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.     

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.     

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.     

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....     

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.     

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.     

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.     

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。     

上海地区种植的21个番茄品种抗细菌性溃疡病的鉴定

番茄溃疡病是由密执安棒形杆菌密执安亚种引起的番茄最具毁灭性病害之一,选育和种植抗耐病品种是防治该病最经济有效的防治手段。本研究收集上海种植的21个番茄品种进行温室育苗,在苗期和成株期分别采用打顶法接种进行抗病性测定。分级调查病情,根据病情指数划分反应型,确定供试品种的抗感类型。在供试的21个番茄品种中,只有欧宝318、粉丽、浙粉202和世纪粉冠王表现为中度感病,其余全为高感品种,没有免疫、抗病或耐病品种。     

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.     

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.     

Use of soil solarization for controlling bacterial canker of tomato in plastic houses in Greece

Soil solarization provided effective control of bacterial canker of tomato in plastic houses. Trials in plastic houses in Preveza County, Greece, during the period 1990–1992 showed that soil solarization (approximately 6 weeks of soil mulching with transparent polyethylene sheets) drastically reduced disease incidence throughout the cropping season. In contrast, soil fumigation with a recommended rate of methyl bromide (70 g/m²) was ineffective. Both wild-type and antibiotic-resistant strains of Clavibacter michiganensis subsp. michiganensis , growing on Nutrient Agar Glycerol (NAG) medium within covered and screwed vials and embedded at various soil depths (5, 15 or 25 cm) before soil solarization, were studied. Weekly sampling of bacteria during treatment showed a sharp decline of populations in the solarized soil compared to the non-treated control plots 4–6 weeks after soil tarping. Populations of marked strains infiltrated into tomato stem segments and buried in the soil decreased significantly after 5–6 weeks of solarization compared to non-treated control plots. The data presented here suggest that soil solarization is useful for the control of bacterial canker of tomato in plastic houses in Greece.     

The use of polymerase chain reaction to detect latent infection of Clavibacter michiganensis subsp. michiganensis in tomato seedlings

A method was developed for routine analysis to detect latent infection of Clavibacter michiganensis subsp. michiganensis in samples from tomato seedling lots, before transplant, using polymerase chain reaction. In samples of 300 stem segments 1 cm long, the sensitivity threshold of the method was estimated at around 1.1 × 10³ corynebacteria or 0.33% of latently infected stems. This method was shown to have a good specificity.     

Effect of Bactericides on Population Sizes and Spread of Clavibacter michiganensis subsp. michiganensis on Tomatoes in the Greenhouse and on Disease Development and Crop Yield in the Field

ABSTRACT Chemical applications, with the exception of mancozeb, reduced population sizes and spread of Clavibacter michiganensis subsp. michiganensis among tomato seedlings in the greenhouse and impacted subsequent plant development and yield in the field. While applications of copper hydroxide, copper hydroxide/mancozeb, copper hydroxide/mancozeb (premixed 12 h before spraying), streptomycin, and streptomycin/copper hydroxide to seedlings in the greenhouse did not differ significantly from the inoculated control, the trend was for these treatments to increase the survival of inoculated transplants in the field in comparison to the inoculated control. In the field, inoculated controls produced yields that were 63% (1995) and 51% (1996) of those produced by uninoculated controls. In both years, with the exception of mancozeb in 1995, all treatments resulted in yields similar to those obtained with the uninoculated control. Plant survival and yield in the field were severely affected when transplants had a pathogen population of >/= x 10(8) CFU/g of tissue. All treatments, with the exception of mancozeb, limited C. michiganensis subsp. michiganensis populations to <5.0 x 10(5). None of the treatments significantly reduced the incidence of fruit spotting compared with that of the inoculated control.     

Comparative dynamics of adherent and nonadherent bacterial populations on maize leaves

ABSTRACT The dynamics of the adherent and nonadherent populations of three bacterial species on maize leaves were examined to identify the extent to which bacteria adhere to leaves and the importance of this adhesion to leaf colonization. Pantoea agglomerans strain BRT98, Clavibacter michiganensis subsp. nebraskensis strain GH2390, and Pseudomonas syringae pv. syringae strain HS191R all rapidly adhered to maize leaves following inoculation, but differed in the percentage of cells that adhered to the leaves. Immediately following inoculation, the percentage of adherent cells was highest for the saprophyte P. agglomerans (8 to 10%) and was much lower for the pathogens C. michiganensis subsp. nebras-kensis and P. syringae pv. syringae (2 to 3 and <1%, respectively), although the results for P. syringae pv. syringae HS191R were based on only one experiment. In the 4 days following inoculation, the percentage of the P. agglomerans populations that adhered to the leaves increased to approximately 70%. Similarly, the percentage of C. michiganensis subsp. nebraskensis and P. syringae pv. syringae cells that resisted removal steadily increased in the days following inoculation, although these increases probably reflected both adherence and localization to endophytic sites. Based on differences in the percentage of cells adhering to several cuticular wax mutants of maize, the rapid adherence of C. michiganensis subsp. nebraskensis cells to maize leaves was influenced by the cuticular wax properties, while the rapid adherence of P. agglomerans was not. Finally, bacterial adherence to leaves was advantageous to P. agglomerans survival and growth on leaves based on the finding that the nonadherent populations of the P. agglomerans strain decreased significantly more than did the adherent populations in the 24 h following inoculation, and increased much less than did the adherent populations over the next 3 days. Similar results with the C. michiganensis subsp. nebraskensis and P. syringae pv. syringae strains indicate that bacterial adherence to leaves, bacterial movement to endophytic sites, or both were advantageous to the survival and growth of these strains on leaves.     

Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.     

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.     

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.     

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     

Two Loci from Lycopersicon hirsutum LA407 Confer Resistance to Strains of Clavibacter michiganensis subsp. michiganensis

ABSTRACT We used molecular markers to identify quantitative trait loci (QTL) that contribute to resistance to bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. Resistance was first identified as a marker-trait association in an inbred backcross (IBC) population derived from crossing Lycopersicon hirsutum accession (LA407) with L. esculentum. Single-marker QTL analysis suggested that at least two loci originating from L. hirsutum LA407, Rcm 2.0 on chromosome 2 and Rcm 5.1 on chromosome 5, contribute to resistance in replicated trials. Two segregating F(2) populations were developed by crossing resistant inbred backcross lines (IBLs) to elite L. esculentum lines and used to confirm QTL associations detected in the IBC population. In these populations, realized heritability estimates were higher for selection based on maximal disease than for selection based on disease progression. Realized heritability in the population carrying Rcm 2.0 was 0.63 and 0.14, respectively, for each selection criteria. Realized heritability estimates were 0.85 for selection based on maximal disease and 0.37 for selection based on disease progression in a population carrying Rcm 5.1. The disease response of F(3) families selected for resistance suggested that both Rcm 2.0 and Rcm 5.1 confer resistance to bacterial strains in the repetitive sequence-based polymerase chain reaction DNA fingerprint classes A and C. Markers linked to Rcm 2.0 explained up to 56% of the total phenotypic variation for resistance in one population, and markers linked to Rcm 5.1 explained up to 73% of the total phenotypic variation for resistance in a separate population.