假鹰爪茎、叶组织内生真菌的分布和rDNA ITS序列系统发育分析

Distribution and Phylogenetic Analysis of Endophytic Fungi in Stems and Leaves of Desmos chinensis Based on Histochemistry and rDNA ITS Sequence

目的研究假鹰爪(Desmos chinensis Lour.)茎、叶组织中内生真菌的分布及其rDNA ITS序列系统发育。方法采用石蜡永久制片法观察鹰爪茎、叶组织显微结构,细胞化学法确定内生真菌的分布,平板分离法分离培养内生真菌,对分离菌株进行rDNA ITS区PCR扩增和测序,通过rDNA ITS序列进行内生真菌的分类鉴定及系统发育分析。结果假鹰爪茎中的内生真菌主要分布在韧皮部薄壁细胞,叶中主要分布主脉上方厚角细胞以内的薄壁细胞、栅栏组织、海绵组织。共分离14株内生真菌,分属于拟茎点霉属(Phomopsis)、刺盘孢属(Colletotrichum)和球座菌属(Guignardia),其中拟茎点霉(P.spp.)占总菌株数的57.1%,其次为胶孢刺盘孢(C.gloeosporioides)和芒果球座菌(G.mangiferae)。结论内生真菌在假鹰爪茎、叶组织中均有分布,无明显组织特异性;rDNA ITS序列系统发育较好的揭示了假鹰爪内生真菌的系统进化关系。

OBJECTIVE To study the distribution and phylogenesis of endophytic fungi in the stems and leaves of Desmos chinen- sis. METHODS Permanent paraffin-cut section, optical microscope photography, and histochemistry were used to observe the distri- bution of endophytic fungi in the stems and leaves of Desmos chinensis. Plate isolation method was used to separate and cultivate the en- dophytie fungi. The genomic DNA of endophytic fungi were extracted, then the rDNA ITS region was amplified using polymerase chain reaction （PCR）. The PCR products were digested by Hae III and Hha I restriction enzymes to determine the species and genotypes, and sequenced according to the digestion genotyping results. Nueleotide sequences of the rDNA ITS of endophytic fungi were used for the identification and phylogenetic analysis. RESULTS Endophytic fungi mainly existed in the parenchyma cells of the phloem in the stems, and in the parenehyma cells, palisade tissue or sponge tissue of the leaves. Fourteen strains of endophytic fungi were isolated from the stems and leaves. Four different genotypes and their sequences were obtained from RFLP analysis and sequencing. These strains were identified as Guignardia mangiferae, Colletotrichum gloeosporioides, and Phomopsis spp. The dominant group of those en- dophytic strains was Phomopsis, represented by 8 strains and accounting for 57. 1% of all. CONCLUSION The endophytic fungi distribute in both stems and leaves of Desmos chinensis without obvious tissue specificity. The result of the phylogenetic analyses with rDNA ITS sequences reveals the phylogenetic relationships of endophytic fungi in Desmos chinensis.