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。     

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.     

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.     

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.     

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.     

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。     

A semiselective medium for the isolation of Acidovorax valerianellae from soil and plant debris

Bacterial black spot of corn-salad ( Valerianella locusta ) caused by Acidovorax valerianellae appeared in France in the beginning of the 1990s. The disease is now widespread in the main area of corn-salad production and is responsible for high depreciation and significant economic losses every year. A semiselective medium is described for the detection of the bacterium in various environments and to identify primary sources of inoculum. The medium TSAV (tryptic soy broth, 3 g L⁻¹, agar 15 g L⁻¹, 5-fluorouracyl 5 mg L⁻¹, novobiocin 5 mg L⁻¹, propiconazole 5 mg L⁻¹) increased chances of obtaining cultures of A. valerianellae from naturally infested soil and from root-debris. Using TSAV, the pathogen was detected in root-debris up to 39 days after the harvest of a diseased crop. As only a few days usually separates harvest from the next sowing date, soil is likely to be an important source of inoculum for the next crop.     

Clavibacter michiganensis ssp. insidiosus in lucerne seeds

Bacterial wilt is a serious disease of Iucerne. Although there are several ways to transmit the pathogen, seed transmission is practically the only way to introduce the bacteria to previously free areas. In this work, naturally infected seed material and commercial seed lots have been screened by a combination of immunofluorescence (IF) staining and dilution plating. Isolates were confirmed by biochemical characterization and finally by artificial infection of lucerne seedlings. For seed lots free from the pathogen, a diagnosis can mostly be achieved within 2 days. The culture step is necessary only if IF staining is not unequivocally negative, and the full procedure has to be run only for a small portion of the samples. The proposed seed assay provides a reliable alternative to the visual inspection of lucerne fields to fulfil the phytosanitary requirements for Clavibacter michiganensis ssp. insidiosus.     

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。     

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.     

Detection of Xanthomonas campestris pv. undulosa infested wheat seeds by combined liquid medium enrichment and ELISA

An enzyme-linked immunosorbent assay (ELISA) was standardized for detecting Xanthomonas campestris pv. undulosa (Xcu) in plant tissues. Antiserum prepared against somatic antigens of Xcu reacted with cells of pathovars undulosa, cerealis, translucens and phleipratensis , but not with other bacterial species belonging to the genera Xanthomonas, Pseudomonas, Agrobacterium, Clavibacter , and Erwinia. The lower limit of detection of pure cultures was 5 × 10³ cfu/ml. A semi-selective enrichment broth (SSEB) improved the recovery of Xcu in cultures mixed with contaminating bacteria commonly found on wheat seeds. In ELISA tests the enriched samples gave two- to three-fold increases in A_405nm readings when viable cells of Xcu were present. By enrichment, X. campestris pathovars undulosa, cerealis, translucens and phleipratensis were detected in samples that originally had less than 5 × 10² cfu/ml. Semi-selective enrichment combined with ELISA (SSEB-ELISA) allowed for determination of the percentages of infestation of wheat seed lots. Potential seedling infection (PSI) of naturally infested wheat seed lots was obtained by growing seed samples in the greenhouse under conditions optimal for disease development. Three methods were evaluated for their capacity to estimate the PSI: ELISA, combined SSEB and ELISA, and direct plating onto semi-selective XTS agar. Percentages of seed infestation determined by combined SSEB and ELISA resulted in a highly significant correlation with the PSI (r = 0·87, P × 005), whereas determinations made by ELISA or direct plating onto XTS did not significantly correlate with the PSI determined in the greenhouse. This test may constitute a convenient tool for fast initial screening of wheat seed lots in wheat certification programmes.     

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.     

Biological control of damping-off of sugar beet by Pseudomonas putida applied to seed pellets

Pseudomonas putida 40RNF applied to seed pellets reduced the occurrence of Pythium damping-off of sugar beet. A density of 6 × 10⁷ 40RNF per pellet reduced Pythium damping-off from 70 to 26% when seeds were sown in artificially infested soil (250 propagules Pythium ultimum per g dry soil). The efficacy of 40RNF was dependent on its density in the seed pellet (in the range 2 × 10⁴–6 × 10⁸ per pellet) and on the number of propagules of Pythium in soil. 40RNF declined to or stabilized at approximately 1 × 10⁶ per pellet 3 days after planting, and this was independent of the inoculum density. This indicated that the crucial steps resulting in damping-off of sugar beet caused by Pythium ultimum must occur within 3–4 days of sowing. 40RNF reduced pericarp colonization by P. ultimum by 43% 48 h after planting and caused a 68% decrease in the number of sporangia of P. ultimum in the surrounding soil (0.0–5.0 mm). P. putida 40RNF also reduced pre and post-emergence damping-off (from 69.5 to 37.5%) caused by indigenous populations of Pythium species in an infested soil and this was as effective as the fungicide hymexazol (69.5 to 40%).     

Seed transmission of Melon necrotic spot virus and efficacy of seed-disinfection treatments

Rates of seed transmission of Melon necrotic spot virus (MNSV) were estimated in seedlings grown from commercial melon ( Cucumis melo ) cv. Galia F₁ seeds. Seedlings at the cotyledon stage and adult plants were assayed for MNSV by DAS-ELISA and RT-PCR. None of the seedling groups tested positive for MNSV by ELISA. The proportion of seedlings infected with MNSV was at least 7 and 8% in seed lots 05 and 06, respectively, as estimated from RT-PCR analysis of grouped seedlings. Fourteen and eight grouped samples (10 seedlings per group), of a total of 200 and 100 seedlings, respectively, grown from infected seeds were MNSV-positive in seed lots 05 and 06, respectively, corresponding to seed-to-seedling transmission rates of 11·3 and 14·8%, respectively. Several seed-disinfection treatments were evaluated for their ability to prevent seed transmission of MNSV. The results suggest that a treatment of 144 h at 70°C can be used to eradicate MNSV in melon seeds without hindering germination.     

A method for detection of seedborne bacterial diseases in tomato seeds

A method for detection and quantitative estimation of tomato seedborne pathogenic bacteria has been developed. It enables detection in a 7 g tomato seed sample of as few as ten colony-forming units per gram tomato seeds of the following seedborne pathogens of tomato:Pseudomonas syringae pv. tomato,Pseudomonas corrugata, Xanthomonas campestris pv.vesicatoria, andClavibacter michiganense subsp.michiganense. With representative seed samples, the method employs dry grinding, weighing, bacterial extraction and quantitative calculation on selective or semi-selective medium. The efficiency of this method was tested by diluting pathogen-free seed lots with naturally or artificially infested tomato seeds. This procedure enables one to determine the minimal threshold of pathogen which can be detected by this method on media, in comparison with the percentage of diseased seedlings developed from the same seed lots in the growth chamber or in the greenhouse.     

Bio-PCR方法检测葫芦科作物种子携带 西瓜嗜酸菌的特异性引物筛选

 由西瓜嗜酸菌(Acidovorax citrulli)引起的细菌性果斑病是一种毁灭性的种传病害,可为害多种葫芦科作物并造成重大经济损失。该病原菌为检疫性有害生物,种子带菌是田间病害发生的最重要初侵染来源,因此,种子健康检测成为病害综合防控过程中的重要环节。Bio-PCR是当前种子携带细菌检测的常用方法,而特异性引物的选择和使用是检测的关键。本研究使用已报道的7对引物对17株西瓜嗜酸菌、10株嗜酸菌属其它种的菌株和6株其它属的植物病原细菌进行了Bio-PCR检测,筛选出对西瓜嗜酸菌特异性最好的引物为SEQID4^m/SEQID5。研究表明:使用该引物对西瓜嗜酸菌MH21纯菌菌悬液的检测限度为10² CFU·mL^-1;在人工添加菌悬液的模拟带菌西瓜种子中,使用ASCM和EBBA两种半选择性培养基结合引物SEQID4^m/SEQID5进行Bio-PCR检测,ASCM对种子中带菌量的检测限度可达到0.01 CFU·g^-1,EBBA对种子中带菌量的检测限度为0.1 CFU·g^-1。     

应用微滴数字PCR同时检测瓜类种子携带果斑病菌和角斑病菌

细菌性果斑病和角斑病是葫芦科作物两大重要细菌病害,病原菌分别为西瓜嗜酸菌Acidovorax citrulli和丁香假单胞菌黄瓜致病变种Pseudomonas syringae pv.lachrymans。两种菌均可通过种子、种苗带菌进行远距离传播。种子检测是预防和控制这两种病害发生的首要环节。本研究应用微滴数字PCR技术(droplet digital PCR,ddPCR)建立了同时检测种子携带西瓜嗜酸菌和丁香假单胞菌的方法。结果显示:两种细菌菌悬液和DNA样品等浓度混合时,ddPCR能同时检测到两种靶标菌的最低混合菌悬液浓度和最低DNA浓度分别为10³ cfu/mL和10^-3 ng/μL,其检测灵敏度是平行测试的real-time PCR方法的10倍;对于非等浓度混合的菌悬液和DNA样品,两种靶标菌菌悬液按浓度比1∶1000(10³∶10⁶ cfu/mL)混合或其DNA浓度比为1∶10000(2.28×10^-3 ng/μL∶22.8 ng/μL)条件下,ddPCR可检测到低浓度的靶标菌,检测灵敏度同样是real-time PCR的10倍。此外,在人工接菌种子测试中,西瓜、甜瓜单粒种子平均带菌量10⁵~10⁶ cfu/粒时,ddPCR方法可检测到带菌率0.2%(n=500)的西瓜、甜瓜种子样品。将分别携带两种菌的种子按比例1∶10混合时ddPCR方法可以准确检出浓度相对低的靶标菌;而使用相同检测引物的real-time PCR检测方法则只能检出西瓜嗜酸菌和丁香假单胞菌带菌率分别为0.2%和2%(n=500)的甜瓜种子混合样品中的西瓜嗜酸菌,未能稳定检出丁香假单胞菌。综上所述,本研究基于ddPCR技术建立了可同时检测两种重要葫芦科种传细菌的方法,检测结果稳定可靠,丰富了当前种传病原细菌的检测技术体系。     

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.     

基于致病基因序列的LAMP方法检测番茄种子携带的溃疡病菌

本研究选取番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis,Cmm)致病岛上的chpC基因的部分序列,作为环介导等温扩增(loop-mediated isothermal amplification,LAMP)靶标片段进行LAMP引物设计。对反应体系优化后进行特异性测定,结果表明供试的89株番茄溃疡病菌中86株检测结果为阳性,3株为阴性,供试的14株非番茄溃疡病菌(其他重要植物病原细菌)均为阴性。检测番茄溃疡病菌菌悬液样品的阈值为4.8×10~5 CFU·mL~(-1),对DNA样品的检测阈值为1.8×10~(-2) ng·μL~(-1),并据此建立了番茄溃疡病菌的LAMP检测方法。将该方法应用于番茄种子携带Cmm的检测,通过提取种子浸提液样品的总DNA,实现了对番茄种子携带Cmm的直接检测。与普通PCR相比,该方法更加快捷简便,不依赖PCR仪等昂贵的仪器设备,可以丰富现有的番茄溃疡病菌分子检测体系,为口岸等检疫部门提供简单易行的检测初筛手段。     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.