Reduced vibriosis mortality in shrimp fed culture fluids from endophytic fungi correlated with Vibrio biofilm inhibition

• Main Author: Imtiyaz F.D.
• Other Authors: Mahidol University
• Format: Article
• Published: 2023
• Subjects: Agricultural and Biological Sciences
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/81440

Luminescent disease and acute hepatopancreatic necrosis disease (AHPND) are deadly shrimp bacterial diseases caused by Vibrio species. These bacteria can form biofilms on the cuticle covering the shrimp stomach and release lethal toxins. Thus, biofilm inhibition is a potential approach to control such diseases. In earlier studies, we found that a few biofilm-inhibiting endophytic fungi from mangrove trees could protect shrimp against Vibrio pathogens. Thus, we isolated 35 new endophytic fungi from two species of mangrove trees (Aegialitis rotundifolia and Bruguera hainesii) and screened their cell-free culture supernatants (CFSs) for inhibition of biofilms by luminescent disease-causing Vibrio harveyi (VH1) and AHPND-causing V. parahaemolyticus (3HP). Using microtiter-plate assays, 25 and 13 of the 35 CFSs screened were found to inhibit biofilms of 3HP and VH1, respectively. In contrast, none of these CFSs inhibited bacterial growth. Four of the CFSs inhibiting 3HP and VH1 were chosen as feed additives to test for efficacy in protecting against the respective diseases. Shrimp were given either CFS-supplemented test feeds or buffer-supplemented control feed for seven days prior to the immersion challenge with the respective pathogens at 106 CFU/ml. Survival was ∼80% in the unchallenged control groups given un-supplemented feeds, while survival with medium-supplemented feed group challenged with 3HP and VH1 was 8.3% and 0%, respectively. With 3HP challenge, all 4 CFSs gave improved survivals (73.7%, 71.6%, 64.1%, and 50.9%), while only 2 CFSs improved survival (52.3% and 45.0%) with VH1 challenge. Using a multigene sequencing approach, the two fungal isolates protecting against both 3HP and VH1 were from the family Didymellaceae, in the genus Leptosphaerulina sp. (MCR00760) and from the family Muyocopronaceae, in the genus Muyocopron (M. laterale, MCR00859). The two fungi that protected against 3HP only were identified as members of the order Hypocreales: one as Nectaria sp. (MCR00774) and the other as an unknown species (MCR00858). The fungal isolate with VH1 biofilm inhibiting activity that failed to protect the shrimp in the feeding experiments was identified as Astrocystis bambusae (MCR00851). Overall, the results revealed that biofilm inhibitors from endophytic fungi have the potential for use as feed supplements to prevent or reduce the severity of bacterial disease infections in shrimp. These biofilm inhibitors may be a prophylactic alternative to antibiotics, especially because they do not inhibit bacterial growth that can lead to the development of escape mutants.