两株铁皮石斛内生真菌对组培苗和原球茎生长的影响

在铁皮石斛内生真菌的研究中,获得了对组培苗的生长呈现不同影响的两株内生真菌DCN-1和DCN-3,为探究其对组培苗和原球茎生长的影响,首先对已分离纯化的菌株DCN-1和菌株DCN-3进行菌株形态和ITS1-5.8S-ITS2区段序列鉴定;采用孢子悬浮液注射和菌丝体附着伤口的方法对铁皮石斛组培苗和原球茎进行接种,观察组培苗的生长情况;参照柯赫氏法则对菌株进行重新分离,并结合切片观察结果验证侵染情况。结果显示:菌株DCN-1和菌株DCN-3分别与Myceliophthora verrucosa、G ibberella moniliformis的相似度最高,其中菌株DCN-1对组培阶段和移栽后的组培苗和原球茎的生长无明显影响,菌株DCN-3会导致组培阶段的植株苗枯萎甚至死亡而对移栽后的组培苗影响不大,但抑制原球茎的生长。徒手组织切片观察及二次分离结果分析显示:两株内生真菌均成功侵染植株体内,并确定相应植株的生长状况与该两株内生真菌的侵染相关。两株内生真菌在在营养较丰富的组织培养条件下对铁皮石斛组培苗和原球茎造成不同的影响,为探究其与铁皮石斛的相互作用和促进铁皮石斛植株生长及有效活性物质产生的复合型菌制剂提供了依据。

Characterization of endophytic fungi from Dendrobium officinale and study of their influence on growth of seedling and protocorm

Two endophytic fungi were isolated from medicinal plant Dendrobium officinale, namely DCN-1 and DCN-3 respectively. To explore the characterizations of two endophytic fungi, the strains which were firstly identified by the morphological and molecular means. Injecting inoculation of spore suspension or adhering mycelium on wounded surface were performed, and observing the growth of seedlings and protocorms. Secondary isolated from seedling after inoculation were done according to Koch's Postulation, and the invasiveness of fungi was verified. The results showed that DCN-1 and DCN-3 were highly similar to Myceliophthora verrucosa and Gibberella moniliformis respectively, according to ITS1-5.8S-ITS2 sequence comparison from GenBank. The transplanted seedlings artificially infected with DCN-1 and DCN-3 were found to grow normally, without obvious symptoms; the seedlings under tissue culture condition, that infected by DCN-3 were found to be obviously influenced, withered or died off, and the growth of protocorms were also notably inhibited, those infected with DCN-1 grew normally. The mycelium could be observed in stem and both fungi were re-isolated from the inoculated plants; it was proved that the symptom on seedling was caused by corresponding fungi. Our work indicated that the two endophytic fungi could give rise to different symptoms under tissue culture conditions of D. Officinale.