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.     

Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation

Streptomycin has been used for decades in Chile to control Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of tomato bacterial canker. The aim of this work was to evaluate streptomycin resistance and to analyse the presence of resistance-related genes in Cmm strains from Chile. A collection of 25 Cmm strains isolated from different localities in central Chile between 1996 and 2015 was analysed. Minimum inhibitory concentration (MIC) of streptomycin was determined. A search of streptomycin resistance-related genes was carried out in Cmm genomes, and the presence of these genes was studied in all Chilean strains using PCR and sequencing techniques. MIC results showed that four of 25 strains were highly sensitive to streptomycin, with MIC values <2 μg mL⁻¹. The remaining 21 strains possessed MIC of streptomycin ≥100 μg mL⁻¹. The strB gene, encoding an aminoglycoside 6-phosphotransferase that inactivates streptomycin, was detected in all Chilean strains, including sensitive and resistant strains. In the 21 resistant strains, a mutation in codon 43 of the rpsL gene was determined, conferring high streptomycin resistance. Interestingly, the four streptomycin-sensitive Cmm strains did not possess this mutation. This study proposes that the continuous use of streptomycin leads to emergence of resistant Cmm strains, challenging researchers to look for novel alternatives to control this plant pathogenic bacterium.     

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.     

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.     

Inhibitory activities of venom alkaloids of Red Imported Fire Ant against Clavibacter michiganensis subsp. michiganensis in vitro and the application of piperidine alkaloids to manage symptom development of bacterial canker on tomato in the greenhouse

Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (CMM) is a highly destructive disease that has caused major economic losses in tomato production worldwide. In seeking disease management alternatives, the inhibitory activity of alkaloids extracted from the Red Imported Fire Ant was studied in the laboratory and the greenhouse. Piperidine and piperideine alkaloids each significantly inhibited CMM growth on nutrient agar plates. The inhibitory activity of piperidine alkaloids was stable at 4 ^° C and 22 ^° C for 12 weeks and at 54 ^° C for 4 weeks. The growth of CMM was negatively correlated with the concentration of piperidine alkaloids in nutrient broth. In the greenhouse, piperidine alkaloids also significantly reduced the symptom development on two tomato cultivars, Better Boy and DRK7018F1. This is the first demonstration that piperidine and piperideine alkaloids from the Red Imported Fire Ant are highly inhibitory against a plant-pathogenic bacterium, viz. CMM. Piperidine alkaloids could provide satisfactory management of CMM bacterial canker on tomato seedlings in the greenhouse. Our findings may lead to the development of a new group of bactericides.     

Virulence and pCM1 plasmid carriage are related to BOX-PCR fingerprint type in strains of Clavibacter michiganensis subsp. michiganensis that cause bacterial wilt and canker of tomato in Argentina

Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato, producing important economic losses worldwide. Its virulence has been related to several putative virulence factors present on a chromosomal pathogenicity island and on plasmids pCM1 and pCM2, in strain NCPPB382. We genotypically characterized a collection of Cmm isolates from the main greenhouse tomato-producing areas of Argentina by BOX-PCR fingerprinting and screened for the presence of genes and plasmids involved in pathogenicity by PCR. In addition, we evaluated in vitro cellulolytic activity and virulence in planta of selected strains. BOX-PCR fingerprinting clustered strains into four groups. Group II was dominant and included the most virulent strains, while Group III was the smallest and had the least virulent strains. All local strains exhibited similar cellulolytic activity. Most of the examined strains carry two plasmids of similar size to those of NCPPB382, although there were strains with one or three plasmids. By PCR amplification of repA, pCM1 was detected only in strains belonging to Group III, which includes local strains closely related to reference strain NCPPB382. All analysed pathogenicity genes were widespread among strains, and so in strains belonging to Groups I and II, celA found on pCM1 in NCPPB382 could be found in the chromosome or in plasmids other than pCM1. This study contributes to a better understanding of the diversity of Cmm genetic profiles and virulence of strains present in Argentina. Such information could be useful for the selection of strains for screening of host resistance and development of resistant tomato varieties.     

Studies on the colonization of axenically grown tomato plants by a GFP-tagged strain of Clavibacter michiganensis subsp. michiganensis

In this study, colonization and disease development of axenically-grown tomato plants by Clavibacter michiganensis subsp. michiganensis (Cmm), the causative agent of bacterial wilt and canker, was investigated. For this, a spontaneous rifampicin resistant strain of Cmm was tagged with a marker that expressed a green fluorescent protein (GFP) in a stable way and which possessed a similar virulence to the parental strain. In vitro plants were drop-inoculated at the stem base and the population dynamics was determined by dilution pour-plating in a selective medium. At 3 h after inoculation, Cmm was already present in low densities in roots, stems and leaves. At 16 dpi, Cmm was found throughout the entire plant in high densities of ca. 10¹⁰ cfu g^?1. Symptoms developed in the in vitro plants typical for Cmm, such as canker, wilting and growth reduction. The presence of Cmm in vascular and parenchymatic tissue of in vitro tomato plants was confirmed by epifluorescence stereo- and confocal laser scanning microscopy. This study showed that in vitro tomato plants can be effectively used for detailed studies on interactions between Cmm and its host, in particular if a GFP-tagged strain of the pathogen is used.     

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.     

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 an in vitro protocol to screen Clavibacter michiganensis subsp. michiganensis pathogenicity in different Solanum species

Clavibacter michiganensis subsp. michiganensis (Cmm) is a quarantine organism in Europe and in many other countries. It is one of the most severe bacterial pathogens affecting tomato. Screening tomato plants for their resistance level to Cmm requires a large amount of space under quarantine conditions and is therefore costly. This project developed a new inoculation protocol on in vitro tomato plants to facilitate a more economic and higher throughput disease screening. A new method using the PathoScreen system was tested to localize green fluorescent protein-tagged Cmm in planta and to quantify the pathogen based on the percentage of corrected GFP (cGFP%). The system was sensitive in detecting the GFP-tagged Cmm in the shoots, but in the roots a high autofluorescence masked detection and thus sensitivity of the assay. The in vitro protocol was tested on several wild relatives of tomato, which were previously screened in a greenhouse assay. The correlation between wilt symptoms in vitro and wilt symptoms in the greenhouse was overall moderate (r = 0.6462). The protocol worked well in differentiating the two parents that were used in the mapping studies. This study shows that the in vitro protocol can be efficiently used for resistance breeding in many tomato genotypes.     

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。     

Clavibacter michiganensis subsp. michiganesis: Tracking strains using their genetic differentiations by ISSR markers in Southern Turkey

There is no available data published related to the dissemination of bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm) and its genetic diversity in Turkey. It is important to control new introduced inoculum sources by commercial seeds and seedlings. Pathogens were identified by morphological features and the identity was confirmed by PCR amplification using a specific primer PSA-4, PSA-R in addition to microbiological tests. ISSR markers showing high polymorphism ratios were selected and used to characterize Cmm strains. The collected strains were classified into different groups on the basis of ISSR-PCR fingerprints, which showed remarkable genetic specificity and diversity not previously identified in Cmm, suggesting that genetic differences are related to dissemination of the pathogen in the region. This is the first ever study carried out on the characterization of Cmm using ISSR. The selected ISSR primers to characterize Cmm can be used to determine genetic differences in further studies.     

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.     

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.     

Pathogenicity and phylogenetic analysis of Clavibacter michiganensis strains associated with tomato plants in Iran

During 2013–2016, 277 tomato fields were surveyed across Iran to monitor the status of bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis. Altogether, 450 plant samples were collected, both with and without symptoms, from which 35 bacterial strains were recovered. These were positive for the PCR test performed using the Clavibacter‐specific primer pair CMR16F1/CMR16R1. Based on the phylogeny of the gyrB gene sequences, 31, three and one of the 35 strains were identified as C. michiganensis, Microbacterium sp. and Agrococcus sp., respectively. The 31 strains of C. michiganensis were further identified as C. michiganensis subsp. michiganensis (23 strains), C. michiganensis subsp. tessellarius (six strains) and Clavibacter spp. (two strains). This was subsequently confirmed by multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, gyrB, ppk, recA and rpoB). In pathogenicity tests, all 23 strains induced wilting symptoms on tomato plants in greenhouse conditions, while no symptoms were observed on eggplant, bell pepper and chili pepper plants. All evaluated pathogenicity determinant genes (celA, pat‐1, tomA, ppaA, chpC and chpG) were detected in 18 out of 31 C. michiganensis strains, using eight specific primer pairs. Estimation of the number of nucleotide differences, sequence similarity matrix and MLSA clustered two peach‐coloured strains (Tom495 and Tom532) separately from all nine previously described subspecies, thereby suggesting these two strains are a new subspecies of C. michiganensis. However, a detailed taxonomic study using multiphased molecular approaches is needed to delineate a formal taxonomic name for these atypical strains.     

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.     

番茄溃疡病菌单克隆抗体的制备与鉴定

为制备并鉴定番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis,Cmm)的单克隆抗体(McAbs),用全菌皮下免疫BALB/C小鼠,采用B细胞杂交瘤技术,经免疫、融合、间接ELISA筛选和克隆等,获得稳定分泌抗体的阳性杂交瘤细胞株,得到了抗番茄溃疡病菌的单克隆抗体。经免疫后获得3株单抗分别为1A4、1C3和1B7,经亚类鉴定分别是IgM、IgG1、IgG1;纯化腹水间接ELISA效价分别为1:3.2×106、1:8.1×105、1:3.2×106;与其他同属不同亚种无交叉反应。结果表明:3株单克隆抗体均具有较高特异性和敏感性,可作为番茄溃疡病菌的检测抗体,其中,1A4的效果最好。番茄溃疡病菌单克隆抗体的获得为进一步研发番茄溃疡病检测试剂盒奠定了基础。     

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。     

A new selective medium for isolation of Clavibacter michiganensis subsp. michiganensis from tomato plants and seed

A new selective and highly sensitive medium was developed for isolation of Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial canker of tomato, from seed and latently infected plants. The new medium (BCT) proved to be superior to all published semiselective media for Cmm and is denoted as selective medium because of (i) its mean plating efficiency, amounting to ≤89% within 7 days for all 30 Cmm strains from different sources tested; (ii) the high selectivity, because accompanying bacterial species occurring on tomato plants and seed or bacteria obtained from culture collections were inhibited to an extent of 98 to 100%; and (iii) the remarkable detection sensitivity. Thus, 8 CFU of Cmm in field plant homogenates containing 12,750 CFU of accompanying saprophytes were detected on BCT. Under these extreme conditions, all of the published semiselective media (D2, KBT, D2ANX, SCM, mSCM, CMM1, mCNS, and EPPO) gave false-negative results. Either some media were rather toxic and Cmm growth was also inhibited or the other, less toxic media allowed growth of high numbers of saprophytes, so that Cmm growth was suppressed. Exclusively, BCT also supported growth of the closely related C. michiganensis subsp. insidiosus, nebraskensis, and tessellarius. The new medium is recommended for Cmm detection in tomato seed, and in symptomless tomato plantlets, to improve disease control of bacterial canker of tomato.