水霉拮抗菌的筛选及其拮抗作用的初步研究

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

为了获得水产动物水霉病病原菌的拮抗微生物,以水霉为靶标,对其拮抗菌进行了筛选,并研究了拮抗作用最强菌株的拮抗活性。实验结果表明:从发生过水霉病害的水体中分离得到了130株细菌,能抑制水霉菌落生长的有三株:LD038、LD057和LD106,其中以LD038的拮抗作用最强,经梅里埃ATB微生物自动鉴定系统鉴定为黏质沙雷氏菌(Serratia marcescens)。培养基对LD038的拮抗活性影响显著,LD038在PDA平板上表现出的拮抗活性最强,大小依次为PDA>BHI>GY>CA>Sabouraud。进一步对菌株LD038体外拮抗活性进行了研究。平板抑制实验表明,LD038能抑制水霉菌丝生长和孢子萌发。琼脂扩散法中,LD038菌落周围产生的抑菌圈直径为20mm,而无菌水对照组中水霉菌丝蔓过滤纸纸片并长至平板边缘;D38菌线间孢子的萌发与孢子距菌线的距离有关,距LD038菌线22mm范围内,孢子的萌发完全被抑制。无菌发酵上清液实验表明,与对照组相比,LD038无菌发酵上清液对孢子的萌发和菌丝生长都表现出显著的抑制作用。其中孢子较菌丝对上清液更为敏感,5倍稀释上清液下的孢子萌发率仅为10%,且萌发后的菌丝生长也同样受到抑制,整个实验过程中菌丝均未形成网状,对照组中萌发形成的菌丝在16h后呈网状。无菌上清原液和2倍稀释上清液中孢子均未萌发。对菌丝而言,仅无菌上清原液能彻底抑制菌丝生长,与对照组相比,2倍和5倍稀释上清液显著延缓了菌丝的生长。当对照组中的菌丝铺满整孔时,2倍和5倍稀释上清液中的菌丝长度分别为0和2.6mm。显微观察表明LD038的无菌发酵上清液导致水霉菌丝形态发生变化,较正常菌丝短而粗大,且出现了黑色原生质聚集。本研究为水霉病害的生物防治提供了一定的理论基础和依据。

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 System for 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, GY and 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 Saprolegnia 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 from the streak of LD038, the inhibition zone was about 22mm in which cyst germination was completely inhibited. 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 hyphae. 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.