Evaluation Of Inhibitory Effect Of Biocontrol Agents Bacillus Isolates On Aquaculture-Conditional Pathogenic Bacteria And Protective Roles In The Culture Of Eirocheir Sinensis, Macrobrachium Nipponnensic And Trionyx Sinensis

Biocontrol agents in the genus Bacillus have been widely applied to protection of livestock and poultry but have not drawn much attention in aquaculture until recently. Methods for evaluating their effects on aquacultured targets are being developed but not standardized. The present study sought to screen Bacillus isolates highly antagonistic to conditional bacterial pathogens Aeromonas spp. commonly occurring in aquaculture, optimize fermentation conditions for a candidate isolate Bacillus Bs01, and evaluate the roles of its preparation in protecting the crab Eirocheir sinensis, the shrimp Macrobrachium nipponnensic and the soft-shelled turtle Trionyx sinensis from bacterial infections under laboratory and greenhouse conditions. We hope our results to facilitate application of biocontrol agents to aquaculture protection in China.Antagonism-based Bacillus screening and optimized culture conditions. Seven Bacillus isolates derived from soil samples were assayed against three species of aquaculture-conditional pathogenic bacteria Aeromonas hydrophila CL99817, A. sobria TL97424 and A. sobria QXS-1 and all exhibited significantly inhibitory effects. The isolate BsOl was found most inhibitory to the bacterial pathogens with overall clear zones being averaged as 14.6 (10.7-21.3) mm in diameters. The isolate Bs03 was secondary to BsOl, yielding an average clear zone of 12.9 (10.7-17.2) mm, followed by the isolates Bs04 and Bs05. The other three Bacillus isolates produced significantly smaller clear zones, ranging from 8.5 to 9.5 mm. The susceptibility of different pathogens to the Bacillus isolates also differed. Aeromonas hydrophila CL99817 was significantly more susceptible (average clear zone 14.9 mm) than A sobria TL97424 and A. sobria (average clear zone less than 10.0 mm). The most antagonistic effect of the isolate Bs01 on the pathogens was not only due to its cell concentrations but also attributed to the metabolites in its liquid cultures because the supernatant excluding bacterial cells remained highly antagonistic to A. hydrophila CL99817, whose growth was inhibited by more than 85% in the supernatant at the regime of 35℃ and pH 7.0. The conditions for fermentation of the isolate Bs01 were optimized as pH 7.0, 40% liquid volume in flask and 35℃. Under such conditions, 24-h incubation based on beef extract - peptone broth plus 0.5% glucose resulted in a culture of 21.6×10~8 CFU/ml, which was 40 times more than that atpH 4.5 (0.58 *108 CFU/ml) and three times more than that at pH 5.5 or 8.5.Protective effect of Bacillus BsOl on Eirocheir sinensis megalopa. A bioassay was conducted to evaluate the protective role of the most antagonistic isolate Bacillus BsOl in the culture of Eirocheir sinensis megalopa. The combinations of three BsOl concentrations (10", 106 and 107 CFU/ml) with a single concentration of A hydrophila CL99817 (106 CFU/ml) in water body were included as three primary treatments plus a blank control (no bacteria). Either Bacillus BsOl or A. hydrophila CL99817 alone was also added at the application rate of 106 CFU/ml. Each of the six treatments was composed of four replicates with each including 20 megalopa. As a result, megalopa mortalities due to bacterial infection developed more rapidly in the pure A. hydrophila CL99817 treatment and the blank control than in other treatments. The treatments with the isolate BsOl delayed the disease-caused deaths and had lower mortalities, obviously varying with the concentration treatments and post-treatment time periods. Moreover, more megalopa individuals (85.1± 14.5%) had successful ecdysis in the pure BsOl treatment than in the blank control (81.4± 14.6%) and the pure A. hydrophila CL99817 treatment (78.4±16.4%).Protection of M. nipponnensic from gill disease by antagonistic Bacillus BsOl. A bioassay including varying Bacillus BsOl concentration treatments (104, 105, 106 and 107 CFU/ml) and one blank control were performed under the same stress of 106 CFU/ml of pathogenic A. hydrophila CL99817. Each treatment consisted of three replicates with each including 30 M. nipponnensic individuals in a glass tank. The resulting time-concentration-mortality data fit well (r2=0.94, F4j5o=196.2, PO.001) to the modified logistic model M = Kl'{l+exp[a+(6o+ b\C+bT,C2)T\), where M was the shrimp mortality due to bacterial infection, Twas the number of days after treatment, and C was the logio-transferred concentration of antagonistia bacteria (CFU/ml) in the water body. The parameter K was interpreted as maximal mortality due to bacterial disease whereas the expression bo+ b\C+ 63C2 depicted the epidemic rate of the disease. The fitted model M = 86.415/{l+exp[2.283-(0.177-0.034C+0.002C2)7]} indicates that the lack of protection by the antagonistic BsOl caused significant faster disease development and thus higher mortalities. At the presence of Bacillus BsOl, however, the shrimps suffered from delayed disease and lower mortalities that apparently depended upon the concentrations of antagonistic bacteria in the water. Based on histopathological examination, the infection by A. hydrophila CL99817 primarily occurred on shrimp gills, which then became filled with blood and swollen so that the respiration by the infected shrimps was difficult. As the infection prolonged, the gill branches ruptured andeventually caused deaths due to respiratory exhaustion. The protection provided by the isolate BsOl to large extent inhibited bacterial infection and development, thus delaying the occurrence of the disease and reducing the shrimp mortalities.Protection of Trionyx sinensis by application of Bacillus BsOl preparation. A pilot trial was conducted in a commercial aquaculture base of the soft-shelled turtle Trionyx sinensis in Jiaxing, Zhejiang during spring and summer 2004. The trial consisted of six treatments in water with varying concentrations of BsOl preparation (106, 105 and 104 CFU/ml) applied once every week or once every three weeks, and one treatment with daily supply of baits containing 109CFU/g plus a blank control. Each treatment was repeated in two pools under greenhouse conditions. Based on 105-day observations, the treatments with the BsOl preparation were somewhat effective in preventing the pool water from acidification, reducing its NO2" and NH3-N concentrations, facilitating the turtle ingestion and growth for higher yields, and enhancing survival. At the stage of transfer from larvae to adults (ca 42 days after treatment), the turtles fed with the BsOl-inclusive baits on average weighted 296.7±16.6 g. Those in the pools applied weekly at the rates of 106, 105 and 104 CFU/ml weighted 289.8±5.3, 291.4±7.3 and 276.6±11.6 g, respectively. These weights were all greater than those in the BsOl treatments applied once every three weeks (281.1±3.4, 282.7± 13.1 and 255.8±0.5 g respectively). In contrast, the turtles in the blank control averagely weighted 245.7±8.6 g only. The results indicate that the BsOl treatments provided conspicuous protection for the turtles during the experiment period and enhance the yield by 5.7-23.2% in comparison to the blank control. The high concentration treatment of the pool water and the bait treatment increased the yield by more than 16%. Moreover, the disease-caused mortalities were lower in the BsOl-inclusive treatments and were minimal at the application rate of 106 CFU/ml.Conclusively, the results based on laboratory bioassays and greenhouse trials highlight the choice of Bacillus BsOl isolate as a candidate biocontrol agent for further development and application in aquaculture. The isolate is characteristic with highly antagonistic effect on common Aeromonas species pathogenic to targets under aquaculture conditions, easy fermentation for high yields and easy colonization in aquaculture environment. More studies are warranted for its further development and application.