Study On The Antifungal Agro-Antibiotic KA08

This study was aimed at inhibiting tobacco brown spot (Alternaria alternata (Freis) Keissler), which has done great harm to tobacco plants. A number of Antagonistic Streptomyces strains, for which this study claims intellectual property rights, were obtained through screening. A systematical study was made of the antagonistic Streptomyces strains with better properties in such aspects as classification and identification, optimization of fermentation conditions, isolation and purification of antagonistic substance, action mechanisms of inhibiting pathogens and controlling disease, and determination of control effects on plant pathogens in greenhouse and the application safety, which led to the development of KA08, a broad-spectrum antifungal agro-antibiotic whose properties are quite different from that of polyoxin. It is of great practical significance in both research and production because currently few effective bio-control agents against plant diseases caused by Alternaria are available and the pathogens have developed antibiotic resistance to polyoxin. The results are as follows:1.371 Streptmyces strains were isolated from 36 soil samples collected from Liaoning, Heilongjiang, Hebei and Shanxi, et al. Out of them, 4 Antagonistic Streptomyces strains with obvious, stable inhibitory effects and extensive inhibition spectrum against different pathogens were screened by assessing the inhibition zone of living body, fermented broth on agar media and the control effect in detached leaves. They are strain 182-2, strain 163,strain 88 and strain 127. Determination of the physical and chemical properties of the fermented broths showed that the 4 strains are all stable in spite of certain differences in their stabilities against temperature, ultraviolet, natural light, acid and alkali, their storage tolerance and subculture. Strain 182-2 has superior capacity of inhibiting pathogens, controlling disease and enduring bad conditions, hence, worth development and application.2.Classical taxonomy and molecular biological taxonomy were adopted in classifying and identifying strain 182-2 and strain 163. Strain 182-2 was identified as Streptomyces cacaoi var. liaoningensis. Strain 163 was identified as Streptomyces microflavus var. liaoningensis. The study reported for the first time the antagonism of Streptomyces microflavus against Alternaria alternata (Freis) Keissler.3.The optimization of the fermentation medium for antibiotic KA08, a secondary metabolite of strain 182-2, was carried out, the result of which was expressed in the following formula: 100g soluble starch, 24.0g peanut powder, 2.0g yester powder, 2.0g NH₄NO₄ ,2.7g CaCO₃, 2.7g NaCl, 1000ml water, at pH7.2. The optimum conditions for fermentation in shake-flask were defined as follows: five-day old strains cultivated on medium for seed broth preparation; inoculation amount, 4.5% (concentration of seed broth being 10⁸cfu/ml); initial pH at 7.0; the volume of broth in a 250ml flask, 40ml; fermentation temperature, 28℃, and fermentation time, 5 days. Examinations of the changes in cell concentration, soluble sugar content, and pH in the metabolic process found that KA08 derives from the basic substance involved in energy metabolism, but the KA08 comes into being at the secondary metabolic stage in stead of the primary one. Meanwhile, the optimization of fermentation can only be realized when the pH is strictly controlled because the antibiotic development and the hypha growth require different optimum pH.4.By means of antibiotic special identification techniques including the paper chromatography in Doskochilova 8 menstruum system and different pH menstruum system, antibiotic KA08 was indicated to be a basic water-soluble substance. It contains several active components. The paper chromatography in Betina menstruum system showed that it has strong polarity, and easily dissolves in water. Its solubility decreases as the polarity of solvents decreases. Based on the analysis of the direction and distance each active component moved in paper electrophoresis, it can be inferred that KA08 contain several strong or weak basic active components and those components belong to antinomycin or streptomycin.In light of these properties and guided by bioassay, isolation and purification of KA08 was conducted adopting such techniques as acidification with oxalic acid, sediment protein with acetone, activated carbon absorption, macroporous resin absorption, ion-exchange column chromatography, Pharmadex LH20 column chromatography and thin-layer chromatography. Five components were obtained. Component Ha has stronger antagonistic activity, and the other four components a, c, d and Be have weaker activities. Determination of the Purity by HPLC showed that component Ha is of low purity, the content of its two major compositions being 62.90% and 17.53% respectively, and that the purity of components a, c, d and Be is 97.53%, 98.79%, 97.64% and 96.06% respectively, all meeting the requirement for structure analysis. The analysis of ultraviolet spectrum and infrared spectrum indicated that components a, c, and d are aminoglycoside substances, and component Be is more identical to nucleosides substance in characteristics. Functional group reaction showed that components Be and d contain aldehyde group and unsaturated bond, component a contain structure of amino acid, peptide or protein, and component c contain unsaturated bond and structure of peptide or protein. Determination of molecular weight by HPLC-MS found that component c has only a molecular weight of 269.0. All factors considered, KA08 differs from the widely used polyoxin, a water-soluble antibiotic originating from S.cacaoi var.asoensis.5.Antibiotic KA08 has obvious inhibition effect on mycelial growth and spore germination and can cause the hypha and the tube to grow in abnormal shapes. The test of antibiosis showed that KA08 has inhibition action to Alternaria alternata, but has little or even no fungicide action. Studies of active mechanism were carried out. The electrical conductivity of cultural filtrate of Alternaria alternata increased obviously after the pathagen was treated with KA08 .The result showed that the protoplasm leaked out from hypha. At the same time, the ergosterol content in mycelia of Alternaria alternata decreased remarkably and MDA content in mycelia and cultural filtrate increased notably. These results proved the action site of KA08 is on the cell membrane. It can check the content of protein in mycelia but has no effect on the chitin in mycelia. Defense-related enzymes, including POD, PPO, PAL, SOD and CAT, had higher activities in tobacco plants pretreated with KA08 than those in tobacco without pretreatment. Pathogenesis-related proteins were induced in tobacco leaves, which indicates that KA08 can induce resistance in translation level. The expressions of NPR1, PR1, and ERF1 were investigated by RT-PCR after treatment with KA08. KA08 treatment could induce the expression of NPR1 and PR1 in tobacco leaves, and the expression of gene PR1 was persistent, which indicated that KA08 could induce salicylate-dependent defense signaling transduction pathway. But ERF1 was not amplified by specific primer.6.Tests of control effects against Alternaria alternata were conducted on tobacco plants in green house, the results of which showed that the KA08 (2.0mg/ml) had a control effect of 60.63%, and slightly damaged those leaves with a cut. Security tests found that antibiotic KA08 transported well in plants. When absorbed through root, at a concentration of 2.0mg/ml or higher, KA08 had slight withering effects on tobacco seedlings at 3-to-4-leave stage, but at a concentration lower than 2.0mg/ml, it worked harmlessly. When sprayed on the leaves at the concentration of 2.0mg/ml or higher, it did no harm to tobacco plants at grown stage and stimulated their growth in some degree.All in all, Antibiotic KA08 is an agro-antagonistic of good properties, the development and application of which are in accordance with the modern concept of biological control in plant protection. It is of great practical value.