SCREENING OF ANTAGONISTIC BACTERIUM STRAIN AGAINST SAPROLEGNIOSIS AND THE PRELIMINARY STUDY OF IN VITRO ANTAGONISTIC ACTIVITY

Pathogenic members of the genus Saprolegnia are the common causative agent of saprolegniosis in aquaculture. In order to find an ideal biological control method for these fungi,the antagonistic bacteria were screened from the pond in which severe outbreak of saprolegniosis occurred. The results showed that 3 out of 130 isolates exhibited in vitro inhibition of Saprolegnia hyphal growth on BHI agar plate inoculated with two parallel streaks of each bacterium. Three strains showed antagonistic effect were named as LD038,LD057 and LD106,respectively. The strain LD038 identified as Serratia marcescens using Automated Microbiology Systemfor the identification of bacteria showed the strongest antagonistc activity among three stains mentioned above. Furthermore,we examined the in vitro antagonism of LD038 against Saprolegnia.Among five media examined,the LD038 exhibited the strongest antagonism using PDA media on which the inhibition zone was about 12mm. The LD038 on CA,GYand BHI media also exhibited antifungal activity,and the inhibition zones ranged from 3mm to 9mm. However,the LD038 on Sabouraud media could not inhibit the growth of Saprolegnia hyphae at all. In addition,the plate assay showed that the LD038 was able to inhibit Sapro legnia hyphal growth on PDA plate using agar diffusion method.And the inhibition zone induced around LD038 colony was about 20mm,whereas Saprolegnia hyphae reached the edge of plate on the controls without the LD038.Moreover,cyst germination of Saprolegnia was also inhibited to a great extent between two parallel streaks of the LD038,and the inhibitory activity was related to the distance fromthe streak of LD038,the inhibition zone was about 22mmin which cyst germination was completely inhibited1 In the broth assay,serial diluted cell-free culture supernatants of the LD038 significantly inhibited Saprolegnia hyphal growth and cyst germination compared with the controls,and Saprolegnia cysts were more sensitive than Saprolegnia hyphal. Saprolegnia cysts were able to germinate in the 1/5 diluted cell-free culture supernatant,but the germination rate was only 10 % in comparison to 96.3 % recorded for the controls. The hyphal growth from germinated cysts was also inhibited.And Saprolegnia hyphae formed hyphal nets in control wells 16 hours later,while the hyphae in the 1/5 diluted cell-free culture supernatant were halted with no branching over the whole experiment. In contrast,the non-diluted and 1/2 diluted cell-free culture supernatants of LD038 completely inhibited the Saprolegnia cyst germination.With respect to Saprolegnia hyphal,only the non-diluted cell-free culture supernatant completely inhibited the hyphal growth,while the 1/2 and 1/5 diluted cell-free culture supernatant postponed Saprolegnia hyphal growth.When the whole control well was confluent with Saprolegnia hyphae,the lengths of hyphae in wells treated with the 1/2 and 1/5 diluted cell-free culture supernatant were 0 and 2.6mm respectively,and Saprolegnia vegetative hyphae began to erupt from agar block in the 1/2 diluted supernatant 20 hours later. Phase-contrast microscopic examination showed that the hyphae in treated groups revealed extensive morphological changes including granulation of the cytoplasm,short and enlargement of hyphae1 On the contrary,the hyphae in the controls were slender containing a clear and granule free cytoplasm. Our study will provide theoretical foundation for biocontrol of saprolegniosis in aquaculture in future.