松树脂溃疡病菌传入中国风险评估及分子生物学研究

【作者】 廖太林；

【导师】 叶建仁； 陈建东；

【作者基本信息】 南京林业大学 ， 森林保护学， 2007， 博士

【摘要】 松树脂溃疡病（Fusarium circinatum Nirenberg & O’Donnell）为国外针叶树上发生的一种危险性林木病害。病害为害可引起寄主树干脂状溃疡、雌花和松果坏死、种子变质以及树苗枯死等症状。该病菌可随种子、苗木、木材、媒介生物以及黏附土壤进行远距离传播。近年来，美国加利福尼亚、日本、南非、智利等地相继发现有该病害为害，松树脂溃疡病在世界各地呈现出迅速扩散蔓延之势。为防止其随疫区货物传入国内，保护中国的森林、环境以及旅游资源，本文对松树脂溃疡病菌生物学特性、传入风险以及分子检测技术进行研究，主要结果如下：（1）松树脂溃疡病菌在6种供试培养基上均能生长，以PDA培养基上生长最好，选择性培养基上生长最差。菌丝适宜生长温度范围为18～27℃，最适温度为24℃。在全黑暗环境下生长最好，光暗交替次之，全光照较差。适宜菌丝生长pH值范围为5～9，最适宜pH为8.0。在基本培养基上，以半乳糖为其最佳碳源，硝酸钠为其最佳氮源，维生素对菌丝生长影响不明显。病菌分生孢子在包括无菌水在内的各种机质中均可萌发，营养以5％葡萄糖为最好。分生孢子在15～35℃、相对湿度85％～100％以及pH值3～12内均可萌发。分生孢子萌发最适温度为25℃、最适pH值为7.0，最适相对湿度为100％。黑暗、氧气充足环境有利于孢子萌发。菌丝、分生孢子致死温度分别为55℃和52℃。（2）遵循系统性、重要性、可操作性以及特殊性原则，本研究建立了由目标层、准则层、指标层组成的外来林木病害风险评估指标体系。应用层次分析法确定评价指标权重数值，提出了外来林木病害风险评估的综合模型及风险划分等级。运用该模型对松树脂溃疡病菌传入中国风险进行了分析，得出风险评估值A=0.8237，表明该病原菌属于传入风险极高类有害生物。上述结果提示松树脂溃疡病菌应列入我国禁止进境的检疫性有害生物名录，口岸应加强其入境检疫。（3）利用AFLP分子标记技术对松树脂溃疡病菌及其近缘种进行了DNA指纹分析，筛选出10对多态性较高的引物。上述10对引物在供试的9种17个菌株中扩增出298条带，其中有多态性带283条，多态性位点百分率为94.97％。每对引物产生的多态性位点百分率在89.29％～100％之间，表明松树脂溃疡病菌及其近缘种间在DNA水平上存在广泛变异。根据AFLP标记结果计算松树脂溃疡病菌及其近缘种间的分子距离，对它们的亲缘关系进行了定量描述。聚类分析结果表明，菌种间的聚类与生物学种分类的结果一致。分析了引物E-AT／M-CAA绘制的镰刀菌AFLP指纹图谱遗传多样性，确定了李瑟组镰力菌各交配群的特异带、差异带，区分了供试的7个交配群。（4）根据GenBank中登录Fusarium属的31种核糖体RNA基因内间隔区（IGS）序列，设计Fusarium属IGS上的一段通用引物G1／G2，可扩增873 bp左右的条带。进一步根据Fusarium属IGS序列差异设计一对引物S1／S2，可特异性从F．circinatum菌株中扩增到一条364 bp左右的条带，而从其他供试菌株中不能扩增出条带，表明该对引物可以将F．circinatum与Fusarium属中的其他种分开。利用上述2对引物组合建立巢式PCR检测体系，能够直接将F．circinatum与其他相似种和相关种分开，且灵敏度可达DNA浓度5×10^-3pg.μl^-1，10个孢子／100μl H₂O。上述结果提示建立的巢式PCR反应体系可望直接应用于口岸对F．circinatum的检测。更多还原

【Abstract】 Pitch canker, caused by Fusarium circinatum Nirenberg & O’Donnell （=F. subglutinans（Wollenweb and Reinking）Nelson, Toussoun, and Marasas f. sp. pini（teleomorph: Gibberellacircinata Nirenberg and O’Donnell））, is a serious disease of conifers: A characteristicsymptom of infection on most hosts is a copious pitchy flow from cankers or necrotic tissue onlimbs, trunks, roots, and cones. Long-range spread of the disease may be connected to themovement of infected seed, seedlings, wood, vector, and soil. More recently, the disease hasbeen introduced into California, South Africa, Japan, and Chile.1) F. circinatum could grow on six different kinds of culture medium which were tested.On PDA plate, its growth was fastest, while on the Fusarium selective medium was slowest.The fungus grew well at 18～27℃, with optimum at 24℃. The treatment of whole darknesscould promote mycelium growth. The fungus also grew well at pH 5～9, with optimum at pH8.0. On the basic culture medium, galactose was the best carbon sources and sodium nitratewas the best nitrogen source. Vitamin did not distinctly affect hypha growth. Conidiosporescould germinate in all media including sterile water, while the optimal medium was 5% glucose.The conidia could germinate at 15～35℃, RH 85%～100% and pH 3～12, with the optimal at 25℃, RH 100% and pH 7.0. Darkness & enough oxygen were good for spores germination. Thelethal temperature for hypha was 55℃, and for spores was 52℃.2) Following the principle of systematicality, essentiality, practicality and specificity, anindex system for risk assessment of exotic forestry disease was established, consisting of 3different layers, i e. objective, criteria, and index layers, and 14 index parameters. In addition,the weight of index parameters was determined by analytic hierarchy process （AHP）, andcomprehensive modeling and risk grading of exotic forestry disease were presented. Pest riskassessment （PRA） of F. circinatum was developed using above modeling. The PRA result was0.8237, and it belongs to very high devastating pest in China. It suggests that F. circinatumshould be listed the quarantine pest in China.3) AFLP fingerprinting analysis of F. circinatum and relative species was carried out. Tenprimer-paris that could generated abundant polymorphism fragments were screened out. Theyamplified 298 nucleotide acid fragments in the seventeen strains of Fusarium spp., 283polymorphism fragments among them. Percentage of polymorphism fragments produced byeach pair of AFLP primer was 94.97% in average, and varied from 89.29%-100%. All thesedata indicated that considerable genetic variation existed among F. circinatum and relativespecies at DNA level. Molecular genetic distances among Fusarium spp. were calculated, andthe relationship among them was described quantitatively. Compared with biological species,the result of cluster analysis were basically similar. Genetic diversity of E-AT/M-CAA AFLPfingerprinting of Fusarium spp. was analyzed, unique and difference bands for each specieswere determined, and all Fusarium section Liseola tested were identified based on the E-AT/M-CAA AFLP fingerprinting.4) The oligonucleotide primers G1/G2 （5’-GCGGTGTCGGTGTGCTTGTA-3’/5’-ACTCACGGCCACCAAACCAC-3’）, derived from the differentiation of intergenic spacer （IGS）regions within Fungi, amplified a single 873 bp product from Fusarium spp.. The IGS DNAsequences of Fusarium spp. were gained from GenBank. Oligonucleotide primers S1/S2（5’-CTTACCTTGGCTCGAGAAGG-3’/5’-CCTACCCTACACCTCTCACT-3’）, derived fromIGS DNA, amplified a product of 364 bp which was unique to F. circinatum. Thenested-PCR using primers G1/G2 and S1/S2 identified F. circinatum from other Fusarium spp..This PCR assay was proved to be highly sensitive with the detection limit of 5×10^-3 pg.μl^-1genomic DNA or 10 spores/100μl H₂O. The result indicates that the nested-PCR couldsuccessfully used to detect the presence of F. circinatum directly from infected plant tissue.更多还原