樹木褐根病快速檢測之研究

褐根病(Brown root rot disease, Brr.)是由 Phellinus noxius 所引起，為好發於溫暖潮濕地區的真菌性病害，可侵染數百種植物並造成植株衰弱死亡，並使木本植物的木材組織腐朽，台灣全島都有發生記錄。褐根病傳染途徑一般是經由樹木根部互相接觸、染病組織移動，或土中帶菌殘體之接觸為主，但少數可藉由子實體產生之擔孢子進行遠距離傳播。現行的褐根病檢測方法有許多種，以檢測標的大致可區分為五大類，即檢測褐根病之罹病植株病徵與病兆、檢測褐根病菌之特定核酸序列(Specific DNA sequences )、檢測褐根病菌特定抗原(Specific antigen)、檢測褐根病菌特定揮發性氣體(VOC)、檢測褐根病菌生理生化特性 (Characteristic of Physiology and Biochemistry)等。常見實用的檢測法，有病徵法(目視檢測病徵兆)、顯微鏡檢、聚合酶鏈鎖反應檢測、恆溫環狀擴增法檢測、酵素連結免疫吸附法檢測、電子鼻檢測、偵測犬檢測、選擇性培養基分離培養、組織染色檢測，一共為九種檢測方式。本研究首先對這 9 種檢測方式進行優勢及劣勢之評比分析，項目包含：偵測樣本範圍、檢測所需時間、專一性、成本、方便性、是否能判斷褐根病菌株是否存活等。結果顯示顯微鏡檢、選擇性培養基、組織染色可分辨樣本中褐根病菌是否存活。這對於藥劑施用與土壤消毒後的檢測是十分重要的。而偵測範圍大者有目試診斷、電子鼻檢測、偵測犬檢測。檢測速度快者有目視診斷、偵測犬檢測、顯微鏡檢、組織染色檢測、電子鼻。檢測專一性高者有 PCR 技術、LAMP技術、 ELISA 技術。檢測成本相對低者有目視診斷、顯微鏡檢。檢測方便性高者則有目視診斷、顯微鏡檢、 LAMP 技術、電子鼻檢測、組織染色檢測。本研究亦進行褐根病菌培養基之改良，發現將 MA+4 中的免賴得提升到原本的兩倍，稱為PN3 者，對木黴菌、青黴菌等嚴重干擾之微生物會有良好的分離效果。又將營養源改為 PDM (1/2MEA +1/2PDA)，則可以讓褐根病菌在選擇性培養基上直接產生具特殊性之皮革化現象。最後新增加入藥劑福賽得、嘉賜銅、甲基多保淨、鏈四環黴素，成為 PDM+8 培養基，發現對細菌有良好的抑制效果，對其他腐朽菌的抑制力也有一定的提升。在專一性及方便性上，都比改良前 MA+4 大為提高。在田間快速鏡檢方面，首先觀察褐根病菌在人工培養基上二至三天時，菌落邊緣的菌絲，會出現具有鈍頭且新生分支與該分支的主幹粗細一致的特徵。另選擇多種染劑，包括食用黃色四號、食用藍色一號、食用綠色 B、食用紅色六號 (Cochineal red A )、甲基藍( Metrhyl blue)、棉藍(Cotton blue)、結晶紫( crystal violet )、孔雀綠(Malachite green)、番紅(Safranin O)等，嘗試進行田間快速鏡檢之染色。結果發現以操作方便性及菌絲染色成效上，仍以選用棉藍為最佳染劑。以此對培養基上的菌落染色可見兩大類菌絲，其一為不易染色而維持黃褐色的骨骼菌絲，另一為易被染色呈現藍色的一般菌絲。又隨著菌落成熟老化，骨骼菌絲數量會上升，而一般菌絲會形成「空洞化菌絲」。以此染劑對田間染病木材樣本之快速鏡檢及染色，發現樣本呈現白腐狀的部位皆可以以棉藍染色，觀察到「空洞化菌絲」，而樣本帶有菌索處的部位，則可以觀察到「骨骼菌絲」及「空洞化菌絲」。

關鍵字

褐根病；有害木層孔菌；褐根病檢測方法；選擇性培養基；顯微鏡檢

並列摘要

Brown root rot disease ( Brr. ) is a tree disease caused by Phellinus noxius in warm and humid areas, including Taiwan. It infects hundreds species of tree plants, often causing tree wood decay, death of living trees, and tree falling all around Taiwan island. Brown root rot disease usually spreads by the underground root-to-root contact, human dissemination of the diseased tissue or seedlings, or the infestation by diseased debris. The basidiocarp and basidiospores may also play roles for long distance dissemination although very rare. At present, the detection of Brr can be classified into the following five categories: Detection or diagnosed by symptoms or signs, detection by its specific DNA sequence, detection by its immunological antigen, detection by the emitted VOC (Volatile organic compounds), and detection by physiological or biochemical characteristics. The current nine practical detection methods include the symptoms and signs diagnosis or called VTA (Visual tree assessment) method, microscopic examination, polymerase chain reaction (PCR) method, LAMP( Loopmediated isothermal amplification )method, ELISA (Enzyme-linked immunosorbent assay), electronic nose method, detective dog method, selective medium method, and histological staining method. In this study the SWOT（ Strengths, weaknesses, opportunities, threats） of these nine methods are analyzed based on criteria such as scale of the detection target, required time, specificity, cost, easiness, and ability to detect the live mycelium, Results showed that only selective medium method, microscopic examination, and histological staining have the ability to detect the live mycelium, and are important for Brr detection after tree injection or soil disinfection. For detecting larger targets, the symptoms and signs diagnosis, electronic nose method, and detective dog method are appropriate. For quick Brr detection, the symptoms and signs diagnosis, microscopic examination, histological staining, and detective dog method are useful. For higher specificity, the PCR, LAMP, and ELISA, are the choices. For lower cost, the symptoms and signs diagnosis, and microscopic examination are suitable. For easiness in the field, the symptoms and signs diagnosis, microscopic examination, LAPM method, electronic nose method, and histological staining, can do the jobs. In this study the previously published selective medium (MA+4), for isolating Brr pathogen, was modified by doubling the benomyl concentration. This named PN3 medium can prevent the interference caused by Trichoderma and Penicillium spp. Then the basal nutrient components of PN3 was modified by replacing the malt extract (ME) with 1/2 ME + 1/2 PDA, named as PDM, for improving the dark patch development of Brr pathogen on PDM directly. Finally new inhibiting biocides such as fosetyl-aluminium, kasugamycin copper, thiophanate-methyl, and tetracycline with streptomycin, were added into PDM+4, making new formula as PDM+8. This PDM+8 selective medium shows higher selective power against the bacteria and other wood rot fungi, increasing its specificity and easiness for detecting the Brr pathogns. The new histological staining methods were also tested out in this study. The Brr mycelium on PN3 or PDM+8 for 2 to 3 days expressed the stunt head and identical branch width, that were specific characteristics of the pathogen. New stains or dyes such as methyl blue, crystal violet, Malachite green, Safranin O, were tested to stain the mycelium within the field tree samples. Results showed that the cotton blue for Brr pathogen performed the best for histological staining jobs. In the cultural medium the Brr pathogen showed two distinguishable hyphae. One is the skeleton hypha which is not stained by cotton blue. Another is the common hypha or arthrospore that can be stained by cotton blue. As the colony become mature or older, some common mycelium become porous or empty within the mycelium, named as porous mycelium, which is a specific characteristic for Brr pathogen. Using the cotton blue to stain the Brr in field tree samples, both skeleton hypha and porous mycelium can be observed under the microscope, making it an easy method for detecting the Brr in the field or in the laboratory.