Isolation Of Antagonistic Endophytes Against Fusariun Graminearum And Study On Antifungal Activity Of The Strains

Endophytes can produce rich biological active substances. Now they are regarded as important microbial resource to screen developing fungicide. In this acticle, endophytes were isolated from 3 plants, including Alopecurus japonicas, Poa pratensis and Cynodon dactylon, the antifungal activity of these endophytes to Fusarium graminearum were determined for screening valuable endophytes and developing biopesticides. The main results are as follows:Sixty-eight strains of endophytes were isolated from 3 kinds of wild gramineous weeds by the methods of the slices of sample and tissue homogenate, of which,31 strains were endophytic fungi,28 strains were endophytic bacteria and 9 strains were endophytic actinomyces. From the experiment of antifungal activity to Fusarium graminearum, we screened 6 active strains, was named KMN-1、KMN-11、KMN-7、KMN-12、CPC-5、 and CPC-3, respectively. The activity of the KMN-1 strain and KMN-11 strain in the experiment of inhibiting mycelia growth against F. graminearum were highest, the value was 71.00% and 85.14%.The antifungal activity of strain KMN-1 and KMN-11 was studied in vitro and vivo bioassay. Strain KMN-1 and KMN-11 has significant antifungal activity against F. graminearum in vitro antagonism test. By the assay of fermentation from strain KMN-1 and strain KMN-11 exhibited antifungal activity, the value of EC50 inhibiting mycelia growth against F. graminearum was 6572.97 mg-L"1 and 5218.34 mg·L-1, respectively. The fermentation filtrate of strain KMN-1 had exhibited control effect against F. graminearum, the protective efficacy and curative efficacy were 54.69% and 57.58% in wheat spikelet test; The protective efficacy and curative efficacy of strain KMN-11 fermentation filtrate were 78.13% and 57.49%. In field experiment, spraying the fermentation filtrate of strain KMN-1 and KMN-11 can also reduce the epidemic degree. The protective efficacy and curative efficacy of strain KMN-1 fermentation filtrate were 52.76% and 57.58%. The protective efficacy and curative efficacy of strain KMN-11 fermentation filtrate were 45.71% and 32.26%.The KMN-1 strain and KMN-11 strain are identified by the morphological characteristics and molecular identification.The results showed that the KMN-1 strain has a high sequence similarity to Alternaria sp. (100% similarity), the characteristics of bacterial colony and microscopic morphology were also similar. As such, we named the KMN-1 strain as Alternaria tenuissima (accession number:KP211537). The KMN-11 strain has a high sequence similarity to Fusarium sp. (99% similarity). Further to determine the EF-la sequence, the result showed that the KMN-11 was distantly related to Fusarium equiseti, and the cultural or morphological characteristics were different. As such, we preliminary named the KMN-11 strain as Fusarium incarnatum(accession number:KP211538).By using the labeling method of green fluorescent protein, colonization of the KMN-11 strain in wheat was studied. The results showed that the transformants which with fluorescent marker was successfully obtained, but strain KMN-1 has poor stability. At the same time, the effects in different conditions for the transformation effect were determined. The results showed that the best time for co-culture KMN-11 strain with Agrobacterium tumefaciens was 20 hours. A. tumefaciens-mediated the transformation of KMN-11 strain were required the presence of AS, and the optimal concentration was 200μg.mL-1. The transformants grew best in the culture dish for pH 7, in this condition, the colony diameter was largest, but excess acid or alkali was not conducive to the growth of transformants.In the test of colonization of the KMN-11 strain in wheat, strain KMN-11 can colonization in wheat for short-term, by using the method of soaking seed, rooting irrigation, spraying and injection. The KMN-11 strain can be detected in wheat after 21 d.Liquid-liquid extraction and thin layer chromatography were used to study the active compound of KMN-11 strain. The results showed that the active compound of KMN-11 strain being extracted from fermentation filtrate. Eight components (Fr.A1-A8) were obtained in the experiment of thin layer chromatography. Among them, components A1, A2, A3 and A5 were found to have the strong inhibiting activity against the spore germination of F. graminearum.4 samples were analysised by high resolution mass spectrometry, the results showed that 4 samples were not pure compound.