草莓酚酸类自毒物质降解菌筛选及对草莓防病促生作用研究
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摘要
本研究从不同土样中筛选到可降解对羟基苯甲酸的菌株;采用琼脂块法测定了所筛选的放线菌对5株草莓根病病原真菌的拮抗作用;研究了模拟土壤条件下所筛选菌株对对羟基苯甲酸的降解能力;对4株放线菌进行了形态及生理生化特征鉴定,获得了5株降解菌的HPLC指纹图谱,并对其中两株放线菌251、252进行了分子生物学鉴定;将降解放线菌菌剂接种到草莓幼苗上,研究其对连作草莓的防病作用及草莓花期、产量和品质的影响。主要结果如下:
1.所筛选到的可降解对羟基苯甲酸的菌株包括3株真菌和7株放线菌,3株真菌分别为黑曲霉、青霉、黄曲霉,放线菌的编号为3、24、25、32、40、42、49。3株真菌中黑曲霉的降解能力最强,在对羟基苯甲酸浓度为4g/L、6 g/L、8 g/L三种情况下培养7天后,降解率为99.5%-99.7%。7株放线菌的降解率均较高。
2.供试降解放线菌对5种草莓病原菌有较强抑制作用。皿内拮抗试验表明:252对病原菌镰刀菌(Fusarium sp.)、尖孢镰刀菌(Fusarium oxysporun)、立枯丝核菌(Rhizoctonia solani)、草莓疫霉(P. fragaride Hickm)有抑制作用,抑菌圈直径分别13、16、14、18mm;251对镰刀菌和草莓疫霉有拮抗作用,其抑菌圈直径分别10、21mm;402对草莓疫霉和立枯丝核菌有拮抗作用,其抑菌圈直径分别为23、11mm;493仅对镰刀菌有拮抗作用且其抑菌圈直径仅为9mm。
3.所筛选的5株菌对对羟基苯甲酸的降解能力从高低依次为黑曲霉、49、252、251、40号菌。40号菌在不添加有机质、加10%及20%有机质时的降解率分别是1.2%、17.0%和29.5%;有机质对其余菌株的降解率无明显影响。黑曲霉在半固态体系中的降解率高于纯固态体系,半固态体系中培养5d、固态体系中培养7d后,黑曲霉对对羟基苯甲酸的降解率分别为77.0%、6.7%。
4.根据放线菌形态与生理生化特征,251、252、40、49均为链霉菌(Streptomyces sp.)。16S rDNA序列分析表明,251、252均为淡紫褐链霉菌,与淡紫褐链霉菌(Streptomyces enissocaesilis)的同源性为100%。
5.采用蘸根接种法研究4种降解放线菌制剂对连作草莓的防病促生作用及对草莓花期、产量、品质的影响,结果表明接种降解放线菌菌剂可降低草莓苗死亡率,相对防效为28.2%-69.2%;使草莓花期提前,促进草莓早结果,其中251、40号菌在挂果初期的结果率较对照分别提高111.0%、206.0%;提高草莓产量,平均株产量增率为4.5%-26.9%,但草莓果实品质下降,其主要指标为草莓果实可滴定酸含量提高,草莓果实糖度、干物质含量、可溶性糖含量、Vc含量下降。品质降低与对照处理受人为影响有关。
The study obtained strains from different soil samples with respect to its ability to degradate p-hydrobenzoic.The antagonistic interaction between degradation actinomycete and 5 strains of pathogenic fungi were tested by the method of agar block. Researched the degradation ability of strains screened under simulated soil condition.Morphological characters, physiological and biochemical characteristics of 4 strains of actinomycete were identified,HPLC fingerprint of 5 degradation strains were obtaided.The molecular identification of two strains 521,252 were carried out.We studied on the biological control disease effect on strawberry seedlings and the influence on strawberry florescence,yield,and fruit quality of agents after inoculating degradation actinomycetes agents into strawberry seedlings.The results are as follows:
1. The screened strains that could degradate the p-hydrobenzoic included 3 strains of fungi which were Aspergillus niger sp.,Aspergillus flavus sp.,Penicillium sp.,respectively and 7 strains of actinomycete numbered 3, 24, 25, 32, 40, 42, 49.The Aspergillus niger sp. had the best degradation ability.When the degradation rate ranged from 99.5%-99.7% under the conditions of 4g/L,6g/L,8g/L p-hydrobenzoic concentration after 7 days.The degradation rate of 7 strains were high.
2. The actinomycetes tested had obvious inhibitive effect on 5 strains strawberry pathogenic fungi.The result of antagonistic test in dish showed: 252 had inhibitive effect on pathogenic fungi Fusarium sp., Fusarium oxysporun, Rhizoctonia solani, P.fragaride Hickm, their width of antimicrobial cycle were 13,16,14,18mm,respectively. 251 had inhibitive effect on Fusarium sp. and P.fragaride Hickm, their width of antimicrobial cycle were 10, 21mm, respectively. 402 had inhibitive effect on P.fragaride Hickm and Rhizoctonia solani, their width of antimicrobial cycle were 23, 11mm, respectively.493 only had inhibitive effect on Fusarium sp. , its width of antimicrobial cycle was merely 9mm.
3. The degradation ability turn from high to low of 5 strains screened is Aspergillus niger sp.,49, 252, 25, 40.The degradation rate of 40 were 1.2%,17.0% and 29.5%, respectively,when without OM,adding 10% OM,adding 20%OM.The OM had no effect on the degradation rates towards p-hydrobenzoic of the rest strains. The degradation rates of Aspergillus niger sp.in semi-soild system was higher than that in soild system.After cultivation in pure soil soild system for 7 days, and semi-soild system 5 days ,the degradation rates of Aspergillus niger sp. towards p-hydrobenzoic were 77.0%,6.7%, respectively.
4 According to actinomycete morphological characters and culture condition,251,252, 40,49 were identified as Streptomyces sp. 16SrDNA sequences analysis showed that 251,252 were Streptomyces Enissocaesilis..Their homologous were 100%.
5. The control disease promotion growth effect on strawberry seedlings and the influence on strawberry florescence, yield, and fruit quality of degradation actinomycetes agents were researched by methods of dipping inoculaition.The results demonstrated that degradation actinomycete agents could decrease death rate of strawberry seedlings, the relative control effect was 28.2%-69.2%.The agents could make flower early and enhance fruiting,the fruiting rate of 251,40 in early stage were higher than that of CK 111.0%, 206.0% seperately.The agents also increase strawberry yield, the increasing rate of average plant product was 14.5%-26.9%. But it make flavor decrease,the major indexs was titratable acid content of strawberry improved, strawberry sugar, dry matter, dissolusble sugar, vitamin C content were lower. The decreased flavor had influence on CK receiving human impact.
1.所筛选到的可降解对羟基苯甲酸的菌株包括3株真菌和7株放线菌,3株真菌分别为黑曲霉、青霉、黄曲霉,放线菌的编号为3、24、25、32、40、42、49。3株真菌中黑曲霉的降解能力最强,在对羟基苯甲酸浓度为4g/L、6 g/L、8 g/L三种情况下培养7天后,降解率为99.5%-99.7%。7株放线菌的降解率均较高。
2.供试降解放线菌对5种草莓病原菌有较强抑制作用。皿内拮抗试验表明:252对病原菌镰刀菌(Fusarium sp.)、尖孢镰刀菌(Fusarium oxysporun)、立枯丝核菌(Rhizoctonia solani)、草莓疫霉(P. fragaride Hickm)有抑制作用,抑菌圈直径分别13、16、14、18mm;251对镰刀菌和草莓疫霉有拮抗作用,其抑菌圈直径分别10、21mm;402对草莓疫霉和立枯丝核菌有拮抗作用,其抑菌圈直径分别为23、11mm;493仅对镰刀菌有拮抗作用且其抑菌圈直径仅为9mm。
3.所筛选的5株菌对对羟基苯甲酸的降解能力从高低依次为黑曲霉、49、252、251、40号菌。40号菌在不添加有机质、加10%及20%有机质时的降解率分别是1.2%、17.0%和29.5%;有机质对其余菌株的降解率无明显影响。黑曲霉在半固态体系中的降解率高于纯固态体系,半固态体系中培养5d、固态体系中培养7d后,黑曲霉对对羟基苯甲酸的降解率分别为77.0%、6.7%。
4.根据放线菌形态与生理生化特征,251、252、40、49均为链霉菌(Streptomyces sp.)。16S rDNA序列分析表明,251、252均为淡紫褐链霉菌,与淡紫褐链霉菌(Streptomyces enissocaesilis)的同源性为100%。
5.采用蘸根接种法研究4种降解放线菌制剂对连作草莓的防病促生作用及对草莓花期、产量、品质的影响,结果表明接种降解放线菌菌剂可降低草莓苗死亡率,相对防效为28.2%-69.2%;使草莓花期提前,促进草莓早结果,其中251、40号菌在挂果初期的结果率较对照分别提高111.0%、206.0%;提高草莓产量,平均株产量增率为4.5%-26.9%,但草莓果实品质下降,其主要指标为草莓果实可滴定酸含量提高,草莓果实糖度、干物质含量、可溶性糖含量、Vc含量下降。品质降低与对照处理受人为影响有关。
The study obtained strains from different soil samples with respect to its ability to degradate p-hydrobenzoic.The antagonistic interaction between degradation actinomycete and 5 strains of pathogenic fungi were tested by the method of agar block. Researched the degradation ability of strains screened under simulated soil condition.Morphological characters, physiological and biochemical characteristics of 4 strains of actinomycete were identified,HPLC fingerprint of 5 degradation strains were obtaided.The molecular identification of two strains 521,252 were carried out.We studied on the biological control disease effect on strawberry seedlings and the influence on strawberry florescence,yield,and fruit quality of agents after inoculating degradation actinomycetes agents into strawberry seedlings.The results are as follows:
1. The screened strains that could degradate the p-hydrobenzoic included 3 strains of fungi which were Aspergillus niger sp.,Aspergillus flavus sp.,Penicillium sp.,respectively and 7 strains of actinomycete numbered 3, 24, 25, 32, 40, 42, 49.The Aspergillus niger sp. had the best degradation ability.When the degradation rate ranged from 99.5%-99.7% under the conditions of 4g/L,6g/L,8g/L p-hydrobenzoic concentration after 7 days.The degradation rate of 7 strains were high.
2. The actinomycetes tested had obvious inhibitive effect on 5 strains strawberry pathogenic fungi.The result of antagonistic test in dish showed: 252 had inhibitive effect on pathogenic fungi Fusarium sp., Fusarium oxysporun, Rhizoctonia solani, P.fragaride Hickm, their width of antimicrobial cycle were 13,16,14,18mm,respectively. 251 had inhibitive effect on Fusarium sp. and P.fragaride Hickm, their width of antimicrobial cycle were 10, 21mm, respectively. 402 had inhibitive effect on P.fragaride Hickm and Rhizoctonia solani, their width of antimicrobial cycle were 23, 11mm, respectively.493 only had inhibitive effect on Fusarium sp. , its width of antimicrobial cycle was merely 9mm.
3. The degradation ability turn from high to low of 5 strains screened is Aspergillus niger sp.,49, 252, 25, 40.The degradation rate of 40 were 1.2%,17.0% and 29.5%, respectively,when without OM,adding 10% OM,adding 20%OM.The OM had no effect on the degradation rates towards p-hydrobenzoic of the rest strains. The degradation rates of Aspergillus niger sp.in semi-soild system was higher than that in soild system.After cultivation in pure soil soild system for 7 days, and semi-soild system 5 days ,the degradation rates of Aspergillus niger sp. towards p-hydrobenzoic were 77.0%,6.7%, respectively.
4 According to actinomycete morphological characters and culture condition,251,252, 40,49 were identified as Streptomyces sp. 16SrDNA sequences analysis showed that 251,252 were Streptomyces Enissocaesilis..Their homologous were 100%.
5. The control disease promotion growth effect on strawberry seedlings and the influence on strawberry florescence, yield, and fruit quality of degradation actinomycetes agents were researched by methods of dipping inoculaition.The results demonstrated that degradation actinomycete agents could decrease death rate of strawberry seedlings, the relative control effect was 28.2%-69.2%.The agents could make flower early and enhance fruiting,the fruiting rate of 251,40 in early stage were higher than that of CK 111.0%, 206.0% seperately.The agents also increase strawberry yield, the increasing rate of average plant product was 14.5%-26.9%. But it make flavor decrease,the major indexs was titratable acid content of strawberry improved, strawberry sugar, dry matter, dissolusble sugar, vitamin C content were lower. The decreased flavor had influence on CK receiving human impact.
引文
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