枯草芽孢杆菌磁场诱变株对采后主要果实病害的控制效果研究
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摘要
通过枯草芽孢杆菌磁场诱变株对采后苹果炭疽病、番茄灰霉病、柑橘青霉病的控制效果试验设计,初步研究了枯草芽孢杆菌磁场诱变株对果实采后主要病害的控制效果及作用机制,同时还对枯草芽孢杆菌的生物学特性进行了研究,本论文的研究结果如下:
1高效拮抗菌筛选
PDA平板拮抗测定表明,有7株磁场诱变拮抗菌的抑菌效果好于未经磁场诱变的枯草芽孢杆菌BSCK,分别是BS0.05-1,BS0.25-4,BS0.25-5,BS0.25-7,BS0.25-1,BSO.10—1,BSl.25。经过显著性分析,BSO.25—1,BSO.10—1,BSl.25,3株拮抗菌为高效拮抗菌株。25℃培养5天后,3株拮抗菌的抑菌效果分别是61.20%,61.59%和61.97%,显著地好于对照BSCK。
2高效拮抗菌对采后果实主要病害的控制效果研究
2.1磁场高效拮抗菌株对苹果炭疽病的控制效果研究
研究表明,20℃和10℃下各处理总体防效均呈现下降趋势,10℃下各处理防效高于20℃下各处理防效,两种储藏温度下先接种拮抗菌的处理防效高于先接种病原菌的处理;20℃时两种接种方式下,均以接种BS1.25的处理防效最高,接种后13天其防效分别为30.45%、14.16%;10℃先接种拮抗菌时,接种BS1.25的处理防效最高,接种后30天其防效为82.46%;先接种病原菌时,以接种BS0.10-1的处理防效最高,其防效达54.39%。
高效拮抗菌对苹果炭疽病的总体防效在10℃、变温储藏、及20℃下依次降低,以储藏在10℃时总体防效最高,20℃时总体防效最低。说明温度在很大程度上影响了枯草芽孢杆菌对苹果炭疽病的控制效果。
2.2磁场高效拮抗菌株对番茄灰霉病的控制效果研究
贮藏温度明显影响拮抗菌在番茄果实上对灰霉病的控病效果。试验结果表明,贮藏在20℃下果实发病较早。先接种拮抗菌时,果实贮藏在20℃下的发病率显著较10℃的低,贮藏6天后,BS1.25在两种温度下的发病率分别是17%和100%。贮藏温度也影响果实病斑直径的发展。先接种拮抗菌的处理,贮藏在20℃下的果实,其病斑直径和防效均显著小于贮藏在10℃下的果实,20℃接种BS1.25的处理第五天其病斑直径为0cm,防效达100%,而在10℃下其病斑直径为0.93cm,防效仅为51.56%。
2.3磁场高效拮抗菌株对柑橘青霉病的控制效果研究
10℃时拮抗菌对青霉病的防效要高于20℃时拮抗菌对青霉病的防效,说明低温抑制了病原菌的生长;先接种拮抗菌的处理防效高于先接种病原菌的处理,可能是由于先接种拮抗菌时,拮抗菌首先抢占伤口处的营养资源,而不利于病原菌生长所致。20℃两种接种方式下,均以接种BS1.25的处理防效最高,其防效分别为44.51%、32.08%。10℃两种接种方式下,各处理防效仍以接种BS1.25的处理防效最高,其防效分别为59.79%、38.10%。
3寄主主要防御酶活性的测定
研究了苹果和番茄果实分别接种炭疽菌(Colletotrichum gloeosporioides)和灰霉菌(Botrytis cinerea Pers.)后,主要防御酶过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)的活性变化。结果表明:苹果和番茄感染病原菌或者被接种拮抗菌均能引起采后果实主要防御酶活性的升高,在贮藏中期均会出现酶活高峰。但酶活的升高是否与寄主的抗性呈正相关还有待近一步的研究。
4枯草芽孢杆菌生物学特性的研究
BSCK菌株和BS1.25菌株在接种的前36小时处于对数生长期,其菌体的生物量在不断增加,在培养36小时后菌量达到峰值;随着培养液PH值由低到高,BSCK菌株和BS1.25菌株的菌体量呈现先升高再下降的趋势;BSCK菌株和Bs1.25菌株在PH值为6至7.5时生长较好,BS1.25菌株在PH值等于6.5时菌体量达到峰值,而BSCK菌株在PH值为7时菌体量达到峰值;BSCK菌株和BS1.25菌株对不同碳源的利用趋势基本相似,当以麦芽糖作为碳源时,两株菌株均生长的较好,其菌体量是其它碳源的2至5倍;而以D-果糖和D-山梨醇作为碳源却很难被两株菌株利用。两株菌株对氮源的利用以酵母粉最好,其次是胰蛋白胨和牛肉浸膏。而无机氮源的效果不理想,尿素和氯化铵作为氮源几乎不被枯草芽孢杆菌所利用。
5枯草芽孢杆菌控病机制的研究
利用枯草芽孢杆菌的不同处理液抑制果实采后主要病害的发展,结果表明:拮抗菌的各处理液对果实采后主要病害有明显的抑制作用,说明枯草芽孢杆菌的活菌体以及分泌的代谢物质具有抑菌活性。拮抗菌BSCK各处理液的抑菌效果要略好于BS1.25,且拮抗菌的菌悬液的抑制效果要好于滤液和高温灭菌液;但接种高温灭菌液处理的果实病斑直径接近对照,有的甚至高于对照,其原因可能是枯草芽孢杆菌分泌的代谢物质热稳定性较差,高温高压下容易失活,而发酵液中又有一部分养分可供病原菌利用所致。
The control effect and mechanism of bacillus subtilis against postharvest fruits diseases have been studied on basis of the test that Botrytis cinerea Pers.,Colletotrichum gloeosporioides and Penicillium italicum were controlled by bacillus subtilis after being radiated in magnetic field.We also studied the biological trait of the bacillus subtilis.The results are as follows:
1.The screening of the high efficiency antagonist
The test showed that 7 strains as magnetic antagonists provided better inhibition of pathogens than others which were not radiated in magnetic field.They are BS0.05-1, BS0.25-4,BS0.25-5,BS0.25-7,BS0.25-1,BS0.10-1 and BS1.25.Among these 7 strains, BS0.25-1,BS0.10-1 and BS 1.25 were high efficiency antagonist after the significant test. And the inhibitory rates of these 3 strains were 61.20%,61.59%,and 61.97%after being stored 5 days at 25℃.And the results are much better than the control.
2.The control effect of high efficiency antagonist on postharvest fruits disease.
2.1 The control effect of high efficiency antagonist on Apple anthracnose
The test showed that no matter at 10℃or 20℃the control effect of all the treatments went down.The control effect at 10℃is better than the one at 20℃.The treatments' control effect which inoculated antagonist first is better than the one which inoculated pathogens first.The treatment that inoculated BS1.25 provided the best control effect,its' control effect of both inoculation is 30.45%and 14.16 at 20℃.The treatment that inoculated BS 1.25 first is the best one at 10℃,its' control effect is 82.46%.
The temperature has a great influence on the control effect.The control effect at 10℃is better than the one at changed temperature.The control effect at 20℃is the lowest.
2.2 The control effect of high efficiency antagonist on Tomato grey mold.
The temperature has a great influence on the control effect.The fruits' incidence rate at 20℃is lower than the rate at 10℃.The incidence rate of the treatment that inoculated BS1.25 is 17%and 100%at the two temperatures.Also the lesion diameter will be also influenced by the temperature.
2.3 The control effect of high efficiency antagonist on Orange penicilliosis.
The control effect at 10℃is better than the control effect at 20℃.It shows that the lower temperature is beneficial for growth of the pathogens.Both kinds of inoculation at 20℃,the treatment that inoculated BS1.25 is the best one,its' control effect is 44.51%and 32.08%.And the same situation occurred at 10℃,its' control effect is 59.79%and 38.10%.
3.Major Defense Enzymes of Host
By means of mensuration the enzyme activity,the changes of activities of peroxidase (POD)、polyphenoloxidase(PPO)and phenylalanine ammonia-lyase(PAL)on apples and oranges were studied.Results showed that the enzyme activities of the hosts raised after inoculation,and during the storage there will be a peak of the enzyme activities.But the relationship between the enzyme activities and the resistance of the host should be studied further more.
4.The study of the biological trait of the bacillus subtilis
The strain of BSCK and BS1.25 are in the logarithm growth time during the first 36 hours.The quantity of the bacterium is at maximum after being cultivated for 36 hours.At the condition of PH=6.5 and PH=7,the strain of BSCK and BS1.25 can grow better severally.Maltose as a source of carbon can be utilized better by the two strains,but D-sorbierite and D-levulose are can't be utilized easily.Yeast is the best nitrogen source, the peptone and jerk cream take second place.
But the salmiac and carbamide can't be utilized by the two strains.
5.The mechanism of bacillus subtilis against postharvest fruits diseases.
Using the different kinds of liquid of bacillus subtilis to control the development of the diseases,the test showed that:the liquid of the antagonist can provide a better control that proved the antagonists themselves and its' metabolite both can control the development of the diseases.And the control effect of BSCK is better than BS1.25.The control effect of the soliquoid is better than filtrate and sterilization.
1高效拮抗菌筛选
PDA平板拮抗测定表明,有7株磁场诱变拮抗菌的抑菌效果好于未经磁场诱变的枯草芽孢杆菌BSCK,分别是BS0.05-1,BS0.25-4,BS0.25-5,BS0.25-7,BS0.25-1,BSO.10—1,BSl.25。经过显著性分析,BSO.25—1,BSO.10—1,BSl.25,3株拮抗菌为高效拮抗菌株。25℃培养5天后,3株拮抗菌的抑菌效果分别是61.20%,61.59%和61.97%,显著地好于对照BSCK。
2高效拮抗菌对采后果实主要病害的控制效果研究
2.1磁场高效拮抗菌株对苹果炭疽病的控制效果研究
研究表明,20℃和10℃下各处理总体防效均呈现下降趋势,10℃下各处理防效高于20℃下各处理防效,两种储藏温度下先接种拮抗菌的处理防效高于先接种病原菌的处理;20℃时两种接种方式下,均以接种BS1.25的处理防效最高,接种后13天其防效分别为30.45%、14.16%;10℃先接种拮抗菌时,接种BS1.25的处理防效最高,接种后30天其防效为82.46%;先接种病原菌时,以接种BS0.10-1的处理防效最高,其防效达54.39%。
高效拮抗菌对苹果炭疽病的总体防效在10℃、变温储藏、及20℃下依次降低,以储藏在10℃时总体防效最高,20℃时总体防效最低。说明温度在很大程度上影响了枯草芽孢杆菌对苹果炭疽病的控制效果。
2.2磁场高效拮抗菌株对番茄灰霉病的控制效果研究
贮藏温度明显影响拮抗菌在番茄果实上对灰霉病的控病效果。试验结果表明,贮藏在20℃下果实发病较早。先接种拮抗菌时,果实贮藏在20℃下的发病率显著较10℃的低,贮藏6天后,BS1.25在两种温度下的发病率分别是17%和100%。贮藏温度也影响果实病斑直径的发展。先接种拮抗菌的处理,贮藏在20℃下的果实,其病斑直径和防效均显著小于贮藏在10℃下的果实,20℃接种BS1.25的处理第五天其病斑直径为0cm,防效达100%,而在10℃下其病斑直径为0.93cm,防效仅为51.56%。
2.3磁场高效拮抗菌株对柑橘青霉病的控制效果研究
10℃时拮抗菌对青霉病的防效要高于20℃时拮抗菌对青霉病的防效,说明低温抑制了病原菌的生长;先接种拮抗菌的处理防效高于先接种病原菌的处理,可能是由于先接种拮抗菌时,拮抗菌首先抢占伤口处的营养资源,而不利于病原菌生长所致。20℃两种接种方式下,均以接种BS1.25的处理防效最高,其防效分别为44.51%、32.08%。10℃两种接种方式下,各处理防效仍以接种BS1.25的处理防效最高,其防效分别为59.79%、38.10%。
3寄主主要防御酶活性的测定
研究了苹果和番茄果实分别接种炭疽菌(Colletotrichum gloeosporioides)和灰霉菌(Botrytis cinerea Pers.)后,主要防御酶过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)的活性变化。结果表明:苹果和番茄感染病原菌或者被接种拮抗菌均能引起采后果实主要防御酶活性的升高,在贮藏中期均会出现酶活高峰。但酶活的升高是否与寄主的抗性呈正相关还有待近一步的研究。
4枯草芽孢杆菌生物学特性的研究
BSCK菌株和BS1.25菌株在接种的前36小时处于对数生长期,其菌体的生物量在不断增加,在培养36小时后菌量达到峰值;随着培养液PH值由低到高,BSCK菌株和BS1.25菌株的菌体量呈现先升高再下降的趋势;BSCK菌株和Bs1.25菌株在PH值为6至7.5时生长较好,BS1.25菌株在PH值等于6.5时菌体量达到峰值,而BSCK菌株在PH值为7时菌体量达到峰值;BSCK菌株和BS1.25菌株对不同碳源的利用趋势基本相似,当以麦芽糖作为碳源时,两株菌株均生长的较好,其菌体量是其它碳源的2至5倍;而以D-果糖和D-山梨醇作为碳源却很难被两株菌株利用。两株菌株对氮源的利用以酵母粉最好,其次是胰蛋白胨和牛肉浸膏。而无机氮源的效果不理想,尿素和氯化铵作为氮源几乎不被枯草芽孢杆菌所利用。
5枯草芽孢杆菌控病机制的研究
利用枯草芽孢杆菌的不同处理液抑制果实采后主要病害的发展,结果表明:拮抗菌的各处理液对果实采后主要病害有明显的抑制作用,说明枯草芽孢杆菌的活菌体以及分泌的代谢物质具有抑菌活性。拮抗菌BSCK各处理液的抑菌效果要略好于BS1.25,且拮抗菌的菌悬液的抑制效果要好于滤液和高温灭菌液;但接种高温灭菌液处理的果实病斑直径接近对照,有的甚至高于对照,其原因可能是枯草芽孢杆菌分泌的代谢物质热稳定性较差,高温高压下容易失活,而发酵液中又有一部分养分可供病原菌利用所致。
The control effect and mechanism of bacillus subtilis against postharvest fruits diseases have been studied on basis of the test that Botrytis cinerea Pers.,Colletotrichum gloeosporioides and Penicillium italicum were controlled by bacillus subtilis after being radiated in magnetic field.We also studied the biological trait of the bacillus subtilis.The results are as follows:
1.The screening of the high efficiency antagonist
The test showed that 7 strains as magnetic antagonists provided better inhibition of pathogens than others which were not radiated in magnetic field.They are BS0.05-1, BS0.25-4,BS0.25-5,BS0.25-7,BS0.25-1,BS0.10-1 and BS1.25.Among these 7 strains, BS0.25-1,BS0.10-1 and BS 1.25 were high efficiency antagonist after the significant test. And the inhibitory rates of these 3 strains were 61.20%,61.59%,and 61.97%after being stored 5 days at 25℃.And the results are much better than the control.
2.The control effect of high efficiency antagonist on postharvest fruits disease.
2.1 The control effect of high efficiency antagonist on Apple anthracnose
The test showed that no matter at 10℃or 20℃the control effect of all the treatments went down.The control effect at 10℃is better than the one at 20℃.The treatments' control effect which inoculated antagonist first is better than the one which inoculated pathogens first.The treatment that inoculated BS1.25 provided the best control effect,its' control effect of both inoculation is 30.45%and 14.16 at 20℃.The treatment that inoculated BS 1.25 first is the best one at 10℃,its' control effect is 82.46%.
The temperature has a great influence on the control effect.The control effect at 10℃is better than the one at changed temperature.The control effect at 20℃is the lowest.
2.2 The control effect of high efficiency antagonist on Tomato grey mold.
The temperature has a great influence on the control effect.The fruits' incidence rate at 20℃is lower than the rate at 10℃.The incidence rate of the treatment that inoculated BS1.25 is 17%and 100%at the two temperatures.Also the lesion diameter will be also influenced by the temperature.
2.3 The control effect of high efficiency antagonist on Orange penicilliosis.
The control effect at 10℃is better than the control effect at 20℃.It shows that the lower temperature is beneficial for growth of the pathogens.Both kinds of inoculation at 20℃,the treatment that inoculated BS1.25 is the best one,its' control effect is 44.51%and 32.08%.And the same situation occurred at 10℃,its' control effect is 59.79%and 38.10%.
3.Major Defense Enzymes of Host
By means of mensuration the enzyme activity,the changes of activities of peroxidase (POD)、polyphenoloxidase(PPO)and phenylalanine ammonia-lyase(PAL)on apples and oranges were studied.Results showed that the enzyme activities of the hosts raised after inoculation,and during the storage there will be a peak of the enzyme activities.But the relationship between the enzyme activities and the resistance of the host should be studied further more.
4.The study of the biological trait of the bacillus subtilis
The strain of BSCK and BS1.25 are in the logarithm growth time during the first 36 hours.The quantity of the bacterium is at maximum after being cultivated for 36 hours.At the condition of PH=6.5 and PH=7,the strain of BSCK and BS1.25 can grow better severally.Maltose as a source of carbon can be utilized better by the two strains,but D-sorbierite and D-levulose are can't be utilized easily.Yeast is the best nitrogen source, the peptone and jerk cream take second place.
But the salmiac and carbamide can't be utilized by the two strains.
5.The mechanism of bacillus subtilis against postharvest fruits diseases.
Using the different kinds of liquid of bacillus subtilis to control the development of the diseases,the test showed that:the liquid of the antagonist can provide a better control that proved the antagonists themselves and its' metabolite both can control the development of the diseases.And the control effect of BSCK is better than BS1.25.The control effect of the soliquoid is better than filtrate and sterilization.
引文
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