抗真菌农用抗生素KA08的研究
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摘要
本文针对近年来烟草生产上危害严重的烟草赤星病(Alternaria alternata(Freis)Keissler),筛选获得了多株具有自主知识产权的拮抗链霉菌,并对具有优良特性的拮抗菌进行了分类鉴定、发酵条件优化、抗菌物质的分离纯化、抑菌防病机理及温室盆栽防效和安全性测定等方面的系统研究,开发出了一种极富研究和应用价值,且性质显著不同于多氧霉素的广谱农用抗生素KA08,对解决生产上有效防治链格孢属真菌病害的生防制剂品种单一、病原菌对多氧霉素的耐药性等问题,具有重要意义。研究结果如下:
1、从采自辽宁、黑龙江、河北、山西等地的36份土样中分离到371株链霉菌。以烟草赤星病菌为靶标,利用活体对峙拮抗、发酵液抑菌活性测定和离体叶片防效试验相结合的多重筛选法,得到4株抑菌效果明显、抑菌谱较广且效果稳定的拮抗链霉菌:菌株182-2、163、88和127。拮抗菌发酵液理化性质测定表明:4菌株对温度、紫外线、自然光、酸碱的耐受性,发酵液的耐贮性和菌株的遗传特性虽有差异,但均较为稳定。其中菌株182-2显示出了良好的抑菌防病效果和优良的稳定性,有开发应用价值。
2、采用经典分类和分子分类相结合的方法,对菌株182-2和菌株163进行了分类鉴定,明确了两菌株的分类地位,菌株182-2为链霉菌属可可链霉菌辽宁变种(Streptomyces cacaoi var.liaoningensis),属灰褐类群;菌株163为链霉菌属细黄链霉菌辽宁变种(Streptomyces microflavus var.liaoningensis),属粉红孢类群,并首次报道了细黄链霉菌对烟草赤星病菌的拮抗作用。
3、通过培养基优化设计,获得了菌株182-2的次生代谢产物抗生素KA08的最佳发酵培养基配方:可溶性淀粉100g,花生饼粉24.0 g,酵母粉2.0 g,NH_4NO_4 2.0 g,CaCO_32.7g,NaCl 2.7 g,水1000ml,pH7.2。抗生素KA08摇床发酵的最佳条件为:制备种子液的菌种种龄为斜面培养5d,接种量4.5%(种子液浓度10~8cfu/ml),初始pH7.0,装液量40ml/三角瓶(250ml),发酵温度28℃,发酵时间5d。对代谢过程中菌体浓度、可溶性糖、pH变化的检测结果表明:抗生素KA08来自能量代谢所用的基质,但其形成是在与初级代谢分开的次级代谢中。同时,抗生素产生和菌丝体生长有各自的最适pH,应分别加以严格控制才能优化发酵过程。
4、通过捷克Doskochilova 8溶剂系统及pH纸层析证明:抗生素KA08为一类碱性水溶性抗生素,含有多个活性组分;Betina溶媒系统纸层析证明:KA08极性大,极易溶于水,且随溶剂极性的减小,溶解度降低。分析纸电泳试验中各组分的移动距离和方向,可知抗生素KA08含有多个强碱或弱碱性活性组分,属于链霉素型或放线菌素型。
根据上述性质,在活性追踪下,采用草酸酸化、丙酮沉淀、活性炭吸附、大孔树脂富集、离子交换柱层析和Pharmadex LH20柱层析精制,以及二次薄层层析分离,对抗生素KA08进行了分离纯化,得到一个抗菌活性较高的组分Ha,四个活性较弱的组分a、c、d和Be。经HPLC纯度检测,组分Ha纯度较低,其两个主要成分的含量分别为62.90%和17.53%;组分a、c、d和Be的纯度分别为97.53%、98.79%、97.64%和96.06%,达到了结构测定的标准。紫外和红外谱图分析表明:组分a、c、d为氨基糖苷类物质,组分Be则更接近于核苷类物质的特点。官能团反应显示:组分Be和d含有醛基和不饱和键;组分a含有氨基酸、肽类或蛋白质结构;组分c含有不饱和键,及肽类或蛋白质结构。液质联用测定表明:组分c是分子量为269.0的小分子物质。总体分析:抗生素KA08不同于生产上广泛使用的可可链霉菌阿索变种产生的多氧霉素。
5、抑菌防病机理研究表明:抗生素KA08能显著抑制病原菌菌丝生长和孢子萌发,并对菌丝和孢子萌发后的芽管有明显致畸作用。抗生试验表明:KA08对赤星病菌主要起抑菌作用,杀菌作用很弱或基本无杀菌效果。抑菌机理研究发现:该物质能导致菌丝体内电解质泄漏,同时,细胞膜上麦角甾醇的含量显著降低,菌丝内和培养液中脂质过氧化产物-丙二醛的含量明显升高,证明其作用位点在病菌细胞膜上。它还可显著抑制菌丝体内蛋白质的含量,但不能通过产生几丁质酶对菌丝细胞壁中的几丁质产生影响。抗生素KA08处理能诱导烟草防御酶系中POD、PPO、PAL、SOD、CAT活性的提高并在一定程度上诱导产生PR蛋白。同时,KA08处理还可以诱导抗病信号通路中NPR1和PR1基因的表达,且PR1基因的表达有持效性,推测其可能启动了水杨酸防御反应信号传导途径。但ERF1基因的表达未被检测到。
6、温室盆栽防效试验证明:采用保护性处理时,抗生素KA08溶液(2.0mg/ml)对烟草赤星病的防效为60.63%,同时,对具有伤口的叶片有轻微损害。安全性测定表明:KA08有较好的体内运转能力。当浓度≥2.0mg/ml,通过根部吸收时,KA08对烟草3～4叶期幼苗有轻微致萎作用;浓度＜2.0mg/ml时表现安全。当浓度≥2.0mg/ml,KA08叶面喷施对成株期植株没有影响,同时具有一定的刺激生长作用。
总之,抗生素KA08是一种具有优良特性的农用抗生素,它的开发和应用符合现代植物保护的生防理念,具有重要的实际应用价值。
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 NH_4NO_4 ,2.7g CaCO_3, 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 10~8cfu/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.
1、从采自辽宁、黑龙江、河北、山西等地的36份土样中分离到371株链霉菌。以烟草赤星病菌为靶标,利用活体对峙拮抗、发酵液抑菌活性测定和离体叶片防效试验相结合的多重筛选法,得到4株抑菌效果明显、抑菌谱较广且效果稳定的拮抗链霉菌:菌株182-2、163、88和127。拮抗菌发酵液理化性质测定表明:4菌株对温度、紫外线、自然光、酸碱的耐受性,发酵液的耐贮性和菌株的遗传特性虽有差异,但均较为稳定。其中菌株182-2显示出了良好的抑菌防病效果和优良的稳定性,有开发应用价值。
2、采用经典分类和分子分类相结合的方法,对菌株182-2和菌株163进行了分类鉴定,明确了两菌株的分类地位,菌株182-2为链霉菌属可可链霉菌辽宁变种(Streptomyces cacaoi var.liaoningensis),属灰褐类群;菌株163为链霉菌属细黄链霉菌辽宁变种(Streptomyces microflavus var.liaoningensis),属粉红孢类群,并首次报道了细黄链霉菌对烟草赤星病菌的拮抗作用。
3、通过培养基优化设计,获得了菌株182-2的次生代谢产物抗生素KA08的最佳发酵培养基配方:可溶性淀粉100g,花生饼粉24.0 g,酵母粉2.0 g,NH_4NO_4 2.0 g,CaCO_32.7g,NaCl 2.7 g,水1000ml,pH7.2。抗生素KA08摇床发酵的最佳条件为:制备种子液的菌种种龄为斜面培养5d,接种量4.5%(种子液浓度10~8cfu/ml),初始pH7.0,装液量40ml/三角瓶(250ml),发酵温度28℃,发酵时间5d。对代谢过程中菌体浓度、可溶性糖、pH变化的检测结果表明:抗生素KA08来自能量代谢所用的基质,但其形成是在与初级代谢分开的次级代谢中。同时,抗生素产生和菌丝体生长有各自的最适pH,应分别加以严格控制才能优化发酵过程。
4、通过捷克Doskochilova 8溶剂系统及pH纸层析证明:抗生素KA08为一类碱性水溶性抗生素,含有多个活性组分;Betina溶媒系统纸层析证明:KA08极性大,极易溶于水,且随溶剂极性的减小,溶解度降低。分析纸电泳试验中各组分的移动距离和方向,可知抗生素KA08含有多个强碱或弱碱性活性组分,属于链霉素型或放线菌素型。
根据上述性质,在活性追踪下,采用草酸酸化、丙酮沉淀、活性炭吸附、大孔树脂富集、离子交换柱层析和Pharmadex LH20柱层析精制,以及二次薄层层析分离,对抗生素KA08进行了分离纯化,得到一个抗菌活性较高的组分Ha,四个活性较弱的组分a、c、d和Be。经HPLC纯度检测,组分Ha纯度较低,其两个主要成分的含量分别为62.90%和17.53%;组分a、c、d和Be的纯度分别为97.53%、98.79%、97.64%和96.06%,达到了结构测定的标准。紫外和红外谱图分析表明:组分a、c、d为氨基糖苷类物质,组分Be则更接近于核苷类物质的特点。官能团反应显示:组分Be和d含有醛基和不饱和键;组分a含有氨基酸、肽类或蛋白质结构;组分c含有不饱和键,及肽类或蛋白质结构。液质联用测定表明:组分c是分子量为269.0的小分子物质。总体分析:抗生素KA08不同于生产上广泛使用的可可链霉菌阿索变种产生的多氧霉素。
5、抑菌防病机理研究表明:抗生素KA08能显著抑制病原菌菌丝生长和孢子萌发,并对菌丝和孢子萌发后的芽管有明显致畸作用。抗生试验表明:KA08对赤星病菌主要起抑菌作用,杀菌作用很弱或基本无杀菌效果。抑菌机理研究发现:该物质能导致菌丝体内电解质泄漏,同时,细胞膜上麦角甾醇的含量显著降低,菌丝内和培养液中脂质过氧化产物-丙二醛的含量明显升高,证明其作用位点在病菌细胞膜上。它还可显著抑制菌丝体内蛋白质的含量,但不能通过产生几丁质酶对菌丝细胞壁中的几丁质产生影响。抗生素KA08处理能诱导烟草防御酶系中POD、PPO、PAL、SOD、CAT活性的提高并在一定程度上诱导产生PR蛋白。同时,KA08处理还可以诱导抗病信号通路中NPR1和PR1基因的表达,且PR1基因的表达有持效性,推测其可能启动了水杨酸防御反应信号传导途径。但ERF1基因的表达未被检测到。
6、温室盆栽防效试验证明:采用保护性处理时,抗生素KA08溶液(2.0mg/ml)对烟草赤星病的防效为60.63%,同时,对具有伤口的叶片有轻微损害。安全性测定表明:KA08有较好的体内运转能力。当浓度≥2.0mg/ml,通过根部吸收时,KA08对烟草3～4叶期幼苗有轻微致萎作用;浓度＜2.0mg/ml时表现安全。当浓度≥2.0mg/ml,KA08叶面喷施对成株期植株没有影响,同时具有一定的刺激生长作用。
总之,抗生素KA08是一种具有优良特性的农用抗生素,它的开发和应用符合现代植物保护的生防理念,具有重要的实际应用价值。
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 NH_4NO_4 ,2.7g CaCO_3, 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 10~8cfu/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.
引文
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