苜蓿小光壳叶斑病抗病品种筛选及其抗病机制研究

Leptosphaerulina leaf spots on alfalfa is a fungal disease induced by Letophaerulina briosiana (Poll.) Graham & Luttrell. It is epidemic widely in Canada and northeast of American which are cool and humid in summer. As one of the important diseases for alfalfa, sometimes it’s more dangerous than Common leaf spot. Our early investigation found that Leptosphaerulina leaf spots on alfalfa was very serious in Heilongjiang province and became one of the main diseases in alfalfa production. So the yield and quality of alfalfa were decreased greatly by it. This study will isolate and identify the L. briosiana from infected alfalfa plants in the field. The molecular biology method for detecting L. briosiana will be developed by analyzing the ITS sequences of L. briosiana.The pathogenic test will be conducted among different cultivars by the isolated strains from the field. One of the eventual goals of this work is to find the resistant and susceptible cultivars, which lay the foundation for further study on identification of molecular markers on resistance loci and disease resistance breeding. Meanwhile the resistance mechanism of alfalfa for L. briosiana will be revealed by detecting the expression level of variety protective enzyme in the plants of resistant and susceptible cultivars. These researches will provides theoretical basis for the control of Leptosphaerulina leaf spots on alfalfa and further study on resistance breeding.

苜蓿小光壳叶斑病是由苜蓿格孢球壳菌（Letophaerulina briosiana (Poll.) Graham & Luttrell）引起的一种真菌病害。其分布较广，在加拿大、美国的东北部和夏季气候较凉爽、潮湿的国家和地区均是紫花苜蓿田重要病害之一，其危害性常超过苜蓿褐斑病。通过本研究小组前期调查发现，目前黑龙江省苜蓿生产田中苜蓿小光壳叶斑病病害情况甚为严重，其已发展为该地区苜蓿田的主要病害之一，极大影响了苜蓿的产量和饲用价值。本研究拟对田间获得的病样进行病原菌菌株分离鉴定。通过ITS序列分析，确立苜蓿小光壳叶斑病分子生物学检测方法。并对我国北方地区常用苜蓿品种质资源进行抗病性鉴定，筛选抗感品种，以期为后续该病抗病位点分子标记、抗病品种选育等方面的深入研究奠定基础。此外，通过对抗感品种感染苜蓿格孢球壳菌（L. briosiana）前后各类保护酶表达水平差异的研究，为抗病机制的揭示提供参

近年来调查发现，苜蓿小光壳叶斑病是黑龙江省分布比较广泛的一种真菌病害，主要侵害苜蓿二、三茬再生草，对苜蓿生产造成严重影响。本研究对该病的病原进行了分离鉴定，设计了其特异性引物，对我国北方地区常用苜蓿品种种质资源进行抗病性评价，筛选抗感品种，并对抗感品种感染病原菌后各生理指标表达水平进行了比较。本研究将为苜蓿小光壳叶斑病抗病育种及抗病机制的研究奠定理论基础，并为病害防控、提高牧草产量和质量提供参考。本研究的主要研究结果如下：.（1）采用形态学和分子生物学技术对从黑龙江省大庆市黑龙江八一农垦大学苜蓿试验田采集到的苜蓿小光壳叶斑病病原菌进行分离鉴定。形态学鉴定结果表明该病原菌属于小光壳属（Leptosphaerulina）；采用提取病原菌菌丝DNA、PCR扩增等分子生物学方法鉴定病原菌，在构建的发育树结果显示该菌与小光壳属的Leptosphaerulina trifolii亲缘关系最近，可信度达100%。两方面结果综合表明：苜蓿小光壳叶斑病的病原菌是Leptosphaerulina trifolii。.（2）根据苜蓿小光壳叶斑病病原菌的rDNA-ITS序列，成功设计出一对具有高度特异性的引物Lt1F/Lt1R。该对引物能特异性地扩增出一条小光壳叶斑病病原菌DNA的230 bp特异性片段，且对病原菌DNA的检测量低至20 fg/µL。设计的引物Lt1F/Lt1R特异性强、灵敏度高，可用于苜蓿小光壳叶斑病的早期诊断。.（3）采用孢子弹射接种方法，以发病率、病情指数为指标，在室内整株盆栽接种小光壳叶斑病，评价了国内外14个苜蓿品种对苜蓿小光壳叶斑病的抗性。结果表明14个苜蓿品种中有1个高抗（HR）品种：‘CW2000’；8个中抗（MR）品种：‘皇冠’、‘北极熊’、‘WL298HQ’、‘SK3010’、‘DS310FY’、‘WL354HQ’、‘龙牧801’和‘百绿皇后’；5个中感（MS）品种：‘敖汉’、‘WL168HQ’、‘TG4’、‘龙牧806’和‘斯贝德’；无高感（HS）品种。.（4）对14个苜蓿品种分别接种病原菌L. trifolii，测定不同品种叶片中的超氧化物歧化酶（SOD）、过氧化氢酶（POD)、丙二醛（MDA）、多酚氧化酶（PPO）、脯氨酸（PRO）和几丁质酶的表达水平。结果表明：无论抗感品种，6个生理指标在接种后均显著高于对照。