北方地区小西葫芦黄花叶病毒的酶联检测和西瓜品种抗病性鉴定

应用DAS-ELISA对分别于1998年在河南、陕西和1999年在河南、陕西、河北和山西采集的葫芦科病毒病样本84份、186份进行小西葫芦黄花叶病毒的检测,表明该病毒在这些地区广泛发生,ZYMV阳性检出率分别为79.8%和57.5%熏在北京和河南临颖县ZYMV发生率较低熏≤23.1%,在其余地区即河北石家庄,河南郑州、开封、孟津,山西运城,陕西西安其阳性检出率在40.0%～92.9%之间。除冬瓜样本没有ZYMV外,西瓜、甜瓜、南瓜、丝瓜、小西葫芦、苦瓜、黄瓜和瓠子均受到ZYMV的侵染。不同地区、年份和作物之间ZYMV阳性检出率存在差异。对18份西瓜品种的抗ZYMV温室人工接种鉴定的试验表明,目前推广的一些品种对ZYMV缺乏抗病性。     

Characterization of phytoplasmas detected in Chinaberry trees with symptoms of leaf yellowing and decline in Bolivia

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in foliage samples from Chinaberry ( Melia azedarach ) trees displaying symptoms of yellowing, little leaf and dieback in Bolivia. A ribosomal coding nuclear DNA (rDNA) product (1·8 kb) was amplified from one or more samples from seven of 17 affected trees by PCR employing phytoplasma-universal rRNA primer pair P1/P7. When P1/P7 products were reamplified using nested rRNA primer pair R16F2n/R16R2, phytoplasmas were detected in at least one sample from 13 of 17 trees with symptoms. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 products indicated that trees CbY1 and CbY17 harboured Mexican periwinkle virescence (16SrXIII)-group and X-disease (16SrIII)-group phytoplasmas, respectively. Identification of two different phytoplasma types was supported by reamplification of P1/P7 products by nested PCR employing X-disease-group-specific rRNA primer pair R16mF2/WXint or stolbur-group-related primer pair fSTOL/rSTOL. These assays selectively amplified rDNA products of 1656 and 579 bp from nine and five trees with symptoms, respectively, of which two trees were coinfected with both phytoplasma types. Phylogenetic analysis of 16S rDNA sequences revealed Chinaberry yellows phytoplasma strain CbY17 to be most similar to the chayote witches'-broom (ChWBIII-Ch10) agent, a previously classified 16SrIII-J subgroup phytoplasma. Strain CbY1 resembled the Mexican periwinkle virescence phytoplasma, a 16SrXIII-group member. The latter strain varied from all known phytoplasmas composing group 16SrXIII. On this basis, strain CbY1 was assigned to a new subgroup, 16SrXIII-C.     

Purification and some properties of Papaya meleira virus, a novel virus infecting papayas in Brazil

'Meleira', or 'sticky disease', is currently the most damaging papaya disease in the mid-eastern Brazilian growing regions. Consistent disease transmission via latex injection, presence of similar isometric particles in the laticiferous vessels of diseased plants, and detection of double-stranded DNA in naturally and experimentally infected papaya trees suggest that a virus is the causal agent. Conclusive evidence for viral aetiology was previously lacking, mostly because every attempt to purify the putative virus from infected papayas had failed. Following the successful purification and partial characterization of the meleira virus, healthy papaya seedlings injected with purified virus particles later developed typical symptoms of the disease. Negatively stained, isometric, full and 'empty' purified virus particles measured 42 and 38 nm, respectively. The viral genome was a single dsRNA molecule of about 12 kbp. Several capsid proteins, ranging in size from 14·4 to 45 kDa, were consistently revealed by PAGE. Papaya meleira virus (PMeV) appears to represent a novel group of viruses, with no known similar counterpart among known plant-, vertebrate-, invertebrate- or prokaryote-infecting viruses.     

Diversity of the coat protein-coding region among Ilarvirus isolates infecting hop in Australia

Coat protein (CP) sequences of 17 Ilarvirus isolates were obtained from hops at three farms in Tasmania, Australia. Phylogenetic analysis of these sequences and additional database sequences indicated several Apple mosaic virus (ApMV) isolate clusters distinct from Prunus necrotic ringspot virus (PNRSV): one containing isolates from apple; one containing a single isolate from almond; a third containing Australian hop isolates of the 'apple' serotype and a German isolate of unknown origin; and a fourth containing Australian hop isolates of the 'intermediate' serotype. Isolates from hop, pear and prune from the Czech Republic either formed a fifth grouping, or were divergent members of the 'intermediate' serotype group. Deduced amino acid (aa) residue differences between the coat proteins of the two hop isolate serotype groups were highlighted as possible regions of serological differentiation. No evidence for coinfection of plants with both serotypes was found. Tests of ApMV-infected hop buds using the Shirofugen flowering cherry assay revealed a possible differentiation of the two strains based on hypersensitivity. Because of serological similarities to PNRSV, these viruses have commonly been reported as strains of PNRSV. However, this study shows ilarviruses from Australian hops are strains of ApMV, but distinct from those infecting Malus spp.     

A new approach to evaluate relative resistance and tolerance of tomato cultivars to begomoviruses causing the tomato yellow leaf curl disease in Spain

A simple procedure to evaluate relative resistance and tolerance of tomato cultivars to the begomoviruses causing tomato yellow leaf curl (TYLC) disease in Spain was developed. To estimate the resistance and tolerance levels of a cultivar, several formulae were developed based on the ratio of infected plants, virus titre (estimated by tissue–print hybridization) and symptom intensity. The formulae were applied to five commercial tomato cultivars (Amoretto, Birloque, Royesta, Tovigreen and Ulises) naturally infected by TYLC viruses. The analyses showed that Ulises, Birloque and Tovigreen exhibited a moderate resistance, and Ulises was also highly tolerant. There was a positive correlation between symptom intensity and virus titre in infected plants, suggesting that the hybridization technique could also be used as an early estimator of tolerance. Finally, molecular hybridization and nucleotide sequence analyses of the begomovirus intergenic region showed that the local TYLC virus population consisted of a single species, Tomato yellow leaf curl virus (TYLCV, formerly TYLCV-Israel), with low genetic variation (nucleotide identity between isolates higher than 97%).     

Rapid detection of Phytophthora cinnamomi using PCR with primers derived from the Lpv putative storage protein genes

Phytophthora cinnamomi is an ecologically and economically important pathogen. In this study, PCR assays were developed with primer pair LPV2 or LPV3 for rapid detection and identification of this organism. Both primer pairs were selected from putative storage protein genes. The specificity of these primer pairs was evaluated against 49 isolates of P. cinnamomi , 102 isolates from 30 other Phytophthora spp., 17 isolates from nine Pythium spp. and 43 isolates of other water moulds, bacteria and true fungi. PCR with both primer pairs amplified the DNA from all isolates of P. cinnamomi regardless of origin. The LPV3 primers showed adequate specificity among all other species tested. The LPV2 primers cross-reacted with some species of Pythium and true fungi, but not with any other Phytophthora species. PCR with the LPV3 primers detected the pathogen at levels of a single chlamydospore or 10 zoospores in repeated tests. The PCR assay was at least 10 times more sensitive than the plating method for detection of the pathogen from artificially infested soilless medium, and, to a lesser extent, from naturally infected plants. PCR with LPV3 primers can be a useful tool for detecting P. cinnamomi from soilless media and plant tissues at ornamental nurseries, whereas the LPV2 primers can be an effective alternative for identification of this species from pure culture. Applications of these assays for detection of P. cinnamomi in other environments were also discussed.     

根结线虫的研究现状

概述了根结线虫的发生分布和传统分类鉴定,以及分子生物学技术(同工酶电泳技术、DNA重组技术、PCR技术)在根结线虫种和生理小种鉴定中的运用及其防治,并对生物防治及抗根结线虫育种、转基因工程在植物线虫学研究领域上的应用前景作了展望。     

一个马铃薯Y病毒山东分离物的分离与鉴定

 从具有典型花叶症状的马铃薯叶片中分离到马铃薯Y病毒(Potato virus Y,PVY)(本文称PVY-SD-TA分离物),扩繁后,提纯病毒,电镜下可观察到700~900 nm×11 nm的病毒粒体,病组织超薄切片观察可见风轮状的内含体结构,寄主反应特性研究表明其能侵染2科13种植物。SDS-PAGE电泳检测病毒编码的外壳蛋白亚基的分子量为33 kDa。以PVY-SD-TA基因组RNA为模板,应用RT-PCR方法和特异引物合成了外壳蛋白基因。对cDNA全序列分析表明,PVY-SD-TA CP基因核苷酸序列与N株系的同源性为96%,与GenBank中登录序列号为AJ390306的O株系分离物的同源性最高,为99%;与国内不同学者报道的PVY中国流行株的同源性分别为96%,97%和98%。通过以上生物学特性和分子水平的研究将PVY-SD-TA鉴定为普通株系(PVY^O株系)。     

利用RT-PCR检测库尔勒香梨苹果茎痘病毒的研究

 以库尔勒香梨新鲜、冷藏、冷冻叶片和皮层为材料,对提取双链RNA (dsRNA)的2种方法和提取总RNA的3种方法进行了分析比较,并对总RNA的提取方法进行了改进,获得了纯度较高、完整性较好的dsRNA和总RNA,在此基础上进行了反转录(RT)和PCR扩增。在国内首次完成了对苹果茎痘病毒(ASPV)的RT-PCR检测,建立了ASPV有效RT-PCR反应体系。用此体系扩增到ASPV一个长约316 bp的片段。实验表明以dsRNA和总RNA为模板均能成功进行RT-PCR检测,且dsRNA优于总RNA。     

引起糖甜菜细菌性叶斑病的萎蔫短小杆菌新致病变种

 1995年在内蒙古临河市新发现了糖甜菜细菌性叶斑病,从病斑所分离的10个细菌菌株经柯赫氏法则验证,均确系该病的病原菌。采用形态观察、表型特征和生理生化特性测定、数值分析、血清学反应、细胞化学成分分析、DNA G+C mol%和DNA-DNA同源性测定进行了鉴定,并与植物病原棒形细菌15个标准菌株进行了比较。该病原菌为革兰氏阳性细菌,不规则短杆状,有一根鞭毛、亚极生或侧生,结合其生理生化特性、细胞化学成分和DNA G+C mol%和DNA-DNA同源性测定结果,认为应属于短小杆菌属(Curtobacterium)的萎蔫短小杆菌(Cur. flaccumfaciens),数值分析也支持这一结论。此外,据血清学反应结果及其对短小杆菌属的其它植物寄主的致病情况,认为该病原菌应是萎蔫短小杆菌种下的一个新的致病变种,定名为Curtobacterium flaccumfaciens pv. beticola pv. nov. Chen et al.,2000(萎蔫短小杆菌糖甜菜致病变种)。