蔬菜病害生防菌的筛选、鉴定及其拮抗物质理化性质研究
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摘要
目前,国内外防治蔬菜病害以化学防治为主。化学农药的大量使用,在控制病害同时也造成了环境污染、农产品有毒物质残留、危害人类健康和病原菌产生抗药性等问题,严重制约了农业的可持续发展。因此,寻找安全、有效的病害防治途径,是当前病害防治研究的重点。研究表明,生物防治是其中一种有效的防治途径。本研究从蔬菜根际土壤中分离筛选到一株具广谱抗性和促生长作用的生防细菌XJ。从菌种的分离筛选、鉴定、摇瓶发酵工艺和拮抗物质理化性质等方面进行了研究。结果如下：
1.采用梯度稀释分离法,从浙江、福建、江西等地采集的160多份蔬菜根际土壤中分离获得712株细菌。以禾谷镰刀菌为指示菌,采用平板对峙法从分离到的712株细菌中筛选获得30株可以抑制禾谷镰刀菌生长的生防菌,其中有10株细菌对病原菌的抑制率达70%以上。采用平皿促生长法从30株生防菌中筛选出9株能促进黄瓜根生长的菌株。
2.采用温室盆栽试验对初筛获得的9株生防菌进行促生试验。结果表明,9株生防菌对黄瓜的生长都有促进作用,其中以AT2的促生长效果最好。通过温室盆栽试验筛选在黄瓜植株上有防病效果的生防菌。试验结果表明,生防菌XJ、DE2、BN8 3个菌株对黄瓜炭疽病都有一定防治效果,其中以菌株XJ的防治效果最好。
3.采用形态特征及生理生化检测和16S rDNA序列分析等方法,对生防菌XJ进行菌种鉴定。形态特征和生理生化鉴定显示,XJ属于枯草芽孢杆菌(Bacillus subtilis)；16S rDNA序列测定和分析结果表明,XJ与枯草芽孢杆菌(B.subtilis)的同源性达99%。因此,两种鉴定方法的结果一致,菌株XJ为枯草芽孢杆菌(B.subtilis)。
4.通过单因素试验和正交试验对菌株XJ的发酵培养基进行优化,结果表明,XJ菌株的发酵培养基的最佳配方为：蔗糖3%,蛋白胨1.5%,黄豆饼粉1%,K2HPO40.4%。同时对菌株XJ的发酵条件进行优化,结果表明XJ菌株最佳发酵条件为：发酵温度是37℃,初始pH值为9.0,接种量为2%,装液量为75mL/300 mL,摇床转速为200 rpm,发酵时间24 h。
5.通过对XJ的拮抗物质的定位研究发现,XJ产生的拮抗物质是胞外物质。通过硫酸铵分级盐析法对XJ产生的拮抗物质进行了提取,发现当硫酸铵饱和度为20%时,沉淀的拮抗活性最大,随着硫酸铵饱和度增加,沉淀的拮抗活性逐渐减弱。当硫酸铵饱和度达到80%时,沉淀基本无拮抗活性,因此,采用60%饱和度的硫酸铵基本可以将活性物质全部沉淀下来。对拮抗物质的理化性质初步研究表明,该拮抗物质在121℃高温处理30 min后,拮抗活性基本不变,121℃高温处理1 h后,拮抗活性下降为67.13%,说明该拮抗物质对高温相对稳定；在pH4-10之间活性稳定,pH小于4,抑菌活性有所下降,pH大于或等于12时,活性完全丧失,说明活性物质在酸性和中性条件下稳定；活性物质对蛋白酶K部分敏感。
At present, chemical control is the major way to prevent vegetables from diseases. The extensive use of chemical pesticides has caused a series of problem, such as environmental pollution, toxic residues in agricultural products, threatening human health and causing pathogen resistance, which has seriously hampered the sustainable development of agriculture. Therefore, finding safer and more effective technique is urgent to solve the above problem. Biological control is one of the most effective technique.
As a biocontrol bacterium XJ, which is isolated from the rhizosphere soil of vegetables, has a broad-spectrum resistance to vegetables diseases and it can also promote plant growth. XJ was studied in the following aspects:isolation and screening, identification, optimization of fermentation technology, physical and chemical properties of antimicrobial substance in the fermented products, etc. Results are as follows:
With gradient diluting soil isolating method,712 bacteria strains were isolated from 162 vegetables rhizosphere soils in Zhejiang, Fujian, Jiangxi and some other provinces.
Using Fusarium graminearum as the tested strain,30 antagonistic strains were obtained from 712 isolated strains by hyphal extension inhibition assay. Inhibitory rate of 10 antagonistic strains against Fusarium graminearum are more than 70%. Using growth-promoting test,9 strains were obtained as the potential biocontrol agents after screening of 30 strains, which promoted the growth of cucumber roots.
In greenhouse test on growth-promoting, the biocontrol agents from 9 strains promoted the growth of cucumber. In greenhouse test of the disease contron, biocontrol potential strains XJ, DE2, BN8 showed antagonistic ability against Colletotrichum lagenarium and XJ showed the strongest effect.
XJ was identified according to its morphological, physiological and biochemical characteristics, as well as 16S rDNA sequence analysis. Morphological characteristics and physiological and biochemical testing results showed that XJ belongs to Bacillus subtilis. The results of 16S rDNA sequencing and analysis showed that the homology was 99% between XJ and Bacillus subtilis. Results were consistent with the two methods. XJ is confirmed to be Bacillus subtilis.
By single factor and orthogonal test, the optimal fermentation medium was screened. The results showed that the liquid medium of XJ fermentation product was constituted by sucrose 3%, peptone 1.5%, soybean flour 1%, K2HPO4 0.4%. Meanwhile, fermentation conditions of strain XJ were optimized. The results showed that the optimal fermentation conditions were:fermentation temperature 37℃, initial pH 9.0, inoculation seed liquid 2%, loading liquid volume 75 mL in 300 mL flasks, shaker rotative velocity 200 rpm and fermentation time 24h.
The results showed that antagonistic substances produced by XJ fermentation product are in the supernant fluid of the culture. Antagonistic substances separated out from ammonium sulfate fractionation. When ammonium sulfate saturation was 20%, the antagonistic activity of the precipitation was the strongest. With the increase of saturation, the antagonistic activity of precipitation was gradually weakened. When the saturation was up to 80%, the precipitation did not show any antagonistic activity. Therefore, All of the active substances could be deposited after adopting 60% saturated solution of ammonium sulfate.
Physical and chemical properties of antagonistic substances were studied. The results showed that the antagonistic activity remained when the antagonistic substances were treated at 121℃for 30 min. The antagonistic activity reduced to 67.13% with 121℃for 1 h treatment. This indicated that the antagonistic substances were thermal stable. The antagonistic substances were also stable in the pH range from 4 to 10. The active substances were partially sensitive to proteinase-K.
1.采用梯度稀释分离法,从浙江、福建、江西等地采集的160多份蔬菜根际土壤中分离获得712株细菌。以禾谷镰刀菌为指示菌,采用平板对峙法从分离到的712株细菌中筛选获得30株可以抑制禾谷镰刀菌生长的生防菌,其中有10株细菌对病原菌的抑制率达70%以上。采用平皿促生长法从30株生防菌中筛选出9株能促进黄瓜根生长的菌株。
2.采用温室盆栽试验对初筛获得的9株生防菌进行促生试验。结果表明,9株生防菌对黄瓜的生长都有促进作用,其中以AT2的促生长效果最好。通过温室盆栽试验筛选在黄瓜植株上有防病效果的生防菌。试验结果表明,生防菌XJ、DE2、BN8 3个菌株对黄瓜炭疽病都有一定防治效果,其中以菌株XJ的防治效果最好。
3.采用形态特征及生理生化检测和16S rDNA序列分析等方法,对生防菌XJ进行菌种鉴定。形态特征和生理生化鉴定显示,XJ属于枯草芽孢杆菌(Bacillus subtilis)；16S rDNA序列测定和分析结果表明,XJ与枯草芽孢杆菌(B.subtilis)的同源性达99%。因此,两种鉴定方法的结果一致,菌株XJ为枯草芽孢杆菌(B.subtilis)。
4.通过单因素试验和正交试验对菌株XJ的发酵培养基进行优化,结果表明,XJ菌株的发酵培养基的最佳配方为：蔗糖3%,蛋白胨1.5%,黄豆饼粉1%,K2HPO40.4%。同时对菌株XJ的发酵条件进行优化,结果表明XJ菌株最佳发酵条件为：发酵温度是37℃,初始pH值为9.0,接种量为2%,装液量为75mL/300 mL,摇床转速为200 rpm,发酵时间24 h。
5.通过对XJ的拮抗物质的定位研究发现,XJ产生的拮抗物质是胞外物质。通过硫酸铵分级盐析法对XJ产生的拮抗物质进行了提取,发现当硫酸铵饱和度为20%时,沉淀的拮抗活性最大,随着硫酸铵饱和度增加,沉淀的拮抗活性逐渐减弱。当硫酸铵饱和度达到80%时,沉淀基本无拮抗活性,因此,采用60%饱和度的硫酸铵基本可以将活性物质全部沉淀下来。对拮抗物质的理化性质初步研究表明,该拮抗物质在121℃高温处理30 min后,拮抗活性基本不变,121℃高温处理1 h后,拮抗活性下降为67.13%,说明该拮抗物质对高温相对稳定；在pH4-10之间活性稳定,pH小于4,抑菌活性有所下降,pH大于或等于12时,活性完全丧失,说明活性物质在酸性和中性条件下稳定；活性物质对蛋白酶K部分敏感。
At present, chemical control is the major way to prevent vegetables from diseases. The extensive use of chemical pesticides has caused a series of problem, such as environmental pollution, toxic residues in agricultural products, threatening human health and causing pathogen resistance, which has seriously hampered the sustainable development of agriculture. Therefore, finding safer and more effective technique is urgent to solve the above problem. Biological control is one of the most effective technique.
As a biocontrol bacterium XJ, which is isolated from the rhizosphere soil of vegetables, has a broad-spectrum resistance to vegetables diseases and it can also promote plant growth. XJ was studied in the following aspects:isolation and screening, identification, optimization of fermentation technology, physical and chemical properties of antimicrobial substance in the fermented products, etc. Results are as follows:
With gradient diluting soil isolating method,712 bacteria strains were isolated from 162 vegetables rhizosphere soils in Zhejiang, Fujian, Jiangxi and some other provinces.
Using Fusarium graminearum as the tested strain,30 antagonistic strains were obtained from 712 isolated strains by hyphal extension inhibition assay. Inhibitory rate of 10 antagonistic strains against Fusarium graminearum are more than 70%. Using growth-promoting test,9 strains were obtained as the potential biocontrol agents after screening of 30 strains, which promoted the growth of cucumber roots.
In greenhouse test on growth-promoting, the biocontrol agents from 9 strains promoted the growth of cucumber. In greenhouse test of the disease contron, biocontrol potential strains XJ, DE2, BN8 showed antagonistic ability against Colletotrichum lagenarium and XJ showed the strongest effect.
XJ was identified according to its morphological, physiological and biochemical characteristics, as well as 16S rDNA sequence analysis. Morphological characteristics and physiological and biochemical testing results showed that XJ belongs to Bacillus subtilis. The results of 16S rDNA sequencing and analysis showed that the homology was 99% between XJ and Bacillus subtilis. Results were consistent with the two methods. XJ is confirmed to be Bacillus subtilis.
By single factor and orthogonal test, the optimal fermentation medium was screened. The results showed that the liquid medium of XJ fermentation product was constituted by sucrose 3%, peptone 1.5%, soybean flour 1%, K2HPO4 0.4%. Meanwhile, fermentation conditions of strain XJ were optimized. The results showed that the optimal fermentation conditions were:fermentation temperature 37℃, initial pH 9.0, inoculation seed liquid 2%, loading liquid volume 75 mL in 300 mL flasks, shaker rotative velocity 200 rpm and fermentation time 24h.
The results showed that antagonistic substances produced by XJ fermentation product are in the supernant fluid of the culture. Antagonistic substances separated out from ammonium sulfate fractionation. When ammonium sulfate saturation was 20%, the antagonistic activity of the precipitation was the strongest. With the increase of saturation, the antagonistic activity of precipitation was gradually weakened. When the saturation was up to 80%, the precipitation did not show any antagonistic activity. Therefore, All of the active substances could be deposited after adopting 60% saturated solution of ammonium sulfate.
Physical and chemical properties of antagonistic substances were studied. The results showed that the antagonistic activity remained when the antagonistic substances were treated at 121℃for 30 min. The antagonistic activity reduced to 67.13% with 121℃for 1 h treatment. This indicated that the antagonistic substances were thermal stable. The antagonistic substances were also stable in the pH range from 4 to 10. The active substances were partially sensitive to proteinase-K.
引文
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