臺灣櫻花流膠病之病原菌分析及生物防治

Abstract

櫻花為薔薇科 (Rosaceae) 李屬 (Prunus) 之落葉樹木，在臺灣多用於景觀栽培及行道樹。櫻花流膠病成因包含病蟲害、生理逆境及機械傷口等因素，其中又以真菌性流膠病為主，罹病時莖部或枝條內部組織褐化，並產生潰瘍、流膠及枝枯病徵，遭受逆境時病徵加劇，嚴重時導致植株死亡。櫻花流膠病普遍被認為由葡萄座腔菌科 (Botryosphaeriaceae) 真菌所引起，已確認之病原菌為Botryosphaeria dothidea、Lasiodiplodia thailandica及Lasiodiplodia theobromae，然而先前研究之採樣地區及樣本數有限，且目前尚缺乏有效的生物防治策略，因此本研究欲釐清臺灣櫻花流膠病之病原菌相，並評估芽孢桿菌 (Bacillus) 對於櫻花流膠病之防治效益。本研究對臺北市國立臺灣大學校園、新北市三芝三生步道及連江縣北竿鄉三處罹病之櫻花進行病原菌相調查，以internal transcribed spacer (ITS)、translation elongation factor 1-α (TEF1)、β-tubulin (TUB2) 及DNA-directed RNA polymerase II second largest subunit (RPB2) 序列進行多基因親緣分析鑑定，共分離到13株Botryosphaeriaceae之真菌，包含B. dothidea (四株)、Botryosphaeria fabicerciana (一株)、Lasiodiplodia chiangraiensis (兩株)、Lasiodiplodia endophytica (一株)、L. theobromae (四株) 及Neofusicoccum parvum (一株)。每個種挑一菌株作為代表以菌絲塊進行創傷接種，確認皆可於山櫻花 (Prunus campanulata Maxim.) 苗莖部造成流膠病徵，並由罹病組織分離出病原菌，完成柯霍氏法則。本研究為B. fabicerciana、L. chiangraiensis、L. endophytica及N. parvum造成櫻花流膠病之首次報導。於馬鈴薯葡萄糖瓊脂培養基上測試流膠病菌於15–35 ℃之菌絲生長速率，發現最適生長溫度為25–30 ℃，與流膠病好發於夏季之田間觀察吻合，且Lasiodiplodia spp. 於35 ℃之生長較其餘菌種良好，顯示該屬具有較佳高溫適應性。本研究評估八株芽孢桿菌菌株於馬鈴薯葡萄糖瓊脂培養基上對各種病原菌之生長抑制能力，其中以Bacillus velezensis B2及PHYRRX8具廣效性抑制效果。選用B. dothidea TP07及L. theobromae TP04於溫室接種山櫻花苗莖部，並利用B. velezensis B2及PHYRRX8進行預防性噴灑及澆灌之防治試驗，定期記錄罹病度並計算病害發展階梯下面積 (area under the disease progress stairs, AUDPS)，於試驗結束後進行組織分離，並收取莖部綠皮層 (phelloderm) 檢測抗氧化酶catalase (CAT)、superoxide dismutase (SOD) 及peroxidase (POD) 活性。結果顯示，噴灑B. velezensis PHYRRX8能顯著降低接種L. theobromae TP04之病害發展階梯下面積、莖部變色率及接種B. dothidea TP07之回分率，噴灑B. velezensis B2則能顯著降低接種L. theobromae TP04之莖部變色率，顯示噴灑Bacillus能有效抑制櫻花流膠病之病害發展。Bacillus施用後CAT、SOD及POD皆無顯著提升，未來可透過分析不同時間點抗氧化酶活性及防禦基因之表現，以全面評估櫻花之誘導抗性機制。本研究釐清臺灣櫻花流膠病相關之Botryosphaeriaceae菌種多樣性，並篩選出能有效減緩病程發展之芽孢桿菌與施用方式，可作為櫻花流膠病整合性防治管理之重要基礎。

Ornamental cherry trees (Prunus spp.; Rosaceae) are deciduous woody plants widely cultivated as ornamental and street trees in Taiwan. Cherry gummosis can be caused by insect, abiotic stresses, mechanical injuries, and pathogens, among which fungal gummosis is considered the predominant cause. The stem and branches of diseased trees typically exhibit internal discoloration, cankers, gummosis, and dieback, and symptoms are often exacerbated under environmental stress, resulting in decline or death. Cherry gummosis is widely attributed to fungi belonging to the family Botryosphaeriaceae, with Botryosphaeria dothidea, Lasiodiplodia thailandica, and Lasiodiplodia theobromae having been identified as causal agents. However, previous studies were limited in terms of sampling locations and sample size, and effective biological control strategies remain lacking. Therefore, this study aimed to elucidate the diversity of pathogens associated with cherry gummosis in Taiwan and evaluate the biocontrol efficacy of Bacillus spp. against this disease. In this study, diseased cherry trees were surveyed at three locations in Taiwan, including the campus of National Taiwan University (Taipei City), Sansheng Trail (Sanzhi District, New Taipei City), and Beigan Township (Lienchiang County). Fungal isolates were identified using multilocus phylogenetic analyses based on sequences of the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1), β-tubulin (TUB2), and DNA-directed RNA polymerase II second largest subunit (RPB2). Thirteen Botryosphaeriaceae isolates were obtained, including four isolates of B. dothidea, one of Botryosphaeria fabicerciana, two of Lasiodiplodia. chiangraiensis, one of Lasiodiplodia endophytica, four of L. theobromae, and one of Neofusicoccum parvum. One representative isolate of each species was selected for pathogenicity tests by wounded inoculation using mycelial plugs. All isolates induced typical gummosis symptoms on stems of Prunus campanulata Maxim. seedlings, and the inoculated fungi were successfully reisolated from symptomatic tissues, thereby fulfilling Koch's postulates. This is the first report of B. fabicerciana, L. chiangraiensis, L. endophytica, and N. parvum as causal agents of cherry gummosis. The effects of temperature on mycelial growth were evaluated on potato dextrose agar at 15–30 ℃. The optimum growth temperature for the gummosis pathogens was determined to be 25–30 ℃, which was consistent with field observations that cherry gummosis occurred most frequently during summer. Notably, Lasiodiplodia spp. exhibited greater growth at 35 °C than the other genera, indicating superior adaptation to high-temperature conditions. Eight Bacillus strains were evaluated for their antagonistic activity against the pathogens on potato dextrose agar. Among them, B. velezensis B2 and PHYRRX8 exhibited broad-spectrum inhibitory activity. B. dothidea TP07 and L. theobromae TP04 were subsequently selected for P. campanulata seedlings in greenhouse assays to evaluate the preventive efficacy of B. velezensis B2 and PHYRRX8 via stem spraying and soil drenching. Disease severity index was assessed periodically, and the area under the disease progress stairs (AUDPS) was calculated. At the end of the experiment, pathogen reisolation was performed, and the stem phelloderm tissues were collected to determine the activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). Spraying of B. velezensis PHYRRX8 significantly reduced the AUDPS and stem discoloration rate in plants inoculated with L. theobromae TP04, as well as the reisolation rate of B. dothidea TP07. Spraying of B. velezensis B2 also significantly reduced the stem discoloration rate in plants inoculated with L. theobromae TP04. The results demonstrate that preventive stem spraying of Bacillus effectively suppress the development of cherry gummosis. However, no significant increases in CAT, SOD, or POD activities were detected following Bacillus application. Future studies should investigate antioxidant enzyme activities and the expression of defense-related gene at different time points to comprehensively evaluate the induced resistance mechanisms in cherry trees. This study clarified the diversity of Botryosphaeriaceae species associated with cherry gummosis in Taiwan and identified Bacillus strains and application methods effective for delaying disease development, laying an important foundation for the integrated management of cherry gummosis.