天然海鱼共附生细菌抗TMV菌株的分离、筛选及鉴定
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摘要
从福建平潭潮间带周边海域采集7种海鱼共10份,选择了6种分离培养基,共获得海洋鱼类共附生细菌119株,将其中的91株海洋细菌液体发酵后,通过叶碟法进行海洋细菌发酵液抑制烟草花叶病毒(Tobacco mosaic virus, TMV)增殖活性的筛选。结果表明,有36株海洋细菌的发酵液对TMV增殖的抑制率超过60%。将这36株海洋细菌发酵液制成粗提物,在1 mg/mL的供试浓度下,有8株海洋细菌抑制TMV增殖的抑制率超过60%,又分别测定这8株海洋细菌粗提物在0.5 mg/mL和0.25 mg/mL抑制TMV增殖的抑制率,其中有3株海洋细菌在0.5 mg/mL时抑制率超过50%,特别是菌株0167B和菌株0110B在0.5 mg/mL时抑制率分别达到66.81%和58.87%,在0.25 mg/mL时抑制率分别达到48.18%和39.19%。
将上述36株活性菌株粗提物在1 mg/mL的供试浓度下,抑制率大于50%的14株菌株液体发酵,发酵液用无菌蒸馏水稀释10倍后,利用半叶法测定其抑制TMV侵染的活性,每株菌重复5次,结果表明,这14株海洋细菌的发酵液平均抑制率都在98%以上,大多数抑制率都是100%。
对5株活性菌株进行了形态鉴定,并对其16S rDNA序列进行了菌株间聚类分析。结果表明,菌株0110B的16S rDNA序列与Bacillus pumilus (EU880529)的16S rDNA序列同源性高达100%,可鉴定为Bacillus pumilus,菌株0111B的16S rDNA序列与Serratia marcescens (AB061685)的16S rDNA序列同源性高达100%,可鉴定为Serratia marcescens,菌株0134B的16S rDNA序列与bacillus subtilis (EU221345)的16S rDNA序列同源性高达100%,可鉴定为bacillus subtilis,菌株0165B的16S rDNA序列与Brevibacterium linens (EU660372)的16S rDNA序列同源性高达100%,可鉴定为Brevibacterium linens,菌株0167B的16S rDNA序列与Arthrobacter sp. (EU231606)的16S rDNA序列同源性高达100%,可鉴定为Arthrobacter sp.。
10 samples of seven marine fish were collected from Pingtan sea area in Fujian provience. 6 different media were used as isolation media. A total of 119 symbiotic and epiphyty bacteria of marine fish were isolated. The fermentation broth of the 91 marine bacteria were used to determine their inhibitory activities Anti-TMV replication by leaf-disk methods. The results showed that a total of 36 marine bacteria were able to inhibit the replication of TMV with the inhibition rate over 60%. The crude extracts of the fermentation broth were acquired and used for further screening test for the 36 marine bacteria. The 8 marine bacteria’s crude extracts at a concentration of 1 mg/mL inhibited the TMV replication with the inhibition rate over 60%. The inhibition rate of the 8 marine bacteria’s crude extracts at the concentration of 0.5 mg/mL and 0.25 mg/mL were tested, respectively. The inhibition rate of the 3 marine bacteria’s crude extracts in the 8 marine bacteria were over 50% at the concentration of 0.5 mg/mL, especially the crude extracts of the two bateria: 0167B and 0110B, showed high inhibition rate. It was 66.81% and 58.87% at the concentration of 0.5 mg/mL, 48.18% and 39.19% at the concentration of 0.25 mg/mL, respectively.
As to the 36 active strains described above whose crude extracts were used to test their inhibition rate Anti-TMV replication at the concentration of 1 mg/mL, the 14 of which showed the inhibition rate higher than 50%. The 14 strains were cultrured with liquid medium subsequently, the fermentation broth were diluted to 10-fold, and then the dilution were used to examine their inhibitory activities Anti-TMV infection by half-leave method. Each strain’s inhibition rate was tested 5 times. The result showed that the mean inhibition rate of all the 14 strains was higher than 98%, most of the 14 stains, inhibition rate Anti-TMV infection were 100%.
Five active strains were identified by morphology characterization. The cluster analysis between identified bacteria and other bacteria from GenBank were deduced from sequences of 16S rDNA. The results showed that 0110B was identified as Bacillus pumilus, 0111B was identified as Serratia marcescens, 0134B was identified as bacillus subtilis, 0165B was identified as Brevibacterium linens, 0167B was identified as Arthrobacter sp..
将上述36株活性菌株粗提物在1 mg/mL的供试浓度下,抑制率大于50%的14株菌株液体发酵,发酵液用无菌蒸馏水稀释10倍后,利用半叶法测定其抑制TMV侵染的活性,每株菌重复5次,结果表明,这14株海洋细菌的发酵液平均抑制率都在98%以上,大多数抑制率都是100%。
对5株活性菌株进行了形态鉴定,并对其16S rDNA序列进行了菌株间聚类分析。结果表明,菌株0110B的16S rDNA序列与Bacillus pumilus (EU880529)的16S rDNA序列同源性高达100%,可鉴定为Bacillus pumilus,菌株0111B的16S rDNA序列与Serratia marcescens (AB061685)的16S rDNA序列同源性高达100%,可鉴定为Serratia marcescens,菌株0134B的16S rDNA序列与bacillus subtilis (EU221345)的16S rDNA序列同源性高达100%,可鉴定为bacillus subtilis,菌株0165B的16S rDNA序列与Brevibacterium linens (EU660372)的16S rDNA序列同源性高达100%,可鉴定为Brevibacterium linens,菌株0167B的16S rDNA序列与Arthrobacter sp. (EU231606)的16S rDNA序列同源性高达100%,可鉴定为Arthrobacter sp.。
10 samples of seven marine fish were collected from Pingtan sea area in Fujian provience. 6 different media were used as isolation media. A total of 119 symbiotic and epiphyty bacteria of marine fish were isolated. The fermentation broth of the 91 marine bacteria were used to determine their inhibitory activities Anti-TMV replication by leaf-disk methods. The results showed that a total of 36 marine bacteria were able to inhibit the replication of TMV with the inhibition rate over 60%. The crude extracts of the fermentation broth were acquired and used for further screening test for the 36 marine bacteria. The 8 marine bacteria’s crude extracts at a concentration of 1 mg/mL inhibited the TMV replication with the inhibition rate over 60%. The inhibition rate of the 8 marine bacteria’s crude extracts at the concentration of 0.5 mg/mL and 0.25 mg/mL were tested, respectively. The inhibition rate of the 3 marine bacteria’s crude extracts in the 8 marine bacteria were over 50% at the concentration of 0.5 mg/mL, especially the crude extracts of the two bateria: 0167B and 0110B, showed high inhibition rate. It was 66.81% and 58.87% at the concentration of 0.5 mg/mL, 48.18% and 39.19% at the concentration of 0.25 mg/mL, respectively.
As to the 36 active strains described above whose crude extracts were used to test their inhibition rate Anti-TMV replication at the concentration of 1 mg/mL, the 14 of which showed the inhibition rate higher than 50%. The 14 strains were cultrured with liquid medium subsequently, the fermentation broth were diluted to 10-fold, and then the dilution were used to examine their inhibitory activities Anti-TMV infection by half-leave method. Each strain’s inhibition rate was tested 5 times. The result showed that the mean inhibition rate of all the 14 strains was higher than 98%, most of the 14 stains, inhibition rate Anti-TMV infection were 100%.
Five active strains were identified by morphology characterization. The cluster analysis between identified bacteria and other bacteria from GenBank were deduced from sequences of 16S rDNA. The results showed that 0110B was identified as Bacillus pumilus, 0111B was identified as Serratia marcescens, 0134B was identified as bacillus subtilis, 0165B was identified as Brevibacterium linens, 0167B was identified as Arthrobacter sp..
引文
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