Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98165
標題: 臺灣芒果炭疽病菌之親緣種與生物學特性
Phylogenetic species and biological characteristics of mango anthracnose pathogens in Taiwan
作者: 林瑋倫
Wei-Lun Lin
關鍵字: 芒果
炭疽病菌
親緣種
內生菌
專一性引子
mango
Colletotrichum
phylogenetic species
endophyte
specific primer
引用: 行政院農業委員會。2016。民國104年農業統計年報。行政院農業委員會出版。362頁。 安寶貞、何明勳、袁秋英、陳文雄。2003。植物保護圖鑑系列10-檬果保護。行政院農業委員會動植物防疫檢疫局。臺北市。250頁。 黃姿碧。1999。應用rDNA內轉錄區間(ITS)序列特性作為炭疽病菌鑑定與親緣關係研究之輔助依據。國立中興大學植物病理學系碩士論文。120頁。 楊秀珠。1998。炭疽病菌之鑑定。檢疫防疫植物病原真菌鑑定研討會專刊 199-232頁。 劉冠霆。2015。篩選及應用拮抗細菌與植物萃取液防治芒果炭疽病。國立中興大學植物病理學系碩士論文。93頁。 Agrios GN, 2005. Plant pathology, 5th edn. Academic, New York Bills GF, 1996. Isolation and analysis of endophytic fungal communities from woody plants.In: Endophytic Fungi in Grasses and Woody Plants: Systematics, Ecology, and Evolution. Redlin SC, Carris LM (eds) APS press, St. Paul, Minnesota, U.S.A. Cannon PF, Damm U, Johnston PR, Weir BS, 2012. Colletotrichum– current status and future directions. Studies in Mycology 73, 181-213. Carbone I, Kohn LM, 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91, 553-556. Chen FY, Lin MT, Chiang MY, 2004. Identification of Colletotrichum species in Taiwan based on the internal transcribed spacer of the ribosomal region. Plant Prot. Bull. 46, 15-26. Crous PW, Groenewald JZ, Risede JM, Hywel-Jones NL, 2004. Calonectria species and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50, 415–430. Damm U, Cannon PF, Woudenberg JHC, Crous PW, 2012a. The Colletotrichum acutatum species complex. Studies in mycology 73, 37-113. Damm U, Cannon PF, Woudenberg JHC, Johnston PR, Weir BS, Tan YP, Shivas RG, Crous PW, 2012b. The Colletotrichum boninense species complex. Studies in mycology 73, 1-36. Daniel HM, Meyer W, 2003. Evaluation of ribosomal RNA and actin gene sequences for the identification of ascomycetous yeasts. International journal of food microbiology 86(1), 61-78. De Silva DD, Ades PK, Crous PW, Taylor PWJ, 2017. Colletotrichum species associated with chili anthracnose in Australia. Plant Pathology 66(2), 254-267. Dodd JC, Estrada A, Jeger MJ, 1992. Epidemiology of Colletotrichum gloeosporioides in Tropics. In: Bailey JA, Jeger MJ, eds. Colletotrichum: Biology, Pathology and Control. CABI, Wallingford, 308-325. Douanla-Meli C, Langer E, Mouafo FT, 2013. Fungal endophyte diversity and community patterns in healthy and yellowing leaves of Citrus limon. Fungal Ecology 6(3), 212-222. Gan P, Nakata N, Suzuki T, Shirasu K, 2017. Markers to differentiate species of anthracnose fungi identify Colletotrichum fructicola as the predominant virulent species in strawberry plants in Chiba Prefecture of Japan. Journal of General Plant Pathology 83(1), 14-22. Garrido C, Carbú M, Fernández‐Acero FJ, Boonham N, Colyer A, Cantoral JM, Budge G, 2009. Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real‐time PCR. Plant Pathology 58(1), 43-51. Glass NL, Donaldson GC, 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61, 1323-1330. Guerber JC, Liu B, Correll JC, Johnston PR, 2003. Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia 95(5), 872-895. Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In Nucleic acids symposium series 41(41), 95-98. Hu MJ, Grabke A, Dowling ME, Holstein HJ, Schnabel G, 2015. Resistance in Colletotrichum siamense from peach and blueberry to thiophanate-methyl and azoxystrobin. Plant Disease 99(6), 806-814. Hyde KD, Cai L, Cannon PF, Crouch JA, Crous PW, Damm U, Goodwin PH, Chen H, Johnston PR, Jones EBG, Liu ZY, McKenzie EHC, Moriwaki J, Noireung P, Pennycook SR, Pfenning LH, Prihastuti H, Sato T, Shivas RG, Tan YP, Taylor PWJ,Weir BS, Yang YL, Zhang JZ, 2009. Colletotrichum—names in current use. Fungal Diversity 39, 147-182. Jayawardena RS, Hyde KD, Damm U, Cai L, Liu M, Li XH et al., 2016. Notes on currently accepted species of Colletotrichum. Mycosphere, 7(8), 1192-1260. Katoh K, Standley DM, 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution 30(4), 772-780. Lima NB, Batista MVDA, De Morais MA, Barbosa MA, Michereff SJ, Hyde KD, Câmara MP, 2013. Five Colletotrichum species are responsible for mango anthracnose in northeastern Brazil. Fungal Diversity 61(1), 75-88. Liu F, Wang M, Damm U, Crous PW, Cai L, 2016. Species boundaries in plant pathogenic fungi: a Colletotrichum case study. BMC evolutionary biology 16(1), 81. Liu F, Weir BS, Damm U, Crous PW, Wang Y, Liu B, Wang M, Zhang M, Cai L, 2015. Unravelling Colletotrichum species associated with Camellia: employing ApMat and GS loci to resolve species in the C. gloeosporioides complex. Persoonia: Molecular Phylogeny and Evolution of Fungi 35, 63-86. López-González RC, Gómez-Cornelio S, Susana C, Garrido E, Oropeza-Mariano O, Heil M, Partida-Martínez LP, 2017. The age of lima bean leaves influences the richness and diversity of the endophytic fungal community, but not the antagonistic effect of endophytes against Colletotrichum lindemuthianum. Fungal Ecology 26, 1-10. Lou PS, Huang YJ, Chung WC, Cheng AS, Chung WH, 2010. Application of PCR-RFLP in detecting benzimidazoles-resistant isolates of Colletotrichum gloeosporioides from mango in Tainan area. Plant Pathol. Bull. 19, 255-260. Mills PR, Sreenivasaprasad S, Brown AE, 1992. Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR. FEMS Microbiology Letters 98(1‐3), 137-143. Mo J, Zhao G, Li Q, Solangi GS, Tang L, Guo T et al., 2018. Identification and Characterization of Colletotrichum Species Associated with Mango Anthracnose in Guangxi, China. Plant Disease, PDIS-09. Mulè G, Susca A, Stea G, Moretti A, 2004. A species-specific PCR assay based on the calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. European Journal of Plant Pathology 110(5-6), 495-502. Nirenberg HI, 1976. Untersuchungen uber die morpholoigische und biologische Differnzierung in der Fusarium-Sektion Liseola. Mitt. Biol. Bundesanst. Land-u. Forstwirtsch. Berlin-Dahlem 169, 1-117. O'Donnell K, Cigelnik E, 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. MoIecular Phylogenetics and Evolution 7, 103-116. Peres NAR, Souza NL, Peever TL, Timmer LW, 2004. Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from citrus. Plant Disease 88, 125-130. Photita W, Lumyong S, Lumyong P, McKenzie EHC, Hyde KD, 2004. Are some endophytes of Musa acuminata latent pathogens?. Fungal Diversity 16, 131-140. Prihastuti H, Cai L, Chen H, McKenzie EHC, Hyde KD, 2009. Characterisation of Colletotrichum species associated with coffee berries in northern Thailand. Fungal Diversity 39, 89-109. Rodriguez RJ, White Jr JF, Arnold AE, Redman ARA, 2009. Fungal endophytes: diversity and functional roles. New phytologist 182(2), 314-330. Sambrook J, Russell DW, 2001. Preparation and analysis of eukaryotic genomic DNA. In: Molecular cloning, a laboratory manual, 3rd ed. Sambrook J, Russell DW, eds, Cold Spring Harbor Laboratory Press pp. 6.1–6.64 Sharma G, Kumar N, Weir BS, Hyde KD, Shenoy BD, 2013. The ApMat marker can resolve Colletotrichum species: a case study with Mangifera indica. Fungal Diversity 61(1), 117-138. Silva DN, Talhinhas P, Várzea V, Cai L, Paulo OS, Batista D, 2012. Application of the Apn2/MAT locus to improve the systematics of the Colletotrichum gloeosporioides complex: an example from coffee (Coffea spp.) hosts. Mycologia, 104(2), 396-409. Talhinhas P, Gonçalves E, Sreenivasaprasad S, Oliveira H, 2015. Virulence diversity of anthracnose pathogens (Colletotrichum acutatum and C. gloeosporioides species complexes) on eight olive cultivars commonly grown in Portugal. European journal of plant pathology 142(1), 73-83. Talhinhas P, Sreenivasaprasad S, Neves-Martins J, Oliveira H, 2005. Molecular and phenotypic analyses reveal association of diverse Colletotrichum acutatum groups and a low level of C. gloeosporioides with olive anthracnose. Applied and Environmental Microbiology 71(6), 2987-2998. Talhinhas P, Sreenivasaprasad S, Neves-Martins J, Oliveira H, 2002. Genetic and morphological characterization of Colletotrichum acutatum causing anthracnose of lupins. Phytopathology 92(9), 986-996. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution 30(12), 2725-2729. Tao G, Liu ZY, Liu F, Gao YH, Cai L, 2013. Endophytic Colletotrichum species from Bletilla ochracea (Orchidaceae), with descriptions of seven new speices. Fungal Diversity 61(1), 139-164. Templeton MD, Rikkerink EHA, Solon SL, Crowhurst RN, 1992. Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata. Gene 122, 225-230. Tuite J, 1969. Plant Pathological Methods – Fungi and Bacteria. Burgess Publ. Col., Minneapolis, Minnesota, 239 pp. Vieira WA, Michereff SJ, de Morais MA, Hyde KD, Câmara MP, 2014. Endophytic species of Colletotrichum associated with mango in northeastern Brazil. Fungal diversity 67(1), 181-202. von Arx JA, 1957. Die arten der gattung Colletotrichum Cda. Phytopathol Z 29, 413-468. Wang YC, Hao XY, Wang L, Xiao B, Wang XC, Yang YJ, 2016. Diverse Colletotrichum species cause anthracnose of tea plants (Camellia sinensis (L.) O. Kuntze) in China. Scientific reports 6, 35287. Weir BS, Johnston PR, Damm U, 2012. The Colletotrichum gloeosporioides species complex. Studies in mycology 73, 115-180. White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications. (Innis MA, Gelfand DH, Sninsky JJ, White TJ eds). Academic Press, New York, 315-322. Wilson D, 1995. Endophyte – the evolution of the term, a clarification of its use and definition. Oikos 73, 247-276.
摘要: 芒果是臺灣重要的熱帶經濟果樹,炭疽病為芒果果實上最主要之病害。在早期的病原菌鑑定中,根據形態的分類與ITS序列將芒果炭疽病菌鑑定為Colletotrichum gloeosporioides與Colletotrichum acutatum兩個種。近年來由多基因鑑定發現許多舊有的種為複合種(species complex),也因此建立許多新種。為了確認目前臺灣芒果上存在的炭疽菌種類,並比較不同種間是否有藥劑感受性、病原性與形態方面的差異,本研究至台南、屏東的芒果產區與其他零星產區,收集愛文、金煌及土芒果等品種,自罹病果實病斑及健康的枝條與花序上上分離到210株炭疽菌株,從中挑選具代表性的97株菌進行分析,包含74株果實病原菌、11株枝條內生菌與12株花序內生菌。其中,屬於C. gloeosporioides species complex與C. acutatum species complex之菌株分別使用ApMat與TUB2基因進行初步分群鑑定,再從各支序群中挑出代表菌株進行多基因親緣關係樹的建構,C. gloeosporioides species complex的菌株使用ACT、CAL、CHS-1、GAPDH及ITS的合併序列建構親緣樹,C. acutatum species complex的菌株使用ACT、CHS-1、GAPDH、HIS3、ITS及TUB2的合併序列建構親緣樹。結果顯示,在果實病原菌中,以C. asianum占78%為優勢種,並包含7種不同的菌落型態,其它還有C. fructicola, C. siamense與C. tropicale等親緣種;在花序內生菌中以C. acutatum species complex內形成的一個獨立支序群,可能為新種(暫稱Casp),有83%之比例,僅有2株為C. asianum;在枝條內生菌中,以C. asianum為最多,占73%,另有2株為Casp,1株為C. sloanei。將上述各種菌以果實接種後確認皆具病原性,且在不同親緣種的致病力試驗中發現,以C. asianum及C. fructicola顯著大於其餘四個親緣種的病斑大小。以芒果葉片接種後則發現,C. asianum可造成較明顯病斑,C. siamense與C. tropicale無法在葉片產生病斑。在藥劑感受性測試中,除了C. tropicale與Casp的菌株對甲基多保淨感受性較高,其餘C. asianum, C. fructicola與C. siamense菌株則有明顯抗藥性;而多數菌株對亞托敏與克收欣有抗藥性;所有菌株則都對得克利、待克利與撲克拉有高度感受性。針對芒果炭疽病菌的主要親緣種,本研究中也設計專一性偵測C. asianum之引子對,引子 Casia-F2 / Casia-R2可成功自C. asianum中增幅出約300 bp之片段,測試其餘12種屬於C. gloeosporioides species complex Musae clade中,與C. asianum相近的不同親緣種之ex-type strains,皆無PCR片段產生。本研究的結果,可提供未來探討親緣種之生物學差異與應用於病原菌生態學之研究。
Mango is one of important tropical fruits in Taiwan. Anthracnose is the primary disease on mango caused by species of Colletotrichum. In the past, mango anthracnose pathogens were classified as Colletotrichum gloeosporioides and Colletotrichum acutatum based on morphological characteristcs or ITS sequence. According to results of multi-gene phylogenetic analysis, C. gloeosporioides and C. acutatum were considered as species complexes.Thus, many new species were established in complexes in these years. The study was aimed to identify phylogenetic species of mango anthracnose pathogens in Taiwan, and to compare their morphology, fungicides sensitivity and pathogenicity. Ninety-seven out of 210 Colletotrichum isolates were collected from diseased fruits (74 isolates), asymptomatic twigs (11 isolates) and peduncles (12 isolates). Isolates of C. gloeosporioides species complex were analyzed with ApMat while isolates of C. acutatum species complex were sequenced with TUB2 to construct phylogenetic trees. Representative isolates from each clade were selected for further identification in multi-genes phylogenetic trees. Isolates of C. gloeosporioides species complex were analyzed with concatenated sequences of ACT, CAL, CHS-1, GAPDH and ITS genes, while isolates of C. acutatum species complex were analyzed with concatenated sequences of ACT, CHS-1, GAPDH, HIS3, ITS and TUB2 genes. In the combined results, C. asianum is a dominant species from diseased fruits with isolation rate of 78% and 7 colony types. In addition, C. fructicola, C. siamense, and C. tropicale were also obtained from fruit anthracnose. In contrast, in peduncle endophytic population, a possible new species of C. acutatum species complex (temporarily named Casp) were dominant with 83% isolation rate, and there were only two C. asianum isolates. The twig endophytic population consisted of C. asianum with 73% isolation rate, two Casp isolates, and one isolate of C. sloanei. In pathogenicity assays, all isolates from diseased fruits, asymptomatic twigs, and peduncles caused anthracnose lesions on mango fruits. In the virulence assays of different phylogenetic species on Irwin mango, C. asianum and C. fructicola caused significantly bigger lesions than other 4 phylogenetic species. On leaf inoculation, C. asianum usually generated lesions, but C. siamense and C. tropicale were not the case. In fungicide sensitivity test, all species except C. tropicale and Casp showed high resistance to thiophanate-methyl and carbendazim. All species were resistant to azoxystrobin and kresoxim-methyl, but sensitive to tebuconazole, difenoconazole and prochloraz. To accelerate the identification of the dominant species, species-specific primers were designed to detect C. asianum. A primer set Casia-F2 / Casia-R2 can specifically amplify a 300 bp fragment from C. asianum, but not from 12 phylogenetic species closely related to C. asianum in Musae clade of C. gloeosporioides species complex. Results in this study may provide the basis for exploring the biological differentiation among phylogenetic species, and for studying the ecology of pathogens.
URI: http://hdl.handle.net/11455/98165
文章公開時間: 2021-08-30
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