榆树黄化病植原体的分子检测与鉴定

 利用植原体16SrRNA基因的通用引物R16rrLF2/R16mR1和R16F2n/R16R2对山东泰山上发生的榆树(Ulmus parvifolia)黄化病感病植株总DNA进行巢式PCR扩增,得到了约1.2kb的特异性片段,从分子水平证实了榆树黄化病的病原(EY-China)为植原体。将扩增到的片段测序,并进行一致性和系统进化树分析。结果表明,该分离物属于植原体榆树黄化组(Candidatus Phytoplasma ulmi),与该组成员16SrRNA序列的一致性均在98.2%以上,其中与16SrV-B亚组中的纸桑丛枝(Paper mulberry wiches'-broom)和枣疯病(Jujube witches'-broom)植原体一致性最高,达到99.4%,在系统进化树中与该亚组成员聚类到同一个分支,说明该分离物属于植原体16SrV-B亚组。本研究首次对在中国引致榆树黄化病的植原体进行了分子检测,并通过核酸序列分析将其鉴定到亚组水平。     

Association of aster yellows subgroup 16SrI-B phytoplasmas with a disease of Rehmannia glutinosa var. purpurea

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra-thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle ( Catharanthus roseus ). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI-B) in naturally infected samples of R. glutinosa var . purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI-B. This is the first report of natural occurrence of phytoplasma-associated disease in R. glutinosa var. purpurea.     

3种检疫性葡萄黄化植原体的快速检测

 葡萄上的植原体病害由于引起叶片黄化而被称为葡萄黄化病。由于这一病害极为严重,葡萄黄化植原体被列为我国的植物检疫对象。其中,葡萄金黄化植原体(16SrV)、维吉尼亚葡萄黄化植原体(16Sr芋) 和澳大利亚葡萄黄化植原体 (16Sr狱) 是引起葡萄黄化病的主要3 个株系,它们导致的病害症状相似,难以区分。本文进行了3 个株系16S rRNA 基因 DNA 序列比对,而后根据同源性相对低的序列设计了43 条特异性引物、103 对引物对组合,对葡萄黄化植原体3 个株系各自的DNA 及混合DNA 进行PCR 扩增,从中筛选出来特异性较强的8 个引物对组合。这些引物对组合,能够同步、特异、快速地检测3 种葡萄黄化植原体。     

桃黄化病植原体昆虫介体的验证及其带毒部位PCR检测

 利用植原体16S rDNA通用引物对采集的北京和天津黄化病桃树总DNA进行巢式PCR检测,证明发病样本的病原为桃黄化病植原体。经过检测昆虫总DNA和经取食过的人工培养液DNA中桃黄化病植原体的16S rDNA,结果表明桃黄化病植原体的有效传播媒介昆虫为桃一点叶蝉。将带毒桃一点叶蝉个体的头部、胸部以及腹部分离,分别在这些部位检测到桃黄化病植原体的16S rDNA,说明桃一点叶蝉的头部、胸部以及腹部都可带毒,表明植原体可从植物汁液进入叶蝉的口针、食道和肠道。     

Increased plant tolerance against chrysanthemum yellows phytoplasma (‘Candidatus Phytoplasma asteris’) following double inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif

The aim of this work was to assess the effects of a combined inoculum of a rhizobacterium and an arbuscular mycorrhizal (AM) fungus on plant responses to phytoplasma infection, and on phytoplasma multiplication and viability in Chrysanthemum carinatum plants infected by chrysanthemum yellows phytoplasma (CY). Combined inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif resulted in some resistance to phytoplasma infection (about 30%), delayed symptom expression in nonresistant plants, improved growth of the aerial part of the infected plants (+68·1%), and altered root morphology (root tip number: +49·9%; branching degree: +82·8%). Combined inoculation with the two beneficial microorganisms did not alter CY multiplication and viability. In inoculated and infected plants, phytoplasma morphology was typical of senescent cells. A more active and efficient root system in double‐inoculated plants probably mediated the effects of the two rhizospheric microorganisms in the infected plants. The practical application of rhizospheric microorganisms for mitigating phytoplasma damage, following evaluation under field conditions, represents an additional tool for the integrated management of phytoplasmosis.     

Detection and characterization of a phytoplasma associated with annual blue grass (Poa annua) white leaf disease in southern Italy

A phytoplasma was detected in annual blue grass (Poa annua L. Fienardo), exhibiting white leaf symptoms, that was grown in the fields near Caserta in southern Italy. Based on restriction fragment length polymorphism analysis of PCR-amplified 16S rDNA sequences, the phytoplasma associated with annual blue grass white leaf disease was identified as a new member of phytoplasma 16S rRNA group XI (16SrXI) (type strain, rice yellow dwarf phytoplasma). The annual blue grass white leaf phytoplasma is most closely related to Bermuda grass white leaf phytoplasma found in Asia. Annul blue grass white leaf and Bermuda grass white leaf phytoplasmas were designated as the third subgroup (16SrXI-C) of group XI. This is the first report that a plant pathogenic phytoplasma belonging to group 16SrXI is present on the European continent.     

Detection and identification of a phytoplasma from lucerne with Australian lucerne yellows disease

Foliar and root symptoms are described for Australian lucerne yellows (ALuY), a disease common in Australian lucerne seed crops. A phytoplasma was detected in plants exhibiting symptoms, but not in symptomless lucerne plants. Oligonucleotide primers specific to the phytoplasma 16S-23S rRNA intergenic spacer region (SR) were used in polymerase chain reaction (PCR) assays on DNA extracted from lucerne plants with and without symptoms. Identical restriction fragment length polymorphism (RFLP) enzyme profiles were obtained for PCR products amplified from 10 yellows-affected lucerne samples. RFLP profiles obtained for four restriction enzymes were different from those of the tomato big bud (TBB) phytoplasma. ALuY phytoplasma PCR products were sequenced to determine phylogeny and were found to fall within the faba bean phyllody phytoplasma group, or phytoplasma group 16srII. Transmission electron microscopy revealed phytoplasmas in the phloem of yellows-affected plant samples, but not in symptomless plant samples. Fungal, bacterial and viral agents in the aetiology of Australian lucerne yellows were ruled out.     

Sensitive Detection of a Phytoplasma Associated with Little Leaf Symptoms in Withania somnifera

Withania somnifera is an important medicinal plant native to the Indian-sub continent. Owing to the presence of a number of precious alkaloids, flavonoids and withanolides, it is widely used in the Indian and African systems of medicines. It is severely affected by phytoplasma present in the sieve tubes of phloem. With a view to micropropagate phytoplasma-free W. somnifera plants, an efficient and effective nested PCR-based system was developed for detection of associated phytoplasmas. Universal primers, designed from the 16S rDNA sequences of phytoplasmas, were applied in direct/nested-PCR. Total DNA extracts from leaf tissues of 33 suspected symptomatic and 11 non-symptomatic plants were subjected to direct PCR. The direct PCR products were subsequently employed as templates in nested PCR. The nested PCR could reamplify direct PCR products yielding a DNA fragment of 1.4 kb. A phytoplasma was detected in all the diseased plants and not from the healthy looking plants. Further, it was sensitive enough to amplify phytoplasma DNA obtained from crude DNA diluted up to 2500 times from naturally infected plants and also from various stages of in vitro-propagated diseased plants. Identical restriction fragment polymorphism enzyme profiles were obtained following restriction enzyme digestion of nested PCR products, obtained from five different plants, by EcoRI, AluI and RsaI restriction endonucleases. The developed nested PCR based system should facilitate indexing of the phytoplasma in different stages of in vitro-generated plants and probably identification of, as yet unknown, hosts and vectors of phytoplasma associated with phytoplasma disease of W. somnifera.     

Production of Monoclonal Antibodies against Apple Proliferation Phytoplasma and their Use in Serological Detection

Two monoclonal antibodies were obtained against the apple proliferation phytoplasma that provide easy, rapid, specific and sensitive serological detection. They reacted specifically by using ELISA and immunofluorescence techniques with apple proliferation-infected periwinkles and apple trees from different regions in northern Italy and Slovenia, but not with several other phytoplasma isolates. We did not observe any monoclonal antibody reaction even using phytoplasmas belonging to the same phylogenetic group such as European stone fruit yellows and pear decline. Two serological techniques, immunofluorescence and ELISA, were compared with DAPI staining and PCR. From July until leaf fall ELISA was as sensitive as PCR but was more rapid and convenient than PCR; immunofluorescence was useful for specific detection of apple proliferation phytoplasma on roots throughout the year. Serological techniques could be conveniently applied in the roots, stems and leaves of apple trees depending on specific phenological stages of the plants.     

上海地区草莓炭疽病病原鉴定

 Seventeen isolates of strawberry anthracnose were obtained from Shanghai suburb. According to the temperature test for mycelial growth, these isolates were divided into two groups:one was strain CMf-04 with optimal temperature of 24℃, the other was including 16 strains with optimal temperature of 28℃, and most of which could produce sexual stage on PSA media, e.g. QPg-961. Conidia of CMf-04 were hyaline, unicellular, straight and fusiform, (12.1-16.4) μm×(3.6-5.4) μm. Conidia of QPg-961 were hyaline, unicellular and ovoid to oblong, (13.0-19.7)μm×(4.1-7.3) μm. On basis of morphologcal, biological characteristics and the sequences of ribosome rDNA ITS, isolate CMf-04 from the strawberry rotten fruits was identified as Colletotrichum acutatum; while all the other isolates from the diseased leaf stalks, runners and root crowns at strawberry plantlet propagation stage were belonged to Colletotrichum gloeosporioides. It proved that C. gloeosporioides was the main pathogen of the strawberry anthracnose in summer strawberry propagation fields in Shanghai and it is of significance for the breeding of resistant strawberry and its control.     

几种药剂对草莓炭疽病的效果

采用菌丝生长速率法测定了48%波尔多液可湿性粉剂、50%咪鲜胺可湿性粉剂、40%福美锌可湿性粉剂和70%代森联干悬浮剂4种药剂对草莓炭疽病菌的室内毒力。结果表明,4种药剂对菌丝生长的EC50的大小顺序为:50%咪鲜胺40%福美锌70%代森联48%波尔多液,50%咪鲜胺的EC50为6.24 mg/L。并选取毒力较好的50%咪鲜胺可湿性粉剂和40%福美锌可湿性粉剂进行了田间药效测定,48%波尔多液可湿性粉剂作为对照。试验发现:50%咪鲜胺可湿性粉剂在田间的效果最好,其800 mg/L处理在田间2次药后7 d的防效达到了80.26%,与对照药剂48%波尔多液可湿性粉剂间存在显著性差异。     

草莓内生细菌的分离及草莓灰霉病菌拮抗菌的筛选鉴定

本研究从健康的草莓植株内分离到54株内生细菌,其中根、茎、叶组织中分离内生细菌种群密度为2.8×101～3.95×104cfu/g鲜重不等。通过对峙试验,得到5株对草莓灰霉病菌有强拮抗作用的菌株,占所分离内生细菌总数的9.26%。来自草莓叶的SL6菌株抑菌效果最佳,抑菌半径达15mm,无菌发酵液对草莓灰霉病菌菌丝的抑制率为97.6%,具有作为生防菌的应用潜能。经形态、生理生化和16SrRNA等分析测定,并通过MEGA4方法构建其16SrDNA系统发育树,将SL6菌株鉴定为枯草芽胞杆菌(Bacillus subtilis)。     

新型生物源农药与化学农药对草莓叶螨的防效对比研究

草莓叶螨是影响北京地区草莓生产的重要害螨,本文对其进行了两种新型生物源农药(藜芦碱、多杀霉素)及化学农药(高效氯氟氰菊酯、联苯菊酯)的防效对比研究。室内毒力测定结果表明:2.5%高效氯氟氰菊酯EC和0.5%藜芦碱SL对朱砂叶螨毒力最高,其LC_(50)分别为2.54mg/L和4.18mg/L;其次是10%联苯菊酯EC和2.5%多杀霉素SC,其LC_(50)分别为13.16mg/L和84.51mg/L。田间试验结果表明:2.5%高效氯氟氰菊酯EC和0.5%藜芦碱SL对草莓叶螨防控效果最好,施药7d后防治效果达80%以上,施药14d后防治效果达82%左右,施药28d后防治效果达84%左右,持效期较长。10%联苯菊酯EC和2.5%多杀霉SC素防控效果欠佳,施药7d后的防治效果为50%左右,具有一定的防治效果;但施药14、21及28d后害虫总数有增长趋势,未表现出持续的抑制效果。与对照区相比,各药剂均表现出对草莓叶螨具有一定的防控作用。高浓度2.5%高效氯氟氰菊酯EC对草莓叶片有轻微药害,在生产中要严格控制其使用剂量;0.5%藜芦碱SL试验处理未对草莓及非靶标生物产生不良影响,安全无药害且防治效果好,在实际生产中具有良好的推广应用价值。     

11个草莓品种对炭疽病的田间抗性表现

在田间自然发病条件下,对种植在北京市昌平区的11个草莓品种的炭疽病发生情况进行了调查和分析比较。结果表明：草莓同一品种的三个部位（叶片、叶柄和匍匐茎）对炭疽病的抗性表现不一致,除‘莫哈维’和‘叙利亚’叶柄的病情指数高于叶片和匍匐茎外,其他品种都是匍匐茎的病情指数最高,‘波特拉’匍匐茎的病情指数高达73。根据叶柄对炭疽病的表现,在所调查的草莓品种中,品种‘森格纳’表现免疫,‘甜查理’、‘阿尔比’和‘圣安德瑞斯’为高抗,‘本尼西亚’和‘波特拉’表现为抗病,‘叙利亚’为高感,其他品种为感病。