運用石蠟切片與掃描式電子顯微鏡技術探討褐根病菌在木本植物之侵染過程

Abstract

褐根病菌 (Phellinus noxius) 主要危害樹木根系及莖基部，使根系腐朽導致樹木萎凋及倒塌死亡。褐根病菌具有生長快速及寄主廣泛的特性，在許多熱帶及亞熱帶國家對行道樹、林木及果樹造成嚴重影響。過去 Nicole 等人 (1995) 利用掃描式及穿透式電子顯微鏡觀察褐根病菌在白樺樹木塊之纏據過程及產生之侵染構造，及 Burcham 等人 (2015) 透過接種帶 reaction zone 之桃花心木木塊，以切片及染色觀察褐根病菌面對植物防禦構造之反應，然而這些僅限於對褐根病菌分解木塊之觀察，至於褐根病菌如何侵染活體植物則仍未明瞭。為克服木本植物組織堅硬造成之顯微觀察困難，本研究透過乙烯二胺 (ethylenediamine) 浸泡以軟化組織、延長滲蠟時間，及使用黏著劑 VECTABOND®處理玻片等方式改良石蠟切片流程。為瞭解褐根病菌在不同樹種上之侵染過程，本研究分別在木本模式植物毛果楊 (Populus trichocarpa)、垂榕 (Ficus benjamina) 及枇杷 (Eriobotrya japonica) 幼苗進行莖部或枝條接種實驗，再透過石蠟切片及掃描式電子顯微鏡觀察，發現褐根病菌主要藉由植物表皮上之傷口及自然開口入侵，接著優先攻擊薄壁細胞導致組織空洞化，再以大量菌絲纏據於皮層部位；另外則會侵入導管進行垂直向之擴散，並藉由射線薄壁細胞進入髓部進一步拓展。為了瞭解侵染後期的現象，本研究自野外收集被自然感染之垂榕、樟樹 (Camphora officinarum) 及南洋杉 (Araucaria cunninghamii) 成樹的根部組織，發現在皮層、導管及髓部除了菌絲之外，也可以觀察到菌絲索的構造及可能形成過程，偶爾亦可見鹿角狀菌 絲。本研究發現褐根病菌雖然被歸屬為木材腐朽菌，但對活體薄壁細胞具高度病原性，其致病機制值得進一步探討。此外，前述優化之石蠟切片技術可應用於探討其他病原微生物侵染木本植物的過程，有助於木本植物組織病理學的發展。

The brown root rot fungus (Phellinus noxius) mainly infects the stem base and roots of trees, causing root rot and wilting of the whole plants. Due to its fast-growing nature and wide host range, P. noxius has caused a serious impact on street, forest, and fruit trees in many tropical and subtropical countries. Previously, Nicole et al. (1995) observed the colonization of P. noxius on birch wood blocks by scanning and transmission electron microscopy. Burcham et al. (2015) inoculated mahogany wood blocks containing the reaction zone with P. noxius and examined how P. noxius responded to plant defense structures through sectioning and staining. However, these are limited to observations of wood decomposition by P. noxius, and how P. noxius infects living plants remains unclear. To overcome the difficulty of microscopic observation caused by the hard tissues of woody plants, this study improved the process of paraffin sectioning by soaking the sample in ethylenediamine to soften the tissue, prolonging the paraffin infiltration time, and treating glass slides with the adhesive agent VECTABOND®. To uncover the infection process of P. noxius on different tree species, the stems or branches of the seedlings of Eriobotrya japonica, Ficus benjamina, and the woody model plant Populus trichocarpa were inoculated with P. noxius and then examined by paraffin sectioning and scanning electron microscopy. Upon infection of the seedlings, P. noxius mainly invaded the epidermis through the wounds and natural openings, followed by quick decomposition of parenchyma cells and the formation of a hollow, which was subsequently occupied by abundant hyphae. The hyphae also invaded the xylem vessels to spread vertically and expanded into the pith through ray parenchyma cells. To investigate the late stage of infection, naturally infected root tissues of Ficus benjamina, Camphora officinarum, and Araucaria cunninghamii trees were collected from the fields and analyzed. The results indicate that, in addition to the hyphae, mycelial cords and their formation were observed in the cortex, xylem vessels, and pith. Occasionally, trichocysts were found as well. This study demonstrates that P. noxius displayed high pathogenicity to living parenchyma cells although it is known as a wood decay fungus. The mechanism underlying fungal pathogenicity is worthy of further exploration. Moreover, this study provides an optimized paraffin sectioning technique for future histopathological research of woody plants infected by other microorganisms.