培养基工艺对哈茨木霉H-13发酵产物及作用效果的研究
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摘要
化肥和农药的过量施用,造成土壤有机质不足、板结、生态结构破坏、导致耕作环境恶化,农产品有机磷、氯超标。根据国际上所提出“可持续农业”,近年来,我国生物肥料的研究和开发利用发展迅速,提出了发展“生态农业”的规划,提倡施用微生物肥料,促进研究和开发,实现改善土壤结构,控制农残超标。
哈茨木霉(Trichoderma harzianum)是木霉属中应用最为广泛的真菌,具有调节作物生长作用,其发酵产物能提高作物相关酶活性和抗衰老作用(如硝酸还原酶活力增强、丙二醛含量降低等),能促进植物对氮、磷、钾的吸收;同时与根际微生物的相互作用,优化土壤的微生物环境,促进植物生长。
本课题属国家863计划项目2003AA241171《哈茨木霉突变株H-13作物生长调节剂的产业化》,本文以N~+注入哈茨木霉后所筛选的突变株(H-13)为发酵菌株,结合黄瓜盆栽和小面积田间实验,测量萌发、生长过程和收获的生物量,采用正交法从温度、pH、培养时间、基质培养基、碳氮源、碳氮比方面研究H-13培养工艺;从培养时间、碳氮源、微量元素方面研究H-13生产赤霉素的液体发酵工艺。
结果表明,pH7、26℃、7d为该菌株最适产孢条件;pH7、28℃、8d最适菌丝生长,该菌株发酵最适培养时间为9d。培养基最适碳氮比为20:1;有机氮源较无机氮源更适于木霉H-13生长,以血红蛋白为氮源的培养基菌丝生长最好、产孢量最大;硝酸盐抑制发酵产物的产生。碳源中单糖利于菌丝生长,以葡萄糖/果糖(1:2)为最佳;双糖则利于产孢,以纤维二糖/麦芽糖(2:1)为最佳。不同碳源产赤霉素量均在0.2-0.3g/L范围内,其中以麦芽糖产赤霉素量最大,达到0.308g/L;从菌丝干重看,纤维二糖利于发酵阶段菌丝的生长。微量元素的种类、数量影响哈茨木霉H-13目的产物的合成,其中MgSO_4·7H_2O对赤霉素含量影响最大,FeSO_4 100mg/L、MgSO_4·7H_2O1g/L、K_2HPO_4 0.4g/L、NaNO_3 6g/L、CaCO_3 0.2 g/L最适于赤霉素的生成,而对菌丝干重影响不明显。
采用发酵产物梯度浓度喷施黄瓜叶面,并通过测定硝酸还原酶、丙二醛、叶绿体色素、过氧化物酶、超氧化物歧化酶(SOD),表明原发酵液浓度稀释100倍时,硝酸还原酶活力为4.807μg/g.h,丙二醛含量仅为4.155μmol/g,叶绿素的含量为1.746 mg/g,超氧物歧化酶活性为341.95μ·gF.W/min,过氧化物酶活性为50μg/(g·min)。
通过发酵液的不同稀释倍数对各时段黄瓜进行喷施,提高黄瓜生物量最佳喷施浓度为原发酵液稀释100倍,最适喷施时段为营养期(移栽至开花);生殖生长期喷施发酵液抑制黄瓜生长,增加黄瓜的死果率;种子发芽率97.33%、发芽势98.8%、苗长5.48cm、根长5.12cm、幼苗干重为8.87mg。移植后的65d左右,株高达79.35cm并开始坐果,比对照组提前一周,结实高峰期长达4周,比对照组延长两周。发酵液的喷施对单株果重量无影响,雌雄比增大,死果率小、坐果数增多是增产的主要因素,幅度高达50.8%。
综上所述,将H-13接入含0.1%血红蛋白,2%纤维二糖/麦芽糖(2:1)的制种培养基中,于pH7、26℃,培养7d,后转入添加2%麦芽糖、0.1%血红蛋白、FeSO_4 100mg/L、MgSO_4.7H_2O 1g/L、K_2HPO_4 0.4g/L、NaNO_3 6/L、CaCO_3 0.2 g/L培养基中发酵9d,将所得发酵产物稀释100倍,在营养生长期喷施植株。采用上述培养基组合发酵,相应产物的浓度在不同生长期的使用参数等工艺路线合理,黄瓜的每亩产量比对照组增加50.8%
The over-used of chemical fertilizer and the pesticide had caused inadequate organic matter,hardening of soil,organism's structure worsened,cultivation environment destroyed,leading to cultivation,the organic phosphorus and chlorine of agricultural product gone beyond standard.In recent years,according to "the sustainable agriculture ",which was brought forward by the international,our country's bio-fertilizer research and exploitation developed fastly,brought up the plan of "eco-agriculture ", microorganism-fertilizer research and development was advocated,which will improvement soil structure and controll residual pesticide beyond standard.
Trichoderma harzianum is the most widely used fungi in Trichoderma,it can adjust crop growth,its fermentation product can increase crop-related enzyme's activity and resist aging effects(such as nitratase's activity increased,MDA's content decreased,etc.).it also can promote plant uptake nitrogen,phosphorus and potassium;Meanwhile it can inhibit proliferation of pathogenic fungi,optimize the soil microbial environment,reduce the incidence of pests and diseases with the rhizosphere microbial interactions,so it can promote plant growth.
This research used H-13,which is selected after N~+ iron implantation Trichoderma harzianum,for the fermentation bacteria.Measure biomasses of bourgeon and growth with a small pot field experiments.Research H-13's culture conditionsfrom temperature,pH, incubation time,substrate media,the source of carbon and nitrogen,the ratio of carbon and nitrogen by orthogonality experiment.Study the liquid fermentation conditions from the incubation time,carbon and nitrogen sources and trace element.
The results showed that the optimum conditions of sporulation and mycelial growth are pH7,26℃,7 days and pH7,28℃,8days,respectively.The optimum fermentation time is 9days.the optimum ratio of carbon and nitrogen is 20:1;organonitrogen is more suitable for H-13 growth than inorganic nitrogen,the best nitrogen of medium for mycelial growth and sporulation is hemoglobin;Nitrate inhibit generation of fermentation products. In Carbon,monosaccharide is good for mycelial growth,glucose/fructose(1:2)as the best; Disaccharide is good for sporulation,cellobiose/maltose(2:1) as the best.the output of GA is within 0.2-0.3g/L in different carbon,maltose is the best for form of GA,the output can get 0.308g/L;according to the dry weight of mycelium,cellobiose is benefit for mycelial growth during the stage of fermentation,the kind and quantity of trace Elements can impact synthesis of fermentation product,MgSO_4.7H_2O is most serious.The optimum conditions for the form of gibberellin are FeSO_4100mg/L,MgSO_4.7H_2O1g/L, K2HPO_40.4g/L,NaNO_36g/L,CaCO30.2 g/L,but this conditions don't impact dry mycelium.
Spraying diffirent concentration of fermentation product on cucumber leaf,measure nitrate reductase,MDA,chloroplast pigment,peroxidase,superoxide dismutase(SOD),the results show that when concentration as 2.4～4.8μg/L,nitrate reductase activity can get 4.807μg/g.h,MDA content is only 4.155μg,The chlorophyll content is 1.746mg/g. Superoxide dismutase activity get 341.95μgF.W/min,Peroxidase activity get 50μg /(g.min).
Spray diffirent dilution multiples of fermentation on different stage of cucumber,the results show that the best conditions for increasing cucumber biomass are the 100 multiple as the best concentraton and trophophase as the best spraying stage,concentration of spraying the best original broth diluted 100 times.When spray fermentation in reproductive stage,it will inhibit growth of cucumber,and increase the death rate of fruit; Seed germination rate is 97.33%,germination potential is 98.8%,the long of plant is 5.48cm,the long of rootis 5.12cm,dry weight of seedling is 8.87mg.After transplantation about 65days,the heightof plant up to 79.35cm and begin fruiting.it is earlier one week than the control group,solid peak for four weeks,it prolong two weeks than the control group.but fermentation did not affect single plant fruit.The major factor of increasing is the sex ratio increased,death rate of fruit decreased and fruiting production increased, the extent of incresing is as high as 50.8%.
To sum up,H-13 inoculate in the culture with the content of 0.1%hemoglobin and cellobiose / maltose(2:1),after culture 7d at pH7、26℃,it was switched into another liquid culture,which 0.1%hemoglobin,sucrose / maltose(2:1)、FeSO_4 100mg/L, MgSO_4.7H_2O 1g/L,K_2HPO_4 0.4g/L,NaNO_3 6g/L and CaCO_3 0.2 g/L in it,then spray the cucumber with the 100 multiples of fermentation dilution at trophophase.With the combination descried above,it is logic for the results of-differents concentrations and increased 50.8%per mu.
哈茨木霉(Trichoderma harzianum)是木霉属中应用最为广泛的真菌,具有调节作物生长作用,其发酵产物能提高作物相关酶活性和抗衰老作用(如硝酸还原酶活力增强、丙二醛含量降低等),能促进植物对氮、磷、钾的吸收;同时与根际微生物的相互作用,优化土壤的微生物环境,促进植物生长。
本课题属国家863计划项目2003AA241171《哈茨木霉突变株H-13作物生长调节剂的产业化》,本文以N~+注入哈茨木霉后所筛选的突变株(H-13)为发酵菌株,结合黄瓜盆栽和小面积田间实验,测量萌发、生长过程和收获的生物量,采用正交法从温度、pH、培养时间、基质培养基、碳氮源、碳氮比方面研究H-13培养工艺;从培养时间、碳氮源、微量元素方面研究H-13生产赤霉素的液体发酵工艺。
结果表明,pH7、26℃、7d为该菌株最适产孢条件;pH7、28℃、8d最适菌丝生长,该菌株发酵最适培养时间为9d。培养基最适碳氮比为20:1;有机氮源较无机氮源更适于木霉H-13生长,以血红蛋白为氮源的培养基菌丝生长最好、产孢量最大;硝酸盐抑制发酵产物的产生。碳源中单糖利于菌丝生长,以葡萄糖/果糖(1:2)为最佳;双糖则利于产孢,以纤维二糖/麦芽糖(2:1)为最佳。不同碳源产赤霉素量均在0.2-0.3g/L范围内,其中以麦芽糖产赤霉素量最大,达到0.308g/L;从菌丝干重看,纤维二糖利于发酵阶段菌丝的生长。微量元素的种类、数量影响哈茨木霉H-13目的产物的合成,其中MgSO_4·7H_2O对赤霉素含量影响最大,FeSO_4 100mg/L、MgSO_4·7H_2O1g/L、K_2HPO_4 0.4g/L、NaNO_3 6g/L、CaCO_3 0.2 g/L最适于赤霉素的生成,而对菌丝干重影响不明显。
采用发酵产物梯度浓度喷施黄瓜叶面,并通过测定硝酸还原酶、丙二醛、叶绿体色素、过氧化物酶、超氧化物歧化酶(SOD),表明原发酵液浓度稀释100倍时,硝酸还原酶活力为4.807μg/g.h,丙二醛含量仅为4.155μmol/g,叶绿素的含量为1.746 mg/g,超氧物歧化酶活性为341.95μ·gF.W/min,过氧化物酶活性为50μg/(g·min)。
通过发酵液的不同稀释倍数对各时段黄瓜进行喷施,提高黄瓜生物量最佳喷施浓度为原发酵液稀释100倍,最适喷施时段为营养期(移栽至开花);生殖生长期喷施发酵液抑制黄瓜生长,增加黄瓜的死果率;种子发芽率97.33%、发芽势98.8%、苗长5.48cm、根长5.12cm、幼苗干重为8.87mg。移植后的65d左右,株高达79.35cm并开始坐果,比对照组提前一周,结实高峰期长达4周,比对照组延长两周。发酵液的喷施对单株果重量无影响,雌雄比增大,死果率小、坐果数增多是增产的主要因素,幅度高达50.8%。
综上所述,将H-13接入含0.1%血红蛋白,2%纤维二糖/麦芽糖(2:1)的制种培养基中,于pH7、26℃,培养7d,后转入添加2%麦芽糖、0.1%血红蛋白、FeSO_4 100mg/L、MgSO_4.7H_2O 1g/L、K_2HPO_4 0.4g/L、NaNO_3 6/L、CaCO_3 0.2 g/L培养基中发酵9d,将所得发酵产物稀释100倍,在营养生长期喷施植株。采用上述培养基组合发酵,相应产物的浓度在不同生长期的使用参数等工艺路线合理,黄瓜的每亩产量比对照组增加50.8%
The over-used of chemical fertilizer and the pesticide had caused inadequate organic matter,hardening of soil,organism's structure worsened,cultivation environment destroyed,leading to cultivation,the organic phosphorus and chlorine of agricultural product gone beyond standard.In recent years,according to "the sustainable agriculture ",which was brought forward by the international,our country's bio-fertilizer research and exploitation developed fastly,brought up the plan of "eco-agriculture ", microorganism-fertilizer research and development was advocated,which will improvement soil structure and controll residual pesticide beyond standard.
Trichoderma harzianum is the most widely used fungi in Trichoderma,it can adjust crop growth,its fermentation product can increase crop-related enzyme's activity and resist aging effects(such as nitratase's activity increased,MDA's content decreased,etc.).it also can promote plant uptake nitrogen,phosphorus and potassium;Meanwhile it can inhibit proliferation of pathogenic fungi,optimize the soil microbial environment,reduce the incidence of pests and diseases with the rhizosphere microbial interactions,so it can promote plant growth.
This research used H-13,which is selected after N~+ iron implantation Trichoderma harzianum,for the fermentation bacteria.Measure biomasses of bourgeon and growth with a small pot field experiments.Research H-13's culture conditionsfrom temperature,pH, incubation time,substrate media,the source of carbon and nitrogen,the ratio of carbon and nitrogen by orthogonality experiment.Study the liquid fermentation conditions from the incubation time,carbon and nitrogen sources and trace element.
The results showed that the optimum conditions of sporulation and mycelial growth are pH7,26℃,7 days and pH7,28℃,8days,respectively.The optimum fermentation time is 9days.the optimum ratio of carbon and nitrogen is 20:1;organonitrogen is more suitable for H-13 growth than inorganic nitrogen,the best nitrogen of medium for mycelial growth and sporulation is hemoglobin;Nitrate inhibit generation of fermentation products. In Carbon,monosaccharide is good for mycelial growth,glucose/fructose(1:2)as the best; Disaccharide is good for sporulation,cellobiose/maltose(2:1) as the best.the output of GA is within 0.2-0.3g/L in different carbon,maltose is the best for form of GA,the output can get 0.308g/L;according to the dry weight of mycelium,cellobiose is benefit for mycelial growth during the stage of fermentation,the kind and quantity of trace Elements can impact synthesis of fermentation product,MgSO_4.7H_2O is most serious.The optimum conditions for the form of gibberellin are FeSO_4100mg/L,MgSO_4.7H_2O1g/L, K2HPO_40.4g/L,NaNO_36g/L,CaCO30.2 g/L,but this conditions don't impact dry mycelium.
Spraying diffirent concentration of fermentation product on cucumber leaf,measure nitrate reductase,MDA,chloroplast pigment,peroxidase,superoxide dismutase(SOD),the results show that when concentration as 2.4～4.8μg/L,nitrate reductase activity can get 4.807μg/g.h,MDA content is only 4.155μg,The chlorophyll content is 1.746mg/g. Superoxide dismutase activity get 341.95μgF.W/min,Peroxidase activity get 50μg /(g.min).
Spray diffirent dilution multiples of fermentation on different stage of cucumber,the results show that the best conditions for increasing cucumber biomass are the 100 multiple as the best concentraton and trophophase as the best spraying stage,concentration of spraying the best original broth diluted 100 times.When spray fermentation in reproductive stage,it will inhibit growth of cucumber,and increase the death rate of fruit; Seed germination rate is 97.33%,germination potential is 98.8%,the long of plant is 5.48cm,the long of rootis 5.12cm,dry weight of seedling is 8.87mg.After transplantation about 65days,the heightof plant up to 79.35cm and begin fruiting.it is earlier one week than the control group,solid peak for four weeks,it prolong two weeks than the control group.but fermentation did not affect single plant fruit.The major factor of increasing is the sex ratio increased,death rate of fruit decreased and fruiting production increased, the extent of incresing is as high as 50.8%.
To sum up,H-13 inoculate in the culture with the content of 0.1%hemoglobin and cellobiose / maltose(2:1),after culture 7d at pH7、26℃,it was switched into another liquid culture,which 0.1%hemoglobin,sucrose / maltose(2:1)、FeSO_4 100mg/L, MgSO_4.7H_2O 1g/L,K_2HPO_4 0.4g/L,NaNO_3 6g/L and CaCO_3 0.2 g/L in it,then spray the cucumber with the 100 multiples of fermentation dilution at trophophase.With the combination descried above,it is logic for the results of-differents concentrations and increased 50.8%per mu.
引文
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