황토로부터 분리한 Paenibacillus 속 세균이 생산하는 지질펩타이드 fengycin의 항진균 제재로의 응용

Abstract

In the present study, potential antifungal activity possessing bacterial strains were isolated from yellow loess soil samples collected from sweet potato cultivating agricultural land, Jeollanam Haenam, Korea. The enrichment culture was performed in Tryptic Soy Agar (TSA) medium. The serially diluted yellow loess soil sample (100 µl) were withdrawn and sprea on the TSA plated and incubated at 30℃ for 24 h. After incubation, F. oxysporum cultured containing yellow loess soil sample spread on the TSA plates and at 30〫℃ for 5day. Totally 14 potential bacterial strains were selected based on the clear zone around the smear. The antifungal activity of the 14 isolated strains were tested against different pathogenic fungal species such as vascular tissue wild causing Fusarium oxysporum, grey mould causing Botrytis cinerea, white mould or watery soft rot and cottony rot causing Sclerotinia sclerotiorum, late blight or water mold causing Phytophthora capsici and Florist’s cyclamen anthracnose causing Colletotrichum gloeosporioides. From the antifungal activity tests against 5 pathogenic fungal species, only one bacterial strain showed higher antifungal activity against all fungal species. This bacterial isolate was selected for further experiments. The potential bacterial strain was further identified by 16S rRNA amplification and nucleotide sequencing. According to the results obtained from the preliminary tests, the strain was tentatively named as Paenibacillus sp. On the basis of 16S rRNA sequence studies, the isolate was found to having 99.52% sequence similarity with Paenibacillus kribbensis AM49. Finally the isolate was named as P. kribbensis CU01. The initial antifungal activity was confirmed by paper disk method using 2 days grown P. kribbensis CU01 in TSB at 30℃. After 2 days incubation, the cells used as antifungal agent against aforementioned pathogenic fungal species. In addition, the competition culture was performed as 1 day grown S. sclerotiorum with P. kribbensis CU01 and pure culture was perfomed as 2 day grown, then incubated under controlled conditions. After incubation, the supernatant was collected and antifungal substance was extracted using chloroform as organic solvent and the extracts were labeled as #1, #2 and #3. All three extracts were used for antifungal activity tests against 5 pathogenic fungal species. After pure culture extracts didn't show antifungal activity. But competition cultured 3 extracts, #1 showed higher antifungal activity. Further, the extract #1 was separated and purified using HPLC equipped with C18 column using a reverse phase flow mode. After analysis, the collected fractions (1 ~ 12) were concentrated and dissolved in desired volume of 99% MeOH. The antifungal activity of this prep samples were tested against all five fungal species using paper disc method. From the results, the fraction Nos. 5, 7, 9 and 10 showed antifungal activity against C. gloeosporioides and F. oxysporum. Among these fractions, the fraction No. 5 and 7 showed higher antifungal activity against F. oxysporum. The effect of antifungal fractions No. 5 & 7 on F. oxysporum hypae was observed in SEM. A significant morphological changes like destruction of hyphae, conidia spores and growth inhibition was noted in 5 days of incubation. For structural elucidation of the antifungal agents obtained in fraction Nos. 5, 7, 9 and 10 were identified by LC/MS. The parental compound LC/MS peaks were obtained molecular weights 1482 m/z, 1464 m/z, 1448 m/z, 1492 m/z from fraction Nos. 5, 7, 9 and 10, respectively. From the generated protonated ion peaks in LC/MS, all the fractions containing the parental antifungal compounds were identified as fengycin. Further experiments such as MALDI-TOF MS/MS are need to complete structural confirmation of fengycin compounds.