Study On Inhibition Mechanism Of New Agricultural Antibiotic 702 On Plant Pathogenic Fungi

A streptomyces strain, which we named "Streptomyces 702" was screened from soil by Applied Microbiology Laboratory of Jiangxi Agricultural University. Research has shown that the new type of polyene macrolide antibiotics - New Agricultural Antibiotic 702 (including Antifungalmycin702 and Fungichromin) produced by Streptomyces 702 has a strong inhibitory effect on a variety of plant pathogenic fungi. It was hopeful to be developed as a novel and highly effective biologic fungicide. In order to further study the inhibition mechanism of New Agricultural Antibiotic 702 on plant pathogenic fungi, we tested the inhibitory effect of New Agricultural Antibiotic 702 on plant pathogenic fungi at different growth stages, and the effects on cell wall, cell membrane, biological macromolecules. Here are the main research methods and results:(1) Inhibitory effects of Antifungalmycin 702 and Fungichromin on Rhizoctonia Solani, Helminthosporium sigmoideum Cav, Magnaporthe grisea and Ustilaginoidea virens were determined by mycelial growth rate method and mycelial dry weight method. The results showed that both of these two antibiotics had strong inhibitory effects on these tested rice pathogenic fungi. The EC50(μg/mL) and EC9o(μg/mL) of Antifungalmycin 702 on these four kinds of rice pathogenic fungi were 7.00-26.71 and 13.20-128.28 respectively. The antifungal activity of Fungichromin was stronger than Antifungalmycin 702, and the EC50 and EC90 were 0.20-2.37 and 3.92-135.54 respectively.(2) In order to explore the inhibitory effects of New Agricultural Antibiotic 702 on rice pathogenic fungi at different growth stages, we tested the effects of New Agricultural Antibiotic 702 on mycelial growth, mycelial morphology, sclerotial formation and germination of Rhizoctonia Solani, and on the mycelial growth, conidial formation and germination, appressorium formation of Magnaporthe grisea. The EC50 and EC90 of New Agricultural Antibiotic 702 on Rhizoctonia Solani were 1.85 and 3.35μg/mL. New Agricultural Antibiotic 702 can increase the mycelial vacuole of Rhizoctonia solani, and the mycelial surface became rough, inclusions increased, the mycelium even became distorted and irregular constriction. Treated with 48.01μg/mL of New Agricultural Antibiotic 702, the formation of sclerotia was delayed 216h compared with control, the formation rate was only 39.21%, and the dry weight was 81.37% less than control, in addition, it can significantly inhibit the germination of sclerotia in 33.51μg/mL. The EC50 and EC90 of New Agricultural Antibiotic 702 on Magnaporthe grisea were 37.01 and 136.21μg/mL. The conidiation of Magnaporthe grisea was only 9.29% of control in 160.08μg/mL of New Agricultural Antibiotic 702, and treated with 20.14μg/mL of New Agricultural Antibiotic 702, the inhibition rate of conidial germination was 95.16%, and the inhibition rate of appressorium formation was up to 100%. We preliminarily explored that New Agricultural Antibiotic 702 had a strong antifungal effect on rice pathogenic fungi at different growth stages.(3) Through the cell wall staining experiments, we found that New Agricultural Antibiotic 702 had a certain impact on Rhizoctonia solani cell wall. It can lead to cell wall of irregular shape, uneven thickness, fuzzy staining, or even no complete cell wall. Further determination of the N-acetyl glucosamine content and chitinase activity showed that, N-acetyl glucosamine content and chitinase activity had a certain degree of increase while treated with New Agricultural Antibiotic 702. It may lead to mycelium leakage of glucose or hinder it’s absorption of glucose, which stimulated the chitinase gene expression, and then hydrolyzed chitin to N-acetyl glucosamine, led to abnormal cell wall growth.(4) In order to further explore the impact of New Agricultural Antibiotic 702 on cell membrane permeability, we determined the conductivity, soluble sugar and protein content of the mycelial culture liquid treated with New Agricultural Antibiotic 702. The results showed that New Agricultural Antibiotic 702 can cause damage to the cell membrane of Rhizoctonia solani. It can increase permeability of cell membrane, resulting in leakage of electrolytes, soluble sugar and protein, the mycelium membrane lipid peroxidation. In addition, New Agricultural Antibiotic 702 can lead to the decline of Rhizoctonia solani ergosterol content. We preliminarily explored that the membrane system is one of the possible action sites of New Agricultural Antibiotic 702. It acted on ergosterol in the plasma membrane, which resulted in membrane permeability increase, inclusion leakage.(5) At last we determined the effects of New Agricultural Antibiotic 702 on biomacromolecule content. We found that New Agricultural Antibiotic 702 had no significant effect on polysaccharide and protein content, but can cause a decline in DNA and RNA content. This may be related to the increased membrane permeability, which led to inclusion leakage, cell metabolism disorder, thereby affecting DNA and RNA synthesis and a series of physiological and biochemical activities.Comprehensive analysis of the above results, we infered that the inhibition mechanism of New Agricultural Antibiotic 702 on plant pathogenic fungi were as follows, New Agricultural Antibiotic 702 may acted on ergosterol in the plasma membrane, damaged the cell membrane, causing leakage of cell contents, and affecting a range of physiological and biochemical activities, which play antifungal effect.