柑橘内生真菌的分离鉴定及其发酵产物对柑橘溃疡病菌的抑制活性

Isolation and identification of endophytic fungi from citrus cultivars and their inhibitory activity against Xanthomonas citri subsp. citri causing citrus canker

关键词:  柑橘内生真菌, 柑橘溃疡病菌, 乙酸乙酯提取物, 最低抑制浓度

Key words:  citrus endophytic fungi, Xanthomonas citri subsp. citri, ethyl acetate extract, minimum inhibitory concentration(MIC)

摘要:

本研究从柑橘抗病品种的健康植株不同组织中分离纯化和鉴定内生真菌,并测定其发酵产物对柑橘溃疡病菌的抑制活性,以明确柑橘抗病品种中内生真菌的组成及其产抗柑橘溃疡病菌活性代谢产物的潜力,为柑橘溃疡病抗菌剂的开发奠定基础。该研究通过组织培养法分离内生真菌,采用形态学和分子生物学方法对其进行鉴定; 基于前期的拮抗预试验结果,选取代表性菌株进行发酵培养,通过乙酸乙酯浸提、真空抽滤、旋转蒸发浓缩制备粗提物; 采用带毒平板涂布法测定不同菌株发酵产物乙酸乙酯提取物对柑橘溃疡病菌的抑制活性。结果表明:(1)共分离得到72株内生真菌,归为2门(Ascomycota、Basidiomycota)、14个属,其中优势属为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、链格孢属(Alternaria)和镰刀菌属(Fusarium)。(2)不同柑橘品种中内生真菌多样性指数为温州蜜柑(桂林)>沙糖桔(桂林)>沙糖桔(梧州)。(3)不同组织中内生真菌多样性变化因地理位置差异而有所不同,采自桂林的温州蜜柑和沙糖桔均为叶片中的内生真菌的多样性高于枝条,而采自梧州的沙糖桔为叶片中的多样性低于枝条,并且采自梧州的柑橘样品与采自桂林的柑橘样品中的内生真菌相似性低。(4)测定了30株内生真菌乙酸乙酯提取物对柑橘溃疡病菌的抑制活性,其中29株菌株表现出不同程度抑制活性。不同柑橘品种中的优势属的MIC介于0.312 5～10 mg·mL^-1之间,特有属的MIC介于0.156～5 mg·mL^-1,共有属镰刀菌属的MIC介于0.312 5～2.5 mg·mL^-1之间。研究结果表明柑橘抗病品种中内生真菌具有丰富多样性,并且其发酵提取物普遍对柑橘溃疡病菌具有抑制作用。特有属抑菌活性总体优于优势属,共有属镰刀菌属在不同柑橘抗病品种中均具有显著抑菌效果。

Abstract:

In this study, to provide a basis for the controlling of citrus canker disease, endophytic fungi from different citrus disease-resistant cultivars were isolated and identified by using a tissue culture method, and the method based on morphology combined with molecular biology, respectively. Representative endophytic fungi were selected based on the antagonism results in pre-experimental, to determine the antibacterial activity with their ethyl acetate extracts from the fermented culture against pathogen Xanthomonas citri subsp. citri, using a toxic medium coating method. The results were as follows:(1)A total of 72 endophytic fungi were obtained from different citrus cultivars and classified into 14 genera. Fungal isolates belong to phylum Ascomycota and Basidiomycota. The frequent genera were Colletotrichum, Guignardia, Alternaria and Fusarium.(2)The diversity indices of endophytic fungi from different citrus as follow: Citrus unshiu Marc.(Guilin)> C. reticulata Blanco cv. Shatangju(Guilin)> C. reticulata Blanco cv. Shatangju(Wuzhou).(3)The diversities indices of endophytic fungi were different, which were isolated from different tissues of citrus associated with different geographical location. The similarity of endophytic fungi in the citrus samples collected from Wuzhou and those from Guilin was low.(4)The antibacterial activity of ethyl acetate extracts from the fermented culture of 30 endophytic fungi were determined against pathogen Xanthomonas citri subsp. citri in vitro assay. Most of their extracts exhibited significant inhibition. Among them, the MIC values of dominant genera, specific genera and shared genus were 0.312 5-10 mg·mL^-1, 0.156-5 mg·mL^-1 and 0.312 5-2.5 mg·mL^-1, respectively. The results indicate that the endophytic fungi in citrus disease-resistant cultivars are rich in diversity, and most of their extracts exhibit inhibition against X. citri subsp. citri. The antibacterial activity of specific genera is generally better than the dominant genera, and all of the strains in the shared genus Fusarium displayed significant inhibition against X. citri subsp. citri.