Identification of an Endophytic Fungus and Its Morphological Effect on Dendrobium officinale
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摘要:
目的 对连城冠豸山上生长较好的野生铁皮石斛进行内生真菌的分离，以获得具有促生效果的菌株，为进一步研究瘤菌根菌属与铁皮石斛共生关系奠定基础。 方法 将连城冠豸山野生铁皮石斛根进行消毒后在PDA培养基上，对长出的内生真菌分离纯化，将一株纯化菌株标记为T11，并通过形态特征与5.8SrDNA-ITS分子生物学方法进行鉴定，并将T11菌株接入液体培养基震荡培养7 d，成菌液后浇灌无菌石斛盆栽苗进行共培养6个月，观察石斛株型的变化。 结果 PDA平板上培养的T11菌株的菌落乳白色，形成同心环，菌丝细胞为双核，有隔，含有椭圆形念珠状细胞，即从形态特征初步鉴定为瘤菌根菌属Epulorhiza sp.，为无性态的胶膜菌Tulasnella calospora。以提取T11菌株DNA为模板进行5.8SrDNA-ITS序列扩增，扩增产物片段长度为630 bp，通过序列比对和系统进化树分析，T11与大花蕙兰植物菌根真菌胶膜菌聚类在同一分支，亲缘关系最为相近，其同源性达到99%。接菌后的石斛苗长势较好，其株高、叶片数和叶宽都显著增加。 结论 从形态特征和分子生物学鉴定得出T11菌株为兰科菌根真菌担子菌的瘤菌根菌属，瘤菌根菌能促进铁皮石斛苗的生长。 -
关键词:
- 铁皮石斛 /
- 内生真菌 /
- 5.8SrDNA-ITS
Abstract:Objective An endophytic fungus from its symbiotic Dendrobium officinale plant was identified and studied for its effect on the morphology of the host. Method The fungus was isolated from the disinfected roots of a wild D. officinale plant grown on top of Mt. Guanzhai in Liancheng county. It was further purified on a PDA medium and labeled as T11 to be identified by morphological and 5.8 SrDNA-ITS molecular biological methods. After a 7 d-culture in a shaking flask, the inoculated liquid was added to the aseptically potted seedlings of D. officinale. Growth of the seedlings was observed for 6 months. Result The milky white T11 colonies on the PDA plate had concentric rings on them. The T11 mycelial cells were binucleate, septate, and elliptical in shape. It was preliminarily identified as an Epulorhiza sp., an asexual plastic film fungus of Tulasnella calospora. Subsequently, the 5.8SrDNA-ITS sequence amplified from the DNA extracted from T11 as template showed the PCR fragment to be 630bp in length. The sequence alignment and phylogenetic tree clustered T11 in the same branch as the T. calospora of Cymbidium, with a 99% genetic homology. With the T11 inoculation, the D. officinale seedlings grew well with significantly increased plant height, leaf count, and leaf width. Conclusion The morphological and molecular biological characteristics of T11 indicated it to be an Epulorhiza sp., the Basidiomycetes of orchidaceae mycorrhizal fungi, which is known to enhance the growth of D. officinale. -
Key words:
- Dendrobium officinale /
- endophytic fungus /
- 5.8 SrDNA-ITS
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图 1 内生真菌T11的形态
注：A为内生真菌的菌落形态; B为菌丝形态。
Figure 1. Morphology of endophytic fungus T11
Note:A:The Colony morphology of endophytic fungi; B:The myceliummorphology.
图 2 PCR扩增产物电泳图片
注：M为DNA Marker；1、2为PCR扩增产物。
Figure 2. Electrophoresis of PCR amplification products
Note:M:DNA Marker; 1, 2:PCR amplification product.
图 3 T11菌株5.8 srDNA-ITS区的核心片段序列
Figure 3. Nucleotide sequence of 5.8srDNA-ITS cord of T11
表 1 T11对铁皮石斛的株高、叶片数和叶宽的影响
Table 1. Effect of T11 on plant height, leaf count and leaf width of D. officinale seedlings
项目Item 3个月 6个月 CK T11 CK T11 株高Stem length/cm 8.34Bb 11.56Aa 11.02 Bb 17.25 Aa 叶片数Leaf number/片 7.83Bb 10.42Aa 10.00 Bb 16.08 Aa 叶宽Leaf width/cm 0.77 Aa 0.82 Aa 0.86 Ab 1.02 Aa 注：同列数据后不同小写字母表示差异显著(P < 0.05)，大写字母表示极显著差异(P < 0.01)。
Note:Date followed different small letters within column indicate significant difference at 0.05 level，different capital letters within column indicate an extremely distinct difference at 0.01 level.
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