in stems left on the soil surface ranged from 23·2 to 24·8 days in the USA, and from 7·8 to 12·3 days in Morocco, whereas the half-life in buried stems ranged from 14·0 to 16·7 days in the USA and from 3·7 to 9·5 days in Morocco. Based on the half-life data, the predicted survival times of C. michiganensis in stems on the soil surface in Ohio, California, Melk Zhar and Aït Melloul would be up to 822, 770, 424 and 261 days, respectively, while the predicted survival times in stems buried in the soil would be 541, 497, 305 and 128 days, respectively. These results show that the survival and half-life of C. michiganensis in plant debris are relatively long and are influenced by both tissue exposure and geographic location.     

不同来源番茄溃疡病菌致病力差异研究

采用打顶法接种、半选择性培养基再分离发病植株中的病原菌,以及特异性PCR验证方法,对来自3个国家9个不同地区的46株番茄溃疡病菌进行了致病性测定,以病情指数评价不同菌株的致病力。结果显示,分离自我国河北滦平县、内蒙古包头市等地的24株菌株的病情指数达到75以上,属于强致病力水平;11株菌株的病情指数为50~75,属于中等致病力;而9株菌株的病情指数为50以下,属于弱致病力;检测同时证实,有2株属于无致病力菌株。强致病力、中等致病力、弱致病力和无致病力菌株占供试菌株总数的比例分别为52.2%、23.9%、19.6%和4.3%,表明供试的46株番茄溃疡病菌存在不同程度的致病力差异。     

Internal methods comparison study and inter‐laboratory study on Clavibacter michiganensis subsp. michiganensis in tomato seeds1

Bacterial canker of tomato is a disease caused by Clavibacter michiganensis subsp. michiganensis, a quarantine bacterium, the spread of which has not been completely controlled in spite of the phytosanitary measures taken within the EPPO region. Since 2008 the French National Laboratory for Plant Health (LNPV) has been working on the assessment of the methods used in laboratories to detect the presence of Clavibacter michiganensis subsp. michiganensis in tomato seeds i.e. dilution plating on semi‐selective media and immunofluorescence. In the 1st stage of the assessment, a methods comparison study was performed with reference strains to determine the performance criteria of the tests in optimal conditions. In the 2nd stage, an inter‐laboratory study on naturally and artificially contaminated seeds was performed with 8 laboratories from 6 European countries. This study demonstrated the strengths and weaknesses of the tests currently in use. Two laboratories took the opportunity the collaborative study offered to evaluate alternative tests: BIO‐PCR and IMS‐plating. These could offer interesting alternatives to optimise the detection procedure for Clavibacter michiganensis subsp. michiganensis on tomato seeds.