臺灣松樹葉部病害之調查：病原鑑定及防治藥劑篩選

Abstract

松樹在臺灣的景觀園藝產業中佔有重要地位，尤其黑松 (Pinus thunbergii) 因高觀賞價值而被廣泛應用。然而黑松葉部病害繁多，對其健康、經濟及觀賞價值造成重大影響。根據前人文獻，臺灣發生之松樹葉震病是由 Lophodermium pinastri 造成，使葉片出現鮮黃及紅棕色條斑；葉枯病則可由包含 Pseudocercospora pini-densiflorae 等多種病原菌造成，使葉片出現深褐色壞死病斑。但上述記載主要基於早期的採集紀錄，除缺乏系統性調查、病原性驗證及有效的防治策略外，部分資訊與近期發表之研究結果抵觸，例如國外研究經接種試驗已證實 L. pinastri 並不具有病原性。為釐清臺灣松樹葉部病害之病原真菌，本研究在新北市、臺北市、桃園市、新竹市、彰化縣及屏東縣進行共 54 次採樣，採集無病徵、疑似葉震病黃化斑 (I 至 V 型)、葉枯病及脫落的枯死松針進行組織分離。結果顯示，Lophodermium spp. 於各型黃化斑幾乎無法檢出 (分離率小於 0.5%)，反而主要自枯死松針中檢出 (分離率 70.2%)，顯示其腐生特性。Lophodermium spp. 分離株經多基因親緣分析鑑定大多為 L. conigenum 及 L. pinastri，此兩物種根據前人文獻皆不具病原性。另一方面，擔子菌 Atractidochium sp. 於嚴重 I 型黃化斑之分離率高達 62.6%，但在無病徵及枯死葉片則低於 3.0%；將其分離株進行多基因親緣分析鑑定為 At. hillariae，為臺灣首次記錄。前人文獻記載 At. hillariae 為德達松 (Pi. taeda) 之葉部內生菌，然而相關研究仍極為稀少。本研究以 At. hillariae 及 Lophodermium spp. 等菌株進行離葉、離枝及田間微創傷菌絲塊接種，發現僅 At. hillariae 能使松針出現黃化及紅棕色條斑，確認 At. hillariae 為臺灣松樹葉震病之病原菌，並建議更名為「Atractidochium 葉斑病」，以凸顯其典型病徵。此外，本研究執行田間罹病度之長期調查，結果顯示 Atractidochium 葉斑病從每年 11 月開始陸續發病至翌年 4 至 5 月達到高峰。為篩選防治藥劑，選擇常用於防治擔子菌果樹葉部病害之殺菌劑，包含滅普寧、氟克殺、百克敏、得克利及克熱淨 (烷苯磺酸鹽)，透過含藥培養基測試菌絲抑制率，結果顯示氟克殺、百克敏、得克利及克熱淨 (烷苯磺酸鹽) 均有良好抑制率 (EC50 值 0.02 ppm 至 0.33 ppm)。挑選抑制效果最佳之百克敏進行田間試驗，發現其對 Atractidochium 葉斑病並無防治效果，可能與施藥時機不當或藥液不易附著在松針表面有關。此外，針對 At. hillariae 之 internal transcribed spacer (ITS) 區段，設計具高專一性及高靈敏度之引子對 Ah3_F/Ah3_R，以建立快速分子檢測技術。本研究亦針對松樹葉枯病進行菌相調查，發現 Pseudocercospora、Alternaria 及 Beltraniella 屬真菌之分離率較高，且三者接種後均能產生類似葉枯病之壞死病徵。本研究針對於臺灣地區黑松上發生之葉斑病及葉枯病，進行田間病害調查、病原菌分離與鑑定、病原性測試、快速分子檢測技術開發、藥劑篩選及評估，提供松樹葉部病害正確診斷及健康管理之依據。

Pine trees play a vital role in Taiwan’s landscape and horticultural industries, with black pine (Pinus thunbergii) being particularly favored for its high ornamental value. However, black pine is highly susceptible to foliar diseases that damage tree health, economic, and ornamental value. According to previous literature, needle cast disease in Taiwan has been attributed to Lophodermium pinastri, which causes yellowish and reddish-brown striped lesions, while needle blight has been linked to multiple pathogens including Pseudocercospora pini-densiflorae, which induces brown necrotic lesions on needles. However, these identifications were primarily based on early collections and lack comprehensive field surveys, pathogenicity tests, and effective control strategies. Moreover, some of the information is inconsistent with the findings from recent research; for example, studies conducted abroad have demonstrated through inoculation that L. pinastri is non-pathogenic. To identify the causal fungi of foliar diseases in pine trees in Taiwan, this study conducted 54 sampling surveys across New Taipei City, Taipei City, Taoyuan City, Hsinchu City, Changhua County, and Pingtung County. Samples were collected from asymptomatic needles, suspected needle cast lesions (types I to V yellowish spot), needle blight lesions, and dead detached needles for tissue isolation. The results showed that Lophodermium spp. were not isolated from any types of the yellowish spot (isolation rate < 0.5%) but were predominantly detected in dead needles on the ground (isolation rate 70.2%), suggesting their saprophytic nature. Multi-locus phylogenetic analysis identified the Lophodermium isolates primarily as L. conigenum and L. pinastri, both of which have been reported in previous literature as non-pathogenic. In contrast, the basidiomycete Atractidochium sp. was isolated at a high frequency from severe type I yellowish spots (isolation rate 62.6%), but at very low frequency from asymptomatic or dead needles (isolation rate < 3.0%). Multi-locus phylogenetic analysis identified these isolates as At. hillariae, representing the first record of this species in Taiwan. At. hillariae had previously been reported as a foliar endophyte in P. taeda, yet studies on this species remain limited. Detached needle, detached twig, and field-based wounded inoculation trials using mycelial discs of At. hillariae, Lophodermium spp. and other fungi revealed that only At. hillariae could cause the yellowish and reddish-brown lesions on needles. These results confirm At. hillariae as the causal agent of needle cast disease in Taiwan. To highlight its characteristic symptoms, it is proposed that the disease be renamed “Atractidochium needle spot disease”. A long-term field survey revealed that the disease onset begins in November and reaches its highest disease severity index between April and May of the following year. To screen for effective control fungicides, five fungicides commonly used against basidiomycete pathogens on foliar disease of fruit trees—tebuconazole, fluxapyroxad, mepronil, pyraclostrobin, and iminoctadine tris (albesilate)—were evaluated in vitro via mycelial growth inhibition assays using fungicide-amended media. Four of the tested fungicides (tebuconazole, fluxapyroxad, pyraclostrobin, and iminoctadine tris [albesilate]) exhibited strong inhibitory effects against At. hillariae, with EC₅₀ values ranging from 0.02 ppm to 0.33 ppm. Among them, pyraclostrobin showed the highest inhibitory efficiency and was therefore selected for subsequent field trials. However, effective disease control was not achieved, likely due to improper application timing or insufficient foliar adherence of the fungicide solution. In parallel, a highly specific and sensitive primer pair, Ah3_F/Ah3_R, was developed based on the internal transcribed spacer (ITS) region of At. hillariae, enabling rapid molecular detection from needle samples. This study also investigated the fungal communities associated with needle blight symptoms. Tissue isolation revealed that species of Pseudocercospora, Alternaria, and Beltraniella were isolated at relatively high frequencies, and all of them could induce necrotic lesions resembling needle blight upon inoculation. This research addressed needle cast and needle blight on Pi. thunbergii through field surveys, pathogen isolation and identification, pathogenicity testing, development of a rapid molecular detection method, and fungicide screening and evaluation. The findings provide a scientific basis for accurate diagnosis and effective management of pine needle diseases.