几种林木病原菌拮抗放线菌的筛选及其活性物质研究
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摘要
本文对采自秦岭火地塘林区的10个土样进行了放线菌的分离,共得到了512株菌株,经过初步分类合并,采用其中的292株进行了对几种常见林木病原菌的拮抗试验,结合发酵液的活性测定和生测治试验得到了一株抑菌谱较广、活性极强的放线菌——268号菌株,并对其做了一系列的研究。主要研究结果如下:
1.以髙氏一号琼脂、天门冬素淀粉琼脂和精氨酸琼脂培养基做为分离培养基,重铬酸钾浓度为150 mg/L,可以较好的分离该土样中的放线菌。
2.各土样的各层链霉菌属均为绝对优势属,其它属分离出的较少,粉红孢类群、金色类群和灰褐类群是大多土样的优势类群。
3.平板初筛结果表明:14、42、45、50、162、189、268及289号菌对多种靶菌均具有较强的拮抗作用。发酵液的活性测定表明:42、189和268号菌株对多种靶菌存在较强的抑制性。预防效果的生物测定结果表明:268号菌株的效果最好,预防效果为64.7%。
4.对268号菌株的发酵液进行了萃取试验,其乙酸乙酯相具有强烈的抑菌活性。乙酸乙酯相的薄层层析试验表明:以体积比10:7的石油醚和丙酮为展开剂可以较好地将活性组分与其他组分分离开。并对268号菌株活性产物的性质进行了初步研究,结果表明:该物质没有挥发性,对酸有着较好的稳定性,对强碱不稳定,对热和紫外线较为稳定。
5.通过单因素试验结合正交试验,对268号菌株的发酵培养基和发酵条件进行了优化,结果表明:最适的发酵培养基配方为:黄豆饼粉10 g/L,蛋白胨3 g/L,葡萄糖10 g/L,NaCl 2.5 g/L,FeSO4 0.01 g/L,pH≈8.0,最适的发酵条件为:菌龄48 h,接种量2%,装液量200 ml,发酵时间6 d,摇床转速150 dr/min。
6.对268号菌株活性产物的抑菌机制进行了初步研究,结果表明:其抑菌活性主要体现在对菌丝的影响上,对杨树溃疡病、苹果腐烂病和杨树腐烂病病原菌的生长抑制作用最强,EC50分别为0.0076 ml/ml、0.0061 ml/ml和0.0043 ml/ml,对镰刀菌和胶孢炭疽菌的EC50分别为0.0531 ml/ml和0.0497 ml/ml。
7.采用经典的鉴定方法,初步将268号菌株定为灰产色链霉菌(Streptomyces griseochromogenes)的一个变种。
512 strains of actinomycetes were separated from the soil collected in 10 samples forest litters in Huoditang Mts . Though elementary classified,292 strains were used for antagonistic experiment. Though test of ferment liquid bioactivity and biological test, N.O 268 strain was screened. Some studies were go along. The main results were as follows:
1.GA,Asa,Aa and K2Cr2O7(150mg/L) were fit into isolation of actinomycetes in this kind soil samples.
2.In every soil samples, streptomyces were dominant genus. In Streptomyces,Roseosporus and Aureus and Griseofuscus were dominant groups.
3.The results of agar plate antagonism experiment showed that: 14 and 42 and 45 and 50 and 162 and 89 and 68 and 289 strains had better bioactivity to many forest pathogenies. By means of test of ferment liquid inhibition, 42 and 189 and 268 strains had better bioactivity to many forest pathogenies. The result of biological test showed that 268 strain had better prevent effect. The prevent effect was 64.7%.
4.Extraction experiment of ferment liquid of N.O 268 strain showed that the Ethyl acetate fraction had best bioactivity. And TLC method was used. The result revealed that the bioactivity was caused by one weaker polarity substance. The characteristics of this bioactive substance were studied. The results showed that this bioactive metabolite did not have volatility. It was acid and heat and UV stable.
5.The bioactivity sustenance had a certain degree inhibition to mycelium. The results of mycelium growth rate showed that the value of EC50 against Dothiorella gregaias and Valsa sordida and Valsa mali were 0.0076ml/ml and 0.0061ml/ml and 0.0043ml/ml respectively. The value of EC50 against Fusarium spp and Colletotrichum gloeosporioides were 0.0531ml/ml and 0.0497ml/ml respectively.
6.It was proved through orthogonal experiments that the optimumcul fermental conditions for producing the antibiotic active substance were soybean cake(10g/L)and peptone(3g/L)and glucose(10g/L) and NaCl(2.5g/L)and FeSO4(0.01g/L) and pH≈8.00 and inoculate with 48 hours seed-age and 2% inocula and culture for 200 ml 6days, 150 dr/min.
7. By means of observation on substrate mycelium, aerial mycelium, spore and rothrix , cultural characteristics,Biochemical and physiological characteristics, NO.268 was primarily identified as a variety of Streptomyces griseochromogenes.
1.以髙氏一号琼脂、天门冬素淀粉琼脂和精氨酸琼脂培养基做为分离培养基,重铬酸钾浓度为150 mg/L,可以较好的分离该土样中的放线菌。
2.各土样的各层链霉菌属均为绝对优势属,其它属分离出的较少,粉红孢类群、金色类群和灰褐类群是大多土样的优势类群。
3.平板初筛结果表明:14、42、45、50、162、189、268及289号菌对多种靶菌均具有较强的拮抗作用。发酵液的活性测定表明:42、189和268号菌株对多种靶菌存在较强的抑制性。预防效果的生物测定结果表明:268号菌株的效果最好,预防效果为64.7%。
4.对268号菌株的发酵液进行了萃取试验,其乙酸乙酯相具有强烈的抑菌活性。乙酸乙酯相的薄层层析试验表明:以体积比10:7的石油醚和丙酮为展开剂可以较好地将活性组分与其他组分分离开。并对268号菌株活性产物的性质进行了初步研究,结果表明:该物质没有挥发性,对酸有着较好的稳定性,对强碱不稳定,对热和紫外线较为稳定。
5.通过单因素试验结合正交试验,对268号菌株的发酵培养基和发酵条件进行了优化,结果表明:最适的发酵培养基配方为:黄豆饼粉10 g/L,蛋白胨3 g/L,葡萄糖10 g/L,NaCl 2.5 g/L,FeSO4 0.01 g/L,pH≈8.0,最适的发酵条件为:菌龄48 h,接种量2%,装液量200 ml,发酵时间6 d,摇床转速150 dr/min。
6.对268号菌株活性产物的抑菌机制进行了初步研究,结果表明:其抑菌活性主要体现在对菌丝的影响上,对杨树溃疡病、苹果腐烂病和杨树腐烂病病原菌的生长抑制作用最强,EC50分别为0.0076 ml/ml、0.0061 ml/ml和0.0043 ml/ml,对镰刀菌和胶孢炭疽菌的EC50分别为0.0531 ml/ml和0.0497 ml/ml。
7.采用经典的鉴定方法,初步将268号菌株定为灰产色链霉菌(Streptomyces griseochromogenes)的一个变种。
512 strains of actinomycetes were separated from the soil collected in 10 samples forest litters in Huoditang Mts . Though elementary classified,292 strains were used for antagonistic experiment. Though test of ferment liquid bioactivity and biological test, N.O 268 strain was screened. Some studies were go along. The main results were as follows:
1.GA,Asa,Aa and K2Cr2O7(150mg/L) were fit into isolation of actinomycetes in this kind soil samples.
2.In every soil samples, streptomyces were dominant genus. In Streptomyces,Roseosporus and Aureus and Griseofuscus were dominant groups.
3.The results of agar plate antagonism experiment showed that: 14 and 42 and 45 and 50 and 162 and 89 and 68 and 289 strains had better bioactivity to many forest pathogenies. By means of test of ferment liquid inhibition, 42 and 189 and 268 strains had better bioactivity to many forest pathogenies. The result of biological test showed that 268 strain had better prevent effect. The prevent effect was 64.7%.
4.Extraction experiment of ferment liquid of N.O 268 strain showed that the Ethyl acetate fraction had best bioactivity. And TLC method was used. The result revealed that the bioactivity was caused by one weaker polarity substance. The characteristics of this bioactive substance were studied. The results showed that this bioactive metabolite did not have volatility. It was acid and heat and UV stable.
5.The bioactivity sustenance had a certain degree inhibition to mycelium. The results of mycelium growth rate showed that the value of EC50 against Dothiorella gregaias and Valsa sordida and Valsa mali were 0.0076ml/ml and 0.0061ml/ml and 0.0043ml/ml respectively. The value of EC50 against Fusarium spp and Colletotrichum gloeosporioides were 0.0531ml/ml and 0.0497ml/ml respectively.
6.It was proved through orthogonal experiments that the optimumcul fermental conditions for producing the antibiotic active substance were soybean cake(10g/L)and peptone(3g/L)and glucose(10g/L) and NaCl(2.5g/L)and FeSO4(0.01g/L) and pH≈8.00 and inoculate with 48 hours seed-age and 2% inocula and culture for 200 ml 6days, 150 dr/min.
7. By means of observation on substrate mycelium, aerial mycelium, spore and rothrix , cultural characteristics,Biochemical and physiological characteristics, NO.268 was primarily identified as a variety of Streptomyces griseochromogenes.
引文
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