Identification of the pathogens causing stem canker on <i>Carya cathayensis</i>

WANG Xuan; MA Liangjin; LÜ Quan; MENG Xianjing; ZHANG Xingyao

doi:10.11833/j.issn.2095-0756.2014.02.012

Volume 31 Issue 2

Mar. 2014

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WANG Xuan, MA Liangjin, LÜ Quan, MENG Xianjing, ZHANG Xingyao. Identification of the pathogens causing stem canker on Carya cathayensis[J]. Journal of Zhejiang A&F University, 2014, 31(2): 238-245. doi: 10.11833/j.issn.2095-0756.2014.02.012

Citation:

WANG Xuan, MA Liangjin, LÜ Quan, MENG Xianjing, ZHANG Xingyao. Identification of the pathogens causing stem canker on Carya cathayensis[J]. Journal of Zhejiang A&F University, 2014, 31(2): 238-245. doi: 10.11833/j.issn.2095-0756.2014.02.012

Identification of the pathogens causing stem canker on Carya cathayensis

doi: 10.11833/j.issn.2095-0756.2014.02.012

1.
School of Forestry and Biotechnology, Zhejiang A & F University, Lin'an 311300, Zhejiang, China
2.
Research Institute of Forestry Ecology, Environment and Protection, Chinese Academy of Forestry, Key Laboratory of Forest Protection of State Forestry Administration, Beijing 100091, China

Received Date: 2013-06-08
Rev Recd Date: 2013-09-18
Publish Date: 2014-04-20

Abstract

Species of the Botryosphaeriaceae family are important pathogens causing cankers and die-back on many woody plants. In previous studies Botryosphaeria dothidea has commonly been reported as a pathogen of Chinese hickory(Carya cathayensis) canker; however, it was unclear whether other species of Botryosphaeriaceae were also related to hickory canker. In one study, several species of Botryosphaeriaceae were isolated from Chinese hickory, and then they were identified based on anamorphic morphology and rDNA Internal Transcribed Spacer(ITS) sequences. A phylogentic analysis indicated that these isolates formed three clades and were identified as B. dothidea, B. fabicercianum, and B. obtusa with isolation rates of B. dothidea (71.42%) predominant and B. fabicercianum and B. obtusa being much lower(14.28%). In pathogenicity testing, all three pathogens caused stem cankers on Chinese hickory but had different degrees of pathogenicity. B. obtusa and B. fabicercianum were first reported as a pathogen of Carya cathayensis canker.
- forest protection,
- cash forestry,
- canker,
- Botryosphaeriaceae,
- Botryosphaeria dothidea,
- phylogeny,
- Carya cathayensis

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References

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[2]	ZHENG Hongbing, SHU Qinglong, DING Zhien. Relationship between nutrient elements in bark and canker of Carya cathayensis[J]. Nonwood For Res, 2007, 25(3):19-23.
[3]	ZHANG Chuanqing, XU Bingchao. First report of canker on Chinese hickory (Carya cathayensis) caused by Botryosphaeria dothidea in China[J]. Plant Dis, 2011, 95(10):1319.
[4]	CHEN Yanlin, LIANG Jun, LÜ Quan, et al. Advances in Botryosphaeriaceae:identification, phylogeny and molecular ecology[J]. Acta Ecol Sin, 2011, 31(11):3197-3207.
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沈阳化工大学材料科学与工程学院沈阳 110142

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山核桃Carya cathayensis隶属于胡桃科Juglandaceae中的山核桃属Carya,是一种著名的干果树种,因其干果富含营养和经济价值较高而得到了广泛栽培^[1]。山核桃干腐病是山核桃生产上一种重要病害,不但影响山核桃的产量,而且会削弱树势,严重时则导致树木过早死亡,并造成重大经济损失^[2]。2011年首次报道山核桃干腐病病原菌为Botryosphaeria dothidea(Moug. ex Fr.) Ces. & De Not,属于子囊菌门葡萄座腔菌科Botryosphaeriaceae的葡萄座腔菌属Botryosphaeria^[3]。葡萄座腔菌科真菌是农业和林业上重要病原菌、内生真菌或潜在的致病菌,主要引起树木溃疡病。葡萄座腔菌属真菌广泛分布于世界各地,而且寄主范围广泛,是森林生态系统中的重要真菌类群^[4]。该菌存在有性型和无性形阶段,其主要形态分类特征为子座、子囊、子囊孢子以及分生孢子的形状、纹饰、颜色、分隔、长宽比、大小及壁厚度等^[5-6]。另外,培养菌落颜色、气生菌丝生长情况及子囊孢子表面超微结构(纹饰)也可用于葡萄座腔菌科真菌的分类和鉴定^[7]。随着分子生物学的快速发展,越来越多的基因序列分析方法应用于葡萄座腔菌科真菌的分类鉴定及系统发育分析,如核糖体小亚基基因(SSU),核糖体大亚基基因(LSU),延长因子α基因(EF1-a),核糖体DNA内转录间隔区(rDNA-ITS),几丁质合酶基因,β微管蛋白(β-tubulin),A-肌动蛋白(A-actin)基因,钙调蛋白(calmodulin)基因等基因^[8]。尤其rDNA-ITS序列是应用最普遍的基因序列,已广泛地应用于很多真菌目、科、属、种等的分类鉴定及系统发育分析。本研究采用形态学特征与与rDNA ITS相结合的方法对分离自中国山核桃的干腐病菌进行了鉴定研究。

1. 材料与方法

1.1. 样品采集和病菌分离

分别从浙江省临安市的昌化镇和横路镇,淳安县,桐庐县和安徽省宁国市等山核桃产区采集干腐病标本(枝条和树干)带回实验室,然后在实验室进行病原菌分离与纯化培养。具体分离方法：首先选取发病枝条和树干,用乙醇对病健交接处的组织进行表面消毒30 s,然后剪成约5.0 mm × 5.0 mm大小的组织块,在体积分数为75.0％的乙醇中浸泡5 s,用无菌水浸洗3次,再用质量分数为1.0%的次氯酸钠浸泡1 min,最后用无菌水清洗3次。用灭菌的滤纸吸干水分,将组织块置于马铃薯葡萄糖琼脂培养基(PDA)平板上,在25 ℃恒温培养箱中进行培养,2 d后挑取菌落边缘的菌丝进行转接培养、纯化,并进行编号和转管保存。

1.2. 致病性测定及病原菌确定

将供试菌株在马铃薯葡萄糖琼脂(PDA)平板上活化培养3~4 d后,用经灭菌的直径为7.0 mm的打孔器打取菌饼。室外选取健康的山核桃,采用丁字型接种方法,将菌饼正面朝向伤口,用已浸湿无菌水脱脂棉保湿。设置重复15个·菌株-1,并设置空白PDA作为对照。接种15 d后观察发病情况,记录不同菌株的病斑数和发病级别,并计算不同菌株的感病指数。将病斑分为3级,15 d后病斑大小在10.0 mm以上的代表数值为“3”,10.0 mm以下5.0 mm以上代表数值的为“2”,5.0 mm以下的代表数值为“1”,不发病的代表数值为“0”。感病指数等于各病级的总代表数值(病斑分级的代表数值与该级标准株数之积)相加,再除以最高一级的代表数值与总株数之积,再乘以100。感病指数越高表示该菌株的致病性越强。对发病病斑进行组织分离,分离得到与接种菌株培养特征一致的菌株确定为该病的病原菌。

1.3. 病原菌形态特征观察

将分离获得的病原菌菌株在PDA平板上培养5 d后,观察菌落培养特性,将菌株接种到由树皮煎汁或松针等制成的培养基上诱导孢子产生。制作徒手切片后,在光学显微镜下观察和测量病原菌分生孢子器、分生孢子梗及分生孢子等特征。根据形态特征进行病原菌种类鉴定。

1.4. 病原菌rDNA-ITS序列扩增与分析

1.4.1. 基因组DNA提取

将病原菌转接到PDA平板上,于25 ℃培养3 d后,刮取约200.0 mg气生菌丝于灭菌后的1.5 mL的离心管中,-20 ℃冰冻过夜,经带研磨杵的电钻研磨破壁后,采用基因组DNA提取试剂盒(北京宝锐通生物科技有限公司)提取真菌基因组DNA。

1.4.2. rDNA-ITS

PCR扩增和纯化采用真菌rDNA-ITS区域通用引物ITS1(5′TCCGTAGGTGAACCTGCGG3′)和ITS4(5′TCCTCCGCTTATTGATATGC 3′)进行聚合酶链式反应(PCR)扩增。PCR反应体系总体积为25.0 μL,包括12.5 μL PCR脱氧核糖核苷三磷酸(dNTP),9.5 μL 双蒸水(ddH2O),引物ITS1/ITS4各1.0 μL,模板DNA 1.0 μL。反应程序：94.0 ℃预变性2 min；94.0 ℃变性30 s；57.3 ℃退火30 s；72.0℃延伸40 s,30个循环；72.0 ℃延伸10 min。经质量浓度为15.0 g·L^-1琼脂糖凝胶电泳检测PCR扩增产物,将条带清晰的PCR扩增产物送交北京宝锐通生物科技有限公司进行双向测序。

1.4.3. 系统发育分析

将测序结果在美国生物技术信息中心(NCBI) GenBank (http://www.ncbi.nlm.gov)中进行同源性比对,下载参比序列,采用clustalX和Mega 5.0软件进行分析比对,采用PAUP 4.0和Mrbayes 3.0软件构建最大简约法(MP)和贝叶斯法(BI)系统发育树。

3. 结论与讨论

本研究从山核桃干腐病发病组织上分离得到了3种葡萄座腔菌属真菌,其中优势菌株为Botryosphaeria dothidea,而且致病性最强；而B. fabicercianum和B. obtusa分离频率较低,致病性较弱。

Botryosphaeria是重要的子囊菌,其无性型包括Diplodia,Dothiorella,Fusicoccum,Lasiodiplodia,Sphaeropsis等^[12],是形态分类中最困难的真菌类群之一。关于山核桃干腐病的研究报道较少,而且关于病原菌种类不明确。杨淑贞等^[13]提出该病病原真菌的有性态为B. fusisporae,无性态为Macrophoma caryae。也有研究认为山核桃溃疡病病原属于半知菌亚门腔胞纲球壳孢科小穴壳菌Dothiorella gregaria,并指出该病的病原与杨树溃疡病和桃树溃疡病的病原相同^[14]。张传清等^[15]认为山核桃干腐病菌为B. dothidea。田甜等^[16]也认为山核桃干腐病病原菌是B. dothidea。本研究结果认为山核桃干腐病菌包括B. dothidea,B. fabicercianum及B. obtusa,但以B. dothidea为优势病菌。

Botryosphaeria是常见的林木干腐和枯梢病菌,尤其B. dothidea是发生最普遍和危害最重的病原菌^[17]。Smith等^[18]报道,B. dothidea在南非引起桉树溃疡病。另外,该菌是桉属Eucalyptus和松属Pinus植物上的内生真菌^[19]。由于Botryosphaeria属真菌在自然条件以无性型最为常见,而且形态特征有限,所以,仅依靠形态特征难于进行种类鉴定。另外,该属真菌在人工培养条件下很难产生分生孢子器,而且耗时长。采用分子生物学技术不失为一种有效方法^[20]。

Reference (20)

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[2]	郑宏兵, 束庆龙, 丁之恩. 山核桃溃疡病与营养元素的关系[J]. 经济林研究, 2007, 25(3): 19-23.	ZHENG Hongbing, SHU Qinglong, DING Zhien. Relationship between nutrient elements in bark and canker of Carya cathayensis[J]. Nonwood For Res, 2007, 25(3): 19-23.
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菌株编号	菌株接收号	GenBank中相似的种类	相似率最高菌株	最高相似率/%	相似菌株接收号
CRY1567	TC527826	Botryosphaeria dothidea	CBS 121484	99	EU650670
CRY1566	TC527822	B. dothidea	CBS116743	99	AY786322
CRY1565	TC527825	B. dothidea	CMW800	99	AY236949
CRY1568	TC527823	B. fabicercianum	CMW24703	100	HQ332195
CRY1569	TC527824	B. fabicercianum	CMW24703	99	HQ332195
CRY1570	TC527827	B. obtusa	CBS119049	99	DQ458889
CRY1571	TC527828	B. obtusa	CBS119049	99	DQ458889

Identification of the pathogens causing stem canker on Carya cathayensis

doi: 10.11833/j.issn.2095-0756.2014.02.012