紫花苜蓿新病毒的分子鉴定及其与苜蓿“丛枝”症状病害关系分析

Medicago sativa L. is an important cultivated forage. In recent years, there are many serious dwarfing alfalfa and a large number of branches growing from their root (witches' broom) appear in 4 to 6 years old alfalfa field in Xinjiang Altay, Tacheng, Changji. The disease rate is 50%-100%, grass production decreased by 50 to 70%. The rate of witches' broom caused by phytoplasma was only 16.8% in the plants with the symptoms, and nine viruses had been detected in the remaining pathogenic plants. but there was no definite virus which could lead to alfalfa witches' broom at home and abroad is reported. In this project, the classification status of alfalfa virus in Xinjiang was determined by small RNA sequencing. The genetic species and population composition of the virus were identified, and the genetic variation and phylogenetic relationship of the strain were analyzed. The relationship between these viruses and the symptom of witches' broom caused by alfalfa virus was analyzed by Real-time quantitative PCR (qPCR). The antiviral resistance of different alfalfa cultivars was studied with potent virus infectious clones. The results will clarify the species of alfalfa in Xinjiang and reveal the relationship between virus and witches' broom symptoms from the molecular level, which can provide scientific basis for the diagnosis, quarantine inspection, Antiviral breeding  and comprehensive control of alfalfa virus disease.

紫花苜蓿是重要的栽培牧草。近年来，在新疆阿勒泰、塔城、昌吉地区4年至6年生紫花苜蓿田间出现严重矮化、根部枝条大量增多（简称丛枝）的植株达50%~100%，草产量下降50~70%。经初步研究确定，具有此症状的植株中由植原体引致的丛枝病的发病率仅为16.8%，在其余发病植株中已检测出9种病毒，但国内外尚无明确可引致紫花苜蓿丛枝症状病害的病原病毒的报道。本项目拟通过小RNA深度测序技术确定新疆苜蓿病毒的分类地位，明确毒源种类和种群组成，分析毒株的遗传变异、系统发育关系；运用实时荧光定量PCR（qPCR）技术确定引起丛枝症状优势苜蓿病毒的类型、组成，分析这些病毒与造成苜蓿丛枝症状病害的关系；构建优势病毒侵染性克隆，开展不同苜蓿品种抗病毒抗性评价研究。研究结果将明确新疆苜蓿病毒的种类，从分子水平揭示病毒与丛枝症状的关系，可为苜蓿病毒病害的诊断、检疫检验、抗病育种和综合防治提供科学依据。

紫花苜蓿是重要的栽培牧草。近年来，在新疆阿勒泰、塔城、昌吉地区4年至6年生紫花苜蓿田间出现严重矮化、根部枝条大量增多（简称丛枝）的植株达50%~100%，草产量下降50~70%。经初步研究确定，具有此症状的植株中由植原体引致的丛枝病的发病率仅为16.8%，在其余发病植株中已检测出9种病毒，但国内外尚无明确可引致紫花苜蓿丛枝症状病害的病原病毒的报道。.本项目按研究计划主要完成了以下研究内容：1）运用RT-PCR技术确定了引起丛枝症状的优势苜蓿病毒主要是苜蓿花叶病毒（Alfalfa mosaic virus，AMV）、苜蓿矮化病毒（Alfalfa dwarf virus，ADV）、菜豆卷叶病毒（Bean leafroll virus，BLRV）和菜豆黄花叶病毒（Bean yellow mosaic virus，BYMV）；2）建立了几种优势苜蓿病毒快速检测的PCR反应体系；3）扩增并克隆了苜蓿的BLRV外壳蛋白基因，运用生物信息学手段对BLRV所对应的蛋白的理化特性以及结构域等进行分析；4）构建了苜蓿花叶病毒（AMV）的侵染性克隆。.通过本项目的实施，明确了新疆苜蓿病毒种类和分布，首次证实菜豆卷叶病毒(BLRV)、苜蓿矮化病毒（ADV)在中国侵染苜蓿，丰富了菜豆卷叶病毒 、苜蓿矮化病毒苜蓿分离物在中国新疆地区基因组序列信息；确定引起苜蓿“丛枝”症状的优势病毒类型、组成；建立了几种有时苜蓿病毒的分子检测技术体系；构建了苜蓿花叶病毒侵染性克隆。研究结果明确了新疆苜蓿病毒的种类，从分子水平揭示病毒与丛枝症状的关系，为苜蓿病毒病害的诊断、检疫检验、抗病育种和综合防治提供科学依据。.本项目执行期间，项目负责人李克梅晋升教授职称，刘琦、王丽丽、代培红3人晋升副教授职称。本项目已培养硕士研究生1名，协助培养硕士研究生2名。在国内学术刊物发表论文4 篇。