目的 对产自于云南的阳春砂仁根内生真菌进行分离、纯培养、解磷功能评价及分类学鉴定。方法 通过PDA和MEA培养基分离砂仁根内生真菌，并纯化培养；利用无机磷源Pikovaskaia’s（PVK）固体和液体培养基筛选具有解磷功能的内生真菌，并通过生长圈直径、生物量、有效磷含量、pH值、磷酸酶活性来分析菌株解磷能力和解磷机制；通过核糖体18 S PCR扩增，对具有解磷效果的菌株进行分子鉴定。结果 研究发现，分离自阳春砂仁根部的24株内生真菌，有10株为深色有隔内生真菌（dark septate endophytes，DSE）。通过难溶性PVK固体培养基筛选发现，共有8株菌可形成解磷生长圈，其中JP-20和JP-23生长直径均大于9 cm，其次为JP-15，生长圈直径为6.06 cm；PVK液体培养基筛选结果显示，菌株JP-23具有较强的解磷效果，生物量显著高于可溶性磷源，液体基质中有效磷含量显著升高，pH明显下降，酸性磷酸酶活性增加。经分子鉴定，初步认定菌株JP-23为枝孢属Cladosporium sp.菌株（GenBank：MK629004）。结论 菌株JP-23可通过调节基质pH值和分泌酸性磷酸酶来分解、吸收难溶性磷源。为研究植物-微生物磷吸收作用机制及砂仁生态种植提供数据支持和理论依据。

Objective To study the characteristics of endophytic fungi separated from the roots of Amomum villosum grown in Jinping County of Yunnan Province, including the culture, evaluation of phosphorus-solubilizing ability and taxonomic identification of target strains. Methods Endophytic fungi in the roots of A. villosum were separated by culturing in the mediums of PDA and MEA, and purely cultured in PDA. The endophytic fungi with phosphorus-solubilizing ability were screened by solid and liquid mediums of Pikovaskaia's (PVK) prepared with inorganic phosphorus source. Then, the phosphorus-solubilizing capacity and reasonable mechanism were analyzed by growth circle, biomass, effective phosphorus content, pH value, and phosphatase activity. Moreover, molecular identification of target strains with the capacity of phosphorus-solubilizing would be carried out by ribosome 18 S PCR amplification. Results The results showed that 24 endophytic fungi were separated from the roots of A. villosum in total, 10 of which were dark septate endophytes (DSE). Eight strains could grow on PVK solid medium and produce phosporus-dissolved growth circle. The growth circle diameters of JP-20 and JP-23 were larger than others, and more than 9 cm, followed by JP-15 with the growth circle of 6.06 cm. Furthermore, it was shown that JP-23 had a strong ability of phosphate-solubilizing due to presenting a high biomass in the PVK liquid medium instead of the medium prepared by soluble phosphorus source. The content of effective phosphorus of JP-23 in PVK liquid medium was significantly increased with an obviously decreasing pH and a sharply rising of acid phosphatase (ACP) activity. Moreover, the strain JP-23 was preliminarily identified as Cladosporium sp. (GenBank:MK629004) by molecular identification. Conclusion An assumption was concluded that strain JP-23 could decompose and use insoluble phosphorus sources by adjusting the pH value and secreting ACP in medium. Our findings would provide data support and theoretical basis for studying the phosphate absorption mechanism of plant-microbial symbiosis system and the ecological plantation of A. villosum.

R282.12

国家自然科学基金资助项目（81560612）；国家重点研发计划“基于群落立体种植的中药材生态农业研究与应用”（2017YFC1700704）；南药协同创新研究中心项目（30270100500）；中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”（2060302）；云南省科学技术厅—云南中医学院应用基础研究联合专项[2017FF116（-019），2017FF117（-014）]；云南省应用基础研究计划面上项目（2019FB122）