苹果轮纹病病原的重新厘定及致病性分化研究

Apple ring rot,putatively caused by Botryosphaeria spp., is an important disease of apple in East Asian countries. The disease etiology is complex and not yet clearly understood,therefore confounding the classification and nomenclature of the pathogen or pathogens involved. For example, several pathogen names, including Botryosphaeria berengeriana，B. berengeriana f. sp. piricola and B. dothidea have been proposed by researchers. This uncertainty has limited further studies of the disease, such as infection sources, epidemiology and disease management techniques. In this project, we will re-delineate the species boundaries of the Botryosphaeria dothidea species complex associated with apple ring rot in East Asian countries, including China, Japan and Korea, based on multi-gene phylogenetics combined with morphology, biology and pathogenic characteristics. It is likely that new taxa will be discovered to reflect the differences found on genetics, pathogenicity and morphology. Molecular markers for PCR identification will be constructed for the new taxa. Concurrently, isolates of Botryosphaeria dothidea sensu lato from conjectural reservoir hosts adjacent to apple orchards will be examined for phylogeny, and pathogenicity tests will be conducted to understand their pathogenicity differentiation on different hosts and to determine their relative importance as infection sources for apple ring rot in natural conditions. The research will lay a foundation for future studies on resistance breeding, disease epidemiology, plant quarantine and sustainable management of apple ring rot.

苹果轮纹病是一种苹果主要病害，由于病原种类混乱不清、侵染来源不明，严重影响了病害流行规律和防治技术研究，成为我国苹果生产健康发展和出口贸易的主要限制因素之一。本研究拟采用多基因核苷酸序列系统发育分析，结合形态学、生物学、致病性特征，对发生于中国、日本、韩国等东亚国家的苹果轮纹病病原进行重新厘定和划分，建立能够反映其遗传、致病性分化、形态变异等异质性的新病原种类或新专化型；建立不同种类或专化型的PCR鉴定体系；同时对苹果园周边寄主相关病原（Botryosphaeria dothidea sensu lato）（广义）进行致病性分化研究，明确其是否为苹果轮纹病的初侵染来源。本研究对于厘清苹果轮纹病病原种类及其分布，明确病害侵染来源具有重要意义，为进一步病害发生规律、抗病育种奠定理论基础，从而为病害可持续性控制和植物检疫提供理论依据及技术支持。

苹果轮纹病在各苹果产区普遍发生，为我国苹果的“三大病害”之一。1907年在日本最早报道。由于葡萄座腔菌属种间界限不清、病原形态的多样性以及缺乏系统的研究等原因，苹果轮纹病病原种类不清、名称混乱，导致不同国家、地区研究结果不一致，甚至矛盾，缺乏可比性。100多年来，轮纹病病原问题一直困扰着植物病理学家。. 本项目通过多基因序列系统学、形态学、致病性、培养特性、寄主范围等研究，获得以下主要结果：明确了我国苹果轮纹病主要由两种病原组成：葡萄座腔菌Botryosphaeria dothidea和粗皮葡萄座腔菌B. kuwatsukai (Hara) G.Y. Sun and E. Tanaka，其中B. kuwatsukai为本研究建立的病原名称新组合；B. dothidea和B. kuwatsukai存在显著种间致病性分化： B. dothidea引起0.7-1.0 mm小型病瘤，B. kuwatsukai引起3-4 mm大型病瘤，B. dothidea寄主范围较宽，B. kuwatsukai仅限于苹果和梨；果园周边寄主的葡萄座腔菌存在明显致病性分化，但他们均能侵染苹果枝干或果实，为苹果轮纹病潜在侵染来源；根据致病性差异将葡萄座腔菌（B. dothidea）划分为7个专化型：苹果专化型、核桃专化型、桃树专化型、杨树专化型、枣树专化型、柳树和柿树专化型；构建了利用SR1R/SR5引物对区分B. dothidea和B. kuwatsukai快速鉴定体系，同时利用37℃下两种病原菌生长速率差异也可以进行菌种的快速鉴定；基于系统发育分析证明粗皮葡萄座腔菌在欧美国家也存在，是世界广布的种类，说明发生在中国等东亚地区的苹果粗皮病不应被列为欧洲及美国的检疫对象。. 本研究对苹果轮纹病发生规律、抗病性鉴定、抗病育种、病害防治等具有重要指导意义，也为扫除我国苹果出口技术壁垒提供了技术支持。