小黄花茶内生真菌的多样性分析及抑菌活性初筛

Diversity analysis of endophytic fungi and preliminary screening of antibacterial activity in Camellia luteoflora

关键词:  小黄花茶, 内生真菌, 分离鉴定, 多样性, 抑菌活性

Key words:  Camellia luteoflora, endophytic fungi, separation and identification, diversity, antibacterial activity

摘要:

为探究小黄花茶内生真菌种类和种群分布规律以及对植物病原真菌的抑制作用,该研究采用组织分离法对小黄花茶内生真菌进行分离纯化,基于形态学和分子生物学进行鉴定并结合统计学分析评价其多样性,再通过平板对峙法筛选出具有抑菌活性的菌株。结果表明:(1)从小黄花茶324份组织块中分离得到内生真菌261株,隶属1门5纲9目22属,其中优势属包括炭疽菌属(Colletotrichum)、间座壳属(Diaporthe)、拟盘多毛孢属(Pestalotiopsis),分离频率分别为21.84%、16.86%、10.34%。(2)研究发现小黄花茶内生真菌在不同季节分布不同,冬季分离出的菌株数量最多,为72株(占27.59%,隶属16个属),春季62株(隶属13个属),夏季59株(隶属15个属),秋季68株(隶属13个属),冬季的香农-维纳指数(H')、辛普森指数(D)、Pielou's均匀度指数(E)和Margalef's丰富度指数(M)最高,春季与冬季内生真菌种类相似性较高,夏季与秋季内生真菌种类相似性较高。(3)小黄花茶内生真菌不同部位分布不同,茎中内生真菌的分布最多,有102株(占39.08%,隶属15个属),根61株(隶属10个属),叶98株(隶属15个属); 茎的H'、D、E、M最高,叶部与茎部内生真菌种类最为相似。(4)平板对峙结果显示,在35株供试内生真菌中,有26株内生真菌至少对1种植物病原真菌有抑制作用,占74.29%,其中CJ-Ⅱ-2、XY-V-3、QY-Ⅱ-4、QJ-Ⅲ-2、DJ-I-2对8种植物病原真菌均有不同程度的抑制作用,XY-V-3对8种植物病原真菌的抑制效果最佳,抑菌率均高于50%,XY-V-3和QJ-Ⅲ-2对2株小黄花茶病原真菌的抑菌率高于50%,具备防治小黄花茶自身病害的潜力。综上所述,小黄花茶内生真菌多样性丰富,部分菌株表现出较好的抑制植物病原真菌的作用,为生物防治产品的研发和小黄花茶病害的防治奠定了基础。

Abstract:

In order to explore the species and population distribution of endophytic fungi in Camellia luteoflora, as well as their inhibitory effects on plant pathogenic fungi, this study used tissue separation method to isolate and purify endophytic fungi in C. luteoflora. These fungi were identified based on morphology and molecular biology, and their diversity was evaluated through statistical analysis. The strains with antibacterial activity were screened out by the plate confrontation method. The results were as follows:(1)A total of 261 strains of endophytic fungi were isolated from 324 C. luteoflora tissue samples, belonging to 1 phylum, 5 classes, 9 orders, and 22 genera. The dominant genera were Colletotrichum, Diaporthe and Pestalotiopsis with isolation frequencies of 21.84%, 16.86% and 10.34%, respectively.(2)The distribution of endophytic fungi in C. luteoflora varied in different seasons. The highest number of strains was isolated in winter(72 strains, accounting for 27.59%, belonging to 16 genera), 62 strains were isolated in spring(belonging to 13 genera), 59 strains were isolated in summer(belonging to 15 genera), and 68 strains were isolated in autumn(belonging to 13 genera). The Shannon-Wiener index(H'), Simpson index(D), Pielou's evenness index(E), and Margalef's richness index(M)were the highest in winter. The similarity of endophytic fungal species between spring and winter was higher, and the similarity between summer and autumn was higher.(3)The distribution of endophytic fungi also varied in different parts of C. luteoflora. The stems had the most abundant endophytic fungi, with 102 strains accounting for 39.08%(belonging to 15 genera), 61 strains were isolated from the roots(belonging to 10 genera), and 98 strains were isolated from the leaves(belonging to 15 genera). The H', D, E, and M were highest in the stems, and the endophytic fungal species in the leaves were most similar to those in the stems.(4)The plate confrontation results showed that among the 35 tested endophytic fungi, 26 endophytic fungi had inhibitory effects on at least one plant pathogenic fungi, accounting for 74.29%, among which CJ-Ⅱ-2, XY-V-3, QY-Ⅱ-4, QJ-Ⅲ-2 and DJ-I-2 had inhibitory effects on eight plant pathogen fungi to varying degrees. XY-V-3 had the best inhibitory effect on eight plant pathogenic fungi, and the inhibitory rate was higher than 50%. XY-V-3 and QJ-Ⅲ-2 have higher inhibitory rate than 50% on two strains of pathogenic fungi in C. luteoflora, which had the potential to control disease of C. luteoflora. To sum up, the diversity of endophytic fungi in C. luteoflora is rich, and some of the strains have higher effect of inhibiting plant pathogenic fungi, which lay a foundation for the research and development of biological control products and the disease control of C. luteoflora.