臺灣茶赤葉枯病菌之鑑定與藥劑感受性調查

Abstract

茶是臺灣重要經濟作物之一，茶赤葉枯病為茶葉生產中重要限制因子，不僅會危害成熟葉造成赤褐色病斑，更會感染嫩芽及嫩葉，造成黑色壞疽病斑而降低產量與成茶品質。茶赤葉枯病一般仰賴化學藥劑進行防治，然而，近十年來不斷有農友反映本病害發生嚴重程度增加及藥效降低。本研究首先調查在臺灣之病原菌相及其發生生態，測試病原菌對目前核准登記使用之殺菌劑之感受性，並對目前主要種植品種進行抗性檢定，以提出針對茶赤葉枯病之整合性防治管理建議。病原菌相調查結果顯示，分離自八個縣市計86處茶園之139株具病原性之炭疽菌菌株，利用形態學及包含真核生物核糖體內轉錄區 (internal transcribed spacer, ITS)、麩醯胺酸合成酶基因 (glutamine synthetase, GS)、Apn2-Mat1-2 intergenic spacer (ApMat)、微管蛋白 (beta-tubulin, TUB2)、肌動蛋白 (actin, ACT)、鈣調蛋白 (calmodulin, CAL) 和甘油醛-3-磷酸脫氫酶 (glyceraldehyde-3-phosphate dehydrogenase genes, GAPDH) 的多基因親緣分析，鑑定為Colletotrichum camelliae (佔所有分離株的 95.6%)、C. fructicola (3.7%) 及C. aenigma (0.7%)，其中C. camelliae在20℃、25℃及30℃下致病力均最強。C. fructicola及C. aenigma是臺灣首次報導之茶樹病原菌，且C. aenigma更為在臺灣首次被報導。針對15種核准登記使用於防治本病害之殺菌劑，測試對臺灣八個主要產茶縣市之37株C. camelliae、三株C. fructicola及一株C. aenigma之菌絲生長抑制率，及對二株C. camelliae、一株C. fructicola及一株C. aenigma之孢子發芽抑制率，結果顯示C. aenigma及C. fructicola對藥劑感受性普遍較C. camelliae菌株高。在登記使用之濃度下，得克利 (tebuconazole)、待克利 (difenoconazole)、扶吉胺 (fluazinam) 及克熱淨 (iminoctadine) 可完全抑制95%測試菌株之菌絲生長，預期在田間具有高防治潛力；免賴得 (benomyl)、甲基多保淨 (thiophanate-methyl)、百克敏 (pyraclostrobin)、嘉賜貝芬 (kasugamycin + carbendazim) 及貝芬四克利 (carbendazim + tetraconazole) 則可完全抑制56–61%以上測試菌株之菌絲生長，具有中等防治潛力；抑制孢子發芽調查中，僅扶吉胺及腈硫醌之登記使用濃度大於其半效應濃度 (median effective concentration, EC50)。C. camelliae菌株對免賴得及甲基多保淨呈敏感性及高抗性兩群，且抗性菌株於各採樣縣市之慣行管理茶區均有發現，進一步分析β-tubulin基因中已知最常發生抗藥性突變之區段，發現高及低感受性菌株僅在第860個核苷酸具有差異，此一點突變造成第198胺基酸由glutamic acid (GAG) 變成alanine (GCG)，符合文獻中造成高度抗藥性之機制。運用本研究新建立之接種方法 (菌絲塊對第3/4葉位的葉片進行離葉傷口接種)，評估33個茶樹品種對三株C. camelliae之抗性，發現 ‘四季春’ 及 ‘柑仔種’ 相對於對照品種 (‘青心烏龍’) 較為耐病，可做為抗病育種之親本。本研究成果可提供之整合管理策略包括：1. 選擇種植如 ‘四季春’ 或 ‘柑仔種’ 之較耐病茶樹品種；2. 在茶樹剛修剪後及嫩葉生長階段，施用保護性殺菌劑以降低病菌之感染；3. 選擇防治效果較佳之化學藥劑 (如得克利、待克利、扶吉胺及克熱淨)、輪用不同作用機制藥劑及使用混合劑，以降低抗藥性突變菌株產生。

Tea [Camellia sinensis (L.) O. Kuntze] is an economically important crop in Taiwan. Brown blight is a key limiting factor for tea production. It causes reddish-brown symptom on mature leaves and black necrotic spots on shoots and tender leaves, which results in reduction of the yield and quality of tea. Brown blight of tea is generally controlled by chemical agents. However, in the past ten years, farmers have continuously reported that the disease severity is increasing and chemical control is ineffective. In this study, we investigated the population composition and ecology of the infectious agents, tested the sensitivity of pathogens to registered fungicides, and screened the resistance of main tea cultivars to the disease. Finally, we proposed effective integrated disease management strategies for farmers. A collection of 139 Colletotrichum isolates obtained from 86 tea plantations in eight cities/counties were identified as Colletotrichum camelliae (95.6% of all isolates), C. fructicola (3.7%), and C. aenigma (0.7%) by morphological characterization and multilocus phylogenic analysis using the ribosomal internal transcribed spacer (ITS), glutamine synthetase gene (GS), the Apn2-Mat1-2 intergenic spacer (ApMat), beta-tubulin (TUB2), actin, calmodulin (ACT), and glyceraldehyde-3-phosphate dehydrogenase genes (GAPDH). Among the three species, C. camelliae was the most pathogenic at 20℃, 25℃, and 30℃. This is the first report of C. fructicola and C. aenigma as tea pathogens in Taiwan, and the first report of C. aenigma in Taiwan. For the 15 fungicides registered for disease control, we tested the inhibition of mycelial growth of 37 isolates of C. camelliae, three isolates of C. fructicola, and one isolate of C. aenigma from eight major tea-producing counties and cities in Taiwan, as well as the inhibition of spore germination of two isolates of C. camelliae, one isolate of C. fructicola, and one isolate of C. aenigma. C. aenigma and C. fructicola were generally more sensitive to fungicides than C. camelliae. At the recommended concentrations, tebuconazole, difenoconazole, fluazinam, and iminoctadine which completely inhibited the mycelial growth of 95% isolates are expected to have high control efficacies in the field. In addition, benomyl, thiophanate-methyl, pyraclostrobin, kasugamycin + carbendazim, and carbendazim + tetraconazole completely inhibited the mycelial growth of 56–61% isolates and considered to have an intermediate potential of control efficacy. Regarding the inhibitory effect on spore germination, only the recommended concentrations of fluazinam and dithianon were greater than the median effective concentration (EC50) values of all isolates. According to the survey of fungicide sensitivity, C. camelliae isolates resistant and highly sensitive to methyl benzimidazole carbamates (MBC) fungicides, benomyl and thiophanate-methyl, were found. The resistant isolates were sampled from conventional tea plantations in all cities/counties. A segment of β-tubulin that is known to have the most frequent fungicide resistance mutations was sequenced. The sensitive and resistant isolates differed only at the 860th nucleotide, which caused the 198th amino acid changed from glutamic acid (GAG) to alanine (GCG). The phenomenon conforms to the mechanism of the emergence of high fungicide resistance in the literature. The newly developed inoculation method (wounded inoculation of detached 3rd/4th tea leaves with mycelial discs) was used to evaluate the resistance of 33 tea cultivars to 3 isolates of C. camelliae. Cutlivars, ‘Shy Jih Chuen’ and ‘Gan Zai Chung’, which showed more tolerance than the control cultivar ‘Chin-shin Oolong’, could be used as the parental candidate for resistance breeding of tea in the future. Based on the findings of this research, the integrated management strategies that can be provided include: 1. planting of disease-tolerant tea cultivar such as ‘Shy Jih Chuen’ or ‘Gan Zai Chung’; 2. preventive application of fungicides immediately after tea pruning and at a young leaf stage; 3. use of effective fungicides (such as tebuconazole, difenoconazole, fluazinam, and iminoctadine), rotation of fungicides with different mode of actions, and use of fungicide mixtures to reduce the emergence of fungicide-resistant mutants.