亞腐霉菌分離與培養技術之建立

Methods on the isolation and cultivation of Pythiogeton species

黃晉興(Jin-Hsing Huang) ； 陳啟予(Chi-Yu Chen)

《Fungal Science》 33卷 (2018/12) Pp. 41-53

摘要

本實驗建立能有效自環境中分離亞腐黴菌之半選擇性培養基，以馬鈴薯葡萄糖瓊脂培養基為基礎，分別添加鏈黴素、撲克拉與免賴得800、1與1 ppm，可減少細菌與真菌的干擾；並藉由亞腐霉菌在此培養基的菌絲及特有菌落形態，可與其它卵菌做出區隔，能在被其他真菌及卵菌生長覆蓋前，順利將標的亞腐霉菌移植純化出來。亞腐黴菌在此培養基菌落呈稀疏狀、菌絲內的原生質無明顯顆粒與空隙，在光學顯微鏡下呈現油亮狀。利用此培養基進行亞腐霉菌的分離，發現有病徵之茭白組織、及浸泡於水中的茭白或水稻之莖葉殘體容易分離得到亞腐霉菌。而從台灣各種作物之水田可分離得不同種之亞腐霉菌，於茭白筍田可分離Pythiogeton abundans、P. microzoosporum、P. oblongilobum、P. puliense、P. ramosum 及P. zizaniae；於水稻田可分離得到P. abundans 及P. ramosum；P.microzoosporum、P. oblongilobum、P. puliense 及P. zizaniae；於水甕菜田可分離得到P. uniforme；於菱角田可分離得到P. paucisporum 及P. proliferatum。上述9 種亞腐霉菌的菌絲皆在裸麥瓊脂生長最佳，除了P. zizaniae 需要在裸麥瓊脂加茭白筍萃取液與組織塊之外，其他8種亞腐霉菌可在裸麥瓊脂繼代培養，而此9種亞腐霉菌在裸麥瓊脂最適生長溫度為28或32 ˚C。無論在瓊脂或液體培養基，除了P. ramosum 之外，其他的供試菌皆不產生氣生菌絲，且菌絲多生長於培養基底部。本研究的結果顯示，亞腐霉菌普遍存在於台灣的水田裏。

關鍵字

亞腐霉菌 ； 半選擇性培養基

並列摘要

A semi-selective medium for isolating Pythiogeton species was established by using PDA as a basal medium supplemented with 800 ppm streptomycin, 1 ppm prochlorate, and 1 ppm benomyl. This medium is designated as modified potato dextrose agar (MPDA) and could efficiently suppress most undesirable microorganisms during the process of isolation. For Pythiogeton growing on MPDA colonies are sparse, and hyphae have no conspicuous granular particles or large vacuoles and appear glassy and refractive under microscope. This type of hyphae is distinct and can be easily recognized on MPDA. The right hyphae of Pythiogeton can be picked up and transferred before other fungi or oomycetes overgrow, which makes the isolation of Pythiogeton efficient. In the isolation test using MPDA, Pythiogeton species can be easily obtained from necrotic tissues of living water bamboo and plant debris of water bamboo and rice. Different Pythiogeton species were isolated from Taiwan paddy fields. Pythiogeton abundans and P. ramosum were isolated from water bamboo and rice; P. microzoosporum, P. oblongilobum, P. puliense and P. zizaniae were isolated from water bamboo; P. paucisporum and P. proliferatum were isolated from water caltrop; P. uniforme was isolated from water convolvulus. When these Pythiogeton species were cultured on agar media, rye sucrose agar (RSA) was the optimal medium for mycelial growth of these Pythiogeton species, except for P. zizaniae, which was optimal on RSA supplemented with both extract and tissues from water bamboo. The optimal temperatures for the mycelial growth of these 9 Pythiogeton species on RSA were 28 ˚C or 32 ˚C. Apart from P. ramosum, Pythiogeton species produced no aerial mycelia on solid and liquid media, and mycelia usually were imbedded within the media. This study revealed that Pythiogeton species are actually common in paddy fields, and could be cultivated once proper approaches are applied.

並列關鍵字

Pythiogeton ； semi-selective medium