Isolation And Antagonistic Mechanism Analyses Of Biocontrol Agents Against Fusarium And Aspergillus Species

Fusarium and Aspergillus are two major pathogens which could infect several important food and oil crops such as wheat, maize, rice, peanut and their product, etc all over the world. These pathogens cause great damage to crop quality and yield, even lead to serious economic losses. They also produce amount of harmful mycotoxins in crops such as trichothecenes and aflatoxins, which result in serious threatens to human and animal health by mis-ingestion, additionally they can lead to cancer or death. China is large agricultural country, the yield and quality of these crops were related with the economic development and the social stability. Hence, the primary work for the crop production and food security was to effectively control Fusarium and Aspergillus.To our knowledge, the methods to control the two pathogens mentioned above were very limited. Natural resistance against them is inadequate and no germplasm exists that provides effective innate resistance to these pathogens. Crop protection against Fusarium or Aspergillus pathogens has relied heavily on chemical control for a long time, which caused severe and undesirable environmental and ecological consequences. More seriously, they also lead to serious pathogen resistance and enhanced mycotoxins production. Hence, the development of newly non-deterious method to control plant disease is of vital importance. In our study, the Fusarium graminearum and Aspergillus flavus were selected as two targets for screening effective microbial biocontrol agents. After tested on microbe isolation, screening, metabolite identification and antifungal mechanism resolution, we got several effective antagonists, identified the predominant antagonistic compounds and elucidated their target on fungal cells, and finally used at pre- and post-harvest against plant pathogens. The main results we obtained were as follows:The main results obtained in biocontrol of Fusarium pathogen.1. Screening of biocontrol agents against Fusarium graminearum. 1809 microbes were isolated from 160 samples collected from 9 provinces in China with different ecosystem. Among these strains, twelve antagonists showed great antifungal activity against Fusarium graminearum in vitro, and used in conidia germination, seedling blight assay and field single head inoculation tests. Finally, three Bacillus spp.(S76-3, DM-3 and DK-1) with highly inhibition rate against conidia germination(97%), seedling blight(46-57%) and wheat head blight(67%-80%) were selected as biocontrol agents for further control and antifungal mechanism study against Fusarium pathogen.2. Biocontrol efficacy of strain S76-3 bacterial bodies and the cell free supernatant. Bacillus amyloliquefaciens S76-3 with best biocontrol efficacy was selected in the test. After fermentation optimization, the optimal medium were obtained(brown sugar 23.3 g/L,bean powder 11.1 g/L,Ca CO3 4.9 g/L,KNO3 8 g/L,KH2PO4 0.5g/L,K2HPO4 1.2 g/L,Mg SO4 ·7H2O 5 g/L,Na Cl 3 g/L), and the bacterial concentration was up to 1010 cfu/ml. The collected bacterial spores mixed with addictive to produce wettable powder(WP) which had high active bacterial concentration to 109 /g. The produced cell free supernatant broth(cfs-broth) and WP of S76-3 were tested in field plot assay. Both the cfs-broth and WP body showed antifungal activity on different wheat cv. in different years. Such as in 2013, the disease index in cfs-broth treatment was reduced by 52%, the mycotoxin reduced by 76%, which were more effective than commercial carbendazim, WP of S76-3 reduced disease index for 13% and mycotoxin concentration for 43%, which were less than carbendazim treatment.3. Structural analyses of antifungal substantce from strain S76-3. Complete genome sequence and PCR amplification proved the existence of lipopeptides genes in strain S76-3. Thin layer chromatography and LC-MS/MS analyses elucidated the production of iturins, plipastatin and surfactin. Further tests conducted with ESI-CID-MS proved that iturin A, plipastatin A/B and surfactin were detected with 2-4 homologues in each.4. Antifungal mechanism tests of identified lipopeptides. Three compounds were collected with HPLC and diluted to series concentrations for antifungal activity and mechanism analyses against Fusarium in vitro. The results elucidated that surfactin had no inhibition activity, iturin A and plipastatin A showed great antifungal activity. Optical and fluorescence microscoy analyses revealed severe morphological and structural changes of conidia and mycelia after treated with iturin A or plipastatin A. Transmission electron microscopy analyses demonstrated that the cell wall and cell membrane were gapped by iturin A and plipastatin A, and finally lead to cell death. The dynamic processes microscopy analyses also proved that iturin A caused distortion and conglobation along hyphae, while plipastatin A caused conglobation in young hyphae and branch tips.The main results obtained in biocontrol of Aspergillus pathogen.1. Screening and identification of antagonist with antifungal volatiles production. Five strains were screened from 100 marine microbes by face-to-face cultured method. They could produce antifungal volatiles and inhibit mycelial growth and conidia germination of Aspergillu flavus. These strains includes one Pseudomonas sp., one Shewanella sp., two Staphylococcus sp. and one Micrococcus sp.. Among these strains, YM8 showed best antifungal activity to 9 important fungal pathogens including Aspergillus spp. and Fusarium spp. with inhibition rate up to 100%. Strain YM8 was selected as potential biocontrol agent for study on inhibition of Aspergillus and mycotoxin, and antifungal mechanism.2. Chemical structure analyses of antifungal volatiles. Gas chromatography mass spectrometry analyses revealed that 15 volatile compounds emitted from strain YM8. These volatiles were from several chemical classes, including aromatics, alkanes, sulfides, enols, oxazole, anthracenes, esters, and phenols. The most abundant compound in the volatile profile was identified as dimethyl trisulfide with relative amount up to 14.195%. Six volatiles with identity over 70% and relative amount over 0.5% were purchased for antifungal test. The result showed that 2, 4-bis(1,1-dimethylethyl)-phenol and dimethyl trisulfide were effective in control Aspergillus. They could completely inhibit mycelial growth and conidial germination at 200 μg/L and 100 μg/L(compound weight to airspace volume), respectively. The other compounds including 2, 4-dimethyl-oxazole, 2-dodecanol, nonane and butylated hydroxytoluene showed limited antifungal activity. Dimethyl trisulfide with great abundance and antifungal activity was vital in strain YM8.3. Inhibition of strain YM8 against Aspergillus flavus on crops. Strain YM8 completely inhibited Aspergillus infection and aflatoxin biosynthesis in maize and peanut samples stored at different water activity(aw) levels. In control treatment, the disease incidence and mycotoxin production were promoted with the increase of aw. Peanuts exhibited disease incidences of 100% and mycotoxins of 54 μg/g at high aw(0.923). Same results were observed in maize samples with disease incidences of 100% and mycotoxins of 5.59 μg/g at aw 0.934. But, YM8 showed great antifungal activity in peanuts and maize samples of three aw, the disease incidence and mycotoxin concentration was 0. Scanning electron microscopy revealed severely damaged conidia and a complete lack of mycelium development and conidiogenesis in YM8 group, the few conidia were severely deformed and irregular shapes with curve and holes on the surface, which lost the infection activity, while in control group abundant mycelium and conidia with regular shade covered on the peanut surface.4. Genomic sequence analyses and Bac library construction of strain YM8. The genome of strain YM8 was 4.5 Mb. The genome was partial digested with Hind III and cloned in E. coli, and 1152 colones(about 60 kb in each) were obtained from the Bac library. After the addition of different amino acids to basic medium, we proved that methionine and cysteine could notabily increase the inhibition activity of strain YM8. The two amino acids containing sulfur element were the main compounds in the production of dimethyl trisulfide, and may be involved in the biosynthesis pathway of dimethyl trisulfide.