海南白蚁共附生放线菌的分离鉴定及其抗热带作物病原真菌活性研究

Isolation and Identification of Symbiotic Actinomycetes from Termites in Hainan and Their Activity Against Tropical Plants Pathogenic Fungi

白蚁(Isoptera)的筑巢和觅食活动主要在土壤及腐烂的木材上进行,所以白蚁极易受到来自环境病原微生物的侵害。因此,白蚁需要依赖共附生微生物菌群形成防御体系,放线菌是其中一个重要的类群,且放线菌能产生具有抗菌活性的次级代谢产物。但是白蚁共附生放线菌能否对热带作物的病原真菌有抑制活性有待研究。为了挖掘在热带农业绿色健康发展中具有应用潜力的海南特色农用放线菌资源,以海南白蚁共生放线菌为研究对象,利用平板分离法从采集的健康白蚁体内分离纯化放线菌,利用平板对峙法筛选生防菌株;通过分子生物学确定其分类地位;平板对峙法研究拮抗菌株的抗菌稳定性、持效性和广谱性;滤纸片法研究抑菌物质的热稳定性。结果从海南白蚁体内分离得到1株拮抗活性较好的放线菌菌株W7,系统发育树分析结果表明该放线菌与链霉菌属(Streptomyces)亲缘关系接近。菌株W7的抗菌活性持久性强,平板接种30 d后仍对植物病原真菌具有抑菌活性,抗菌稳定性好;菌株W7抑菌谱广,对16株植物病原真菌有不同程度的抑菌活性,其中对芒果胶孢霉(Colletotrichum gloeosporioides Penz.)、香蕉炭疽霉(Colletotrichum musae)、辣椒棒孢霉(Corynespora cassiicola)、豇豆瓜果腐霉[Pythium aphanidermatum(Edson)Fitzp]抑制作用较强,对芦笋拟茎点霉(Phomopsis asparagi)、荔枝疫霉(Peronophythora litchi)抑制作用较弱;菌株W7发酵粗提物有较好的热稳定性,但是抑菌率会随着水浴温度的增高而降低。本文首次对海南白蚁共附生放线菌进行了分离鉴定,并从中筛选得到1株具有抑制多种热带作物病原真菌活性的生防菌株,为高效生防菌剂的研发提供新资源。

The nesting and foraging activities of termites are mainly carried out in soil and rotting wood,where many pathogenic microorganisms live.However,termites are extremely vulnerable to these environmental pathogenic microorganisms.Therefore,termites need to rely on their mutualistic symbiotic microflora to form a defense system.Species of actinomycetes are one of the important groups in termite’s symbiotic microflora,which can produce diverse structures of secondary metabolites with antibacterial and antifungal activity.Whether the actinomycetes in termites can inhibit the pathogenic fungi of tropical crops remains to be studied.In order to explore the characteristic actinomycetes with potential application in the healthy development of tropical agriculture in Hainan,the actinomycetes were isolated and purified by the plate separation method from the healthy termites collected in the tropical plants garden of Chinese Academy of Tropical Agricultural Sciences in Haikou.Biocontrol strains were screened against 16 species of plant pathogenic fungi including Colletotrichum gloeosporioides Penz.,Colletotrichum musae,Colletotrichum australianum,Phyllosticta capitalensis,Neopestalotiopsis clavispora,Gilbertella persicaria,Lasiodiplodia theobromae,Phomopsis caricae-papayae Fetrak&Cif.,Phomopsis asparagi,Thielaviopsis paradoxa,Corynespora cassiicola,Corynespora sp.,Pythium aphanidermatum(Edson)Fitzp and Peronophythora litchi by the growth plate-confrontation method.Identification of bioactive strain was based on the phylogenetic tree analysis constructed with 16S rRNA gene sequence.Growth plate-confrontation was directly used to investigate the antimicrobial stability,persistence and bacteriostatic spectrum for the strain and the filtering paper method was used to study the heat stability of antimicrobial substances for the crude extract of the bioactive strain.As a result,one strain of actinomycete(W7)with the best biocontrol activity was isolated from termites.Phylogenetic tree analysis showed that the strain W7 was closely related to Streptomyces genus.Most importantly,the bioactivity tests showed that the strain W7 had strong antifungal persistence and antifungal stability,even after cultivating for 30 d,it still had better antifungal against the tested plant pathogenic fungi.The strain W7 had inhibitory activity against 16 pathogenic fungi.It had strong inhibitory activity against plant pathogenic fungi C.musae,C.cassiicola,P.aphanidermatum(Edson)Fitzp,with strong antibacterial persistence and antibacterial stability.The strain also displayed weak antibacterial activity on plant pathogenic fungi P.asparagi,P.litchi.The fermentation extract showed good heat stability towards the mango pathogenic fungi C.gloeosporioides Penz.,but the inhibition rate would decrease with the increase of water bath temperature.This is the first paper to investigate the termite-associated actinomycetes in Hainan.Bioactivity evaluation revealed termite-associated actinomycetes could be good resources for discovering biocontrol agent to control crop disease.