地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

Selective detection of Pseudomonas syringae pv. tomato using dot blot hybridization and real-time PCR

Two rapid detection methods based on dot blot hybridization with a nonradioactive DNA probe and molecular beacon-PCR were developed for the specific detection of Pseudomonas syringae pv . tomato , the causal agent of bacterial speck of tomato. A 1378 bp DNA fragment (Acc. No. AM039892), obtained from the extension of a 255 bp fragment generated by a RAPD protocol, was used to find a suitable combination of primers specific for the tomato pathovar. A 138 bp fragment from the genome of P. syringae pv. tomato DC 3000 was used as DNA probe. In dot blots of DNA extracted from either pure cultures or artificially contaminated seeds washes, the probe recognized specifically the tomato pathovar. A molecular beacon was designed from the same region for the specific detection and quantification of P. syringae pv . tomato by real-time PCR. A highly significant correlation was observed between the amount of target DNA and the cycle threshold (Ct). Using a fast protocol for DNA extraction, from pure cultures and from washes of artificially contaminated seeds, the limit of detection was about 1 × 10² CFU. The diagnostic tools developed proved highly specific for P. syringae pv. tomato and simple to use. They can therefore be applied to large-scale testing of tomato seeds and seedlings for the assessment of their phytosanitary condition in nurseries.     

Co-operational PCR coupled with dot blot hybridization for detection and 16SrX grouping of phytoplasmas

A protocol based on Co-operational PCR has been successfully applied to the detection of phytoplasmas. A triprimer reaction coupled with hybridization using general and specific probes permitted detection of ' Candidatus Phytoplasma mali', ' Ca . Phytoplasma prunorum' and ' Ca . Phytoplasma pyri', and their identification as members of 16S ribosomal quarantine group X. The sensitivity of this method was at least one hundred times greater than conventional PCR and similar to that achieved by nested PCR and real-time PCR. The method was validated by testing field samples collected from Malus , Prunus and Pyrus spp. and Olea europaea and compared with seven phytoplasmas maintained in Catharanthus roseus .     

植物对卵菌的非寄主抗病性

 卵菌是多分枝的群体,包括60多种疫霉菌、多个活体营养的霜霉菌和100多种腐霉菌,其中许多是植物病原菌。     

大豆疫霉菌氧化固醇结合蛋白在大肠杆菌中的表达、纯化及活性鉴定

氟噻唑吡乙酮 (oxathiapiprolin) 是目前生产上防治植物病原卵菌病害的高活性杀菌剂,其靶标被认为是氧化固醇结合蛋白 (oxysterol-binding protein, OSBP)。氧化固醇结合蛋白及其相关蛋白 (OSBP-related proteins, ORPs) 是一种脂质转运蛋白,保守存在于多个物种中。虽然其他部分物种的该蛋白三维结构已经被解析,然而尚未见有关植物病原卵菌中该蛋白异源表达及纯化的报道,因而限制了该蛋白的结构药理学研究及基于靶标的新型杀菌剂开发。本文旨在建立大豆疫霉菌 (Phytophthora sojae, Ps) 氧化固醇结合蛋白Psosbp的异源表达、纯化及活性鉴定体系。研究选取与人源OSBP序列保守的OSBP相关结构域 (oxysterol-binding protein-related domain, ORD),构建适于在大肠杆菌Escherichia coli中表达的质粒并通过异丙基-β-D-硫代半乳糖苷 (IPTG) 诱导表达,使用亲和层析及凝胶过滤层析对目的蛋白进行纯化,使用SDS-PAGE及Western blot对蛋白进行种类鉴定,分别使用Tycho™ NT.6及微量热泳动技术对蛋白进行结构完整性及活性鉴定。结果表明,选取了Psosbp蛋白序列的第600位至967位氨基酸,即Psosbp(600-967) 作为Psosbp的ORD结构域,构建了表达质粒pET28a-MBP-TEV-Psosbp(600-967)-His6。IPTG可诱导目的蛋白的表达且蛋白均为可溶状态,使用Ni-NTA琼脂糖树脂对蛋白进行亲和纯化,并基于分子质量大小,通过分子排阻色谱进一步分离获得了纯度较高的重组蛋白MBP-TEV-Psosbp(600-967)-His6。Tycho™ NT.6测定结果表明,重组蛋白具有完整的蛋白质高级结构;微量热泳动结果表明,氟噻唑吡乙酮可以与重组蛋白结合,说明所纯化出的Psosbp蛋白具有生物活性。综上,本文建立了植物病原卵菌的氧化固醇结合蛋白异源表达纯化及活性鉴定体系,并直接证实了抑制剂氟噻唑吡乙酮可结合氧化固醇结合蛋白。     

Taxonomic study of plant pathogenic oomycetes

Journal of General Plant Pathology -     

Identification,pathogenicity and community dynamics of fungi and oomycetes associated with pea root rot in northern France

The pea root rot complex is a major concern for green pea production worldwide. This study aimed at characterizing its composition and dynamics throughout a cropping season in northern France. To this end, fungi and oomycetes were isolated from green pea plant roots with symptoms sampled at the flowering stage in 22 fields in 2017, and at the pea emergence, elongation and flowering stages in two fields in 2018. Out of 646 isolates collected, 317 were identified using molecular markers. Fusarium oxysporum, F. solani and F. redolens were highly predominant. Pathogenicity tests separated the isolates into four aggressiveness groups. F. solani isolates were the most aggressive. Phylogenetic analysis of their TEF1 sequences showed that they mainly belonged to the F. pisi lineage, and that F. oxysporum isolates were genetically close to isolates from the UK that did not belong to the forma specialis pisi. In addition, several Clonostachys rhizophaga isolates are reported for the first time to cause pea root rot. The oomycetes were rarely found and were represented by a few Pythium spp. isolates. Lastly, this study shows that the fungal and oomycete communities associated with pea root rot change during the cropping season. The level of dissimilarity of the root-rot-associated communities decreased throughout the cropping season towards a more similar composition at the flowering stage, dominated by F. solani, F. oxysporum and F. redolens. The proportion of nonpathogenic to weakly pathogenic isolates decreased progressively during the growing season in favour of moderately to highly pathogenic isolates.     

应用DB-EIA技术快速检测甘蔗宿根矮化病研究

就甘蔗宿根矮化病DB-EIA检测技术、检测灵敏度、样品蔗汁保存方式及保存时间进行研究。结果表明DB-EIA检测甘蔗宿根矮化病具有操作简单、高效、重复性好、灵敏度高、对蔗汁新鲜度要求不高等优点。该方法检测灵敏度为1～1/10(稀释倍数);蔗汁保存于4℃或-20℃条件下3d,对检测结果无显著影响,-20℃条件下保存45d,大部分样品检测结果不受影响,少部分样品出现假阴性。     

卵菌与真菌Argonaute家族基因的生物信息学分析

 Argonaute蛋白广泛存在于真核生物与原核生物中,可在非编码小RNA或DNA的引导下,对完全匹配或部分匹配的靶标进行切割、翻译抑制或染色体修饰。本研究利用生物信息学对127种卵菌与真菌的基因组进行分析,旨在了解各个物种中AGO家族基因的数量、蛋白结构域、进化关系及转录模式等。结果发现,大部分卵菌与真菌的基因组中(51%)含有2个AGO基因,而疫霉菌和壶菌等平均含有4个以上。卵菌与真菌的AGO基因在进化上相互独立,多拷贝AGO基因可能是通过基因复制形成;大部分AGO基因具有6个可预测的功能域(即:N端、Linker 1、PAZ、Linker 2、MID和PIWI),并且在PAZ和PIWI功能域上,与核酸5'和3'端结合及与催化活性相关的氨基酸位点整体相对保守,仅个别位点存在一定的差异。侵染大豆过程中,大豆疫霉和终极腐霉的两对同源AGO基因具有保守的表达模式,且基因表达水平相对较高,可能具有相似的生物学功能。上述结果将为深入解析AGO介导的RNA干扰机制及生物学功能奠定基础。     

卵菌与真菌Argonaute家族基因的生物信息学分析

Argonaute蛋白广泛存在于真核生物与原核生物中,可在非编码小RNA或DNA的引导下,对完全匹配或部分匹配的靶标进行切割、翻译抑制或染色体修饰。本研究利用生物信息学对127种卵菌与真菌的基因组进行分析,旨在了解各个物种中AGO家族基因的数量、蛋白结构域、进化关系及转录模式等。结果发现,大部分卵菌与真菌的基因组中(51%)含有2个AGO基因,而疫霉菌和壶菌等平均含有4个以上。卵菌与真菌的AGO基因在进化上相互独立,多拷贝AGO基因可能是通过基因复制形成;大部分AGO基因具有6个可预测的功能域(即:N端、Linker 1、PAZ、Linker 2、MID和PIWI),并且在PAZ和PIWI功能域上,与核酸5’和3’端结合及与催化活性相关的氨基酸位点整体相对保守,仅个别位点存在一定的差异。侵染大豆过程中,大豆疫霉和终极腐霉的两对同源AGO基因具有保守的表达模式,且基因表达水平相对较高,可能具有相似的生物学功能。上述结果将为深入解析AGO介导的RNA干扰机制及生物学功能奠定基础。     

甘肃省马铃薯炭疽病的鉴定及室内药剂筛选

在甘肃省马铃薯主要种植区的田间和贮藏库中分别采集不同时期发病植株和块茎,进行症状描述,并进行病原菌分离鉴定和致病性测定,以及室内药剂筛选。结果表明,该病害在茎秆上形成褐色长条斑;叶片上症状多不明显;薯块上病斑近圆形或不规则形,略下陷,逐渐褐色腐烂;在发病部位形成分生孢子盘;马铃薯炭疽病菌菌丝无色至浅褐色,有隔膜;分生孢子盘黑褐色,其上着生有分隔和顶部渐尖的刚毛;分生孢子棒状,无色;分生孢子梗无色,具分隔,紧密排列在分生孢子盘上;通过ITS序列同源性分析,与已报道的Colletotrichum coccodes相似性达100%。基于病原形态特征和ITS序列分析结果,鉴定该病原菌为球炭疽菌(Colletotrichum coccodes),其引起的病害为马铃薯炭疽病。经室内毒力测定,甲基硫菌灵、苯醚甲环唑、丙环唑.苯醚甲环唑、苯醚甲环唑.嘧菌酯、肟菌.戊唑醇、咪鲜胺锰盐和噻霉酮对马铃薯炭疽病菌的EC50小于10,理论上对该病害有较好的控制作用。     

Investigation on the transformation of fluchloralin by soil fungi: Identification of some metabolites

Fluchloralin [N-(2-chloroethyl)-α,α,α,-trifluoro-2,6-dinitro-N-propyl-p-toluidine] was transformed by two common soil fungi, Aspergillus flavus Link and Fusarium solani (Mart.) Sacc. in pure culture. The fungi were isolated from enrichment cultures of an analogous herbicidal compound, pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine)]. Transformation of fluchloralin by these fungi resulted in the formation of several metabolites, 13 of which were identified. Besides the known mechanisms of microbial transformation of dinitroanilines, methylation of the anilino nitrogen and direct elimination of a nitro group at 2 position from the aromatic ring without any further substitution were also observed.     

Identification by suppression subtractive hybridization and expression analysis of Arabidopsis thaliana putative defence genes during Orobanche ramosa infection

A suppression subtractive hybridization strategy was used to identify genes that were induced 2 h after infection of Arabidopsis thaliana by broomrape (Orobanche ramosa) seedlings. Among these genes, the expression of twelve was analysed from the first hour to the seventh day of this compatible interaction by cDNA blot analyses. The twelve genes showed a transient expression, which occurred in seven cases as early as the first or second hour of interaction and ended 2 or 3 h later. Most of the proteins encoded by these genes are already known to be involved in different A. thaliana response pathways to pathogen attack. The first two genes to be induced (Rc kinase and ACaM5) encoded a receptor and a calmodulin, and could be involved in signal transduction. The two following genes encoding a sucrose carrier (SUC1) and a protein with a pectin methylesterase inhibitor domain were found to be overexpressed; their roles are consistent with plant defence emergence. The gst1, gst11 and peptide methionine sulfoxide reductase genes, which were turned on early, are known to play a role in the detoxification of reactive oxygen species. The accumulation of mRNAs for lox1, a latex allergen and a myrosinase binding protein could be related to a jasmonate dependent pathway, while genes for a class III peroxidase and a caffeoyl-CoA 3-O-methyltransferase, both likely to be involved in cell wall reinforcement, were also upregulated.     

Proteomic studies of phytopathogenic fungi,oomycetes and their interactions with hosts

Proteomics, the systematic analysis of the proteome, is a powerful tool in the post-genomic era. Proteomics studies have examined global changes in proteomes of phytopathogenic fungi, oomycetes and their hosts during compatible or incompatible interactions. This article compiles proteomics reports in order to decipher the molecular mechanisms underlying fungal development (infection-related morphogenesis), fungal or oomycete—host plant interactions, and phytopathogenesis.     

Identification by suppression subtractive hybridization and expression analysis of Medicago truncatula putative defence genes in response to Orobanche crenata parasitization

Crenate broomrape (Orobanche crenata) is the major constraint for pea and faba bean production in the Mediterranean region. In this study, a systematic sequencing of expressed sequence tags (ESTs) was chosen to obtain a first global picture of the assembly of genes involved in defence response. A cDNA-library was established by suppression subtractive hybridization in the model legume Medicago truncatula infected by O. crenata in order to identify a large number of host plant ESTs. Eighty-one presumably up-regulated genes were identified and classified in functional categories. EST-annotations showed homologies to a number of well-characterized genes. Most of the proteins encoded by these genes, are already known in defence in M. truncatula, such as genes related to the JA pathway or involved in cell wall modifications. A notable number of the ESTs, however, were derived from novel genes not matching entries of the large-scale M. truncatula sequences collections. Expression analyses by quantitative RT-PCR of 20 genes corresponding to different functional categories showed high expression levels, supporting their involvement in the defence response.     

European collaborative studies for the validation of PCR-based detection tests targeting regulated fungi and oomycetes

In 2007, the mycology unit of the French plant protection laboratory (LNPV-UMAF) organized and launched four collaborative studies for the validation of detection protocols targeting the regulated oomycetes Phytophthora ramorum , P. fragariae / P . rubi , Plasmopara halstedii and the fungus Monilia fructicola . The participants were recruited through the European Mycological Network (EMN). All four protocols were based on species-specific PCR tests already published in the scientific literature and, except for Pl. halstedii , combined a detection test and a confirmation of detection test. For each target organism, we evaluated the performance of protocols, i.e. accuracy, qualitative repeatability and qualitative reproducibility, by a statistical analysis of the results obtained by the 16 participant laboratories with a series of 10 blinded samples. As demonstrated by the collaborative trials results, all four detection protocols were shown to be fit for the purpose of regulatory compliance. The collaborative trial appears a powerful tool to evaluate the performance of a detection method, and is of special interest to laboratories employing a quality assurance system.     

Identification of genes associated with phytoplasma resistance through suppressive subtraction hybridization in Chinese jujube

Jujube witches' broom (JWB) is a destructive disease for Chinese jujube caused by phytoplasma. A suppression subtractive hybridization library of resistant cultivar ‘Xingguang’ was constructed under phytoplasma stress to identify genes related to JWB resistance. 77 of 200 unique expressed sequence tags had significant sequence homologies and were classified into 10 functional groups. The most abundant group was disease/defense (20.8%), which was consistent with the phytoplasma stress. These differentially expressed genes provide the groundwork for addressing the plant–phytoplasma interaction. Meanwhile, the expression of five selected genes (TLP, PR10, HSP70, ERF, kinase-related protein) was confirmed to upregulate at different infection periods.     

基于小RNA深度测序技术鉴定侵染我国部分省市草莓种苗的病毒种类

随着草莓保护地栽培面积的增加和无性繁殖种苗的繁殖与调运,草莓病毒病的发生与流行日益严重。为明确侵染我国部分省市草莓种苗的病毒种类,应用小RNA深度测序技术进行检测,并利用RT-PCR技术对结果进行验证及序列分析。结果表明,从来自我国7省市的41株具有典型病毒病症状的草莓种苗样品中检测到草莓斑驳病毒strawberry mottle virus (SMoV)、草莓镶脉病毒strawberry vein banding virus(SVBV)和草莓轻型黄边病毒strawberry mild yellow edge virus (SMYEV)3种。SMoV、SVBV和SMYEV的检出率分别为34.1%、24.4%和2.4%。选取不同产地草莓种苗上检出的不同病毒进行部分序列测定和分析,获得了3个SMoV分离物(四川分离物schhy13、辽宁分离物lnhy23和河北分离物hbhy28)的部分RNA1 3′端非编码区606 bp核苷酸序列,其一致性为98.12%～99.34%。测定并获得了5个SVBV分离物(辽宁分离物lnhy15、lnhy17、lnhy24、河北分离物hbhy28和陕西分离物sh...     

基于巢式反转录-聚合酶链式反应技术检测葡萄病毒A和葡萄病毒B

正皱木复合病在世界范围内普遍发生,是引起葡萄木质部畸形的一类病害的统称,在沙地葡萄、Kober5BB和LN33品种上表现茎痘、茎沟和栓皮等症状,其中葡萄病毒A(Grapevine virus A,GVA)与Kober5BB品种上出现茎沟症状有关,而葡萄病毒B(Grapevine virus B,GVB)与LN33品种上出现栓皮症状有关。GVA和GVB属葡萄病毒属,主要通过嫁