摘 要

杉木（Cunninghamia lanceolata）是我国重要的速生树种，而杉木炭疽病成为杉木上重要的常发性病害，危害杉木的针叶、枝梢甚至是茎部，炭疽病病原生长速度快，能够通过空气、雨水和昆虫等媒介传播，极易从伤口、皮孔、气孔和自然孔口侵入，某些年份会在局部地区爆发流行，带来巨大的经济损失。

本研究从江苏，福建，广西等8个省份的杉木主要栽培区的炭疽病叶片材料分离获得64个菌株。根据形态特征，68.25%菌株被鉴定为Colletotrichum gossypii、7个菌株被鉴定为C. fragariae，C. gloeosporioides和C. tropicale各4株，C. karstii有2株、C. musae、C. truncatum、C. caricae各仅有1株，借助引物ITS1/ITS4、PCR分析等手段充分证明，形态分析的各类真菌均符合其各自的特征。

8类炭疽菌菌株在菌落颜色、形态、菌丝日均生长速率、分生孢子的形状与大小、附着胞形态以及相同时间内孢子萌发率等方面均具有明显差异，甚至同种炭疽菌种内也存在着形态差异，具有一定的种内多样性。

基于ITS基因序列构建系统发育树，来自8个省份样地的34株杉木炭疽病菌以较高的支持率聚为6个进化分支，其中20个菌株与C. gossypii模式菌株聚为一支（Ⅰ），说明其在炭疽病菌中的优势地位，有2株与C.theobromicola模式菌株聚为一支（Ⅱ），SM1#C等8株聚为一支（Ⅴ），并且分为两个亚组，SM1#C与C. gloeosporioides聚为一组，其余与C. fructicola聚为一组，有2株单独聚为一支（Ⅵ），这一类群与其他类群亲缘关系较远，总体来说，杉木炭疽菌具有物种多样性。

8类炭疽菌菌株在杉木离体叶片上经人工接种均可以造成叶片发病，引起典型的杉木炭疽病的症状特征。不同菌株致病力也存在差异，分为强、中、弱三类。C. gossypii、C.fragariae、C.gloeosporioides属于致病性强的菌种，同种菌株对于不同部位的叶片致病力不同，对于嫩叶危害更大。

关键词：杉木炭疽病；病原菌；鉴定；多样性

Genetic Diversity of Anthracnose Pathogenies in Main Culture Areas of Cunninghamia lanceolata

ABSTRACT

Cunninghamia lanceolata is an important fast-growing species of trees in China, and its anthracnose has became an important frequent disease, which can do harm to Cunninghamia lanceolata’s needle leaves, branchlets, even stem. Colletotrichum has fast growth, can be spread by air, rain, incent and so on and it is easy to invade plants by wound, lenticel, stoma and natural orifice. It often bursts and is popular in some parts area in some years with bringing heavy economic losses.

64 fungus were separated, purified, and cultivated from diseased needle leaves which are from the main cultivated areas in Jiangsu, Fujian, Guangxi, Zhejiang, Jinagxi and Guizhou and so on. According to morphological identification on them, 68.25% fungus were classified into Colletotrichum gossypii. 7 fungus were identified as C. fragariae,, There are 4 fungus in the part of C. gloeosporioides as well as C. tropicale. C. karstii has 2 fungus, While C. musae, C.truncatum, C. caricae all only has 1 fungus. All these fungus were in accord with festure of Colletotrichum by primer ITS1/ITS4 and PCR.

It is obvious that there are difference between 8 types of Colletotrichum on fungus’ color, shape, growth rate, conidium’s shape and size, appressorium’s shape and germinaation of conidium. Even that situation was same in one type of Colletotrichum which means intraspecific diversity.

This research constructs the phylogenetic tree of Colletotrichum by ITS and 34 fungus were divided to 6 evolutionary branches from 8 provinces. 20 fungus gathered with C. gossypii model (Ⅰ), and that means the C. gossypii occupies a position of prominence among Colletotrichum. There are 2 fungus gathering with C.theobromicola model (Ⅱ). 8 fungus gathered into one type which has two groups. 2 fungus were divided to one group and this group’s genetic relationship is far from others. In a word, Colletotrichum in China is multiple.

All 8 types Colletotrichum can cause disease whose symptom tally with anthracnose on isolated needle leaves of Cunninghamia lanceolata by artificial inoculation. It is different in pathogenicity which were divided into strong, medium and weak. C. gossypii, C.fragariae, C.gloeosporioides are strong on pathogenicity. If the leaves from different position, the pathogenicity will be different, this research promised that it do more harm to tender leaves.

Key Word: anthracnose of Cunninghamia lanceolata, pathogenies fungus, identification, genetic diversity

目 录

1 研究概述 1

1.1 研究目的与意义 1

1.2 杉木发展现状 1

1.3 杉木炭疽病的研究进展 1

1.3.1杉木炭疽病的症状 1

1.3.2杉木炭疽病的病原 2

1.4炭疽菌的研究概况 3

1.4.1炭疽菌属的建立和分类地位 3

1.4.2炭疽菌检测技术 3

1.4.3 炭疽菌分类研究进展 4

2 材料与方法 6

2.1 实验材料 6

2.1.1 供试材料 6

2.1.2 供试培养基 6

2.2 实验方法 6

2.2.1 样品采集 6

2.2.2 病原菌分离与纯化 6

2.2.3 病原菌形态学观察 7

2.2.3.1 菌落形态观察与菌丝生长速率测定 7

2.2.3.2 菌种分生孢子、附着胞观察与萌发率统计 7

2.2.4 分子生物学鉴定 7

2.2.4.1 基因组DNA的提取及PCR扩增 7

2.2.4.2 基因序列比对和系统发育树构建 7

2.2.5 致病性鉴定 8

3 结果分析 9

3.1杉木炭疽病菌纯化结果与鉴定 9

3.2 杉木炭疽病菌形态观察 12

3.2.1 杉木炭疽病菌菌落形态与菌丝生长速率 12