| Hybrid bamboo blight caused by Arthrinium phaeospermum was discovered in hybrid bamboo growing area in Sichuan, which could cause the plant to wilt or death. Based on the principal of biological control, this paper showed the isolation and screening of antagonistic actinomycetes from healthy hybrid bamboo which collected from diseased area. In order to prove the biocontrol ability of antagonistic strain, some studies were made, including optimization of fermentation conditions and test of antimicrobial spectrum and control efficiency. Furthermore, the primary antimicrobial substance from fermentation filtrate was purified. The mechanism of antifungal protein to induce resistance of plant and inhibit pathogens was researched. And the partial coding sequence of antifungal protein was cloned by using molecular biological techniques. The results are listed as follows:1. Exploring the microecological distribution and antimicrobial activity of actinomycetes in hybrid bamboo could provide microbial resources for biocontrol of hybrid bamboo blight. Epiphytic and endophytic actinomycetes of healthy hybrid bamboo were separately isolated by tissue washing and homogenate. Plate confrontation culture was used as preliminary screening, and lesion control in excised tissues and field trial were used as secondary screening for the the most effective biocontrol strains. The taxonomic identification of biocontrol strains was studied through their morphological, physiological, biochemical characteristics, with sequencing of16S rDNA genes and phylogenetic analysis. The results showed that96strains of actinomycetes were isolated from hybrid bamboo,93.75%of which were affiliated to eight groups of Streptomyces, the rest were affiliated to Micronnospora and Nocardia. Strains YSSPG3showed the best inhibitory effect, the GN32followed, the control efficiency of both were significantly higher than other strains on excised twigs and leaves. In the field, Strain YSSPG3had better control efficiency against hybrid bamboo blight than strain GN32, reached80.08%in leaves and81.02%in twigs. Strains YSSPG3and GN32were identified as Streptomyces purpeofuscus and Streptomyces californicus respectively.2. The nutrition combination and culture conditions of fermentation for S. purpeofuscus YSSPG3were optimized by experiments of one-factor-at-a-time design and orthogonal array, and the metabolic parameters under optimized culturing condition were tested, all for improving the content of the antimicrobial substance in the fermentation broth and laying a good foundation for productive practice. Results showed that liquid fermentation medium was screened out through optimization experiment, with ingredients of glucose5g, glycerol5g, soybean powder3g, peptone5g, NaCl2.5g, CaCC>22g, distilled water1000ml, the optimal condition being initial pH7.0, inoculum volume of spore suspension (108cfu/ml)1%(V/V), media volume100ml/300ml, stirring speed140r/min, fermented for4days at25℃. The strain YSSPG3produced alkali during the cultivation. The biomass and the antibiotic activity of optimal fermentation medium was the maximizing at96h. The content of total sugar, reducing sugar and amino nitrogen decreased gradually after36h, a sharp decline appeared during the logarithmic growth phase and stationary phase (72-96h).3. In order to confirm the biocontrol potential of S. purpeofuscus YSSPG3for plant diseases, the effect and control efficacy of the optimal fermentation filtrate on A. phaeospermum, the pathogen of hybrid bamboo blight, were tested by conidial germination, mycelial growth as well as lesion control in excised tissues and in field trial. Results showed that the optimal fermentation filtrate had inhibitory effect on conidial germination and mycelial growth. It was found that the germ tube and mycelium of A. phaeospermum exhibited obviously malformation after treated with the cell-free fermentation filtrate. The development of lesions were inhibited remarkably on excised twigs and leaves which were treated with the pure or10times dilution of cell-free fermentation filtrate and inoculated the mycelia cake, the control efficiency of4d was more than70%. In the field trails, the control efficiency of spreading the pure or5times dilution of cell-free fermentation filtrate reached80%, which was significantly higher than the50%carbendazim (30mg/mL) and70%thiophanate methyl (30mg/mL)(P<0.01).4. To clarify the nature of the antimicrobial substance from fermentation filtrate, the antifungal protein was finally purified by ammonium sulfate precipitating and column chromatography analyzing, with a molecular mass of5075.619Da and isoelectric point (pI) of6.77, and lacking the activity of hydrolytic enzyme. The inhibitory activity of the antifungal protein would decreased distinctly at the higher temperature (≥60℃) and in the condition of acidity (pH<7), and a long ultraviolet radiation time (≥12), but tolerant to proteinase K, trypsin and chloroform. The N-terminal sequence between the antifungal protein and a kind of chitin-binding protein AFP1from S. Tendae Tu901was of higher homology in81.00%。5. To reveal the anti-disease mechanism of antifungal protein AMP to hybrid bamboo blight comprehensively, hybrid bamboo seedlings were treated with different concentrations of antifungal protein by spraying to foliage or soaking root, and inoculated with A. phaeospermum or not. The effects of antifungal protein on disease index, the content of MDA and souble protein, and major defense-related enzymes activities were investigated. The results showed that the content of MDA and souble protein, defense-related enzymes activities such as superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), catalase (CAT), phenylalanine amonnialyase (PAL), endochitinase and β-1,3-glucanase had different responses to the disease resistance of hybrid bamboo induced by treating with10times,20times,50times, and100times dilution of antifungal protein mother liquid (360.56μg/mL antifungal protein as the mother liquid). The change of the content of MDA and souble protein, and enzymes activities treated with both inducement and challenge inoculation were faster than those only treated with inducement in the plants. The activities of SOD, PPO, POD, PPO, CAT, PAL, endochitinase and [3-1,3-glucanase with different concentrations of antifungal protein inducement and challenge inoculation or not had the positive correlations with the concentration of antifungal protein. The changes over time and increased range at the same period of POD, PPO, CAT activities induced by soaking root were greater than the spraying treatment, but [3-1,3-glucanase activity to the contrary; There was no significant difference of SOD, PAL and endochitinase among two inducement methods. The correlation analysis showed that the PPO, POD, CAT and β-1,3-glucanase activity induced by spraying antifungal protein had significant correlation with infection index; SOD, PAL, endochitinase and β-1,3-glucanase activity induced by soaking root with antifungal protein had significant correlation with infection index. It suggested that the defense-related enzymes activities might have internal association with infection index.6. To investigate the antifungal effects of antifungal protein AMP on A. phaeospermum and reveal its antifungal mechanism, the ultrastructure of normal and treated mycelial was observed by scanning electron microscope and transmission electron microscope. The permeability, the lipid peroxidation and the content of souble protein were tested by conductivity meter and spectrophotometer. The composition of cell wall were analyzed with Infrared Spectrum. And the genome DNA was detected by agarose gel electrophoresis. The results showed that the antifungal protein on pathogen effective EC50was0.132uμg/mL, MIC was3.606μg/mL, MBC was18.028μg/mL. The inhibition rate of mycelial growth and spore germination was more than80%when antifungal protein concentrations reached1.202μg/mL. The studies revealed that antifungal protein not only inhibited the hyphal growth and spore germination but also caused a series of marked morphological and structural alterations. These changes included irregular swelling, severe malformations of germ tubes and hyphae, and considerable thickening of the hyphal cell walls, particularly at the hyphal tip region. Under transmission electron microscope, the protoplasm of the treated mycelia cell became inhomogeneous and vacuolization. The permeability and lipid peroxidation of the treated mycelia cell increased obviously, but the content of souble protein showed a marked decrease. Antifungal protein didn’t harm the DNA from A. phaeospermum. The detection of chitin indicated that the change in the structure of the cell wall. The absorption peak of the hydroxyl, carbon-hydrogen bond was attenuated while the (3-glucosidic bond, carbon-oxygen bond, and alcoholichydroxyl absorption was enhanced. There was no obvious change in glucan chromatogram to the contrast.7. According to the N-terminal amino acid residues sequence of antifungal protein AMP and the homology sequence, the primers were synthesized. The sequence of223bp nuclear acids was amplified by using the chromosome DNA of S. purpeofuscus YSSPG3as the template with PCR technique. Comparing with chitin-binding protein from S. clavuligerus ATCC27064and S. Tendae Tu901, the sequence homology for nuclear acids were83.4%and79.8%respectively. The deduced amino acid sequence was identical with the known25amino acid residues sequence on N-termial of antifungal protein AMP, and showed78.1%homology compared to chitin-binding protein from S. clavuligerus ATCC27064and S. Tendae Tu901. |
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