摘要
植原体,原称类菌原体,是一类无细胞壁,存在于植物韧皮部筛管细胞中的植物病原原核生物。寄主范围广,对许多经济作物造成严重的危害。本研究对海南的植原体病害进行了系统的调查研究,从分子水平鉴定了16种植原体病害的病原,明确了他们的分类地位,对海南全省植原体病害的多样性有了全面的掌握,利用实时荧光PCR技术对槟榔黄化病,建立起了一套快速、灵敏、准确、实用的病原检测方法,对该病害的流行和防治起到监控的作用。主要研究结果如下:
(1)本研究首次对海南岛内植原体病害进行了系统的调查,利用植原体通用引物R16mF2/R16mR1和R16F2n/ R16R2对采集自海南岛内18个市县的疑似植原体感染的61个样品进行了检测,从50株植物样品中检测到植原体的存在。通过Blast程序搜索、进化树构建及iPhyClassifier分析共鉴定出槟榔黄化、柱花草丛枝、柱花草黄化、花生丛枝、猪屎豆丛枝、长春花小叶、长春花变叶、长春花黄化、刺芫荽丛枝、假臭草丛枝、木豆丛枝、卵叶山蚂蝗丛枝、野茼蒿变叶、银胶菊丛枝病、竹丛枝和苦楝丛枝等16种植原体病害,在分类上分别属于16SrI-B亚组、16SrI-B**亚组16SrⅠ-G亚组、16SrⅠ-S亚组、16SrII-A亚组、16Sr II-A**亚组、16Sr II-M亚组和16Sr V-H亚组成员,其中16SrⅠ-G亚组、16SrⅠ-S亚组、16Sr II-M亚组和16Sr V-H亚组是以新的亚组形式提出的,丰富了植原体病害的多样性。
(2)鉴定出的16种植原体病害,广泛分布于海南省境内的18个市县,其中以花生丛枝组(16Sr II)和翠菊黄化组(16SrⅠ)为主流株系,榆树黄化组(16SrⅤ)呈零星发生,16Sr II植原体在检测的豆科和菊科植物上都有发生。
(3)对海南岛内槟榔黄化病进行了系统的调查,目前槟榔黄化病在海南屯昌、定安、琼海、万宁、陵水、保亭、三亚等市县已有严重发生,全省染病槟榔面积已达4万亩以上,早期得病的槟榔园已全园摧毁,颗粒无收;重病园发病率甚至达到90%,减产78%-80%;部分槟榔园发病率在10%-30%之间。此病在海南正处于迅速扩展蔓延的阶段。
(4)首次从分子水平确定了海南槟榔黄化病是由翠菊黄化组(16SrⅠ组)中的G亚组植原体引起的,暂时命名为在病原鉴定的基础上,根据植原体的16S rDNA保守区,设计了1组用于检测槟榔黄化病植原体的实时荧光PCR引物/TaqMan MGB探针,建立了槟榔黄化病植原体的实时荧光PCR快速检测体系。该方法对未展开的槟榔花苞组织及心叶(砍伐表现症状的病株取样)检出几率可以达到100%和80%;对疑似染病槟榔的第2、3、5片叶片(不砍伐表现症状的病株,只取部分叶片)的检出几率分别达到26.7%、46.7%和60.0%。为国内外首次报槟榔黄化植原体(Arecanut yellow leaf,AYL),道。
Phytoplasmas, formerly called mycoplasma-like organisms, were cell wall-less pathogenic prokaryotes and resided in the sieve cell of plant phloem tissue, which caused diseases in hundreds of economic plant species worldwide. The report here investigated and studied phytoplasma diseases in Hainan systematically. We identified pathogens of 16 phytoplasma disease on molecular level and cleared the taxonomy of these pathogens which helped us to aware of the diversity of phytoplasma diseases in Hainan. Real-time PCR was done to Arecanut yellow leaf disease which caused serious damages to Arecanut production, the second pillar production only to natural rubber in Hainan. A set of simple, rapid and reliable detection system for arecanut yellow leaf disease was established to monitor the epidemic and control of the disease. This research made a comprehensive and systemic investigation on phytoplasma diseases in Hainan for the first time. Using phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/ R16R2, phytoplasmas were detected in 50 of 61 plant samples from 18 counties in Hainan province by polymerase chain reaction (PCR). Based on sequences comparison of Blast program in GenBank, construction of phylogenetic tree and analysis of iPhyClassifier, 16 phytoplasma diseases have been identified. They were Stylosanthes witches’-broom, Stylosanthes yellows, Crotalaria witches'-broom, Peanut witches'-broom, Periwinkle little leaf, Eryngium foetidum witches’-broom, Praxelis clematidea Witches'-Broom, Common parthenium witches'-broom, Gynura crepidioides phyllody, Desmodium ovalifolium witches'-broom, Bamboo witches'-broom and Chinaberry witches'-broom. These pathogens classified into 6 subgroup as follow, 16SrI-B subgroup, 16SrI-B** subgroup, 16SrⅠ-G subgroup, 16SrⅠ-S subgroup, 16SrII-A subgroup, 16Sr II-A** subgroup, 16Sr II-M subgroup and 16Sr V-H subgroup, among which 16SrⅠ-G subgroup, 16SrⅠ-S subgroup, 16Sr II-M subgroup and 16Sr V-H subgroup was proposed as novel strains, highly enriched the diversity of phytoplasma diseases. The 16 phytoplasma diseases identified here distributed in 18 counties in Hainan. Peanut witches'-broom(16Sr II)and Aster yellows(16SrⅠ)were the main strains while Elm yellows(16SrⅤ)sporadically. 16Sr II phytoplasma were detected in all the legumina and compositae samples Based on a systematic investigation into arecanut yellow leaf disease in Hainan, the disease has been epidemic in Tunchang, Ding’an, Qionghai, Wanning, Lingshui, Baoting, Sanya, et al. in Hainan and caused serious consequences. Infected area of arecanut in the province has reached over 3 million mu. Early infected orchards have already destroyed, and crop failure. In seriously diseased orchards, the incidence of illness up to 90%, cut 78% -80%, or even never produced. Incidence has already reached 10-30% in some orchards. The disease is spreading rapidly in Hainan.
We identified the phytoplasma caused arecanut yellow leaf disease in Hainan belongs to Aster yellows group at the molecular level for the first time and generated a new subgroup 16SrⅠ-G.. . On the basis The phytoplasma was temporarily called Arecanut yellow leaf (AYL) of the pathogen identified, a TaqMan MGB probe was designed for detection of arecanut yellow leaf disease according to the conservative region of AYL phytoplasma 16S rDNA gene. A real-time fluorescence PCR detection system for arecanut yellow leaf disease was established. The detecting accuracy for unexpanded inflorescence and unexpanded leaf came up to 100% and 80%, respectively. The detecting accuracy came up to 26.7%, 46.7 and 60% respectively for 2nd leaf, 3rd leaf and 5rd leaf.
引文
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