一种大连细叶早熟禾内生真菌的形态学和系统发育学研究

Morphological and phylogenetic properties of an endophyte from Poa angustifolia grown in Dalian, Liaoning

【目的】鉴定分离自辽宁省大连市细叶早熟禾中的内生真菌。【方法】在辽宁省大连市的黄金山和白玉山采集细叶早熟禾(Poa awgws啡L.),通过显微镜观察植物是否含菌,统计样品中内生真菌的检出率;从含菌样品中分离内生真菌,纯化后在马铃薯葡萄糖琼脂(PDA)培养基上培养3周,测定分离菌株的形态学特征;运用IQS法提取分离菌株的总DNA,利用特异性引物妒增分离菌株的P-Tubulin(tubB)和Translation elongation factor 1-a(碎4)基因片段,利用DNAssist 2.2和ClustalX 1.81软件进行序列的多重比对和分析,运行PAUP 4 beta 10、MrModeltest 2.3选择合适的模型,用MrBayes 3.1.2构建最大似然树,分析分离菌株的系统发育学特征。【结果】共釆集植物样品262个,其中内生真菌的检出率达57.3%;根据分离地点和菌落特征筛选和纯化了10个分离菌株,选用其中6个分离菌株进行形态学特征的调查,发现分离菌株呈典型的Neotyphodium属形态学特征,进而和其他宿主为早熟禾属植物的内生真菌进行比较,发现分离菌株的特征与Epichlo谷liyangensis相近,但分生抱子较小[(5.3±0.3)|jmx(2.9±0.2)Mm],生长速度稍慢(每周8.7±1.4 mm);基于5个分离菌株tubB和tefA片段序列的系统发育学特征显示,它们的如65和对^序列分别为单拷贝,并与五.中的第二拷贝聚为一类。【结论】分离菌株在原始宿主植物上没有形成子座,分离菌株的形态学特征和系统发育学特征均与五./zyawge/ts/j?有明显差异。兼顾其它早熟禾属植物内生真菌的宿主植物的种类和地理分布特征,这些菌株有可能属于一个新的内生真菌类群。

[Objective] Ten endophytic strains obtained from Poa angustifolia grown in mountains of Huangjin and Baiyu in Liaoning Province were identified. [Methods] Asymptomatic P. angustifolia samples were collected. Plant culms from each sample were stained by Rose Bengal （1%） for 5 minutes. Hyphae were detected under microscope and the infection level was calculated. The fungal strains were isolated, purified and incubated to observe the morphological properties. Fungal genomic DNA samples were extracted by IQS DNA extraction method. Fragments of ^-tubulin （tubB） and translation elongation factor 1-a （tefA） genes were amplified by specific PCR primers for isolated strains. Sequences of tubB and tefA were aligned and analyzed by DNAssist 2.2 and ClustalX 1.81, the model was selected by PAUP 4 beta 10 and MrModeltest 2.3. The phylogenetic characteristic of these strains were analyzed through the maximum likelihood tree performed by MrBayes 3.1.2. [Results] Total of 262 Poa angustifolia samples were collected, 57.3% of the plants were infected while no stroma was discovered on the culms. Ten endophytic strains were isolated and purified from these plants samples. Six strains were used to observe the morphological properties. Each of them was identical with those of epichloid endophytes. Comparing with other endophytes from Poa spp. plants, morphological characteristics of the strain were similar to E. liyangensis except smaller conidia and slower growth rate. Five strains were confirmed to possess single alleles of tubB and tefA. Phylogenetic trees based on tubB and tefA fragments respectively revealed that these 5 strains clustered with the second alleles of E. liyangensis. [Conclusion] These results indicated significant differences between these strains and E. liyangensis. Based on the host species and geographic distribution of other endophytes from Poa spp. plants, they might be a new taxonomic group of epichloid endophyte.