Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds

Plasmopara halstedii , the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii . The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µ L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes.     

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.     

向日葵茎溃疡病菌RPA检测方法的建立

向日葵茎溃疡病菌(Diaporthe helianthi)是我国进境植物检疫性病原真菌.本研究针对D.helianthi的cal基因保守序列设计特异性引物,建立该病菌的重组酶聚合酶扩增检测技术(RPA)方法,并对其特异性、灵敏度及适用性进行评价.结果 显示,建立的RPA方法特异性强,只有3个D.helianthi样品能...     

菜豆种子普通细菌性疫病菌检测

 由Xanthomonas axonopodis pv. phaseoli和Xanthomonas fuscans subsp. fuscans引起的菜豆普通细菌性疫病是严重影响菜豆生产的限制因子之一,可造成严重的产量和品种损失。染菌种子是病原菌传播的主要途径。本研究对5个菜豆主要产区的60份菜豆种子样品进行普通细菌性疫病菌检测。在MT选择性培养基上有36份种子样品浸提液检测到目标病原菌, 种子样品带菌量为2.49×10²~5.20×10⁷ CFU/粒。选择36个分离物接种感病品种“英国红”植株,所有分离物均引起接种植株发病。特异PCR检测结果表明,有20个分离物为X. fuscans subsp. fuscans,16个分离物为X. axonopodis pv. phaseoli。试验结果表明,我国一些菜豆主产区商业种植和研究用种子多数污染普通细菌性疫病菌,建议建立无菌种子生产区和加强种子管理,以有效控制病害发生。     

Colonization of citrus seed coats by 'Candidatus Liberibacter asiaticus': implications for seed transmission of the bacterium

Huanglongbing is an economically damaging disease of citrus associated with infection by 'Candidatus Liberibacter asiaticus'. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of 'Ca. Liberibacter asiaticus' DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from 'Sanguenelli' sweet orange and 'Conners' grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from 'Sanguenelli' and from 'Conners' fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of 'Sanguenelli' and 'Conners', respectively. Small amounts of pathogen DNA were detected in 10% of 'Sanguenelli' seedlings grown in the greenhouse, but in none of 204 extracts from 'Conners' seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of 'Sanguenelli' and 'Conners' which were germinated on agar, and in 5% of 'Sanguenelli' but in none of 164 'Conners' seedlings which grew from these seeds on agar. No pathogen DNA was detected in 'Ridge Pineapple' tissue at 3 months post-grafting onto 'Sanguenelli' seedlings, even when pathogen DNA had been detected initially in the 'Sanguenelli' seedling. Though the apparent colonization of 'Conners' seeds was more extensive and nearly uniform compared with 'Sanguenelli' seeds, no pathogen DNA was detected in 'Conners' seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.     

Inter-isolate variation for virulence in Plasmopara halstedii (sunflower downy mildew) from Hungary

A total of 82 isolates of Plasmopara halstedii , collected from all production areas of Hungary between 1976 and 1993, were assessed for their virulence pattern on a standard set of sunflower differentials under glasshouse conditions. The isolates were classified into six pathogenic races each representing a particular virulence phenotype. From 1976 until 1988 all the isolates were found to be virulent only on sunflowers possessing no known resistance genes, thus classified as race 1. There was an apparent shift in the virulence of the P. halstedii population collected after 1988. Six races (1, 2, 3, 4, 8 and 9) were identified among the 45 samples collected between 1989 and 1993, with races 1 and 3 predominant, at a frequency of 35% each. While the increase in race virulence is undoubtedly due to selection imposed by resistant hybrids, the origin of the new races is unknown. Whether new races have arisen from the indigenous P. halstedii population, or whether they have been imported from abroad, can only be reliably determined by DNA techniques, such as fingerprinting.     

Rapid detection of Fusarium oxysporum f. sp. lactucae on soil,lettuce seeds and plants using loop‐mediated isothermal amplification

Fusarium oxysporum f. sp. lactucae (FOL) is a soil‐ and seedborne pathogen and the causal agent of fusarium wilt on lettuce. Four races have been identified within FOL, with different worldwide distribution. Several molecular techniques have been used to detect and identify this pathogen; however, not all of them have the optimal characteristics in terms of sensitivity to perform FOL detection in plant and seed material. A loop‐mediated isothermal amplification (LAMP) assay was developed based on the sequence‐characterized amplified region (SCAR) obtained in a previous rapid amplification of polymorphic DNA (RAPD) study. The LAMP assay has been validated according to the EPPO standard PM7/98. The LAMP assay was tested with lettuce seeds, soil and plant material, and can be used successfully to amplify DNA from each of these matrices. In seed lots artificially inoculated with FOL, the detection limit of the LAMP test was 0.004% infected seed.     

Transient expression of gfp in the obligate biotrophic oomycete Plasmopara halstedii using electroporation and a mechanoperforation method

Studying plant biotrophic oomycetes is difficult, because they depend on living plant cells for nutrition, which necessitates investigations in planta . Internal optical labelling is a powerful tool for analysing intra- and interspecific interactions. Different approaches are described for establishing a gfp -transformation system for Plasmopara halstedii , the causal agent of sunflower downy mildew. A vector containing gfp and the oomycete-specific ham34 regulators from Bremia lactucae was constructed for transformation experiments. Particle bombardment of infected sunflower cotyledons during different developmental stages of the pathogen did not result in successful transformation. In contrast, transient gfp expression in sporangia was achieved using electroporation. However, gfp expression was lost during the subsequent round of infection. A novel transformation method for biotrophic organisms in planta employing mechanoperforation – so-called Löchern–resulted in sporangiophores of P. halstedii transiently expressing gfp and emerging distant from the site of transformation. The new technique is advantageous compared with others as the transformed hyphae can recover during their vegetative growth, before reproductive structures occur. This first step lays an important foundation for further investigations of obligate biotrophic organisms.     

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。     

哈蜜瓜种子带细菌性果斑病菌检测技术的研究

哈蜜瓜细菌性果斑病是世界性的检疫对象之一,主要以种子带菌进行远距离传播,本文通过温室育苗、室内分离和PCR(聚合酶链式反应)技术对哈蜜瓜种子带菌情况和主要带菌部位进行了检验,结果表明:3种方法都可以对种子带菌情况进行检测,分离检测和PCR检测还可以确定种子的带菌部位,PCR技术具有特异性强、灵敏度高、检测时间短等特点.另外检测结果也表明种子带菌以种皮为主,种仁的带菌量较少.     

A quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds

Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR‐based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5^.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.     

Reliable detection of the fungal pathogenfusarium oxysporum f.sp.albedinis, causal agent of bayoud disease of date palm, using molecular techniques

Bayoud, caused by the soilborne fungusFusarium oxysporum f.sp.albedinis (FOA), is the most serious disease of date palm. Since the disease is located in the North African countries of Morocco and Algeria, and advancing steadily eastwards, the ultimate goal is to prevent spread of the pathogen to other date-growing areas in the region and farther afield. Molecular diagnostic techniques have been developed for detection of FOA. In view of the fact that the fungus does not exist in Israel, DNA of FOA was obtained to determine the reliability of these methods for diagnostic purposes. Random amplified polymorphic DNA was not reliable enough for differentiation between FOA and various pathogenic and saprophyticFusarium isolates. However, the polymerase chain reaction utilizing FOA-specific primers was accurate and enabled amplification of a unique band specific to FOA DNA alone, and not that of the other tested pathogenic and saprophyticFusaria. The availability of a rapid and reliable diagnostic tool for detection of FOA will enable the Plant Protection and Inspection Services of the Israel Ministry of Agriculture to test date palm tissue for the presence of the pathogen. Contribution no. 513/00 from the Inst. of Plant Protection, ARO, The Volcani Center, Bet Dagan, Israel.     

Real-time PCR identification and detection of Fuscoporia torulosa in Quercus ilex

Fuscoporia torulosa is the causal agent of white alveolar wood decay on several species including a large number of forest trees. Early detection of the fungus is essential to identify diseased trees before spread occurs to healthy plants. However, current detection methods based on isolation from infected tissues on semi-selective media are laborious, time consuming and require expertise in identifying the pathogen after isolation. In the present study, a rapid and reliable Scorpion-PCR based molecular method to identify and detect F. torulosa in planta was developed in a highly polymorphic portion of the internal transcribed spacer (ITS) regions. Specificity of primers and probe was assessed by means of both BLAST analyses and by using genomic DNA from 131 F. torulosa isolates and 43 other fungi and oomycetes from different hosts and geographic areas. In Scorpion-PCR the limit of detection was 1 pg of total DNA and a high correlation ( r ² = 0·996) was achieved between target DNA quantity and cycle threshold (Ct). Real-time PCR combined with effective procedures for DNA extraction enabled the detection of F. torulosa from naturally infected tissues of oaks with and without fruit bodies in approximately 6 h. Comparative testing showed that detection of F. torulosa in wood samples is more sensitive and reliable with real-time PCR than with conventional isolation.     

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.     

Strategies to prevent spread of Leptosphaeria maculans (phoma stem canker) onto oilseed rape crops in China; costs and benefits

Field experiments in Europe have shown that Chinese cultivars of winter oilseed rape ( Brassica napus ) are very susceptible to the pathogen Leptosphaeria maculans (cause of phoma stem canker). Climatic and agronomic conditions in China are suitable for L. maculans since the closely related but less damaging pathogen L. biglobosa occurs on the winter and spring oilseed rape crops there. Major gene resistance to L. maculans is not durable; when introduced into commercial oilseed rape cultivars it is rapidly rendered ineffective by changes in the pathogen population. The threat to Chinese oilseed rape production from L. maculans is illustrated by the way in which L. maculans has spread into other areas of the world where previously only L. biglobosa was present, such as Canada and Poland. Models were developed to describe the spread (in space and time) of L. maculans across Alberta province, Canada, based on survey data collected over a 15-year period. These models were used to estimate the potential spread of L. maculans across the Yangtze river oilseed rape growing areas of China and its associated costs. Short-term strategies to prevent occurrence of severe phoma stem canker epidemics in China include training of extension workers to recognise symptoms of the disease and use of PCR-based diagnostics to detect the pathogen on imported seed. Long-term strategies include the introduction of durable resistance to L. maculans into Chinese oilseed rape cultivars as a component of an integrated disease management programme. The costs of such strategies in relation to costs of a phoma stem canker epidemic are discussed.     

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。     

实时荧光定量PCR法检测十字花科细菌性黑斑病菌

为有效防控我国的检疫性有害生物十字花科细菌性黑斑病菌Pseudomonas syringae pv.maculicola在国内的传播与蔓延,通过设计1对特异性引物3539,利用132株靶标和非靶标菌为模板进行PCR扩增,建立了实时荧光定量PCR法,并进行了模拟种子带菌试验。结果显示,引物3539为只针对十字花科细菌性黑斑病菌扩增出的特异性产物;在模拟种子带菌检测中,常规PCR对菌悬液的检测限为10~5CFU/m L,实时荧光定量PCR的检测限为10~3CFU/m L,其中10~8CFU/m L菌液的Ct值最低,为22.90,10~3CFU/m L菌液的Ct值最高,为35.73,且不同浓度菌液间的Ct值均有显著差异;不同带菌率模拟种子的检测结果表明,常规PCR和实时荧光定量PCR能检测到的带菌率分别为0.5%和0.1%。研究表明,实时荧光定量PCR法不仅可用于病种的检测,也可用于病害的早期诊断。     

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.     

Mycelial growth rate and toxin production in the seed pathogen Pyrenophora semeniperda: resource trade‐offs and temporally varying selection

Pyrenophora semeniperda, an important pathogen in Bromus tectorum seed banks in semi‐arid western North America, exhibits >4‐fold variation in mycelial growth rate. Host seeds exhibit seasonal changes in dormancy that affect the risk of pathogen‐caused mortality. The hypothesis tested is that contrasting seed dormancy phenotypes select for contrasting strategies for increasing pathogen fitness, and that increased fitness on nondormant seeds involves a resource trade‐off between toxin production and growth. The strategy for successfully attacking rapidly germinating nondormant seeds at high inoculum loads in autumn involves increased post‐infection aggressiveness to prevent seed escape through germination. An earlier study demonstrated that slow‐growing strains caused higher mortality than faster‐growing strains on nondormant host seeds at high inoculum loads. In this study, production of the toxin cytochalasin B was significantly higher in slower‐growing strains, and was induced only in seeds or in seed‐constituent‐containing media. Its production was reduced in vivo by Bromus tectorum seeds, suggesting direct involvement in pathogenesis on seeds. Fast‐growing strains caused significantly higher mortality than slow‐growing strains at low inoculum loads on dormant seeds, which apparently have resistance that is overcome at high loads or through rapid mycelial proliferation. In a co‐inoculation study, the fast‐growing isolate produced 3 × more stromata than the slow‐growing isolate on dormant seeds, whereas the slow‐growing isolate was twice as successful on nondormant seeds. These results provide evidence that mycelial growth rate variation and associated variation in cytochalasin B production represent a trade‐off maintained through temporally varying selection resulting from seasonal variation in host seed dormancy status.