重要虫生真菌莱氏野村菌芽生孢子的形态发生

Morphogenesis of blastospores of Nomuraea rileyi, an important entomogenous fungus

经对比,萨氏麦芽糖-酵母浸粉培养液(SMY)较适合制备莱氏野村菌Nomuraea rileyi芽生孢子。以菌株Nr09接种该培养基,在130r/min、25℃全光照下震荡培养,观察芽生孢子在不同时期发育的形态变化。结果表明,分生孢子萌发产生芽管;24h萌发率为42%,36h时达84%。芽管迅速伸长形成菌丝。在48h前芽生孢子通过菌丝顶端及分枝末端缢缩的方式形成,后期则主要通过芽生孢子继续出芽的方式大量形成,78h芽生孢子数量达到最大值。芽生孢子的形成方式可为一端、两端或多端芽殖。芽生孢子的形成过程可分为5个阶段:(I)分生孢子膨大期;(II)芽管萌发期;(III)芽管延长期;(IV)芽生孢子形成初期;(V)芽生孢子指数生长期。30h后培养液中有少量草酸钙结晶出现并逐渐增多。到84h时有31%的芽生孢子细胞内液泡聚集增大,表明芽生孢子已开始进入衰老阶段。使用指数生长期制备的Nr09芽生孢子进行几丁质酶基因转化,转化效率达79个转化子/μgDNA。

Sabouraud-Maltose-Yeast extract liquid medium （SMY） was optimal for preparation of the blastospores of the entomopathogenic fungus Nomuraea rileyi, and the medium was used for morphogenesis study of the Nr09 strain of the fungus by incubation at 130r/min, 25℃, and illumination for 24h. The result showed that the conidia of N. rileyi produced germ tubes and developed into hyphae, with germination ratio at 42% in 24h after inoculation and 84% in 36h. Blastospores formed by the mode of constriction of hyphal terminals within 48h, and largely multiplied by successive budding of the spores, reaching the maximum in 78h.Budding patterns could be unipolar, bipolar, or multipolar. In summary, five developmental stages could be defined： （I） conidium swelling, （II） emergence of germ tubes, （III） elongation of germ tubes, （IV） initiation of blastospores, and （V） exponential growth of blastospores. Some crystals of calcium oxalate were produced by the fungus after incubation in 30h. By 84h, big vacuoles developed in the cells of 31% blastospores, suggesting the beginning of senescence. The blastospores of Nr09 prepared at exponential phase were used for transformation of an exogenous chitinase gene, Bbchit1, with a transformation frequency up to 79 transformants per μg DNA.