目的 對(duì)產(chǎn)自于云南的陽(yáng)春砂仁根內(nèi)生真菌進(jìn)行分離、純培養(yǎng)、解磷功能評(píng)價(jià)及分類學(xué)鑒定。方法 通過PDA和MEA培養(yǎng)基分離砂仁根內(nèi)生真菌，并純化培養(yǎng)；利用無機(jī)磷源Pikovaskaia’s（PVK）固體和液體培養(yǎng)基篩選具有解磷功能的內(nèi)生真菌，并通過生長(zhǎng)圈直徑、生物量、有效磷含量、pH值、磷酸酶活性來分析菌株解磷能力和解磷機(jī)制；通過核糖體18 S PCR擴(kuò)增，對(duì)具有解磷效果的菌株進(jìn)行分子鑒定。結(jié)果 研究發(fā)現(xiàn)，分離自陽(yáng)春砂仁根部的24株內(nèi)生真菌，有10株為深色有隔內(nèi)生真菌（dark septate endophytes，DSE）。通過難溶性PVK固體培養(yǎng)基篩選發(fā)現(xiàn)，共有8株菌可形成解磷生長(zhǎng)圈，其中JP-20和JP-23生長(zhǎng)直徑均大于9 cm，其次為JP-15，生長(zhǎng)圈直徑為6.06 cm；PVK液體培養(yǎng)基篩選結(jié)果顯示，菌株JP-23具有較強(qiáng)的解磷效果，生物量顯著高于可溶性磷源，液體基質(zhì)中有效磷含量顯著升高，pH明顯下降，酸性磷酸酶活性增加。經(jīng)分子鑒定，初步認(rèn)定菌株JP-23為枝孢屬Cladosporium sp.菌株（GenBank：MK629004）。結(jié)論 菌株JP-23可通過調(diào)節(jié)基質(zhì)pH值和分泌酸性磷酸酶來分解、吸收難溶性磷源。為研究植物-微生物磷吸收作用機(jī)制及砂仁生態(tài)種植提供數(shù)據(jù)支持和理論依據(jù)。

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

國(guó)家自然科學(xué)基金資助項(xiàng)目（81560612）；國(guó)家重點(diǎn)研發(fā)計(jì)劃“基于群落立體種植的中藥材生態(tài)農(nóng)業(yè)研究與應(yīng)用”（2017YFC1700704）；南藥協(xié)同創(chuàng)新研究中心項(xiàng)目（30270100500）；中央本級(jí)重大增減支項(xiàng)目“名貴中藥資源可持續(xù)利用能力建設(shè)項(xiàng)目”（2060302）；云南省科學(xué)技術(shù)廳—云南中醫(yī)學(xué)院應(yīng)用基礎(chǔ)研究聯(lián)合專項(xiàng)[2017FF116（-019），2017FF117（-014）]；云南省應(yīng)用基礎(chǔ)研究計(jì)劃面上項(xiàng)目（2019FB122）