玉米大斑病生防放线菌的筛选鉴定及液体发酵的研究
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摘要
玉米大斑病(Northern corn leaf blight)是由大斑刚毛座腔菌[Setosphaeriaturcica (Luttrell) Leonard and Suggs]引起的一种严重的叶枯性病害，是世界玉米产区分布较广、危害较重的主要病害之一。目前，玉米大斑病菌的防治以抗病品种的培育为主，辅以一定的栽培管理和化学防治，但是化学药剂防效不稳定，且易污染环境。因此，利用安全高效的生防菌防治玉米大斑病具有重要的现实意义。
本研究从吉林省洮南、人参地和长白山自然保护区采集土样，采用稀释涂布法分离放线菌，共获得527株。以玉米纹枯病菌、玉米大斑病菌、玉米灰斑病菌、稻瘟病菌、大豆核盘菌、尖孢镰刀菌、人参锈腐病菌、柑橘炭疽病菌为靶标菌，采用平板对峙法进行初筛，获得108株拮抗放线菌，选择抑菌效果最好的一株放线菌BZ45，采用抑制菌丝生长速率法、牛津杯法、抑制孢子萌发法对其发酵滤液的活性进行测定。结果发现，菌株BZ45对玉米纹枯病菌、玉米大斑病菌、玉米灰斑病菌、稻瘟病菌、大豆核盘菌、尖孢镰刀菌、人参锈腐病菌、柑橘炭疽病菌均有拮抗作用，抑菌圈直径大小为31.00~46.60mm；菌株BZ45的发酵滤液对8种病原真菌菌丝均有抑制作用，抑菌率为10.48%~75.51%，其对玉米大斑病菌的抑菌圈直径为23.80mm，对玉米大斑病菌孢子萌发有较强抑制作用。
经过培养特征、形态特征、生理生化特性、16SrDNA序列测定及系统发育的分析，将菌株BZ45鉴定为壮观链霉菌（Streptomyces spectabilis）。
通过单因素试验和正交设计试验，确定其最佳发酵配方和培养条件为：果糖1.5%、蛋白胨3.0%、KH_2PO_40.1%、NaCl0.04%、CaCO_30.1%，起始pH为7.2，种子液接种量为10%，250mL装瓶量50mL，培养温度为28℃，200r/min，摇瓶培养4d。
利用特异性引物扩增的方法，检测到放线菌BZ45基因组中存在聚酮合酶I型（PKS I）基因、非核糖体多肽合成酶(NRPS)基因，不含有聚酮合酶II型（PKS II）基因。产生的次生代谢产物有挥发性和扩散性，产生的酶有蛋白酶，还产生铁载体，不产生IAA和纤维素酶。
The northern corn leaf blight (NCLB) is a serious foliar wilt disease of maize inmany corn belts. It is incited by the ascomycete fungus Setosphaeria turcica (Luttrell)Leonard and Suggs, with its Asexual phase Exserohilum turcicum (Passerini) Leonardand Suggs. NCLB is mainly controlled by resistant cultivars, while cultivationmanagement and chemicals are the complement. But Chemicals control effect is notstable, and easy to pollute the environment. So use of safety and efficient antagonisticmicrobe is one of the most important.
In this study, actinomycetes were isolated by Pour Plate method from soil collectedfrom Taonan, Panax ginseng C. A. Mey. planting area and Changbai Mountain NaturalReserve in Jilin, and gain527strains.108antagonistic actinomycetes againstRhizoctonia solani, Setosphaeria turcica, Cercospora zeaemaydis, Pyricularia oryzae,Sclerotinia sclerotiorum, Fusarium oxysporum, Cylindrocarpon destructans,Colletotrichum gloeosporioides were selected by using confrontation culture method.From them strain BZ45possessed prominent bioactivity and were further studied. Thefermentation filtrates of BZ45were determined by suppression of mycelial growthmethod, cylinder plate method and inhibition on the spore germination in vitro. It wasdemonstrated to be antagonistic against Rhizoctonia solani, Setosphaeria turcica,Cercospora zeaemaydis, Pyricularia oryzae,Sclerotinia sclerotiorum, Fusariumoxysporum, Cylindrocarpon destructans, Colletotrichum gloeosporioides withdiameters of inhibition zones of31.00~46.60mm. The fermentation filtrates of strainsBZ45had antagonistic activity to the mycelial growth of8plant pathogens,too.Inhibition rate of fermentation filtrates was10.48%~75.51%. The fermentationfiltrates have higher inhibition on the mycelial growth of Setosphaeria turcica withzones of inhibition of23.80mm. The fermentation filtrates have higher inhibition onthe spore germination of Setosphaeria turcica.
Strain BZ45were identified as Streptomyces spectabilis by morphological andcultural traits, physio-biochemical characteristics and16S rDNA sequence andphylogenetic analysis.
By using single-factor test and orthogonal test,the optimum fermentation mediumcomponent and condition of strain BZ45were cultured in1.5%fructose,3%peptone,0.1%KH_2PO_4,0.04%NaCl,0.1%CaCO_3, the initial pH of7.2, liquidvolume50ml in250ml,at28℃,200r/min, inoculation volume10%and shaked for4d.
Strain BZ45have genes of type I polyketide synthases (PKSI), nonribosomalpeptide synthase (NRPS) by PCR, don’t have type II polyketide synthases (PKSII).Itproduce volatile and diffusible metabolite. It produces protease, also producessiderophore, does not produce the IAA and cellulose enzyme.
本研究从吉林省洮南、人参地和长白山自然保护区采集土样，采用稀释涂布法分离放线菌，共获得527株。以玉米纹枯病菌、玉米大斑病菌、玉米灰斑病菌、稻瘟病菌、大豆核盘菌、尖孢镰刀菌、人参锈腐病菌、柑橘炭疽病菌为靶标菌，采用平板对峙法进行初筛，获得108株拮抗放线菌，选择抑菌效果最好的一株放线菌BZ45，采用抑制菌丝生长速率法、牛津杯法、抑制孢子萌发法对其发酵滤液的活性进行测定。结果发现，菌株BZ45对玉米纹枯病菌、玉米大斑病菌、玉米灰斑病菌、稻瘟病菌、大豆核盘菌、尖孢镰刀菌、人参锈腐病菌、柑橘炭疽病菌均有拮抗作用，抑菌圈直径大小为31.00~46.60mm；菌株BZ45的发酵滤液对8种病原真菌菌丝均有抑制作用，抑菌率为10.48%~75.51%，其对玉米大斑病菌的抑菌圈直径为23.80mm，对玉米大斑病菌孢子萌发有较强抑制作用。
经过培养特征、形态特征、生理生化特性、16SrDNA序列测定及系统发育的分析，将菌株BZ45鉴定为壮观链霉菌（Streptomyces spectabilis）。
通过单因素试验和正交设计试验，确定其最佳发酵配方和培养条件为：果糖1.5%、蛋白胨3.0%、KH_2PO_40.1%、NaCl0.04%、CaCO_30.1%，起始pH为7.2，种子液接种量为10%，250mL装瓶量50mL，培养温度为28℃，200r/min，摇瓶培养4d。
利用特异性引物扩增的方法，检测到放线菌BZ45基因组中存在聚酮合酶I型（PKS I）基因、非核糖体多肽合成酶(NRPS)基因，不含有聚酮合酶II型（PKS II）基因。产生的次生代谢产物有挥发性和扩散性，产生的酶有蛋白酶，还产生铁载体，不产生IAA和纤维素酶。
The northern corn leaf blight (NCLB) is a serious foliar wilt disease of maize inmany corn belts. It is incited by the ascomycete fungus Setosphaeria turcica (Luttrell)Leonard and Suggs, with its Asexual phase Exserohilum turcicum (Passerini) Leonardand Suggs. NCLB is mainly controlled by resistant cultivars, while cultivationmanagement and chemicals are the complement. But Chemicals control effect is notstable, and easy to pollute the environment. So use of safety and efficient antagonisticmicrobe is one of the most important.
In this study, actinomycetes were isolated by Pour Plate method from soil collectedfrom Taonan, Panax ginseng C. A. Mey. planting area and Changbai Mountain NaturalReserve in Jilin, and gain527strains.108antagonistic actinomycetes againstRhizoctonia solani, Setosphaeria turcica, Cercospora zeaemaydis, Pyricularia oryzae,Sclerotinia sclerotiorum, Fusarium oxysporum, Cylindrocarpon destructans,Colletotrichum gloeosporioides were selected by using confrontation culture method.From them strain BZ45possessed prominent bioactivity and were further studied. Thefermentation filtrates of BZ45were determined by suppression of mycelial growthmethod, cylinder plate method and inhibition on the spore germination in vitro. It wasdemonstrated to be antagonistic against Rhizoctonia solani, Setosphaeria turcica,Cercospora zeaemaydis, Pyricularia oryzae,Sclerotinia sclerotiorum, Fusariumoxysporum, Cylindrocarpon destructans, Colletotrichum gloeosporioides withdiameters of inhibition zones of31.00~46.60mm. The fermentation filtrates of strainsBZ45had antagonistic activity to the mycelial growth of8plant pathogens,too.Inhibition rate of fermentation filtrates was10.48%~75.51%. The fermentationfiltrates have higher inhibition on the mycelial growth of Setosphaeria turcica withzones of inhibition of23.80mm. The fermentation filtrates have higher inhibition onthe spore germination of Setosphaeria turcica.
Strain BZ45were identified as Streptomyces spectabilis by morphological andcultural traits, physio-biochemical characteristics and16S rDNA sequence andphylogenetic analysis.
By using single-factor test and orthogonal test,the optimum fermentation mediumcomponent and condition of strain BZ45were cultured in1.5%fructose,3%peptone,0.1%KH_2PO_4,0.04%NaCl,0.1%CaCO_3, the initial pH of7.2, liquidvolume50ml in250ml,at28℃,200r/min, inoculation volume10%and shaked for4d.
Strain BZ45have genes of type I polyketide synthases (PKSI), nonribosomalpeptide synthase (NRPS) by PCR, don’t have type II polyketide synthases (PKSII).Itproduce volatile and diffusible metabolite. It produces protease, also producessiderophore, does not produce the IAA and cellulose enzyme.
引文
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