杨叶肖槿内生真菌多样性和抑菌活性研究

Diversity,antifungal and antibacterial activity of endophytic fungi from Thespesia populnea

【目的】对半红树药用植物杨叶肖槿(Thespesia populnea)内生真菌的多样性及其抑菌活性进行研究,为开发生态友好型抗菌剂提供参考依据。【方法】采用形态学和分子生物学相结合的方法,对广西北部湾沿海杨叶肖槿茎、叶和树皮组织中的内生真菌进行分离鉴定。选用从柿树、核桃、荔枝中分离的炭疽菌(Colletotrichum)、香蕉枯萎病菌尖孢镰刀菌古巴专化型FOC1(Fusarium oxysporum f.sp.cubense 1)和FOC4(Fusarium oxysporum f.sp.cubense 4)、黄瓜瓜链格孢菌(Alternaria cucumerina)、白骨壤内生真菌链格孢菌(Alternaria alternata)和罗非鱼海豚链球菌(Streptococcus iniae)为指示菌,通过平板对峙法和滤纸片法对杨叶肖槿内生真菌提取物进行抗真菌和抗细菌活性筛选。【结果】从杨叶肖槿中共分离纯化出60株内真生菌,分属于5个属25个种。其中,炭疽菌属(Colletotrichum)和间座壳属(Diaporthe)为优势菌属,分离率为48.33%和26.67%;其余菌属分别是葡萄座腔霉属(Botryospha⁃eria)、亚隔孢壳属(Didymella)和小隐孢壳属(Cryptosporella),对应的分离率为15.00%、6.70%和3.33%。从杨叶肖槿树皮组织分离到的菌株最多,共分离到4个属23株菌,占总分离量的38.33%;其次为叶片组织(31.67%)和茎组织(30.00%)。发现12株内生真菌与NCBI数据库已公布ITS rDNA序列相似性低于95.00%,为潜在新种。抑菌结果表明,炭疽菌J1⁃20对8种指示菌均有较强的抑菌活性,对柿树炭疽菌、核桃炭疽菌、荔枝炭疽菌、香蕉枯萎病菌FOC1和FOC4、黄瓜瓜链格孢菌、白骨壤链格孢菌的抑菌率分别为39.39%、25.00%、19.10%、32.43%、28.20%、89.00%和74.19%,对罗非鱼海豚链球菌的抑菌圈为21.00 mm。【结论】杨叶肖槿内生真菌多样性丰富,分布较均匀。其中J1⁃20菌株对8种指示菌具有广谱抑菌性,有较好的开发潜力。

【Objective】To provide a reference for finding eco⁃friendly antibacterial agents,the diversity of endophytic fungi in the medicinal plant Thespesia populnea in mangroves and their antifungal and antibacterial activities were studied.【Method】Endophytic fungi were isolated and purified from the stem,leaf and bark tissues of the medicinal plant T.populnea in Guangxi Beibu Gulf mangroves,and identified by a combination of morphology and molecular biology.Colletotrichum from Diospyros kaki,Juglans regia,Litchi chinensis,and Fusarium oxysporum f.sp.cubense 1(FOC1),Fusarium oxysporum f.sp.cubense 4(FOC4),and Alternaria cucumerina from Cucumis sativus,as well as en⁃dophytic fungus Alternaria alternata from Avicennia marina,and Streptococcus iniae from Oreochromis niloticus,were selected as indicator organisms.The antifungal and antibacterial activities of endophytic fungal extracts were screened by plate confrontation method and filter paper method.【Result】A total of 60 strains of endophytic fungi were isolated and purified from T.populnea,belonging to 5 genera and 25 species.Among them,the genera Colletotrichum and Diaporthe were dominant,with the isolation rates of 48.33%and 26.67%,respectively.The rest were Botryosphaeria,Didymella and Cryptosporella,with isolation rates of 15.00%,6.70%and 3.33%respectively.The most strains were isolated from the bark tissue of T.populnea,with a total of 4 genera and 23 strains,accounting for 38.33%of the total isolation a⁃mount,followed by leaf tissue(31.67%)and stem tissue(30.00%).In addition,12 endophytic fungi were found to have ITS rDNA sequence similarity of less than 95.00%with NCBI database,indicating that they might be potential new species.The results of antifungal and antibacterial activities indicated that J1⁃20 had strong antifungal and antibacterial activities against the 8 selected indicator organisms.The antifungal rates against Colletotrichum of D.kaki,Colletotrichum of J.regia,Colletotrichum of L.chinensis,FOC1 and FOC4,A.cucumerina from C.sativus,A.alternata from A.marina,were 39.39%,25.00%,19.10%,32.43%,28.20%,89.00%and 74.19%,respectively.Meanwhile,the inhibitory zone of J1⁃20 against S.iniae was 21.00 mm.【Conclusion】The endophytic fungi in T.populnea have rich diversity and relatively uniform distribution.Among them,J1⁃20 has broad⁃spectrum antifungal and antibacterial activities against 8 indicator organisms and has good development potentials.