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.     

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.     

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。     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Development of a tomato plant resistant to Clavibacter michiganensis using the endolysin gene of bacteriophage CMP1 as a transgene

The bacteriophage CMP1 endolysin gene (lys), encoding, a peptidase that was shown to effectively reduce Clavibacter michiganensis by specifically hydrolysing its murein, was transferred into tomato plants by Agrobacterium‐mediated transformation. The presence of the gene was verified by PCR and the gene product was confirmed in immunoblots and stably expressed over three generations. Transgenic tomato plants did not show disease symptoms after infection with C. michiganensis subsp. michiganensis, despite the fact that small amounts of bacteria could still be identified in xylem sap and leaf extracts, although in significantly reduced amounts.     

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。