山东牛心柿炭疽病菌的分离鉴定及致病性

doi:10.11707/j.1001-7488.20150416

摘要/Abstract

摘要：

【目的】对侵染牛心柿引起炭疽病的病原菌进行分离鉴定,分析其致病性,初步构建其侵染谱,为柿树病害的病原诊断和防治提供理论依据和技术支持。【方法】通过组织分离法和单孢分离法从感病牛心柿的果实、叶片和嫩梢中分离病原菌,根据菌落和孢子形态特征及其rDNA-ITS 序列分析进行种类鉴定,采用以菌饼进行伤口和非伤口接种、孢子悬浮液进行喷雾接种的离体接种试验分析其致病性。【结果】从感病牛心柿果实、叶片和嫩梢中分离获得9个病原菌分离物,菌落及孢子形态特征显示均为炭疽菌。以真菌转录间隔区通用引物ITS6和ITS4为引物,以菌丝总DNA为模板,扩增获得病原菌rDNA-ITS基因片段,经rDNA-ITS 序列分析和系统进化树的构建,将该病原物鉴定为柿树炭疽菌,将其rDNA-ITS基因序列提交到GenBank数据库(基因登录号KF010811)。牛心柿炭疽病菌和次郎炭疽病菌对不同品种柿树的致病性相同,均无差异性,对果实的致病性比叶片强; 接种7天后,叶片无病斑或病斑较小,菌饼无伤口接种几乎不发病,在叶片上不产生病斑,果实上病斑较小; 不同的接种方式在果实和叶片上产生的病斑大小为: 菌饼伤口接种 > 孢子悬浮液接种 > 菌饼无伤口接种; 牛心柿炭疽病菌和次郎炭疽菌对桃、苹果、梨等果树的叶片和果实均不致病。【结论】柿炭疽病菌具有一定的专性寄生性,对除柿树外的其他果树不致病,且在遗传上有一定的保守性,因地理距离产生的变异很小,这些特性对于柿炭疽病的防治具有重要意义,在一定程度上可以避免或削弱因病菌的遗传变异而产生的抗药性,从而有利于炭疽病的防治。在柿树有伤口时最易侵染致病,雨天或高湿度的情况下经孢子侵染致病的可能性居其次。在田间管理时注意避免造成柿树伤口感染,或者避免在下雨天气进行修剪,可最大程度地减轻或消除柿树炭疽病菌的侵染以及柿炭疽病的发生和流行,从而在一定程度上解决该病流行而造成的减产问题。

关键词: 牛心柿, 柿树炭疽病, 病原分离与鉴定, rDNA-ITS序列分析, 离体接种试验

Abstract:

【Objective】 This study aims to isolate and identify a pathogen which infects the persimmon plants and causes persimmon anthracnose, then investigate the pathogenicity so as to analyze its infect spectrum, and provide the theoretical basis and technical assistance for the pathogenic diagnosis and disease control of persimmon anthracnose in persimmon productivity.【Methods】 The pathogen was isolated from anthracnose lesions on beef heart persimmon fruits, leaves and twigs in the persimmon orchards in Zhujiapo town, Linju county, Zhuge town, Yishui county and Wanjishan experimental station of Shandong Institute of Pomology with tissue separation and single spore separation, and identified by morphological characteristics and rDNA-ITS sequence analysis. The pathogenicity was investigated via in vitro inoculation test by disk-wound inoculation, disk-no-wound inoculation and spore-suspension inoculation. 【Results】 Nine isolates were obtained from persimmon fruits, leaves and twigs via tissue separation and single spore separation. The analysis of morphological characteristics showed that the 9 isolates belong to genus Colletotrichum. Then rDNA-ITS sequence analysis, via PCR amplification with the universal primers of ITS6 and ITS4, showed that the 9 isolates obtained were in full accord with the morphological observation, and the nucleotide sequence of rDNA-ITS gene (GenBank accession No. KF010811) shared 100%, 100% and 99.8% identity with that of anthracnose pathogens on Jiro persimmon (JQ957543), 'Wuheshi' persimmon in Zhejiang (AY787483) and New Zealand isolate (GQ329690) respectively. The phylogenetic analysis based on ITS sequences showed that beef heart persimmon isolate was located at the same branch of phylogenetic tree with the isolates of Jiro persimmon (JQ957543), 'Wuheshi' persimmon in Zhejiang (AY787483, AY791890) and New Zealand persimmon (GQ329687, GQ329688 and GQ329690), which indicated that the pathogen causing the anthracnose disease of beef heart persimmon could be identified as Colletotrichum horii. The in vitro inoculation test showed that there was equal pathogenicity on different persimmon cultivars between 'Niuxinshi' isolate and 'Cilang' isolate of C.horii, and they have no pathogenicity on peach, apple and pear. On the 7th day after inoculation, the lesions on persimmon fruits were much larger than that on leaves, there were no symptoms on leaves via disk-no-wound inoculation, and lesions were small via spore suspension inoculation. The lesion size on fruits and leaves via different inoculation was inthe following order of:disk-wound inoculation > spore-suspension inoculation > disk-no-wound inoculation. 【Conclusions】C.horii possessed a certain degree of obligate parasitic and genetic conservatism, with no pathogenicity to other fruit trees besides persimmon and little variation due to the geographic distance. These characteristics were significant for the control of persimmon anthracnose, which could play an important role in avoiding and weakening the resistance to chemicals due to the genetic variation of the pathogen, and thus facilitate the control of persimmon anthracnose, and guarantee quality of fruits and increase persimmon productivity. The wound infection by the pathogen will occurred most easily, followed by spore infecton in raining days or under the high humidity condition, which indicated that the pathogen infection, the occurrence and popularity of persimmon anthracnose will be avoided or eliminated largely by reducing persimmon wound in the field management and avoiding pruning in raining days. Thus, the measures would to some extent solve the problem of lost production in persimmon due to the popularity of persimmon anthracnose.

Key words: beef heart persimmon, persimmon anthracnose, pathogen isolation and identification, rDNA-ITS sequence analysis, in vitro inoculation

中图分类号:

S736.15

余贤美, 侯长明, 王洁, 王海荣, 安淼, 艾呈祥. 山东牛心柿炭疽病菌的分离鉴定及致病性[J]. 林业科学, 2015, 51(4): 126-133.

Yu Xianmei, Hou Changming, Wang Jie, Wang Hairong, An Miao, Ai Chengxiang. Identification of Pathogen Causing Beef Heart Persimmon Anthracnose in Shandong and Its Pathogenicity[J]. Scientia Silvae Sinicae, 2015, 51(4): 126-133.

参考文献

邓维萍, 杨敏, 杜飞, 等. 2013. 云南葡萄产区葡萄炭疽病病原鉴定及致病力分析. 植物保护学报, 40(1):61-67.
(Deng W L, Yang M, Du F, et al. 2013. Identification of the pathogen causing grape anthracnose in Yunnan and its pathogenicity. Acta Phytophylacica Sinica, 40(1):61-67.[in Chinese])
方仲达. 1998. 植病研究方法.3版. 北京: 中国农业出版社.
(Fang Z D. 1998. Plant disease research methods. 3^rd ed. Beijing: China Agriculture Press.[in Chinese])
刘开启, 牟惠芳, 刘凤英. 1988. 柿炭疽病的研究. 山东农业大学学报, 19: 69-71.
(Liu K Q, Mu H F, Liu F Y. 1988. Studies on persimmon anthracnose (Gloeosporium Kaki Hori). Journal of Shandong Agricultural University, 19: 69-71.[in Chinese])
余贤美, 侯长明, 王洁, 等. 2014. 次郞甜柿炭疽病菌的分离鉴定及其rDNA-ITS 序列分析. 经济林研究, 32(1): 45-50.
(Yu X M, Hou C M, Wang J, et al. 2014. Isolation, identification and rDNA-ITS sequence analysis of pathogen of Jiro persimmon anthracnose. Nonwood Forest Research, 32(1): 45-50.[in Chinese])
张敬泽, 徐同, 何黎平. 2005. 浙江无核柿炭疽病病菌鉴定及附着胞形成过程中的核相变化. 菌物学报, 24(1): 116-122.
(Zhang J Z, Xu T, He L P. 2005. Anthracnose pathogen on Diospyros kaki cv. Wuheshi and its nuclear behavior in process of appressorium formation. Mycosystema, 24(1): 116-122.[in Chinese])
Arx J A Von. 1957. Die Ariten der Gattung Colletotrichum Cda. Phytopathologische Zeitschrift, 29: 413-468.
Arx J A Von. 1970. A revision of fungi classified as Gloeosporium. Bibliotheca Mycologica, 24: 1-203.
Cai L, Hyde K D, Taylor P W J, et al. 2009. A polyphasic approach for studying Colletotrichum. Fungal Diversity, 39:183-204.
Cannon P F, Buddie A G, Bridge P D. 2008. The typification of Colletotrichum gloeosporioides. Mycotaxon, 104: 189-204.
Du Sh N, Bai G Sh, Zhang Sh J, et al. 2010. Identification and control of persimmon anthracnose. Plant Disease and Pests, 1(1): 40-42.
Gardes M, Bruns T D. 1996. ITS-RFLP matching for identification of fungi// Clapp J P. Species diagnostics protocol. Totowa, New Jersey: Humana Press.
Hori S. 1910a. Kaki no Shinbyogai Tansobyo. Engei no Tomo, 6(1): 58-61.
Hori S. 1910b. Kaki no Shinbyogai Tansobyo. Engei no Tomo, 6(2): 21-24.
Hu S C, Li M, Wang J G, et al. 2006. IFB-Lactam-1, a natural compound with anti-Gloeosporium kaki Hori activity. Acta Crystallogr. Section E: Struct Rep Online, 62: 5777-5778.
Hyde K D, Cai L, Cannon P F, et al. 2009. Colletotrichum-names in current use. Fungal Diversity, 39: 147-182.
Ito S. 1911. Gloeosporiose of the Japanese persimmon. Botanic Magazine (Tokyo), 25: 197-202.
Kitagawa H, Glucina P. 1984. Persimmon culture in New Zealand. DSIR Information Series 159. Science Information Publishing Centre: Wellington, New Zealand.
Kwon J H, Kim J, Chio O, et al. 2013. Anthracnose caused by Colletotrichum horii on sweet persimmon in Korea: Dissemination of conidia and disease development. Journal of Phytopathology,161(7-8):497-502.
Lee J H, Han K S, Lee S C, et al. 2004. Early detection of epiphytic anthracnose inoculum on phyllosphere of Disopyros kaki var. domestica. Plant Pathology Journal, 20: 247- 251.
Maffei L. 1921. Una malattia delle foglie del "Kaki" dovuta al Colletotrichum kaki m. sp. Rivista di Patologia Vegetale, 11: 116-117.
Shenoy B D, Jeewon R, Lam W H, et al. 2007. Morpho-molecular characterization and epitypification of Colletotrichum capsici (Glomerallaceae, Sordariomycetes), the causative agent of anthracnose in chilli. Fungal Diversity, 27: 197-211.
Than P P, Prihastuti H, Phoulivong S, et al. 2008. Review: Chilli anthracnose disease caused by Colletotrichum species. Journal of Zhejiang University, 9:764-778.
Weir B S, Johnston P R. 2010. Characterisation and neotypification of Gloeosporium kaki Hori as Colletotrichum horii nom. nov. Mycotaxon, 111: 209-219.
White T J, Bruns T, Lee S. 1990.Analysis of phytogenetic relationships by amplification and direct sequencing of ribosomal RNA genes// Innis M A, Gelfand D H, Sninsky J J, et al. PCR protocols: A guide to methods and applications. New York: Academic Press.
Xie L, Zhang J Z, Cai L, et al. 2010. Biology of Colletotrichum horii, the causal agent of persimmon anthracnose. Mycology, 1(4): 242-253.
Yonemori K, Honsho S, Kanzaki S, et al. 2008. Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species. Tree Genetics and Genomics, 4: 149-158.
Zhang J Z. 2008. Anthracnose of persimmon caused by Colletotrichum gloeosporioides in China. Asian and Australasian Journal of Plant Science and Biotechnology, 2(2): 50-54.