马铃薯青枯病病菌PMA-PCR活菌检测体系优化与初步应用

应用叠氮溴化丙锭(PMA)与PCR技术相结合,建立一种有效快速检测包括活的非可培养状态(Viable But Non-Culturable,VBNC)在内的马铃薯青枯菌活菌的方法。根据青枯菌lpxC基因序列设计特异性引物lpxC5a/6a,探究不同PMA浓度、曝光时间对浓度为1×10~9 cfu/m L的热致死菌悬液中死亡菌体抑制效果的影响,确定PMA最佳处理方案,对采自云南地区的19份田间土样中的包括VBNC状态在内的青枯菌活菌进行检测。结果显示,引物lpx C5a/6a可特异性扩增青枯菌而产生304 bp的条带。当样品中PMA终浓度为40μmol/L,650 W卤素灯曝光15 min,PMA可有效抑制死亡菌体的扩增,而对包括VBNC状态在内的青枯菌活菌的扩增没有影响。在19份田间土样检测中,有7份土样检测出存在青枯活菌。     

Pathogenic characters of Japanese potato strains of Ralstonia solanacearum

Ralstonia solanacearum (Rs) strains in phylotypes I and IV isolated from potato in Japan were investigated for pathogenicity on potato, tomato, eggplant, Solanum integrifolium, tobacco, groundnut, and pumpkin. The strains were divided into 17 types based on differences in their pathogenicity on the tested plants. Particularly, the pathogenicity of most phylotype I strains on eggplant was distinctly different from that of the phylotype IV strains. When nine potato varieties (included two breeding lines) were inoculated with several Rs strains, phylotype IV strains were highly virulent on the breeding lines that are regarded as resistant to phylotype I strains.     

Test performance study for detection of Ralstonia solanacearum and Clavibacter sepedonicus in potato tubers with TaqMan PCR

A test performance study (TPS) was organized in 2018 with ten official testing laboratories to evaluate the performance of different real-time PCR tests for the detection of Clavibacter sepedonicus and/or Ralstonia solanacearum in potato tubers. Participants were sent spiked potato extracts with low (0.8–1.2 × 10⁴ cfu mL^-1), medium (1.6–2.4 × 10⁵ cfu mL^-1) and high (1.6–2.4 × 10⁷ cfu mL^-1) bacterial loads, DNA extracts thereof and heel-end cores from symptomatic potato tubers. The four real-time PCR tests in this TPS for detection of C. sepedonicus were considered fit for purpose as principal screening methods. Two real-time PCRs in this TPS were considered fit for purpose as principal screening methods for detection of R. solanacearum. A third real-time PCR missed 23% of the DNA samples from low-level R. solanacearum spikes and is considered not fit for purpose as a principal screening method. Correct identification of spiked samples was lower when DNA extraction from the spiked samples was performed by the participating laboratories, highlighting the importance of appropriate DNA extraction protocols.     

Comparison of the effectiveness of five extraction methods for Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum from potato tubers

In the EU Control Directives, the recommended extraction procedure for testing potatoes for Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum comprises incubation followed by differential centrifugation. This method can be qualified as complex because of the number of different steps required. This study evaluates five different extraction methods for each bacterium from both a technical point of view and for the quality of the results. Results showed that in the case of C. m. sepedonicus the clarification step should be avoided. The incubation/shaking method with three subsamples gives at least as satisfactory results as the official EU procedure. It also has other advantages, facilitating immunofluorescence readings due to the reduced quantity of plant debris, and improving the speed and the reliability of the analysis.     

PMA-qPCR定量检测青枯菌活菌方法的建立

本文将叠氮溴化丙锭(PMA)与荧光实时定量PCR技术相结合,建立了一种适于青枯菌不同小种菌株活细胞精准、快速检测的PMA-qPCR方法。通过单因素变化试验对PMA预处理反应体系中的各参数进行优化,确立了PMA终浓度为15 ng/μL,黑暗孵育时间为10 min,曝光时间为5 min的PMA预处理体系。试验结果表明,当活菌比例大于10%,PMA-qPCR的测定结果均在理论活菌数相对应的95%置信区间内。检测灵敏度测试结果显示,该方法适用于活菌数在5.0×10~2～5.0×10~8 cfu/mL范围内菌悬液的检测。本文建立的PMA-qPCR方法可在一定范围内有效去除青枯菌死菌的干扰,定量检测出活菌数量,研究结果可为植物细菌性青枯病的流行规律研究提供新的技术支撑。     

Rapid field detection of Ralstonia solanacearum in infected tomato and potato plants using the Staphylococcus aureus slide agglutination test

Ralstonia solanacearum is the major cause of bacterial wilt, which affects over 200 species of plants, many of economic importance. Current methods for detection and identification of the pathogen rely on isolation on semi-selective media followed by a combination of serological and molecular techniques and plant inoculation. Such methods are time-consuming, and require extensive laboratory facilities and skilled personnel. A reliable and rapid screening technique, which could be applied in the field, would reduce the number of samples submitted for laboratory testing and provide a vital component in disease control measures. A programme for the control of R. solanacearum has been introduced in Portugal and a Staphylococcus aureus slide agglutination test was used directly on tomato and potato plants in the field and on bacterial cultures under laboratory conditions. The results obtained show that this technique can play a major role in control programmes by providing a sensitive tool for the rapid detection of the pathogen.     

Highly sensitive detection of Ralstonia solanacearum in latently infected potato tubers by post-enrichment enzyme-linked immunosorbent assay on nitrocellulose membrane

A post-enrichment enzyme-linked immunosorbent assay (ELISA) on nitrocellulose membrane (NCM-ELISA) is described for the detection of Ralstonia solanacearum in latently infected potato tubers. The polyclonal antiserum specificity was significantly improved by adsorption with cross-reacting bacteria. The detection efficiency after enrichment was compared with those of nucleic acid spot hybridization (NASH), double-antibody-sandwich immunoassay (DAS-ELISA) and plating on modified Kelman's medium. After 48 h of incubation of the tuber extracts with modified SMSA broth at 30°C, sensitivities of post-enrichment NCM-ELISA, DAS-ELISA and NASH were similar. As few as 10cells mL⁻¹ were detected in either inoculated or naturally infected tuber extracts. Of 255 field samples, no cross-reactivity of NCM-ELISA was observed. Post-enrichment NCM-ELISA thus provides a reliable and sensitive low-cost method that is rapid and easy to use, making it suitable for assessing susceptibility of breeding lines to bacterial wilt, ecological studies and seed quality control in developing countries.     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.     

Survival of Ralstonia solanacearum and Ralstonia pseudosolanacearum in drain water

The survival in drain water of two strains of Ralstonia solanacearum and three strains of Ralstonia pseudosolanacearum, including two strains able to cause wilt in roses, was determined. Water draining from drip‐irrigated rock wool mats on which roses were grown was supplemented with the pathogen and survival was monitored at 4, 12, 20 and 28°C for up to 112 days. All strains were able to survive for at least 112 days in drain water at 12 and 20°C, but at 4°C maximum survival was 56 days. At 28°C, the survival period was strain dependent, but was at least 56 days. Populations declined gradually in non‐sterile drain water to a low level (maximum 100 cfu mL^?1 after 112 days). In sterile drain water (autoclaved prior to addition of populations), no or only a limited decline in populations was found at 112 days, dependent on strain and temperature. Drain water that tested negative for Ralstonia in the dilution plating assay was tested for the presence of cells in a viable but non‐culturable state (VBNCs). Tomato plants were inoculated, but no symptoms developed, and plants sampled 22 days post‐inoculation were negative in a plating assay. Therefore, no indications were found that VBNCs were present.     

Development of a standard method for detection of potato spindle tuber viroid in potato plants

A standard test method for detecting viroids was designed, to be applied on imported plant material, for which a zero-tolerance exists in the Netherlands towards potato spindle tuber viroid (PSTV).Partial purification of nucleic acids after homogenizing leaf material with a Polytron homogenizer, followed by increasing the viroid concentration by inoculation of an intermediate tomato host, and complete purification of the small nucleic acids from the tops of these plants, followed by polyacrylamide gelectrophoretic analysis, proved successful. With this procedure, now used as a standard method, more samples could be handled than with other methods tested.Desalting by Sephadex filtration proved to be superior to dialysis. An attempt to develop a serological test for PSTV failed. Albinism, induced in PSTV-infected tomato plants by certain environmental conditions, was not of diagnostic value.

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Samenvatting Voor het viroïde, dat de aardappelspindelknolziekte veroorzaakt (ASKV) geldt in Nederland een nultolerantie. Al het geïmporteerde aardappelmateriaal wordt daarom getoetst op het voorkomen van het viroïde. Voor dat doel is een betrouwbare en, zo mogelijk, ook snelle toets noodzakelijk, die niet alleen secundaire infecties maar ook jonge, primaire infecties kan aantonen.Een standaardmethode, die werd ontwikkeld, bleek zeer betrouwbaar, hoewel niet snel. Zij toont meer infecties aan dan snellere methoden die in het buitenland beschreven zijn. De toets omvat de volgende stappen: 2–5 g bladmateriaal wordt vermalen en op het homogenaat wordt een eenvoudige nucleïnezuurextractie en-concentratie toegepast. Dit preparaat wordt gebruikt om vier jonge tomatezaailingen te inoculeren. Door deze zaailingen 4 weken onder optimale omstandigheden te houden wordt het eventueel aanwezige viroïde vermeerderd. Geeft tenminste één van de tomateplanten symptomen, dan wordt het oorspronkelijke monster ziek verklaard. Vertoont geen van de vier tomateplanten symptomen dan wordt een nucleïnezuurextractie uitgevoerd van de topjes van deze planten. Kleine nucleïnezuurmoleculen worden geïsoleerd, geconcentreerd en tenslotte geanaliseerd met behulp van polyacrylamide gelelektroforese.Om overdracht van het ene naar het andere monster te voorkomen werd voor het vermalen gebruik gemaakt van verwisselbare schachten bij de Polytron homogenisator.Ontzouten van de nucleïnezuurextracten met Sephadexfiltratie gaf betere resultaten en was sneller uitvoerbaar dan dialyse.Pogingen om een specifiek antiserum tegen ASKV te maken zijn niet gelukt. Onder onze omstandigheden was het ook niet mogelijk om op een betrouwbare manier albinisme in geïnfecteerde planten te induceren als middel om infecties met ASKV op te sporen.

     

辣椒青枯病拮抗菌株的筛选及田间防效的测定

用抑菌圈－定殖力双重测定法筛选出6个定殖密度大于10^5cfu/g根，抑菌能力亦较强，同时又极易于人工培养的菌株，作为青枯病的潜在生防菌株。温室试验，防病效果分别达83.4%(J2)、91.6%(J3)、58.4%(FH17）、75.0%(BB11)、66.6％（BT4）和41.6%（BT6），增产效果为21.3%-110.1%，其中以J3增产效果最为显著，BB11次之。1996年在南京和淮阴两地的辣椒青枯病小区进行试验，移栽40d后各菌株平均防效分别为：66.0%(J2)、61.6%(J3)、58.2%(FH17)、62.4%(BB11)、72.8%(BT4)和37.1%(BT6);平均增产效果为45.7%(J2)、76.5%(J3)、32.4%(FH17)、78.5%(BB11)、29.3%(BT4)和19.0%(BT6)。     

Specific detection of Ralstonia solanacearum 16S rRNA sequences by AmpliDet RNA

The potential of AmpliDet RNA for specific detection of Ralstonia solanacearum in potato tuber samples and surface water was demonstrated. AmpliDet RNA is a procedure based on nucleic acid sequence based amplification (NASBA) of RNA sequences and homogeneous real time detection of NASBA amplicons with a molecular beacon. The procedure is carried out in sealed tubes, thus reducing the risks for carry-over contamination. AmpliDet RNA enabled reliable detection of specific 16S rRNA sequences of R. solanacearum in total RNA extracts from potato tuber samples in 90min at a level of 10 cells per reaction, equivalent to ca. 10⁴cellsml^–1 of sample. In surface water, AmpliDet RNA allowed detection of R. solanacearum at a level of 10cfuml^–1, after concentrating bacteria from 200ml of surface water into 1ml of surface water by centrifugation.All strains of R. solanacearum and a strain of R. syzygii were positive in AmpliDet RNA, but not other (related) bacterial species. Ralstonia solanacearum (race 3, biovar 2) could be detected reliably in 18 naturally infected potato tuber samples containing varying concentrations of cells. Ninety-one negative tuber samples, from which no R. solanacearum was isolated, were tested in AmpliDet RNA, including 23 samples containing bacteria (cross-) reacting with antibodies against R. solanacearum in immunofluorescence (IF) cell-staining. Only one negative sample, containing high numbers of IF-positive cells, was positive in AmpliDet RNA.     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

Performance of real‐time PCR and immunofluorescence for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing

This paper describes a comparison study of test methods and supports the use of real‐time polymerase chain reaction (PCR) for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing. These 2 bacteria are quarantine organisms under European Union (EU) regulatory control and testing for (latent) infections of these bacteria in seed potatoes is mandatory. Real‐time PCR tests were performed on 276 routine potato tuber samples, including samples infected with either C. michiganensis subsp. sepedonicus or R. solanacearum, and the performance of these real‐time PCR tests was compared with that of immunofluorescence (IF). Real‐time PCR tests, using different primer sets and extraction and PCR protocols, proved to be sensitive and specific for the detection of C. michiganensis subsp. sepedonicus and R. solanacearum in potato tubers in routine testing, and performed at least as well as IF. Real‐time PCR is a good addition to the detection protocols as laid down in EU regulations (EU Council Directives 2006/56/EC and 2006/63/EC).     

Integrated approach for detection of nonculturable cells of Ralstonia solanacearum in asymptomatic Pelargonium spp. cuttings

Ralstonia solanacearum (biovar 2, race 3) is a soil and water-borne pathogen that causes serious diseases in several solanaceous hosts. It can also infect geranium plants, posing an important threat to their culture when latently infected cuttings are imported from countries where the pathogen is endemic. R. solanacearum can be present in very low numbers in asymptomatic geranium cuttings, and/or in a particular stressed physiological state that escapes direct isolation on the solid media usually employed. Consequently, an integrated protocol has been developed to analyze asymptomatic geranium cuttings routinely. The first screening tests include isolation and co-operational-polymerase chain reaction (Co-PCR), based on the simultaneous and co-operational action of three primers from 16S rRNA of R. solanacearum. This method was selected as the most sensitive one, able to detect only 1 cell/ml including nonculturable cells. When isolation is negative but Co-PCR is positive, the bioassay in tomato plants is proposed, since stressed bacterial cells or those present in low numbers that do not grow on solid media can be recovered from inoculated tomato plants and retain pathogenicity. This methodology has been demonstrated to be useful and has allowed us to assess the relevance of the physiological status of bacterial cells and its implications in detection. It also reveals the risk of introducing R. solanacearum through asymptomatic geranium material when relying only on bacterial isolation.     

Diversity of Ralstonia solanacearum Infecting Eggplant in the Philippines

ABSTRACT The diversity of Ralstonia solanacearum strains isolated from eggplant (Solanum melongena) grown in five provinces of the Philippine island group of Luzon was assessed using a recently described hierarchical system. All strains keyed to race 1, biovar 3 or 4. Phylotype-specific multiplex polymerase chain reaction (PCR) indicated that, like most other strains of Asian origin, all the strains in our Philippine collection belong to phylotype I. Taxometric and phylogenetic analyses of partial endoglucanase gene sequences of strains from this collection and those previously deposited into GenBank revealed at least four subgroups among the otherwise monophyletic phylotype I strains. Nucleotide polymorphisms within each subgroup were infrequent and, among the subgroups identified in this study, variation was always <1.3%, indicating that the large majority of strains could be assigned to a single sequevar. Genomic DNA fingerprinting using enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed additional fine-scale genetic variation that was consistent with the endogluconase sequence data. Whole-pattern and band-based analyses of the genomic fingerprint data revealed four and eight distinct genotypes, respectively, within our collection. Eggplant from infested fields in different provinces tended to harbor mixed populations of ERIC genotypes, with the predominant genotype varying by location.     

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.     

False positive Ralstonia solanacearum real‐time PCR results in routine potato tuber samples caused by Advenella species: adaptations to avoid these cross‐reactions

Several real‐time PCR tests for the detection of Ralstonia solanacearum have been developed in recent years. Only the RS primer‐probe system, developed by Weller et al., detects all phylotypes of R. solanacearum in one test. The Saxon State Company for Environment and Agriculture (BfUL) has been using this real‐time PCR test since 2012 for routine testing of potato samples for R. solanacearum. Since the introduction of this test in the laboratory, samples were analysed which were suspected to be false positives [as they gave negative results in other standard tests of the European Union (EU) Commission Directive 2006/63/EC]. Advenella kashmirensis was identified as the cause of selected false positive samples. Inclusion of the three other known Advenella species revealed that this genus could be responsible for false positive results. Because of the rising number of these cases over the last years, two different modifications of the original test from Weller et al. were evaluated independently. It was possible to reduce false positive events, caused by Advenella species by 95% in retested potato tuber samples by using a shortened RS‐primer set in the first adaption of the RS primer‐probe system of Weller et al. The combination of the original RS primer‐set, published by Weller et al., with the RSP‐55T MGB‐probe, published recently in Vreeburg et al., in a second adaption, eliminated false positive events completely.     

Comparison between ELISA and bio tin-la belled probes from cloned cDNA of potato virus X for the detection of virus in crude tuber extracts

Diseases caused by potato virus X (PVX) are of significant agronomic importance, and early detection is vital. Biotinylated DNA probes were prepared by random-primed labelling from cDNA inserts and used for the detection of PVX in crude extracts of infected tubers. The minimum detection level in these extracts is in the order of femtograms of PVX RNA. Comparison with ELISA showed that the hybridization method is 100–250 times more sensitive. Probes prepared by this method are highly specific for target RNA even in crude tuber extracts.     

Situation of Ralstonia solanacearum in the EPPO region in 1997

In the early 1990s, Ralstonia solanacearum , an EPPO A2 quarantine pest which previously occurred only sporadically in some of the southern Member Countries, caused a number of outbreaks of potato brown rot over a wider area in the EPPO region. The National Plant Protection Organizations of the EPPO Member Countries concerned have taken action to eradicate the pest, and in particular the European Union has taken joint action through a Control Directive. Eradication has been successful in many cases, but is complicated by the fact that R. solanacearum can persist on wild Solanum dulcamara and be dispersed by water. EPPO has collected information from its Member Countries through the period of the outbreaks and eradication, and presents in this article an overall view and a detailed country-by-country situation.