Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection

The vascular pathogen Clavibacter michiganensis subsp. michiganensis is responsible for bacterial wilt and canker of tomato. Pathogenicity of this bacterium is dependent on plasmid-borne virulence factors and serine proteases located on the chromosomal chp/tomA pathogenicity island (PAI). In this study, colonization patterns and movement of C. michiganensis subsp. michiganensis during tomato infection was examined using a green fluorescent protein (GFP)-labeled strain. A plasmid expressing GFP in C. michiganensis subsp. michiganensis was constructed and found to be stable in planta for at least 1 month. Confocal laser-scanning microscopy (CLSM) of inoculated stems showed that the pathogen extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem. Acropetal movement of the wild-type strain C. michiganensis subsp. michiganensis NCPPB382 (Cmm382) in tomato resulted in an extensive systemic colonization of the whole plant reaching the apical region after 15 days, whereas Cmm100 (lacking the plasmids pCM1 and pCM2) or Cmm27 (lacking the chp/tomA PAI) remained confined to the area surrounding of the inoculation site. Cmm382 formed biofilm-like structures composed of large bacterial aggregates on the interior of xylem walls as observed by CLSM and scanning electron microscopy. These findings suggest that virulence factors located on the chp/tomA PAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates.     

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。     

Colonization of tomato seedlings by bioluminescent Clavibacter michiganensis subsp. michiganensis under different humidity regimes

Tomato bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis, is transmitted by infected or infested seed and mechanically from plant to plant. Wounds occurring during seedling production and crop maintenance facilitate the dissemination of the pathogen. However, the effects of environmental factors on C. michiganensis subsp. michiganensis translocation and growth as an endophyte have not been fully elucidated. A virulent, stable, constitutively bioluminescent C. michiganensis subsp. michiganensis strain BL-Cmm 17 coupled with an in vivo imaging system allowed visualization of the C. michiganensis subsp. michiganensis colonization process in tomato seedlings in real time. The dynamics of bacterial infection in seedlings through wounds were compared under low (45%) and high (83%) relative humidity. Bacteria multiplied rapidly in cotyledon petioles remaining after clip inoculation and moved in the stem toward both root and shoot. Luminescent signals were also observed in tomato seedling roots over time, and root development was reduced in inoculated plants maintained under both humidity regimes. Wilting was more severe in seedlings under high-humidity regimes. A strong positive correlation between light intensity and bacterial population in planta suggests that bioluminescent C. michiganensis subsp. michiganensis strains will be useful in evaluating the efficacy of bactericides and host resistance.     

Bacterial canker of tomatoes — histological characterization of cultivar resistance and seed transmission

Journal of Plant Diseases and Protection - Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial canker in tomatoes representing a significant threat to worldwide tomato production....     

Silencing of host basal defense response-related gene expression increases susceptibility of Nicotiana benthamiana to Clavibacter michiganensis subsp. michiganensis

Clavibacter michiganensis subsp. michiganensis is an actinomycete, causing bacterial wilt and canker disease of tomato (Solanum lycopersicum). We used virus-induced gene silencing (VIGS) to identify genes playing a role in host basal defense response to C. michiganensis subsp. michiganensis infection using Nicotiana benthamiana as a model plant. A preliminary VIGS screen comprising 160 genes from tomato known to be involved in defense-related signaling identified a set of 14 genes whose suppression led to altered host-pathogen interactions. Expression of each of these genes and three additional targets was then suppressed in larger-scale VIGS experiments and the effect of silencing on development of wilt disease symptoms and bacterial growth during an N. benthamiana-C. michiganensis subsp. michiganensis compatible interaction was determined. Disease susceptibility and in planta bacterial population size were enhanced by silencing genes encoding N. benthamiana homologs of ubiquitin activating enzyme, snakin-2, extensin-like protein, divinyl ether synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase 2, and Pto-like kinase. The identification of genes having a role in the host basal defense-response to C. michiganensis subsp. michiganensis advances our understanding of the plant responses activated by C. michiganensis subsp. michiganensis and raises possibilities for devising novel and effective molecular strategies to control bacterial canker and wilt in tomato.     

番茄细菌性溃疡病菌的实时荧光PCR检测

 由Clavibacter michiganensis subsp.michiganensis(Cmm)引起的番茄细菌性溃疡病是一种严重危害番茄生产的种传细菌性病害。根据ITS序列多态性设计引物及TaqMan探针进行实时荧光PCR检测的结果表明,这组引物一探针能检测出所有供试的Cmm菌,对照菌均未检测到荧光信号。用接种但未显示症状的番茄苗叶片及人工处理的带菌种子提取的核酸作为模板,均能检测到病菌,其检测灵敏度比常规PCR高约100倍。实验中不需病原菌的分离培养及PCR的后续处理。该方法快速、简便、安全、准确,适用于出入境检验检疫及种子、种苗健康检测领域。     

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.     

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。     

Induction of the viable but nonculturable state in Clavibacter michiganensis subsp. michiganensis and in planta resuscitation of the cells on tomato seedlings

Bacterial canker of tomato is an economically important seedborne disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm). Copper‐based bactericides and seed treatment with hydrochloric acid are commonly used for bacterial canker management. Recent studies have shown that some bacteria can enter a viable but nonculturable (VBNC) state, and fail to form colonies on microbiological agar media. Bacteria in the VBNC state can recover their culturability when returned to favourable conditions. This study reports the induction of the VBNC state in Cmm by CuSO₄ and low pH, and resuscitation of VBNC cells on tomato seedlings. Flow cytometry using the nucleic acid dyes SYTO 9 and propidium iodide, combined with agar plating, was used to assess VBNC cell counts. It was demonstrated that CuSO₄ and low pH induced the VBNC state in Cmm and the rate of induction increased with copper ion concentration and acidity. Pathogenicity tests showed that some of the VBNC cells induced by CuSO₄ retained their ability to colonize tomato seedlings but failed to produce typical bacterial canker symptoms by 2 months post‐inoculation. This was probably due to low levels of resuscitation of VBNC Cmm cells resulting in low levels of initial inoculum. This study has improved understanding of the VBNC state of Gram‐positive phytopathogenic bacteria. Most importantly, because copper‐based chemicals and low pH conditions are used for disease management, induction of the VBNC state and subsequent resuscitation of Cmm cells on tomato seedlings may limit pathogen detection by culture‐based assays yet present a risk for disease development in the field.     

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。     

Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds

Several seed extraction procedures, used for detection of Clavibacter michiganensis ssp. michiganensis ( Cmm ) in naturally infected and artificially infested tomato seed lots were evaluated. Extraction methods that included grinding the seeds were significantly better at detecting the pathogen in three different seed lots than methods that used only soaking. The detection threshold of Cmm in relation to seed sample size was determined by adding naturally infected seeds into samples of three different sizes. Cmm was detected by agar plating assay, on three media (CNS, mSCM, D₂ANX), and by direct PCR from seeds and Bio-PCR (bacteria cultured on agar media prior to PCR). In samples of 10 000 seeds containing one infected seed, Cmm could be detected only by Bio-PCR and in only one replicate out of five. In samples containing five or 10 infected seeds per 10 000 seeds, three of five and five of five replicates, respectively, were detected by the three detection methods. In samples of 5000 seeds, one infected seed could be detected in all five replicates only after adding a concentration step. A high correlation ( R ² = 0·9448) between artificially infested seeds and the disease incidence was found. Seed lots infested with less than 58 colony-forming units (CFU) per g did not cause disease under glasshouse conditions, whereas lots with about 1000 CFU g⁻¹ caused disease in 78 plants out of 2000.     

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.     

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.     

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.     

Ingress of Clavibacter michiganensis subsp. michiganensis into Tomato Leaves Through Hydathodes

ABSTRACT Hydathodes of tomato leaves served as extremely efficient infection courts for the bacterial canker pathogen, Clavibacter michiganensis subsp. michiganensis. Chlorotic lesions developed at the tips of leaflet lobes about 2 weeks after inoculation of guttation droplets. Lesions expanded along the leaflet margins and became necrotic. Movement of C. michiganensis subsp. michiganensis from the inoculated leaflet into the rachis was slow and erratic. Histological observations revealed that pathogen populations first developed within large intercellular spaces lying beneath the stomata, which serve as water pores in tomato hydathodes. Bacteria were first observed within vessels of the large marginal fimbriate veins 7 days after inoculation. By 14 days after inoculation, large populations could be seen within the vessels; and by 21 days after inoculation, tissue collapse was widespread and masses of bacteria could be seen in the intercellular spaces and within necrotic cells.     

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.     

Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment. However, only little headway has so far been made in developing biocontrol strategies against C. michiganensis subsp. michiganensis. To address this knowledge gap, we investigated the antagonistic capacity of PCA, produced by Pseudomonas sp. LBUM223, and DAPG and HCN, both produced by Pseudomonas sp. LBUM300, on C. michiganensis subsp. michiganensis under in vitro and in planta conditions. Nonsynthesizing isogenic mutants of the producer strains were also developed to further dissect the role of each individual metabolite on C. michiganensis subsp. michiganensis biological control. Novel specific quantitative polymerase chain reaction TaqMan assays allowed quantification of C. michiganensis subsp. michiganensis in tomato plants and rhizospheric soil. Pseudomonas spp. LBUM223 and LBUM300 significantly repressed C. michiganensis subsp. michiganensis growth in vitro, while their respective nonproducing mutants showed less or no significant antagonistic activity. In planta, only Pseudomonas sp. LBUM300 was capable of significantly reducing disease development and C. michiganensis subsp. michiganensis rhizospheric population, suggesting that the production of both DAPG and HCN was involved. In summary, simultaneous DAPG/HCN production by Pseudomonas sp. LBUM300 shows great potential for controlling bacterial canker of tomato.     

Induction of defence-related enzymes and resistance by the plant activator acibenzolar-S-methyl in tomato seedlings against bacterial canker caused by Clavibacter michiganensis ssp. michiganensis

Using tomato seedlings, the plant defence activator acibenzolar-S-methyl (benzo-[1,2,3]-thiadiazole-7-carbothioic acid-S-methyl ester, ASM; Bion 50 WG) was assayed for its ability to induce resistance against Clavibacter michiganensis ssp. michiganensis , the causal agent of bacterial canker of tomato. In ASM-pretreated plants, reduction in disease severity (up to 76·3%) was correlated with lower bacterial growth (up to 68·2% lower) during the time course of infection. To understand the possible mechanism of action of ASM, alterations in the activities of peroxidase (POX) and chitinase were assessed as markers of resistance. The enhanced resistance of ASM-treated plants was associated with significant increases in the activities of POX and chitinase     

中国北方番茄细菌性溃疡病菌的rep-PCR基因指纹图谱分析

对来自全国若干省市的番茄细菌性溃疡病菌(Clavibacter michiganensis subsp.michiganensis)的33个菌株进行rep-PCR分析。结果表明,用引物BOX分别扩增出6～15条多态性条带,条带大多数集中在500～2800bp之间;用引物ERIC扩增的条带不清晰,可能是反应条件不适合,也可能是其不适合对番茄细菌性溃疡病菌进行多态性分析;BOX-PCR表明番茄细菌性溃疡病菌菌株具有丰富的遗传多态性和较大的遗传变异,对产生的指纹图谱进行分析:在遗传距离为0.18时,测试的33个菌株可划分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ和Ⅶ等7个遗传相似组群,其中Ⅵ组群包含的菌株最多。研究还表明番茄细菌性溃疡病菌的遗传分群与菌株的地理来源关系密切,但与该病的发病年份上没有必然联系,这也从另一侧面说明了该病害是土壤带菌引起的。     

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.