臺灣樹木褐根病子實體及其模式植物之研究

Abstract

自2007年至2018年間於臺灣本島及外島進行樹木褐根病之調查，診斷確認感染褐根病 (Phellinus noxius (Corner) G. H. Cunningham) 之病株樣本數量為2,287件；其中在田間較少被發現之褐根病子實體的病株，樣本數量為164件，在這164件案例中，有33種新寄主為田間首次發現樹木褐根病子實體之紀錄。大部分之樹木褐根病子實體為不顯眼、平伏的形式，有些則是托架形態。子實體在臺灣本島及外島都有出現，於臺灣中部及南部出現頻率較高，通常分布於海拔200至300公尺間，另隨著海拔上升至800公尺而逐漸減少。比較34個擔孢子單孢分離株培養於馬鈴薯瓊脂培養基(PDA, potato dextrose agar) 及燕麥抽出物瓊脂培養基 (MEA, malt extract agar medium) 的生長速率差異性，結果顯示培養於PDA上的34株經統計分析共有3個不同等級的生長速率，而在MEA上的則有4個不同等級的生長速率。以上證明褐根病菌的子實體及擔孢子，可增加族群基因之多樣性，有助於樹木褐根病的長距離傳播。 本研究另採集自榕樹之新鮮褐根病子實體中，將所收集之擔孢子置於雨水等不同溶液中，進行擔孢子發芽實驗，結果發現擔孢子在每一種溶液中的發芽率皆超過50%。本研究嘗試以105-106 spores/mL 的擔孢子濃度直接接種在四種不同樹苗的基部，結果皆未出現病徵或病兆，表示無法接種成功。但以105-106 spores/mL 之擔孢子懸浮液，先接種於臺灣櫸木邊材切片，置於室溫下經2個月的觀察，發現有部分的臺灣櫸木邊材切片，可以觀察到褐根病菌感染其上，並形成褐色網紋及菌絲面。將此些褐根病菌成功感染的臺灣櫸木片作為接種源，接種於臺灣櫸木的樹苗，經歷3個月的觀察，可以接種成功，證明褐根病菌擔孢子具有間接感染之能力。本研究自2007年起，在田間2287案例中總共發現了13個褐根病是屬於樹幹直接感染或枝條直接感染的案例，這也間接證明了擔孢子確實具有傳播感染能力。因為颱風於臺灣發生十分頻繁，颱風的侵害常會造成樹木產生傷口，推測擔孢子可以經由此些傷口侵染樹木，達成長距離傳播之結果。 過去在褐根病的實驗中，為確認樹木褐根病菌之病原性，其接種實驗普遍有發病緩慢之問題，甚至有接種二年仍不見接種部位出現病徵或病兆之情況。因此，本研究乃經篩選、測試，選擇出極為敏感之馬拉巴栗 (Pachira macrocarpa (Cham. & Schl.) Schl.) 盆栽樹苗，做為樹木褐根病之接種模式植物。接種方法是以電鑽對莖基部鑽孔，並以接種小麥燕麥粒菌種者發病最快。利用此一簡單快速的方法，馬拉巴栗模式植物可於接種後1至2週內產生病徵，其外部病徵主要為莖腐、樹皮凹陷、褐化、葉片黃化及植株萎凋，莖部之內部病徵則包括組織水浸狀及褐化。以小麥燕麥粒菌種當接種源所產生病徵之效果，較以木屑培養基當接種源之效果好。以馬拉巴栗作為樹木褐根病接種模式植物，符合模式植物之標準，包括「1. 高敏感度，2. 植物材料容易取得，3. 操作容易，4. 病徵容易表現，5. 病徵具有專一性」等。利用此種模式植物，將可用以快速病原性檢測、研究藥劑之藥物動力學，以及各種藥劑對褐根病之防治效能。

Since 2007, a comprehensive survey of the occurrence of brown root rot disease has been conducted in Taiwan. The rare basidiocarps of the pathogen Phellinus noxius (Corner) Cunningham were also investigated throughout Taiwan and on outer islands. To 2018, a total of 2,287 disease cases of brown root rot were recorded, and 164 cases were found with basidiocarps on diseased trees. These 164 cases included 33 newly discovered tree hosts on which were borne the fruiting body of P. noxius. Most of the basidiocarps were of the flat type which is inconspicuous, while some belong to the bracket type. Generally, basidiocarps were found in most counties of Taiwan and on the outer islands, but they exhibited higher frequencies in the central and southern parts of Taiwan. They tended to occur more at elevations of 200~300 m in the hills, but gradually disappeared at the elevation increased to 800 m. To compare variations in growth rates, 34 single-basidiospore isolates were obtained and grown on both potato dextrose agar (PDA) and malt extract agar medium (MEA). Results showed three levels of significant growth differences on PDA, and four levels of differences on MEA, indicating growth rate variations among these sexual basidiospores. The abundance of P. noxius basidiocarps in nature suggests that genetically variable basidiospores are involved in long‐distance dispersal of this fungus which is responsible for serious tree diseases. Fresh basidiospores were collected from live basidiocarps growing on a small-leafed banyan (Ficus microcarpa Linn. f. ), and the basidiospores were rinsed out. The germination experiment revealed that they could germinate in all kinds of liquids including rainwater, with germination rates usually exceeding 50%. The direct inoculation of basidiospore suspensions at concentration of 105~106 spores/mL on four tree species was consistently unsuccessful. The same spore suspension was used to inoculate wood chips from Taiwan zelkova (Zelkova formosana Hayata) sapwood. After 2 months, some of the trees had been colonized by basidiospores, showing dark-brown line or network symptoms. Using these colonized wood chips, we successfully inoculated wounded roots of Taiwan zelkova seedlings within 3 months, proving the virulence and role of basidiospores in nature. Since 2007, 13 cases of twig or stem infection by P. noxius were found in the field, confirming transmission by basidiospores. In Taiwan, due to the high frequency of typhoon damage to trees, abundant stem wounds should act as important sites for basidiospore deposition and infection. This could facilitate the long-distance dispersal of this disease in nature. Previously, pathogenicity tests for suspected brown root rot pathogen were not easy to conduct and took a very long time. For these reasons, we tried to find a very sensitive plant, the Malabar chestnut (Pachira macrocarpa (Cham. & Schlecht.) Walp.), to use as a bioassay or model plant for this pathogen. The inoculation method with an electric drill followed by insertion of wheat-oat culture inoculum makes this bioassay with Malabar chestnut very easy to conduct. With this simple, rapid method, model plants expressed infection symptoms within 1~2 weeks. External symptoms consist of stem rot, sunken bark, browning, and leaf chlorosis and wilting, while internal symptoms included water-soaking and browning of stem tissues. Inoculation from wheat-oat medium performed the best, much better than the enriched sawdust medium. In conclusion, the Malabar chestnut model as a disease bioassay plant fulfilled the following criteria: (a) high sensitivity; (b) high availability of plant materials; (c) ease of operation; (d) rapid expression of symptoms; and (e) exhibition of specific symptoms. On the basis of this model plant, new fungicides for controlling brown root rot disease were injected into the stem of Malabar chestnut, and the inoculum was simultaneously inoculated to study their control efficacy and fungicidal dynamics in the plant. This plant model can be utilized for the rapid screening of fungicides effective against brown root rot disease.