甘蔗赤条病菌巢式PCR检测

甘蔗赤条病是由燕麦食酸菌燕麦亚种(Acidovorax avenae subsp.avenae,Aaa)引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术,根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物,建立Aaa巢式PCR检测方法。结果表明,建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A.oryzae具有特异性,可扩增出454 bp目的条带,对近缘种德氏食酸菌A.delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L.xyli subsp.xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板,巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL,是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测,阳性检出率分别为100.0%和28.6%,表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。     

甘蔗宿根矮化病菌多克隆抗体的制备

 甘蔗宿根矮化病（Ratoon stunting disase,RSD）是甘蔗生产中最为严重的细菌病害之一,常导致感病品种新植蔗减产10%～15%,宿根蔗减产20%～25%,在干旱的情况下感病品种的宿根蔗产量损失可达60%^［1］。RSD病原菌为 Leifsonia xyli subsp.xyli（Lxx）,寄生于甘蔗木质部导管内,革兰氏阳性^［2］,体外分离培养非常困难。该病害自从1944 年首次在澳大利亚昆士兰州的甘蔗品种Q28 上发现以来,在世界各产区普遍发生,现已广泛分布于各甘蔗种植区。该菌主要通过带菌种茎和砍收工具传播^［3］,已感病的蔗株又无明显的外部症状,从而导致该病害无意识地传播,造成病害蔓延,对甘蔗生产危害极大。甘蔗宿根矮化病的检测方法主要有形态学、血清学、分子生物学等方法,血清学由于具有操作简便、准确性高、灵敏度好,同时能够处理大量样品而在国外被广泛采用。目前国内所用的RSD血清全部依赖于国外进口,检测成本高,使得血清学方法无法在国内普及。为此我们分离纯化了RSD的病原菌并制备了RSD的多克隆抗体,为甘蔗宿根矮化病敏感、稳定和快捷的检测提供了必要的保证。     

Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by Multiplex PCR with Coamplification of Host DNA

A new multiplex PCR assay was developed for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers. The assay combines two different tests in one reaction mixture. First, a highly specific and sensitive detection of the pathogen and second, an indicator test for successful amplification (internal PCR control), which monitors potentially false-negative PCR results, caused by inhibition of the PCR. For the simultaneous amplification of two different targets in one reaction mixture, a mix of two different primer sets was used. For the detection of C. michiganensis subsp. sepedonicus, a pathogen-specific primer set PSA-1/PSA-R was used, based on the intergenic spacer region of the 16S–23S rRNA genes of C. michiganensis subsp. sepedonicus. For the simultaneous amplification of the internal PCR control, the plant-specific primer set NS-7-F/NS-8-R was employed, permitting amplification of target sequence from plant DNA present in DNA extractions from potato core fluid. The applicability of the multiplex PCR was verified in 3500 composite samples of 200 seed potato tubers from 143 different cultivars in a survey for C. michiganensis subsp. sepedonicus by parallel testing using immunofluorescence, a bioassay in eggplant seedlings and multiplex PCR.     

宿根矮化病菌对甘蔗品质及茎、叶超微结构的影响

 甘蔗宿根矮化病(Ratoon Stunting Disease, RSD)是由Leifsonia xyli subsp.xyli (Lxx)引起的,是目前世界所有植蔗地区危害性极大的病害之一。本实验以甘蔗品种新台糖22号(ROC22)健康植株为对照,感染RSD植株为处理,观察和测定RSD侵染甘蔗引起的蔗株农艺性状、蔗糖分以及茎、叶超微结构的变化。结果表明:(1)感染RSD种茎的出苗率比对照减少2.94个百分点;株高比对照下降28.85 cm;茎径比对照减少0.28 cm;节间长度比对照减短3.50 cm;单茎重比对照低0.36 kg。(2)感染RSD植株的蔗糖分低于对照0.9个百分点(绝对值)。(3)利用透射电镜技术对感染RSD植株茎、叶细胞超微结构进行观察表明,叶片叶肉细胞、维管束鞘细胞及茎细胞内的细胞器及细胞核都发生了明显的病理变化。与健康叶片相比,叶绿体变形,叶绿体基质片层大部分消解,基粒结构消失,叶绿体外膜和内膜剥离。线粒体形态异常,有的肿大、内嵴模糊,严重者内嵴消失并空泡化,仅剩未被消解的残骸;细胞核形态变为不规则,核膜破裂,染色质分布不均匀,呈降解状态。在感染RSD甘蔗茎维管束导管细胞内积累有大量的电子致密物质,细胞壁有不同程度的溶解和断裂,这可能和RSD病原细菌侵染有关。以上结果表明:RSD侵染甘蔗后,可能导致光合效率下降,对水分和营养物质的运输能力降低,从而导致甘蔗品质和产量的降低。     

16S nested-PCR技术检测玉米细菌性枯萎病菌

 玉米细菌性枯萎病是玉米上的重要种传病害,病原菌为Pantoea stewartii subsp.stewartii。本研究设计了16S通用引物,扩增该病菌及其近似种的16S rDNA,通过序列测定和分析,针对该病菌设计了特异性引物,采用nested-PCR技术,能够准确地区别该病菌及其近似种,检测的灵敏度在DNA水平上达到10^-3 pg级,检测活菌则达到2 cfu。检测人工污染的玉米种子时,不受种子提取液中其它物质的干扰,灵敏度依然达到2 cfu。     

甘蔗白叶病植原体巢式PCR检测方法的建立及初步应用

甘蔗白叶病(sugarcane white leaf,SCWL)是由植原体引起的重要甘蔗病害[1],广泛分布在印度、泰国等许多国家[1,2].我国甘蔗产区的栽培品种也有SCWL的发生[3].甘蔗是无性繁殖作物,植原体可通过繁殖种苗进行传播,台湾斑纹叶蝉(Matsumuratetlix hiroglyhious)通过咬食感染甘蔗植株的韧皮部可引起该病害[4].     

甘蔗宿根矮化病菌实时荧光定量PCR检测方法的建立

 甘蔗宿根矮化病是由Leifsonia xyli subsp. xyli (Lxx)引起的一种世界性甘蔗细菌病害。根据Lxx的Pat1基因保守序列,设计并合成了一对特异性引物Pat1F (5′-GGTTCCATTGCTTACCGATT-3′)/Pat1R(5′-CAAGTTTCGACAGGAACAGC-3′),和一条TaqMan探针(FAM-5′-CCACGGCTACGTCAATTCGGG-3′-TAMRA),建立了一种特异性强、灵敏度高的甘蔗宿根矮化病菌实时荧光定量PCR检测方法。结果表明,本研究建立的实时荧光定量PCR方法,对Lxx的检测最低下限为10² copies·μL^-1。应用实时荧光定量PCR与常规PCR方法对14个甘蔗品种进行Lxx检测,阳性检出率分别为86%和43%,表明实时荧光定量PCR比常规PCR检测方法具有更高的灵敏度。研究结果为甘蔗宿根矮化病的诊断、田间发生动态监测、脱毒健康种苗检测及品种/材料交换检疫检测提供了新技术支撑。     

Bacterial brown stripe of rice in soil-less culture system caused by <Emphasis Type="Italic">Acidovorax avenae</Emphasis> subsp. <Emphasis Type="Italic">avenae</Emphasis> in China

In 2010, the outbreak of a disease with symptoms similar to bacterial brown stripe was observed in rice seedlings planted in a perlite culture system in China. The causal bacterium was identified as Acidovorax avenae subsp. avenae based on its biochemical and physiological characteristics, cellular fatty acid composition, Biolog data, specific PCR detection and 16S rRNA gene sequence analysis. The bacterial isolates caused similar symptoms after inoculation of rice seedlings. This report is the first of bacterial brown stripe of rice in a soil-less culture system caused by A. avenae subsp. avenae in China.     

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction

Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 10³ culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.     

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。     

Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10–100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month ( P  < 0·001), but not by cultivar ( P  = 0·394). The new assays were shown to be useful tools for rapid detection and identification of these pathogens and to allow rapid and accurate assessment of the casual agents of anthracnose in strawberry.     

广西南宁甘蔗赤条病调查及分子检测

2020年在广西南宁市金光农场发现'桂糖58'和'柳城05-136'疑似感染甘蔗赤条病,为明确其病原,本研究对不同甘蔗品种进行了发病率调查,并采集病样进行了PCR检测分析.田间调查结果表明:不同品种自然发病率不同,'桂糖58'发病率为29％～52.33％,发病严重田块平均发病率为49.67％,发病中等田块平均发病率为3...     

Use of the DNA sequence of the intergenic spacer region between the 16S and 23S rRNA genes for the identification of Clavibacter michiganensis subsp. insidiosus at the molecular level

A study has been performed to identify Clavibacter michiganensis subsp. insidiosus at the molecular level, using the polymerase chain reaction (PCR) technique with oligonucleotide primers based on specific sequence recognition of the intergenic spacer region between the 16S and 23S rRNA genes. The pair of primers was designed on the basis of available DNA sequence data for that region in C. m. insidiosus and other bacteria. Using this pair of primers, a large amount of an amplified DNA fragment of 218 bp in length was obtained from C. m. insidiosus. The specificity of this amplification was proved by PCR analysis, using the above-mentioned pair of primers and templates from different bacteria, some related to C. m. insidiosus. The PCR products were analysed using agarose gel electrophoresis.     

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.     

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of <Emphasis Type="Italic">Acidovorax citrulli</Emphasis>, the bacterial pathogen of watermelon fruit blotch

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.     

苜蓿萎蔫病菌TaqMan探针实时荧光PCR检测方法的建立

苜蓿萎蔫病菌是我国对外检疫性二类有害生物，目前国内尚无发生6在出入境捡验检疫中主要是采用生物学和血清学方法进行检测，劳动强度大，耗费时间长。根据苜蓿萎蔫病菌与其它细菌菌株16SrDNA序列差异，设计出对苜蓿萎蔫病菌具有稳定点突交特异性探针，利用该探针对棒形杆菌属4个种及其它属细菌进行了实时荧光PCR检测实验。结果表明，只有苜蓿萎蔫病菌能检测到荧光信号，其它细菌没有荧光产生。该方法特异性强，灵敏度高，能检测到21．4fg质粒DNA，比常规PCR灵敏100倍，而且整个过程只需要2～3h。该方法可有效地应用于进出境病原菌检测之中。     

Isolation and genetic characterization of Acidovorax avenae from red stripe infected sugarcane in Northwestern Argentina

Red stripe is a bacterial disease of sugarcane causing important economic losses in Argentina that affects 30 % of the milling stems and consequently the juice quality. In this study, sugarcane leaves exhibiting red stripe symptoms were sampled in the 2008–09 growing season from 13 different sugarcane producing areas of Tucumán and Salta (northwest of Argentina). To achieve the identification and characterization of the causal agent of red stripe, bacterial isolation was performed. Species-specific PCR using Oaf1/Oar1 primers allowed the amplification of a fragment of 550 bp from approximately 50 % of the isolates; 16S rDNA sequences analysis displayed a similarity greater than 99 % with Acidovorax avenae subsp. avenae. By means of RAPD-PCR the presence of at least four different biotypes among the analyzed isolates was detected. Results of pathogenicity test allowed us to confirm A. avenae subsp. avenae as the pathogenic agent for red stripe. This study constitutes the first report on the identification and molecular characterization of this plant pathogen from the Argentina sugarcane production areas. The genetic diversity observed among A. avenae is an important factor to be considered to improve an accurate diagnosis and/or the selection of sugarcane tolerant clones.     

New subspecies-specific polymerase chain reaction-based assay for the detection of Acidovorax avenae subsp. citrulli

New subspecies-specific primers for the detection of Acidovorax avenae subsp. citrulli ( Aac ), the seedborne bacterium which causes bacterial fruit blotch of cucurbits, were designed based on PCR fragments obtained in ERIC- and BOX-PCR profiles of Aac strains. PCR with both primer sets identified 30 strains from different locations and hosts, but did not amplify DNA from other closely related species and subspecies assessed, with the exception of DNA of one strain of A. avenae subsp. avenae , which was amplified by one of the primer sets, named BX-S. This primer set, based on a BOX-PCR fragment, performed under high-stringency conditions without losing its detection sensitivity. The primers were also evaluated for their ability to detect the pathogen in contaminated watermelon and melon seed samples. The BX-S primers facilitated the detection of the pathogen from washings of 5000-seed samples with 0·02% infestation. This primer set was also assessed for detection using immunomagnetic separation polymerase chain reaction (IMS-PCR) and was shown to be as sensitive as a previously described primer set (AACF2/R3), detecting 0·02% infestation in seed samples. This highly specific and sensitive primer set could be used to improve PCR-based detection of this important pathogen.     

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.