放线菌BM10菌株的生物学初步研究及诱变选育
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摘要
放线菌(Actinomycetes)BM10菌株是由本实验室于2005年从海南霸王岭原始森林土壤中分离筛选得到的一株生防菌,其发酵液无菌滤液对香蕉枯萎病菌1、4号小种(F. oxysporum f.sp. cubense race1/race4,Focr1/4)、黄瓜枯萎病菌(F. oxysporum f.sp cucumerinum)、西瓜枯萎病菌(F. oxysporum f.sp.niveum)、辣椒枯萎病菌(F. oxysporium Schl )、辣椒疫霉病菌( Phytophthora capsici )、香蕉炭疽病菌[Colletotrichum musae(Berk.et Curt.)Arx]、香蕉黑星病菌[Phyllosticta musarum (Cke.)Petr]等多种重要植物病原真菌具有强烈抑制作用,其作用机理是产生抗生素。本项研究对放线菌BM10菌株产生抗生素的类别、发酵液中抗生素的稳定性、动物毒性、培养条件及分类地位等生物学特性进行了初步研究。以BM10为出发菌株,以香蕉枯萎镰刀菌4号小种为测试菌,进行了紫外线(UV)、微波(MW)、亚硝基胍(NTG)、硫酸二乙酯(DES)和氯化锂(LiCl)等诱变剂的诱变效应、最佳诱变剂种类、最佳诱变剂量和最佳诱变处理时间等研究,并以“氯化锂+紫外线”及“紫外线+亚硝基胍”等为复合诱变剂对BM10菌株进行了第2、3轮诱变选育。其主要研究结果如下:
1.BM10菌株发酵液浓缩150倍后呈褐色膏状,具有糖浆香甜味。经乙酸乙酯萃取后的抗生素粗提物分为水溶性和脂溶性两类。水溶性和脂溶性粗提物均对辣椒疫霉菌具有强抑菌活性,但只有水溶性粗提物对香蕉枯萎镰刀菌4号小种有强抑菌活性。
2.稳定性研究结果表明,BM10菌株发酵液在自然光照8h、紫外线辐射2h、100℃加热1h、pH2至pH12处理24h及常温下贮存6个月等条件下,其抑菌活性保持稳定。
3.16S rDNA测序和聚类分析结果表明,BM10菌株与链霉菌属的序列一致性达99.57-99.68%,说明BM10菌株属于链霉菌属(Streptomyces sp.)。
4.抗生素产生的基础条件研究结果表明,BM10菌株最适产孢培养基为黄豆粉琼脂培养基,最适发酵培养基为黄豆粉培养液,产抗生素高峰在发酵96h。在250mL三角瓶中发酵培养液的最适装液量为50mL,最适接菌量为5%,发酵周期为96h,摇床转速200 r/min。
5.小白鼠毒性试验初步研究结果表明,小白鼠经BM10发酵上清液灌胃一周后,体重无明显变化,行动、取食正常;经BM10饲喂1个月后,小白鼠变得兴奋,互相追逐撕咬,彼此经常打架,3个月后表现如饲喂1个月后,体重无明显变化。
6.研究分析了5种诱变剂处理BM10菌株的诱变效应,结果表明,在这五种诱变剂中,亚硝基胍的诱变效应最强,依次是紫外线、氯化锂、硫酸二乙酯和微波,微波的诱变效应最弱。初步明确了BM10菌株诱变处理的最佳诱变剂为亚硝基胍,最佳处理剂量为1mg/mLNTG处理45min。
7.对第一次诱变处理的BM10菌株进行初筛和摇瓶发酵复筛,筛选出6株抑菌活性强的突变菌株:L0.2-89、W250-45、N1-60-20、U90-82、N1.5-30-1和N1-45-9,其对Focr4的抑菌效果分别比出发菌株BM10提高了29.47%、33.93%、35.72%、35.72%、38.84%和45.65%。
8.以第一次诱变筛选出的抑菌活性最高的突变菌株N1-45-9为出发菌株,进行“LiCl+UV”复合诱变处理,经初筛和复筛,筛选出1株强抑菌活性突变菌株LU68,其对Focr4的抑菌效果比N1-45-9提高了3.51%,比原始出发菌株BM10提高了46.71%。
9.以LU68为出发菌株,进行“UV+NTG”复合诱变处理,经初筛和复筛后,获得产素水平较高的5株突变菌株:UN82、UN71、UN78、UN84和UN83,其对Focr4的抑菌效果分别比出发菌株LU68提高了5.18%、5.60%、6.07%、8.99%和12.26%,分别比原始出发菌株BM10提高了48.33%、48.75%、49.22%、52.14%和55.41%。
Actinomycetes strain BM10 was a biological control agent isolated from the virgin forest soil of Bawangling nature reserve of Hainan Province, China by our laboratory in 2005. The filtrate of fermentation liquid of Actinomycetes BM10 is strongly inhibitory in vitro to many important pathogens such as Fusarium oxyspoum f.sp.cubense race 4 and race 1(Focr4/Focr1), F. oxysporum f.sp. niveum, F. oxysporum f.sp. cucumerinum, F. oxysporium Schl, Phytophthora capsici, Colletotrichum musae and Phyllosticta musarum, etc. when grown on culture plates by antibiotics produced. In this study, the sort of antibiotics produced by BM10, stability of antibiotics in fermentation liquid, toxicity to mouse, fermentation condition and identification, etc. BM10 were preliminary exploited. The mutation effects to BM10 of some mutants such as ultraviolet, microwave, nitrosoguanidine(NTG), Diethyl sulfate (DES) and LiCl, were studied. Then antibiotics high-yield breeding of BM10 was carried out by using complex mutagenic methods when the optimal mutagen, optimal dosage and optimal treatment time were cleared. The preliminary results were as follows:
1. The concentrated liquid was brownish ointment and had fragrant and sweet taste of syrup after concentrated 150 times. The crude extract of antibiotics BM10 were classified as hydrosoluble and liposoluble active ingredients after extracted by ethyl acetate, and two have strongly inhibit effects against Phytophthora capsici, but only the part of hydrosoluble ingredients have inhibit effect against Focr 4.
2. The result of stability research indicate that the fermentation liquid of BM10 when maintained at exposed to sunlight for 8 hours or ultraviolet for 2 hours, or at 100℃water bath for 1 hours, in a pH2 to pH12 condition for 24 hours, or at room temperature for 6 months can retain its original potency.
3. The sequence analysis result of 16S rDNA and cluster anslysis showed that the BM10 is 99.57-99.68% homologous with 10 Streptomyces strains in GenBank databases and indicated that BM10 strain belongs to Streptomyces genus.
4. The results of basic conditions of antibiotic produced research showed that soybean flour culture medium was the optimum culture medium for BM10 strain growth and spore-produce and the soybean flour liquid culture medium is also the optimum culture medium for BM10 fermentation. The antibiotic yield reached the peak when cultured for 96 hours. The optimum liquid culture volume was 50ml in 250ml Erlenmeyer flask, and the optimum inoculum amount was 5% of the culture liquid volume, and the optimum rotation speed was 200r/min.The maximum antibiotic productivity appeared after cultured for 108 hours under these optimal conditions.
5. The results of toxicity research on BM10 showed that no acute-toxicity to mouse when pour BM10 fermentation supernatant to mouse’stomach with 1mL per mouse for a week, and these mouse’s body weight didn’t have obvious change, action and take food were normal. After feed mouse with 1mL BM10 fermentation supernatant per mouse for a month, these mouse became excited, hunted, worried and fight each other often. But After feed for three months, these mouse act come back normal as usual, and their weights were no obvious change.
6. The mutagenic effect of five mutagens on BM10 were tested, and the results showed the strongest mutagen is NTG , then are UV, LiCl, DES and microwave in turn. The results also showed the optimal mutagen was NTG, its optimal dosage was about 1 mg/mL.
7. Six antibiotic high-yield mutant of BM10 were screened out in first mutation breeding. They are L0.2-89、W250-45、N1-60-20、U90-82、N1.5-30-1 and N1-45-9, and their inhibitory effects against Focr4 increased 29.47%, 33.93%, 35.72%, 35.72%, 38.84% and 45.65%, respectively, compared with BM10 strain.
8. The mutant strain N1-45-9 was chosen to carry out second complex mutation by method of treated with 0.2%LiCl plus UV. The results of fermentation screen showed that only one high antibiotic yield mutant strain LU68 was screened out. It’s inhibitory effects increased 3.51% compared with N1-45-9 and increased 46.71% compared with BM10.
9. The mutant strain LU68 was chosen to carry out third complex mutation breeding by method of treated with UV plus NTG. The results of fermentation screening showed that five antibiotic high-yield mutant strains: UN82、UN71、UN78、UN84 and UN83 were screened out. Their inhibitory effects were increased by 5.18%、5.60%、6.07%、8.99% and 12.26%, respectively, compared with LU68, and increased by 48.33%,48.75%,49.22%,52.14% and 55.41%, compared with BM10.
1.BM10菌株发酵液浓缩150倍后呈褐色膏状,具有糖浆香甜味。经乙酸乙酯萃取后的抗生素粗提物分为水溶性和脂溶性两类。水溶性和脂溶性粗提物均对辣椒疫霉菌具有强抑菌活性,但只有水溶性粗提物对香蕉枯萎镰刀菌4号小种有强抑菌活性。
2.稳定性研究结果表明,BM10菌株发酵液在自然光照8h、紫外线辐射2h、100℃加热1h、pH2至pH12处理24h及常温下贮存6个月等条件下,其抑菌活性保持稳定。
3.16S rDNA测序和聚类分析结果表明,BM10菌株与链霉菌属的序列一致性达99.57-99.68%,说明BM10菌株属于链霉菌属(Streptomyces sp.)。
4.抗生素产生的基础条件研究结果表明,BM10菌株最适产孢培养基为黄豆粉琼脂培养基,最适发酵培养基为黄豆粉培养液,产抗生素高峰在发酵96h。在250mL三角瓶中发酵培养液的最适装液量为50mL,最适接菌量为5%,发酵周期为96h,摇床转速200 r/min。
5.小白鼠毒性试验初步研究结果表明,小白鼠经BM10发酵上清液灌胃一周后,体重无明显变化,行动、取食正常;经BM10饲喂1个月后,小白鼠变得兴奋,互相追逐撕咬,彼此经常打架,3个月后表现如饲喂1个月后,体重无明显变化。
6.研究分析了5种诱变剂处理BM10菌株的诱变效应,结果表明,在这五种诱变剂中,亚硝基胍的诱变效应最强,依次是紫外线、氯化锂、硫酸二乙酯和微波,微波的诱变效应最弱。初步明确了BM10菌株诱变处理的最佳诱变剂为亚硝基胍,最佳处理剂量为1mg/mLNTG处理45min。
7.对第一次诱变处理的BM10菌株进行初筛和摇瓶发酵复筛,筛选出6株抑菌活性强的突变菌株:L0.2-89、W250-45、N1-60-20、U90-82、N1.5-30-1和N1-45-9,其对Focr4的抑菌效果分别比出发菌株BM10提高了29.47%、33.93%、35.72%、35.72%、38.84%和45.65%。
8.以第一次诱变筛选出的抑菌活性最高的突变菌株N1-45-9为出发菌株,进行“LiCl+UV”复合诱变处理,经初筛和复筛,筛选出1株强抑菌活性突变菌株LU68,其对Focr4的抑菌效果比N1-45-9提高了3.51%,比原始出发菌株BM10提高了46.71%。
9.以LU68为出发菌株,进行“UV+NTG”复合诱变处理,经初筛和复筛后,获得产素水平较高的5株突变菌株:UN82、UN71、UN78、UN84和UN83,其对Focr4的抑菌效果分别比出发菌株LU68提高了5.18%、5.60%、6.07%、8.99%和12.26%,分别比原始出发菌株BM10提高了48.33%、48.75%、49.22%、52.14%和55.41%。
Actinomycetes strain BM10 was a biological control agent isolated from the virgin forest soil of Bawangling nature reserve of Hainan Province, China by our laboratory in 2005. The filtrate of fermentation liquid of Actinomycetes BM10 is strongly inhibitory in vitro to many important pathogens such as Fusarium oxyspoum f.sp.cubense race 4 and race 1(Focr4/Focr1), F. oxysporum f.sp. niveum, F. oxysporum f.sp. cucumerinum, F. oxysporium Schl, Phytophthora capsici, Colletotrichum musae and Phyllosticta musarum, etc. when grown on culture plates by antibiotics produced. In this study, the sort of antibiotics produced by BM10, stability of antibiotics in fermentation liquid, toxicity to mouse, fermentation condition and identification, etc. BM10 were preliminary exploited. The mutation effects to BM10 of some mutants such as ultraviolet, microwave, nitrosoguanidine(NTG), Diethyl sulfate (DES) and LiCl, were studied. Then antibiotics high-yield breeding of BM10 was carried out by using complex mutagenic methods when the optimal mutagen, optimal dosage and optimal treatment time were cleared. The preliminary results were as follows:
1. The concentrated liquid was brownish ointment and had fragrant and sweet taste of syrup after concentrated 150 times. The crude extract of antibiotics BM10 were classified as hydrosoluble and liposoluble active ingredients after extracted by ethyl acetate, and two have strongly inhibit effects against Phytophthora capsici, but only the part of hydrosoluble ingredients have inhibit effect against Focr 4.
2. The result of stability research indicate that the fermentation liquid of BM10 when maintained at exposed to sunlight for 8 hours or ultraviolet for 2 hours, or at 100℃water bath for 1 hours, in a pH2 to pH12 condition for 24 hours, or at room temperature for 6 months can retain its original potency.
3. The sequence analysis result of 16S rDNA and cluster anslysis showed that the BM10 is 99.57-99.68% homologous with 10 Streptomyces strains in GenBank databases and indicated that BM10 strain belongs to Streptomyces genus.
4. The results of basic conditions of antibiotic produced research showed that soybean flour culture medium was the optimum culture medium for BM10 strain growth and spore-produce and the soybean flour liquid culture medium is also the optimum culture medium for BM10 fermentation. The antibiotic yield reached the peak when cultured for 96 hours. The optimum liquid culture volume was 50ml in 250ml Erlenmeyer flask, and the optimum inoculum amount was 5% of the culture liquid volume, and the optimum rotation speed was 200r/min.The maximum antibiotic productivity appeared after cultured for 108 hours under these optimal conditions.
5. The results of toxicity research on BM10 showed that no acute-toxicity to mouse when pour BM10 fermentation supernatant to mouse’stomach with 1mL per mouse for a week, and these mouse’s body weight didn’t have obvious change, action and take food were normal. After feed mouse with 1mL BM10 fermentation supernatant per mouse for a month, these mouse became excited, hunted, worried and fight each other often. But After feed for three months, these mouse act come back normal as usual, and their weights were no obvious change.
6. The mutagenic effect of five mutagens on BM10 were tested, and the results showed the strongest mutagen is NTG , then are UV, LiCl, DES and microwave in turn. The results also showed the optimal mutagen was NTG, its optimal dosage was about 1 mg/mL.
7. Six antibiotic high-yield mutant of BM10 were screened out in first mutation breeding. They are L0.2-89、W250-45、N1-60-20、U90-82、N1.5-30-1 and N1-45-9, and their inhibitory effects against Focr4 increased 29.47%, 33.93%, 35.72%, 35.72%, 38.84% and 45.65%, respectively, compared with BM10 strain.
8. The mutant strain N1-45-9 was chosen to carry out second complex mutation by method of treated with 0.2%LiCl plus UV. The results of fermentation screen showed that only one high antibiotic yield mutant strain LU68 was screened out. It’s inhibitory effects increased 3.51% compared with N1-45-9 and increased 46.71% compared with BM10.
9. The mutant strain LU68 was chosen to carry out third complex mutation breeding by method of treated with UV plus NTG. The results of fermentation screening showed that five antibiotic high-yield mutant strains: UN82、UN71、UN78、UN84 and UN83 were screened out. Their inhibitory effects were increased by 5.18%、5.60%、6.07%、8.99% and 12.26%, respectively, compared with LU68, and increased by 48.33%,48.75%,49.22%,52.14% and 55.41%, compared with BM10.
引文
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