香蕉叶斑病病原菌芭蕉新拟盘多毛孢的鉴定

doi:10.16420/j.issn.0513-353x.2021-1115

摘要/Abstract

摘要：

在广东省茂名市香蕉产区进行病害调查时发现一种为害较为严重的香蕉新发叶斑病害。根据柯赫氏法则对其进行病原菌分离及致病性检测，并根据形态学特征和ITS、β-tubulin和tef1序列分析对该病原菌进行鉴定。研究结果表明：分离得到的病原菌菌株MM3-2z9能够侵染香蕉叶片，并能诱发与田间类似的症状。其菌落在PDA培养基上呈白色，产生明显的同心轮纹；分生孢子纺锤形，具4个隔膜，中间3个细胞异色，顶细胞无色，锥形，附属丝2 ~ 4根（多为3根），基部细胞有1根附属丝，中生，无色。序列同源性分析显示MM3-2z9菌株3个单孢菌株的ITS、β-tubulin和tef1序列与芭蕉新拟盘多毛孢（Neopestalotiopsis musae）菌株相应序列的一致性都高于98%，3片段串联构建系统发育树的结果表明，菌株MM3-2z9与芭蕉新拟盘多毛孢（N. musae）亲缘关系最近。基于形态学与系统发育树分析结果，鉴定菌株MM3-2z9为芭蕉新拟盘多毛孢（N. musae）。该病原菌还可快速侵染大蕉和粉蕉‘粉杂1号’的叶片。

关键词: 香蕉, 叶斑病, 病原菌鉴定, 芭蕉新拟盘多毛孢

Abstract:

A new emerging banana leaf spot disease was found during a disease survey in the banana-producing area of Maoming City，Guangdong Province. The pathogen was isolated and tested for pathogenicity according to Koch's postulate，and the pathogen was identified based on morphological characteristics and sequence analyses of ITS，β-tubulin and tef1 genes. The results showed that strain MM3-2z9 could infect banana leaves and induce similar symptoms as in the field. The colony of strain MM3-2z9 on the PDA medium was white，producing obvious concentric ring patterns. The conidia were fusoid with 4-septate，three median cells versicoloured，apical cell hyaline，pyramidal，with two to four filiform apical appendages（mostly three），and basal cell with appendage single was centric and hyaline. Sequence homology analysis indicated that the ITS，β-tubulin and tef1 sequences of three single-conidial strains of MM3-2z9 were more than 98% identical to the corresponding sequences of Neopestalotiopsis musae stains. The phylogenetic tree constructed by the three fragments showed that strain MM3-2z9 was most closely related to N. musae. Therefore，strain MM3-2z9 was identified as N. musae based on morphological analysis and phylogenetic tree analysis. In addition to damaging banana（Musa AAA Cavendish）leaves，the pathogen could also quickly infect the leaves of M. × paradisiaca ABB and Musa ABB Pisang Awak‘Fenza 1’.

Key words: banana, leaf spot disease, pathogen identification, Neopestalotiopsis musae

中图分类号:

S668.1

梁嘉莉, 吴启松, 陈广全, 张荣, 徐春香, 冯淑杰. 香蕉叶斑病病原菌芭蕉新拟盘多毛孢的鉴定[J]. 园艺学报, 2023, 50(2): 410-420.

LIANG Jiali, WU Qisong, CHEN Guangquan, ZHANG Rong, XU Chunxiang, FENG Shujie. Identification of the Neopestalotiopsis musae Pathogen of Banana Leaf Spot Disease[J]. Acta Horticulturae Sinica, 2023, 50(2): 410-420.

图/表 8

表1 本研究中所用引物信息

Table1 Primer information used in this study

基因 Gene	引物名称 Primer name	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature
ITS	ITS4	TCCTCCGCTTATTGATATGC GC	57
ITS	ITS5	GGAAGTAAAAGTCGTAACAAGG
β-tubulin	BT2A	GGTAACCAAATCGGTGCTTTC	58
β-tubulin	BT2B	ACCCTCAGTGTAGTGACCCTTGGC
Tef1	EF1-688F	CGGTCACTTGATCTACAAGTGC	59
Tef1	EF1-986R	TACTTGAAGGAACCCTTACC

图1 香蕉新叶斑病的田间早期（A、B）和后期（C）症状

Fig. 1 Early（A，B）and late（C）symptoms of new leaf spot of banana in the field

图2 病原菌MM3-2z9的3个单孢菌株接种巴西蕉叶片后（11 d）的发病症状

Fig. 2 Symptoms of banana leaves inoculated with three monospora strains of MM3-2z9（11 days）

图3 病原菌MM3-2z9在大蕉和粉蕉接种7 d后的发病症状

Fig. 3 Pathogenic symptoms of MM3-2z9 in Musa × paradisiaca ABB and Musa ABB Pisang Awak inoculated for seven days

图4 香蕉新叶斑病菌MM3-2z9的形态特征 A、B：PDA培养基上菌落的正、反面；C：分生孢子团；D：产孢细胞及初生分生孢子；E：产孢细胞；F ~ H：分生孢子。

Fig. 4 Morphological characteristics of pathogenic bacteria A，B：Positive and negative sides of colony on PDA medium；C：Conidia；D：Sporulation cells and primary conidia；E：Sporulation cell；F-H：Conidia.

图5 MM3-2z9的3个单孢菌株（A、B、C）ITS（ITS4/ITS5）扩增条带的电泳检测

Fig. 5 Electrophoretic detection of ITS（ITS4/ITS5）amplified bands of three monospore strains（A，B，C）of MM3-2z9

图6 基于ITS序列采用NJ法构建的分子系统发育树分支节点处显示NJ树的分支节点自展值。下同。

Fig. 6 Molecular phylogenetic tree constructed by NJ method based on tandem of ITS sequence Branch node displays the branch node self-expansion value of NJ tree. The same below.

图7 基于串联ITS、β-butulin、tef1序列采用NJ法构建的分子系统发育树

Fig. 7 Molecular phylogenetic tree constructed by NJ method based on tandem of ITS，β-butulin，and tef1 sequences

参考文献 33

[1]	Alves A, Crous P W, Correia A, Phillips A J L. 2008. Morphological and molecular data reveal cryptic species in Lasiodiplodia theobromae. Fungal Diversity, 28:1-13.
[2]	Carbone I, Kohn L M. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91 (3):553-556. doi: 10.1080/00275514.1999.12061051 URL
[3]	Crous P W, Summerell B A, Swart L, Denman S, Groenewald J Z. 2011. Fungal pathogens of Proteaceae. Persoonia Molecular Phylogeny & Evolution of Fungi, 27 (1):20-45.
[4]	Crous P W, Wingfield M J, Guarro J, Cheewangkoon R, van der Bank M, Swart W J, Stchigel A M, Cano-Lira J F, Roux J, Madrid H, Damm U, Wood A R, Shuttleworth L A, Hodges C S, Munster M, Jesús Yáñez-Morales M de, Zúñiga-Estrada L, Cruywagen E M, Hoog G S de, Silvera C, Najafzadeh J, Davison E M, Davison P J N., Barrett M D, Barrett R L, Manamgoda D S, Minnis A M, Kleczewski N M, Flory S L, Castlebury L A, Clay K, Hyde K D, Maússe-Sitoe S N D, Shuaifei Chen, Lechat C, Hairaud M, Lesage-Meessen L, Pawłowska J, Wilk M, Śliwińska-Wyrzychowska A, Mętrak M, Wrzosek M, Pavlic-Zupanc D, Maleme H M, Slippers B, Mac Cormack W P, Archuby D I, Grünwald N J, Tellería M T, Dueñas M, Martín M P, Marincowitz S, Beer Z W der, Perez C A, Gené J, Marin-Felix Y, Groenewald J Z. 2013. Fungal Planet description sheets:154-213. Persoonia, 31:188-296. doi: 10.3767/003158513X675925 pmid: 24761043
[5]	Fang Zhong-da. 1998. Research methods of plant diseases. Beijing: China Agriculture Press. (in Chinese)
	方中达. 1998. 植病研究方法. 北京: 中国农业出版社.
[6]	Felsenstein J. 1985. Confidence limits on phylogenies:an approach using the bootstrap. Evolution, 39 (4):783-791. doi: 10.1111/j.1558-5646.1985.tb00420.x pmid: 28561359
[7]	Fu Gang, Pan Lian-fu, Du Chan-juan, Yan Wei-hong. 2015. Description of banana diseases in Guangxi from 2011 to 2013. Guangxi Plant Protection, 28 (1):1-5. (in Chinese)
	付岗, 潘连富, 杜婵娟, 晏卫红. 2015. 2011—2013年广西香蕉病害记述. 广西植保, 28 (1):1-5.
[8]	Fu Yan-ping, Qi Yan-xiang, Xie Yi-xian, Peng Jun, Zeng Fan-yun, Zhang Xin. Identification and biological characteristics of the pathogen causing cordana leaf spot of banana in Hainan Province. Acta Tropical Biology, 12 (1):63-71. (in Chinese)
	扶艳萍, 漆艳香, 谢艺贤, 彭军, 曾凡云, 张欣. 2021. 香蕉灰纹病的病原鉴定及其生物学特性. 热带生物学报, 12 (1):63-71.
[9]	Glass N L, Donaldson G C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology, 61 (4):1323-1330. doi: 10.1128/aem.61.4.1323-1330.1995 pmid: 7747954
[10]	He Chun-ping, Zheng Xiao-lan, Li Rui, Wu Wei-huai, Yu Xian-mei, Ke Xing-ye, Zheng Fu-cong. 2010. Identification and biological characters of Rambutan gray spot disease. Journal of Fruit Science, 27 (2):270-274. (in Chinese)
	贺春萍, 郑肖兰, 李锐, 吴伟怀, 余闲美, 柯兴业, 郑服丛. 2010. 红毛丹灰斑病病原菌鉴定及生物学特性研究. 果树学报, 27 (2):270-274,321.
[11]	Huang S L, Yan B, Wei J G, Yan W H, Cen Z L, Yang T. 2007. First report of plantain zonate leaf spot caused by Pestalotiopsis menezesiana in China. Australasian Plant Disease Notes, 2 (1):61-62.
[12]	Hyde K D, Hongsanan S, Jeewon R, Bhat D J, McKenzie E H C, Jones E B G, Phookamsak R, Ariyawansa H A, Boonmee S, Qi Z, Abdel-Aziz F A, Abdel-Wahab M A, Banmai S, Chomnunti P, Bao-Kai C, Daranagama D A, Das K, Dayarathne M C, Silva N I d, Dissanayake A J, Doilom M, Ekanayaka A H, Gibertoni T B, Góes-Neto A, Shi-Ke H, Jayasiri S C, Jayawardena R S, Konta S, Lee H B, Wen-Jing L, Chuan-Gen L, Jian-Kui L, Yong-Zhong L, Zong-Long L, Manawasinghe I S, Manimohan P, Mapook A, Niskanen T, Norphanphoun C, Papizadeh M, Perera R H, Phukhamsakda C, Richter C, Santiago A L C M d A, Drechsler-Santos E R, Senanayake I C, Tanaka K, Tennakoon T M D S, Thambugala K M, Qing T, Tibpromma S, Thongbai B, Vizzini A, Wanasinghe D N, Wijayawardene N N, Hai-Xia W, Jing Y, Xiang-Yu Z, Huang Z, Jin-Feng Z, Bulgakov T S, Camporesi E, Bahkali A H, Amoozegar M A, LiAraujo-Neta L S, Ammirati J F, Baghela A, Bhatt R P, Bojantchev D, Buyck B, Silva G A d, Lima C L F d, Oliveira R J V d, Souza C A F d, Yu-Cheng D, Dima B, Duong T T, Ercole E, Mafalda-Freire F, Ghosh A, Hashimoto A, Kamolhan S, Ji-Chuan K, Karunarathna S C, Kirk P M, Kytövuori I, Lantieri A, Liimatainen K, Zuo-Yi L, Xing-Zhong L, Lücking R, Medardi G, Mortimer P E, Nguyen T T T, Promputtha I, Raj K N A, Reck M A, Lumyong S, Shahzadeh-Fazeli S A, Stadler M, Soudi M R, Hong-Yan S, Takahashi T, Tangthirasunun N, Uniyal P, Yong W, Ting-Chi W, Jian-Chu X, Zhong-Kai Z, Yong-Chang Z, Jun-Liang Z, Lin Z. 2016. Fungal diversity notes 367-490:taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity, 80 (1):1-270. doi: 10.1007/s13225-016-0373-x URL
[13]	Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16 (2):111-120. doi: 10.1007/BF01731581 pmid: 7463489
[14]	Kumar S, Stecher G, Tamura K. 2015. MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33:1870-1874. doi: 10.1093/molbev/msw054 URL
[15]	Li Xiao-na, Zhang Hui-jian, Zeng Xiao-hong, Li Yi-ping, Zeng An-yi, Xie Long-lian, Dong Ding-chao, Zeng Xiao-fen. 2018. Progress in research on the prevention and control of banana leaf spot in the world. China Tropical Agriculture, 80 (1):34-36. (in Chinese)
	李晓娜, 张慧坚, 曾小红, 李一萍, 曾安逸, 谢龙莲, 董定超, 曾筱芬. 2018. 世界香蕉叶斑病防治研究进展. 中国热带农业, 80 (1):34-36.
[16]	Lin Shan-hai, Huang Si-liang, Qin Li-ping, Fu Gang, Cen Zhen-lu, Xie Ling. 2011. Investigations on the fungal populations associated with Banana leaf spot diseases in Guangxi. Plant Protection, 37 (5):181-184. (in Chinese)
	林善海, 黄思良, 覃丽萍, 付岗, 岑贞陆, 谢玲. 2011. 广西香蕉真菌性叶斑病病原菌种群结构分析. 植物保护, 37 (5):181-184.
[17]	Maharachchikumbura S S N, Guo L D, Cai L. 2012. A multi-locus backbone tree for Pestalotiopsis,with a polyphasic characterization of 14 new species. Fungal Diversity, 56 (1):95-129. doi: 10.1007/s13225-012-0198-1 URL
[18]	Maharachchikumbura S S N, Guo L D, Chukeatirote E. 2011. Pestalotiopsis:morphology,phylogeny,biochemistry and diversity. Fungal Diversity, 50 (1):167-187. doi: 10.1007/s13225-011-0125-x URL
[19]	Maharachchikumbura S S N, Hyde K D, Groenewald J Z. 2014. Pestalotiopsis revisited. Studies in Mycology, 79 (11):121-186. doi: 10.1016/j.simyco.2014.09.005 URL
[20]	O’Donnell K, Cigelnik E. 1997. Two divergent intragenomic rDNA ITS 2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution, 7 (1):103-116. doi: 10.1006/mpev.1996.0376 URL
[21]	Qi Pei-kun. 2000. The history of fungal diseases of fruit trees in Guangdong. Beijing: China Agriculture Press. (in Chinese)
	戚佩坤. 2000. 广东果树真菌病害志. 北京: 中国农业出版社.
[22]	Qi Yan-xiang, Zhang Xin, Ding Zhao-jian, Zeng Fan-yun, Peng Jun, Xie Yi-xian. 2019. A list of diseases on banana(Musa spp.). Journal of Tropical Agricultural Sciences, 39 (1):58-69. (in Chinese)
	漆艳香, 张欣, 丁兆建, 曾凡云, 彭军, 谢艺贤. 2019. 香蕉病害名录. 热带农业科学, 39 (1):58-69.
[23]	Qi Ying-bo. 2020. Identification and basic research of blueberry new diseases caused by Pestalotiopsis kenyana[M. D. Dissertation]. Shenyang: Shenyang Agricultural University. (in Chinese)
	齐鹰博. 2020. 类拟盘菌危害蓝莓的新纪录种发现及病害防治基础研究[硕士论文]. 沈阳: 沈阳农业大学.
[24]	Saitou N, Nei M. 1987. The neighbor-joining method:a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4:406-425. doi: 10.1093/oxfordjournals.molbev.a040454 pmid: 3447015
[25]	Sang Li-wei. 2007. The survey on the banana fungus diseases in Hainan island and the research on the biological characteristics of Curvularia lunata [M. D. Dissertation]. Hannan: South China University of Tropical Agriculture. (in Chinese)
	桑利伟. 2007. 海南岛香蕉真菌病害调查及弯孢霉的生物学特性研究[硕士论文]. 海南: 华南热带农业大学.
[26]	Sang Li-wei, Zheng Fu-cong. 2006. Main diseases of banana in China and its control. Journal of Anhui Agricultural Sciences, 34 (9):1841-1845. (in Chinese)
	桑利伟, 郑服丛. 2006. 我国香蕉的主要病害及防治. 安徽农业科学, 34 (9):1841-1845.
[27]	Shi Ling-bo, Li Yuan, Fei Nuo-ya, Fu Jun-fan, Yan Xue-rui. 2017. Identification of the Pestalotiopsis pathogen of Blueberry leaf spot. South China Fruits, 46 (1):24-28. (in Chinese)
	石凌波, 李媛, 费诺亚, 傅俊范, 严雪瑞. 2017. 蓝莓拟盘多毛孢叶斑病病原菌鉴定. 中国南方果树, 46 (1):24-28.
[28]	Song Li-sha, Wei Ji-guang, Yun Chao-guang, Yang Xiu-hao, Luo Ji-tong, Huang Song-dian. 2016. Survey of species diversity in the endophytic fungal genus Pestalotiopsis in Liangfengjiang national forest park,Guangxi,China. Forestry Science, 52 (7):87-95. (in Chinese)
	宋利沙, 韦继光, 云朝光, 杨秀好, 罗基同, 黄松殿. 2016. 广西良凤江国家森林公园植物内生拟盘多毛孢属真菌种类多样性调查. 林业科学, 52 (7):87-95.
[29]	Wang Zhen-zhong, Li Xue-wen, Sun Zheng-xiang, Jiang Yan-quan, Liao Lin-feng. 2006. Pathogenic identification of a new banana disease caused by Phytophthora sp. Guangdong Agricultural Sciences,(4):50-52. (in Chinese)
	王振中, 李学文, 孙正祥, 姜彦全, 廖林凤. 2006. 一种疫霉菌引起的香蕉新病害的病原初步鉴定. 广东农业科学,(4):50-52.
[30]	White T J, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols,A Guide to Methods and Application,(1):315-322.
[31]	Yang Xingyu, Xu Linbing, Wu Yuanli, Qiu Diyang, Fan Linlin, Huang Bingzhi. 2022. Genome(ABBB)identification of a hybrid banana cultivar‘Fenza 1’. Acta Horticulturae Sinica, 49 (9):1991-1997. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0514
	杨兴玉, 许林兵, 吴元立, 邱迪洋, 范琳琳, 黄秉智. 2022. 香蕉杂交种‘粉杂1号’ABBB基因组鉴定. 园艺学报, 49 (9):1991-1997. doi: 10.16420/j.issn.0513-353x.2021-0514
[32]	Zhang Jia-xiang. 2002. Identification of Pestaiotiopsis spp. from southern China and study on the species-recognizing characters[M. D. Dissertation]. Hangzhou: Zhejiang University. (in Chinese)
	张家祥. 2002. 中国南方拟盘多毛孢属(Pestalotiopsis)真菌及其分种性状的研究[硕士论文]. 杭州: 浙江大学.
[33]	Zheng Qing-wei. 2020. Identification and comprehensive control strategy of banana leaf spot disease. Pesticide Market Information, 675 (12):59-60. (in Chinese)
	郑庆伟. 2020. 香蕉叶斑病的鉴别与综合防治策略. 农药市场信息, 675 (12):59-60.