柑橘采后病害生防菌YS-1的筛选和YS-1粗提物的抑菌机理及性质研究
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摘要
在世界140个国家和地区都种植有柑橘,其中主要集中在中国、美国、巴西以及地中海沿岸的一些国家,是全球第一大水果。中国的柑橘栽培面积和产量均占世界首位,是柑橘第一大生产国。在生物技术研究方面,我国紧跟国际步伐,部分研究还处于领先水平。但在柑橘采后病害的防治研究方面,我国落后于美国、巴西、西班牙等国,原因主要是起步较晚,重视不够。所幸的是,随着人们对食品安全意识和环境保护意识的增强,我国正在加强这方面的力量配备和研究开发力度,期望在未来几年会有所突破。
本文以江西的两大特色柑橘品种--新余蜜橘和南丰蜜橘为试验材料,筛选到了对柑橘采后危害最为严重的两大真菌性病害(青霉病和绿霉病)抑制作用强的微生物拮抗菌(命名为YS-1),并对其离体和活体抑菌效果进行了研究；为了探讨菌株YS-1的发酵工艺,本文采用单因子和正交设计相结合的试验方法对菌株YS-1发酵产抗菌物质的培养条件进行了优化；为探索抗菌物质的分离纯化条件,对菌株YS-1的发酵液预处理条件的选择以及该菌株的抗菌物质进行了理化特性的研究,以期为后续抗菌物质的分离纯化研究提供理论基础。本文主要研究结果如下：
1.首先从分离、筛选着手,尝试了从不同途径,如土壤、果表、叶表及果实伤口处等分离菌株,采用离体初筛和活体复筛的方法,最终从金橘果表筛选到了一株拮抗作用较强的菌株YS-1。经形态学、生理生化、16S rDNA及rpoB基因等多重鉴定,确定该菌株属类芽孢杆菌(Paenibacillus brasilensis),序列登录号为：JQ677085。
2.通过研究菌株YS-1对柑橘青霉病菌和绿霉病菌的抑制效果,发现离体平板上,菌株YS-1的菌悬液和上清液都对这两种病原菌表现出拮抗作用,且菌悬液对绿霉病菌的抑制效果好于上清液,而上清液表现出对青霉病菌有更好的抑制效果。在活体上菌株YS-1对青霉病菌的抑制效果,上清液的抑菌效果比菌悬液的要好,且两者之问有显著差异,上清液处理过的果实发病率仅为33.74%,病斑直径为4.71mm。
3.抑菌机理研究试验发现,菌株YS-1的抑菌机理既可能与营养和空间的竟争有关,也与菌体抗生素的分泌有关,菌体本身也产生直接抑菌作用。通过对菌株YS-1抑菌谱的研究,发现其除了对柑橘青霉病原菌和绿霉病原菌表现出较强的拮抗作用外,对柑橘采后其它真菌性病害如柑橘炭疽病菌和柑橘酸腐病菌也有抑制作用,且对柑橘采后细菌性病害如柑橘溃疡病菌也有较强的抑制作用。
4.对菌株YS-1产活性物质的生物效价进行了测定,得出计算生物效价的公式,并通过单因子试验和正交设计试验相结合的方法获得拮抗菌YS-1产生物活性物质的最佳培养基配方和发酵最优条件参数。研究结果为：由抑菌圈计算生物效价的公式为Y=10(x+10.695)/8.904xn；优化后的培养基配方为可溶性淀粉7.0%,酵母提取物2.5%,NaCl0.6%, MgSO40.2%, K2HPO40.2%;优化后的培养条件为接种量4%、发酵温度30℃、发酵初始pH为6.0,培养时间为72h；确定了最佳碳源为可溶性淀粉,最佳氮源为酵母提取物。
5.探讨了发酵液的预处理方法,本文通过采用加热法和调酸法对菌株YS-1的发酵液进行预处理,得出将发酵液的pH调为3.0进行调酸处理,80℃下加热处理15min为最佳预处理条件。
6.对菌株YS-1的抗菌物质在不同溶剂中的溶解性进行了试验,推测该粗提物中的抑菌物质是一种极性较大的水溶性物质。
7.本文还对菌株YS-1抗菌物质的稳定性进行了试验研究,发现该物质能耐高温,耐酸及紫外照射,在121℃下灭菌20min, pH为3.0和紫外照射24h后抑菌活性均保持较好。对氯仿敏感,低温和常温下耐贮藏。说明菌株YS-1所产生的抗菌物质有较好的稳定性。
Citrus spp., the largest fruit crops worldwide, are cultivated in140countries and regions, mainly in China, Brazil, USA and Mediterranean countries. China occupies a prominent position in citrus industry due to the largest area and yield. We keep pace with the world in the aspect of biotechnology, part of the research in the leading level. But we are behind the USA, Brazil, Spain and other countries in the biocontrol of postharvest diseases of citrus mainly because of late starting and no enough attention. Fortunately, we are strengthening the power equipment and research efforts about the aspect along with the public's growing concern for food safety and environmental protection. We expect that the research will be a breakthrough in the next few years.
A strain of bacteria (named YS-1) that has high biocontrol efficacy on postharvest diseases of blue mold and green mold on citrus fruits was isolated and identified to be Paenibacillus brasilensis. The efficacy of YS-1for the controlling of postharvest diseases of citrus in vitro and in vivo, the optimization of the culture conditions of YS-1, the conditions selection of YS-1fermentation broth pretreatment were studied. The solubility and stability of antimicrobial substance produced from YS-1were also studied. The results are as followed:
1. A total of191isolates from the soil, the surface of fruits and leaves, the wound of fruits were screened for antagonism against Penicillium italicum and Penicillum digitatum. Bacterial YS-1having the highest biocontrol efficacy was isolated from the surface of kumquat fruit by intial selection and the farther selection. It was identified as Paenibacillus brasilensis by the Biolog microbial identification system combined with16S rDNA sequence analysis, biochemical and physiologic characteristics and rpoB sequence analysis. The Gen Bank is JQ677085.
2. There was inhibition of fermentation supernatant and cell suspension of strain YS-1on the pathogens of blue mold and green mold. The inhibition of the cell suspension on Penicillum digitatum showed significant advantages compared with the supernatant of strain YS-1, nevertheless the efficacy of the supernatant for the controlling of postharvest Penicillium italicum was better than that of the cell suspension. There was significant difference between the two treatments in vivo test, and the fruit disease incidence and lesion diameter were33.74%and4.71mm, respectively.
3. The antibacterial mechanism of strain YS-1were investigated, the results showed that it was concerned with not only the secretion of antibiotics but also the competition of nutrition and space. In addition, the strain YS-1also had a direct inhibitory effect. The antagonism spectrum of YS-1showed that the strain YS-1inhibited not only fungal diseases such as blue mold, green mold and anthracnose but also bacterial diseases such as citrus canker.
4. Biological potency of active substances produced by strain YS-1was measured and calculated from the inhibition diameter. And the formula is: Y=l0(x+I0.695)/8.904. Optimization of culture medium and fermentation conditions was studied through the combination of one-factor-at-a-time test and orthogonal design test. The optimal medium was consisted of soluble starch7%, yeast extract2.5%, NaCl0.6%, MgSO40.2%, K2HPO40.2%; the optimal fermentation conditions was4%inoculum, fermentation temperature30℃, initial pH of fermentation6.0, culture time was72h; the best carbon source and nitrogen source was soluble starch and yeast extract, respectively.
5. The methods of fermentation broth pretreatment were discussed by heating and adjustment acidity. The results showed that the suitable pretreatment methods were adjustment the pH of the fermentation broth to3.0and heat treatment for15min at80℃.
6. The solubility of the antagonistic substances of the strain YS-1in different solvents were studied. The result showed that the material is a kind of water-soluble substances being of high polarity.
7. The stability of YS-1antagonistic substances was studied. The results showed that there was resistance to high temperature, acid and ultraviolet radiation. The antagonistic substances showed better antagonistic activity at121℃for20min, pH low to3.0, Ultraviolet irradiation for24h and low temperature storage for a month. But it was sensitive to chloroform.
本文以江西的两大特色柑橘品种--新余蜜橘和南丰蜜橘为试验材料,筛选到了对柑橘采后危害最为严重的两大真菌性病害(青霉病和绿霉病)抑制作用强的微生物拮抗菌(命名为YS-1),并对其离体和活体抑菌效果进行了研究；为了探讨菌株YS-1的发酵工艺,本文采用单因子和正交设计相结合的试验方法对菌株YS-1发酵产抗菌物质的培养条件进行了优化；为探索抗菌物质的分离纯化条件,对菌株YS-1的发酵液预处理条件的选择以及该菌株的抗菌物质进行了理化特性的研究,以期为后续抗菌物质的分离纯化研究提供理论基础。本文主要研究结果如下：
1.首先从分离、筛选着手,尝试了从不同途径,如土壤、果表、叶表及果实伤口处等分离菌株,采用离体初筛和活体复筛的方法,最终从金橘果表筛选到了一株拮抗作用较强的菌株YS-1。经形态学、生理生化、16S rDNA及rpoB基因等多重鉴定,确定该菌株属类芽孢杆菌(Paenibacillus brasilensis),序列登录号为：JQ677085。
2.通过研究菌株YS-1对柑橘青霉病菌和绿霉病菌的抑制效果,发现离体平板上,菌株YS-1的菌悬液和上清液都对这两种病原菌表现出拮抗作用,且菌悬液对绿霉病菌的抑制效果好于上清液,而上清液表现出对青霉病菌有更好的抑制效果。在活体上菌株YS-1对青霉病菌的抑制效果,上清液的抑菌效果比菌悬液的要好,且两者之问有显著差异,上清液处理过的果实发病率仅为33.74%,病斑直径为4.71mm。
3.抑菌机理研究试验发现,菌株YS-1的抑菌机理既可能与营养和空间的竟争有关,也与菌体抗生素的分泌有关,菌体本身也产生直接抑菌作用。通过对菌株YS-1抑菌谱的研究,发现其除了对柑橘青霉病原菌和绿霉病原菌表现出较强的拮抗作用外,对柑橘采后其它真菌性病害如柑橘炭疽病菌和柑橘酸腐病菌也有抑制作用,且对柑橘采后细菌性病害如柑橘溃疡病菌也有较强的抑制作用。
4.对菌株YS-1产活性物质的生物效价进行了测定,得出计算生物效价的公式,并通过单因子试验和正交设计试验相结合的方法获得拮抗菌YS-1产生物活性物质的最佳培养基配方和发酵最优条件参数。研究结果为：由抑菌圈计算生物效价的公式为Y=10(x+10.695)/8.904xn；优化后的培养基配方为可溶性淀粉7.0%,酵母提取物2.5%,NaCl0.6%, MgSO40.2%, K2HPO40.2%;优化后的培养条件为接种量4%、发酵温度30℃、发酵初始pH为6.0,培养时间为72h；确定了最佳碳源为可溶性淀粉,最佳氮源为酵母提取物。
5.探讨了发酵液的预处理方法,本文通过采用加热法和调酸法对菌株YS-1的发酵液进行预处理,得出将发酵液的pH调为3.0进行调酸处理,80℃下加热处理15min为最佳预处理条件。
6.对菌株YS-1的抗菌物质在不同溶剂中的溶解性进行了试验,推测该粗提物中的抑菌物质是一种极性较大的水溶性物质。
7.本文还对菌株YS-1抗菌物质的稳定性进行了试验研究,发现该物质能耐高温,耐酸及紫外照射,在121℃下灭菌20min, pH为3.0和紫外照射24h后抑菌活性均保持较好。对氯仿敏感,低温和常温下耐贮藏。说明菌株YS-1所产生的抗菌物质有较好的稳定性。
Citrus spp., the largest fruit crops worldwide, are cultivated in140countries and regions, mainly in China, Brazil, USA and Mediterranean countries. China occupies a prominent position in citrus industry due to the largest area and yield. We keep pace with the world in the aspect of biotechnology, part of the research in the leading level. But we are behind the USA, Brazil, Spain and other countries in the biocontrol of postharvest diseases of citrus mainly because of late starting and no enough attention. Fortunately, we are strengthening the power equipment and research efforts about the aspect along with the public's growing concern for food safety and environmental protection. We expect that the research will be a breakthrough in the next few years.
A strain of bacteria (named YS-1) that has high biocontrol efficacy on postharvest diseases of blue mold and green mold on citrus fruits was isolated and identified to be Paenibacillus brasilensis. The efficacy of YS-1for the controlling of postharvest diseases of citrus in vitro and in vivo, the optimization of the culture conditions of YS-1, the conditions selection of YS-1fermentation broth pretreatment were studied. The solubility and stability of antimicrobial substance produced from YS-1were also studied. The results are as followed:
1. A total of191isolates from the soil, the surface of fruits and leaves, the wound of fruits were screened for antagonism against Penicillium italicum and Penicillum digitatum. Bacterial YS-1having the highest biocontrol efficacy was isolated from the surface of kumquat fruit by intial selection and the farther selection. It was identified as Paenibacillus brasilensis by the Biolog microbial identification system combined with16S rDNA sequence analysis, biochemical and physiologic characteristics and rpoB sequence analysis. The Gen Bank is JQ677085.
2. There was inhibition of fermentation supernatant and cell suspension of strain YS-1on the pathogens of blue mold and green mold. The inhibition of the cell suspension on Penicillum digitatum showed significant advantages compared with the supernatant of strain YS-1, nevertheless the efficacy of the supernatant for the controlling of postharvest Penicillium italicum was better than that of the cell suspension. There was significant difference between the two treatments in vivo test, and the fruit disease incidence and lesion diameter were33.74%and4.71mm, respectively.
3. The antibacterial mechanism of strain YS-1were investigated, the results showed that it was concerned with not only the secretion of antibiotics but also the competition of nutrition and space. In addition, the strain YS-1also had a direct inhibitory effect. The antagonism spectrum of YS-1showed that the strain YS-1inhibited not only fungal diseases such as blue mold, green mold and anthracnose but also bacterial diseases such as citrus canker.
4. Biological potency of active substances produced by strain YS-1was measured and calculated from the inhibition diameter. And the formula is: Y=l0(x+I0.695)/8.904. Optimization of culture medium and fermentation conditions was studied through the combination of one-factor-at-a-time test and orthogonal design test. The optimal medium was consisted of soluble starch7%, yeast extract2.5%, NaCl0.6%, MgSO40.2%, K2HPO40.2%; the optimal fermentation conditions was4%inoculum, fermentation temperature30℃, initial pH of fermentation6.0, culture time was72h; the best carbon source and nitrogen source was soluble starch and yeast extract, respectively.
5. The methods of fermentation broth pretreatment were discussed by heating and adjustment acidity. The results showed that the suitable pretreatment methods were adjustment the pH of the fermentation broth to3.0and heat treatment for15min at80℃.
6. The solubility of the antagonistic substances of the strain YS-1in different solvents were studied. The result showed that the material is a kind of water-soluble substances being of high polarity.
7. The stability of YS-1antagonistic substances was studied. The results showed that there was resistance to high temperature, acid and ultraviolet radiation. The antagonistic substances showed better antagonistic activity at121℃for20min, pH low to3.0, Ultraviolet irradiation for24h and low temperature storage for a month. But it was sensitive to chloroform.
引文
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