紫菜腐霉拮抗菌的筛选和鉴定

Screening and identification of antagonistic bacteria against Pythium causing red rot disease in Neopyropia

关键词:  腐霉  拮抗菌  假交替单胞菌  生物防治

Key words:  Pythium  Antagonistic bacteria  Pseudoalteromonas  Biocontrol

摘要:

生物防治广泛用于农作物的病害防治，该方法在藻类病害防控方面尚未有相关的报道。腐霉(Pythium sp.)是引起紫菜(Neopyropia)赤腐病(red rot disease)的主要病原，本研究的目的是筛选和鉴定对紫菜腐霉有拮抗能力的细菌。从养殖藻类及其养殖环境中分离鉴定了385株细菌，通过平板对峙法筛选到9株对腐霉有拮抗作用的细菌，进一步通过含毒介质法筛选到3株胞外产物对腐霉具有抑制活性的细菌(P3、P6和P19)。3株拮抗菌对8株腐霉均有拮抗活性，对腐霉生长的抑制率分别为52.09%~97.95% (P3)、26.81%~78.04% (P6)、10.47%~41.91% (P19)。通过16S rRNA鉴定和多位点序列进化分析(16S rRNA-dnaA-dnaN-recA)，将P3和P6鉴定为杀鱼假交替单胞菌(Pseudoalteromonas piscicida)，P19鉴定为解肽假交替单胞菌(Pseudoalteromonas peptidolytica)。本研究筛选得到的拮抗菌为下一步建立紫菜赤腐病的生物防治方法奠定了基础。

Abstract:

China is the largest producer of Neopyropia yezoensis, ranking first in the world for cultivation area and yield production. In N. yezoensis production, diseases occur frequently every year due to increased farming density, environmental deterioration, and germplasm degeneration, resulting in serious economic losses to farmers. Red rot disease is caused by Pythium sp. and is one of the most common diseases during N. yezoensis farming, leading to empty nets and harvest loss. Air-dry, cold storage, and acid wash are common methods to counteract red rot disease in N. yezoensis farming. These physical or chemical disinfection methods, however, are not completely effective, and some have serious consequences. For example, refrigeration equipment and space will greatly increase costs, and acid wash treatments can cause environmental pollution. Although research has attempted to select or cultivate disease-resistant strains of laver, there remains no laver strain completely immune to red rot disease. Biocontrol is an effective method that is widely used in disease control of land crops. Biocontrol is potentially an environment-friendly and effective control method for macroalgal diseases. However, limited information exists on biocontrol in macroalgal diseases. During the growth and development of macroalgae, a variety of metabolites are produced on their surfaces, which provide suitable substrates for microbial colonization. The microbial community attached to the surface of algae is highly diverse and can produce many kinds of biologically active compounds. These compounds not only play a major role in normal morphology, growth, and development of algae, but also have antibacterial, antiviral, antiparasitic, and other activities to protect the host from harmful organisms. Therefore, the epiphytic microorganisms of algae provide good sources of microorganisms for biological screening. This study aimed to screen and identify bacteria with antagonistic ability towards Pythium sp.. A total of 385 bacterial strains, isolated from farming algae and their culturing environments, were screened. In the first round of screening, the plate confrontation method was used and repeated twice and confirmed that nine strains had antagonistic effects on the growth of Pythium sp.. The diameter of the bacteriostatic zone was approximately 1.65–16.54 mm. In the second round of screening, three strains (assigned as P3, P6, and P19) were further investigated using the toxic medium method for inhibitory activities in their extracellular products. Repeated experiments showed that the bacteriostatic rate was approximately 20.04%–30.09%. The antibacterial spectrum was determined by the plate confrontation method. Strains P3, P6, and P19 all had antagonistic effects on the eight tested strains of Pythium preserved in our laboratory. The inhibition rates reached 52.09%–97.95% for P3, 26.81%–78.04% for P6, 10.47%–41.91% for P19, respectively. The Pythium hyphae on the confrontation edge were further investigated by lactic acid phenol cotton blue staining. When compared with Pythium hyphae in a control group, the density and color of Pythium hyphae against strains P3 and P19 became sparse and lighter. There were no significant changes in Pythium hyphae against strain P6. Strains P3 and P6 were identified as Pseudoalteromonas piscicida, and P19 as P. peptidolytica, based on 16S rRNA gene identification and multilocus sequences analysis of 16S rRNA-dnaA-dnaN-recA. The bacterial strains of P3, P6, and P19 had significant antagonistic capabilities against the pathogenic Pythium strains. This indicates they are potential biocontrol probiotics for the control of red rot disease in N. yezoensis. The present study provides the foundation for research on the evaluation and application of antagonistic bacterial strains in the biocontrol of red rot disease of N. yezoensis.