刺参“腐皮综合征”颉颃菌的筛选及鉴定

Screening and identification of antagonistic bacteria for skin ulcerative syndrome in Apostichopus japonicus

为了防治刺参“腐皮综合征”,筛选微生物制剂菌株,试验采用对峙培养法以灿烂弧菌和假交替单胞菌为指示菌,进行了颉颃微生物筛选,并用形态学结合16SrDNA序列分析法进行菌株鉴定,对优良颉颃菌进行了凝集试验和攻毒试验。结果表明：共分离筛选到10株对2种病原菌具有较高颉颃作用的细菌;经形态学鉴定和16SrDNA序列同源性分析,9株为芽孢杆菌属,1株为节杆菌属;菌株CN101、HN96、SY38和SE88不产生溶血素,浓度为1×109cfu/mL以下时,对刺参幼苗安全;4株菌在20小时时对灿烂弧菌和假交替单胞菌的凝集率分别在73.50%和70.40%以上,自凝集能力均在76.25%以上。对灿烂弧菌的抑菌圈直径在11.5~20.0mm之间,对假交替单胞菌的抑菌圈直径在10.0~17.0mm之间。说明CN101、HN96、SY38和SE88对2种病原菌均具有高颉颃性和凝集性,并对刺参安全,可用于开发微生物制剂防治刺参“腐皮综合征”。

To prevent and treat skin ulcerative syndrome in Apostichopus japonicas, and screen the strains for microbial agents, antagonistic microbes were screened by confrontation culture method using Vibrio splendidus （ V. splendidus ） and Pseudoalteromonas ifaciens （P. ifaciens ） as indicator bacteria. The antagonistic bacteria were identified by morphology combined with 16S rDNA sequence analysis. The agglutination and challenge tests were carried out for excellent antagonistic bacteria. The results showed that a total of 10 strains with higher antagonistic effect a- gainst V. splendidus and P. ifaciens were isolated and screened, and then used for morphological identification and homologous analysis of 16S rDNA sequence, thereinto, 9 strains belonged to the genus Bacillus, and 1 strain belonged to the genus Arthrobacter; CN101, HN96, SY38 and SE88 strains did not produce hemolysin, and safe concentration was less than 1 × 10^9 efu/mL to juvenile Apostichopus japonicus. Moreover, the agglutination rates of the four strains against V. splendidus and P. ifaciens at 20 h were more than 73.50 % and 70.40%, and their auto - ag- glutination rate was more than 76.25 %, respectively. The diameters of the inhibition zones of the four strains against V. splendidus were from 11.5 mm to 20.0 mm, and the diameters of the inhibition zones against P. ifacieus were from 10.0 mm to 17.0 mm. The results indicate that the CN101, HN96, SY38 and SE88 strains have high antagonism and agglutination against V. splendidus and P. ifaciens, and are safe for Apos- tichopus japonicus, and can be used for developing microbial agents to prevent and treat skin ulcerative syndrome in Apostichopus japonicus.