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Shewanella putrefaciens has been recognized as an emerging important pathogen in aquaculture. However, scarce information is available on the characterization and microbial control of S. putrefaciens as a causal agent of hepatopancreas necrosis disease in Chinese mitten crab Eriocheir sinensis. In this study, a multi-resistant S. putrefaciens isolate (DZ-A) was identified as a causal pathogen of hepatopancreas necrosis disease in Chinese mitten crabs. It showed a lethal dose (LD50) of 2.20 × 105 CFU ml-1 in Chinese mitten crabs, and multiple resistance to aminoglycoside, chloramphenicol, macrolide, penicillin, peptide, and tetracycline antimicrobials. In addition, Bdellovibrio powder exhibited a significant antibacterial effect against the pathogenic S. putrefaciens, and conferred significant protection to challenged Chinese mitten crabs with relative percentage survivals of 80.00% to 93.33% via significant improvement in their immune response and antioxidant capability. The findings of this study provide valuable insights into the phenotypic characterization and biological control of pathogenic S. putrefaciens in Chinese mitten crabs.

Shewanella putrefaciens is a vital bacterial pathogen implicated in serious diseases in Chinese mitten crab Eriocheir sinensis. Yet the use of probiotics to improve the defense ability of E. sinensis against S. putrefaciens infection remains poorly understood. In the present study, the protective effect of dietary R. sphaeroides against S. putrefaciens infection in E. sinensis was evaluated through antioxidant capability, immune response, and survival under bacterial challenge assays, and its protective mechanism was further explored using a combination of intestinal flora and metabolome assays. Our results indicated that dietary R. sphaeroides could significantly improve immunity and antioxidant ability of Chinese mitten crabs, thereby strengthening their disease resistance with the relative percentage survival of 81.09% against S. putrefaciens. In addition, dietary R. sphaeroides could significantly alter the intestinal microbial composition and intestinal metabolism of crabs, causing not only the reduction of potential threatening pathogen load but also the increase of differential metabolites in tryptophan metabolism, pyrimidine metabolism, and glycerophospholipid metabolism. Furthermore, the regulation of differential metabolites such as N-Acetylserotonin positively correlated with beneficial Rhodobacter could be a potential protection strategy for Shewanella infection. To the best of our knowledge, this is the first study to illustrate the protective effect and mechanism of R. sphaeroides supplementation to protect E. sinensis against S. putrefaciens infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Excessive nitrogen (N) discharged in water is a major cause of eutrophication and other severe environmental issues. Biological N removal via heterotrophic nitrification and aerobic denitrification (HN-AD) has drawn particular attention, owing to the merit of concurrent nitrification and denitrification inside one cell. However, the mechanisms underlying N transformation during HN-AD remain unclear. In the present study, the HN-AD strain Pseudomonas sp. Y15 (Y15) was isolated to explore the N distribution and flow, based on stoichiometry and energetics. The total N removal efficiency by Y15 increased linearly with C/N ratio (in the range of 5-15) to ∼96.8%. Of this, ∼32.2% and ∼64.6% were transformed into gas-N and biomass-N, respectively. A new intracellular N metabolic bypass (NO → NO 2 ) was found, to address the substantial gaseous N production during HN-AD. Concering energetics, the large portion of the biomass-N is ascribed to the synthesis of the amino acids that consume low energy. Finally, two novel stoichiometric equations for different N sources were proposed, to describe the overall HN-AD process. This study deepens the fundamental knowledge on HN-AD bacteria and enlightens their use in treating N-contaminated wastewater., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liang Shen reports financial support was provided by National Natural Science Foundation of China. Liang Shen reports financial support was provided by Natural Science Foundation of Fujian Province of China., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A coupled system comprised of a biofilm electrode reactor (BER) and a manganese ore substrate microbial fuel cell-constructed wetland (MFC-CW) system was used to remove co-exposed antibiotic and Zn (II), as well as simultaneously reduce copies of antibiotic resistance genes (ARGs) in the current study. In this system, BER primarily reduced the concentrations of antibiotics and Zn (II), and the effluent was used as the input to the MFC-CW, thereby providing electricity to BER. Co-exposure to a high concentration of Zn (II) decreased the relative abundances (RAs) of ARGs in the BER effluent, whereas the remaining sub-lethal concentration of Zn (II) increased the RAs of ARGs in the MFC-CW effluent. Even though the absolute copies of ARGs in the effluents increased during co-exposure, the total number of target ARG copies in the effluent of MFC-CW was significantly lower than that of BER. Moreover, BER pre-treatment eliminated most of Zn (II), which improved the electrical power generation characteristic of the MFC-CW unit. Correspondingly, the bacterial community and the ARGs hosts were analyzed to demonstrate the mechanism. In conclusion, the coupled system demonstrates significant potential to reduce antibiotics, Zn (II) and environmental risks posed by ARGs., Competing Interests: Conflicts of interest There are no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Rhodobacter probiotics are considered as good alternatives to antibiotics for aquaculture. Yet the beneficial effects of Rhodobacter on Chinese mitten crab Eriocheir sinensis are still unclear, and more functions of Rhodobacter supplementation need to be clarified. In this study, a 60-day feeding trial was performed to investigate the protective effects of R. azotoformans against citrobacteriosis in E. sinensis by growth performance, serum immunity, hepatopancreatic antioxidant capability, intestinal flora, and resistance to Citrobacter freundii challenge assays. The results showed that R. azotoformans supplementation significantly and dose-dependently increased weight gain and specific growth rate as well as activities of serum immune and hepatopancreatic antioxidant enzymes, leading to notable improvement in the growth performance, serum immunity and hepatopancreatic antioxidant status of E. sinensis. Besides, R. azotoformans supplementation significantly enhanced intestinal microbial abundance and diversity in E. sinensis, and conferred significant protection of the crabs against C. freundii challenge with seven-day survival rates of 70.0%-100.0%. To the best of our knowledge, this is the first study to reveal the protective effects of R. azotoformans against citrobacteriosis in E. sinensis., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Providencia rettgeri has been recognized as a zoonotic pathogen of humans and aquaculture animals and has become a global public health concern. However, scarce information is available on the characterization of pathogenic P. rettgeri from kuruma shrimp Marsupenaeus japonicus. In the present study, a P. rettgeri isolate (KM4) was confirmed as a causative agent of red leg disease in cultured M. japonicus, which showed a median lethal dose (LD 50 ) value of 5.01 × 10 5 CFU·ml -1 and had multiple resistances to aminoglycosides, sulfonamides, and tetracycline antimicrobials used in aquaculture. In addition, the whole genome of isolate KM4 was sequenced and found to consist of a single circular chromosome of 4,378,712 bp and a circular plasmid of 171,394 bp. The genome sequence analysis further revealed the presence of potential virulence and antibiotic resistance genes in isolate KM4, which probably rendered this isolate particularly virulent. To our knowledge, this is the first study to characterize P. rettgeri pathogens from kuruma shrimp infected with red leg disease. The findings of this study can provide novel insights into the presence and distribution of pathogenicity-associated genes in shrimp-pathogenic P. rettgeri., (© 2022 Wiley-VCH GmbH.)

Citrobacter freundii is an important bacterial pathogen that causes serious diseases in Chinese mitten crab Eriocheir sinensis. However, scarce information is available on the use of Bacillus licheniformis to protect E. sinensis from C. freundii infection by improving the immunity, antioxidant ability and intestinal flora. In the present study, a 60-day feeding trial was conducted to examine the potential effects of dietary supplementation with antagonistic B. licheniformis on the non-specific immunity, antioxidant capability, intestinal flora and resistance of E. sinensis to C. freundii infection. The results indicated that dietary supplementation of B. licheniformis could boost non-specific immunity and antioxidant capability of E. sinensis in a significant dose-dependent manner respectively by increasing serum lysozyme, superoxide dismutase, alkaline phosphatase, and catalase activities and hepatopancreatic superoxide dismutase, catalase, acid phosphatase activities. In addition, crabs fed diets with B. licheniformis displayed increased composition and diversity of the intestinal flora, and exhibited significant defense against C. freundii infection with the relative percentage survivals ranging from 61.9% to 100.0% for seven days. To our knowledge, this is the first report of antagonistic B. licheniformis as a supplement in Chinese mitten crab feed to effectively resist C. freundii infection by improving the non-specific immunity, antioxidant status and intestinal flora., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Bacterial spot, which is caused by several Xanthomonas species, is an economically important disease in tomato (Solanum lycopersicum). Great efforts have been made for the identification of resistant sources and the genetic analysis of resistance. However, the development of resistant commercial varieties is slow due to the existence of multiple species of the pathogen and a poor understanding of the resistance mechanism in tomato. The current study revealed that the Rx4 gene encodes a nucleotide-binding leucine-rich repeat protein in the wild tomato species Solanum pimpinellifolium and specifically recognizes and confers a hypersensitive response (HR) to Xanthomonas euvesicatoria pv. perforans race T3 expressing the AvrXv3 avirulence protein. Complementation of the Rx4 gene in the susceptible tomato line Ohio 88119 using a transgenic approach resulted in HR, whereas knockout of the gene through CRISPR/Cas9 editing in resistant lines Hawaii 7981 and PI 128216 led to non-HR to race T3. Transcription of Rx4 was not induced by the presence of race T3. Furthermore, the Rx4 protein did not show physical interaction with AvrXv3 but interacted with SGT1-1 and RAR1. Virus-induced gene silencing of SGT1-1 and RAR1 in the resistant line PI128216 suppressed the HR to race T3. Taken together, our study confirms Rx4 is the gene conferring the HR to bacterial spot race T3 and reveals the potential roles of SGT1-1 and RAR1 as signals in the Rx4-mediated HR. This discovery represents a step forward in our understanding of the mechanism of resistance to bacterial spot in tomato and may have important implications for understanding plant-bacterial interactions., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Aeromonas hydrophila is a well-known bacterial pathogen associated with mass mortalities in aquaculture. Yet, few reports are available on whiteleg shrimp-pathogenic A. hydrophila . In the present study, a virulent isolate WS05 was confirmed as a causative agent of diseased freshwater-cultured whiteleg shrimp and showed a mean lethal dose (LD 50 ) value of 4.8 × 10 4 CFU mL -1 . It was identified phenotypically and molecularly as an A. hydrophila strain, and exhibited susceptibility to several veterinary antibiotics extensively used in aquaculture, including cotrimoxazole, doxycycline, florfenicol, neomycin, and tetracycline. In view of the strongest inhibition zone of florfenicol against isolate WS05, the synergistic effect of the combinations of florfenicol and herb extracts was further evaluated, and the result indicated that Punica granatum extract was a potential synergist of florfenicol against isolate WS05 and the fractional inhibitory concentration index (FICI) for the florfenicol- P. granatum extract was calculated as 0.31. When combined with 7.81 mg mL -1 P. granatum extract, the minimum inhibitory concentration (MIC) of florfenicol against isolate WS05 was reduced from 0.50 to 0.03 mg L -1 , and its activity against isolate WS05 was also enhanced with a significant reduction of ≥3.61 log in cell density after 24 h of treatment compared with that in the single drug treatment. In addition, the protective effect was potentiated by the combination of florfenicol and P. granatum extract, with a cumulative mortality of 36.66% ( p < 0.05) and 33.33% ( p < 0.05) lower than that in the single treatment with florfenicol and P. granatum extract after the challenge with isolate WS05 for seven days. As far as we know, this is the first study to describe whiteleg shrimp-pathogenic A. hydrophila and suggest P. granatum extract as a potential synergist of florfenicol against the A. hydrophila pathogen.

Liquid preparations of bdellovibrios are currently commercialized as water quality improvers to control bacterial pathogens in whiteleg shrimp Penaeus vannamei . However, the efficacy of these liquid preparations is significantly impaired due to a dramatic loss of viable cells during long-term room temperature storage. Thus, new formulations of bdellovibrios are greatly needed for high-stablility room-temperature storage. In the present study, the encapsulated powder of Bdellovibrio sp. strain F16 was prepared using spray drying with 20 g L -1 gelatin as the coating material under a spray flow of 750 L h -1 , a feed rate of 12 mL min -1 , and an air inlet temperature of 140 °C. It was found to have a cell density of 5.4 × 10 7 PFU g -1 and to have spherical microparticles with a wrinkled surface and a diameter of 3 μm to 12 μm. In addition, the encapsulated Bdellovibrio powder presented good storage stability with its cell density still remaining at 3.5 × 10 7 PFU g -1 after 120 days of room-temperature storage; it was safe for freshwater-farmed whiteleg shrimp with an LD 50 over 1200 mg L -1 , and it exhibited significant antibacterial and protective effects at 0.8 mg L -1 against shrimp-pathogenic vibrios. To our knowledge, this is the first report on a promising Bdellovibrio powder against shrimp vibrios with high stable room-temperature storage.

The use of bdellovibrios has been regarded as an alternative to control multidrug-resistant pathogens and fish bacteriosis. However, scarce information is available on the potential of bdellovibrios in the presence of copper sulfate, which is an algicide widely used to treat cyanobacterial blooms in aquaculture. In the present study, the effects of copper sulfate at sublethal and lethal levels (0.1 and 1.0 mg·L -1 ) on Bdellovibrio sp. strain BDF-H16 were evaluated. The growth of Bdellovibrio sp. strain BDF-H16 was significantly promoted by both concentrations of copper sulfate, but less so by the lethal concentration. The bacteriolysis of gibel carp-pathogenic Aeromonas hydrophila by Bdellovibrio sp. strain BDF-H16 was also stimulated by copper sulfate in both solid and liquid media. However, Bdellovibrio sp. strain BDF-H16 with 0.1 mg·L -1 copper sulfate clearly inhibited infection of gibel carps by A. hydrophila better than Bdellovibrio sp. strain BDF-H16 with 1.0 mg·L -1 copper sulfate did.

Vibriosis has become a major global economic problem in freshwater-farmed whiteleg shrimp (Penaeus vannamei). The prevention and control of vibriosis are now priority research topics. In this study, a pathogenic strain (QH) was isolated from vibriosis-infected freshwater-farmed P. vannamei that resulted in leg yellowing and was identified as a Vibrio cholerae isolate through phylogenetic analysis and the API 32GN system. A phylogenetic tree that was constructed using the neighbor-joining method further confirmed the QH isolate as a V. cholerae strain. A virulent outer membrane protein (ompU) gene was found to be present in the QH isolate, which further confirmed its pathogenicity. In addition, Bdellovibrio bacteriovorus conferred significant protection against V. cholerae: B. bacteriovorus exhibited significant bacteriolytic effects on the V. cholerae pathogen, possessed a wide prey range that included Vibrio pathogens, and displayed a positive protective efficacy against experimental V. cholerae infection in P. vannamei. To the best of our knowledge, this is the first report of the control of shrimp pathogen V. cholerae with B. bacteriovorus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Carotenoid content is the primary determinant of fruit color that affects nutritional value and appearance in tomato. Phytoene synthase (PSY) is the key regulatory enzyme in the carotenoid biosynthesis pathway. Absent function of PSY1 in tomato fruit results in yellow flesh phenotype. We, here, report that two different transcripts, a wild-type (Psy1) and a chimeric mRNA (Psy1/Unknown), exist in a yellow-fruited tomato accession PI 114490. Psy1/Unknown is generated by joining exons from two different genes, Psy1 and an unknown gene, transcribed using both complementary DNA strands. The Psy1 shows low expression in the fruit of PI 114490, while the expression of Psy1/Unknown in the fruit of PI 114490 shows the same pattern as Psy1 in red fruit. The PSY1/Unknown has a lower function than PSY1 in a bacterial expression system. Coincidence of one single-nucleotide polymorphism (SNP) in the fourth intron and one simple sequence repeat (SSR) with 19 AT repeats in the downstream sequence of Psy1 gene with Psy1/Unknown in a set of yellow-fruited tomato lines indicates that Psy1/Unknown might be caused by the SNP and/or SSR. One possible explanation of these observations is trans-splicing. Severely reduced Psy1 transcript caused by Psy1/Unknown results in low accumulation of carotenoid and yellow flesh in PI 114490.

Bacteriosis has become a major economic problem in the farming of the Pacific white shrimp Penaeus vannamei. However, no definitive data are available about Proteus penneri infection in cultured P. vannamei and its control. In this study, a virulent strain NC was isolated from diseased P. vannamei suffering from red body disease and identified as a P. penneri isolate through phylogenetic analysis and ATB 32GN system. A phylogenetic constructed tree using the neighbour-joining method identified the NC isolate as a P. penneri strain. In addition, Bdellovibrio bacteriovorus conferred significant protection against P. penneri: it exhibited significant bacteriolytic effects on the pathogenic P. penneri, had a wide prey range towards Proteus pathogens, and displayed a good protective efficacy on experimental P. penneri infection in P. vannamei. To the best of our knowledge, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus.

The use of Bacillus probiotics has been demonstrated as a promising method in the biocontrol of bacterial diseases in aquaculture. However, the molecular antibacterial mechanism of Bacillus still remains unclear. In order to explore the antibacterial mechanism of the potential antagonistic Bacillus amyloliquefaciens strain G1, comparative proteomics between B. amyloliquefaciens strain G1 and its non-antagonistic mutant strain was investigated. The 2-dimensional electrophoresis gel maps of their total extracted proteins were described and 42 different proteins were found to be highly expressed in strain G1 in comparison with those in the mutant strain. 35 of these up-regulated proteins were successfully identified using MALDI-TOF-TOF MS and databank analysis, and their biological functions were analyzed through the KEGG database. The increased expression of these proteins suggested that high levels of energy metabolism, biosynthesis and stress resistance could play important roles in strain G1's antagonism. To our knowledge, this is the first report on the proteins involved in the antagonism mechanism of B. amyloliquefaciens using a proteomic approach and the proteomic data also contribute to a better understanding of the molecular basis for the antagonism of B. amyloliquefaciens.

Saprolegnia species have been implicated for significant fungal infections of both living and dead fish as well as their eggs. In the present study, an oomycete water mould (strain HP) isolated from yellow catfish (Peleobagrus fulvidraco) eggs suffering from saprolegniosis was characterized both morphologically and from ITS sequence data. It was initially identified as a Saprolegnia sp. isolate based on its morphological features. The constructed phylogenetic tree using neighbour joining method indicated that the HP strain was closely related to Saprolegnia ferax strain Arg4S (GenBank accession no. GQ119935), that had previously been isolated from farming water samples in Argentina. In addition, the zoospore numbers of strain HP were markedly influenced by a variety of environmental variables including temperature, pH, formalin and dithiocyano-methane. Its zoospore formation was optimal at 20 °C and pH 7, could be well inhibited by formalin and dithiocyano-methane above 5 mg/L and 0.25 mg/L, respectively. To our knowledge, this is the first report on the S. ferax infection in the hatching yellow catfish eggs.

Recent studies have revealed that the use of bdellovibrios is an alternative to control bacteriosis. However, no bdellovibrios are available against Aeromonas hydrophila infections in sturgeons. In the present study, a potential Bdellovibrio strain F16 was isolated from sturgeon gut samples, using a sturgeon-pathogenic A. hydrophila as the prey bacterium. It was initially identified as a Bdellovibrio strain using morphological characteristics and specific PCR amplification, and confirmed to be Bdellovibrio sp. strain ETB (GenBank Accession No. DQ302728) and Bdellovibrio bacteriovorus strain SRA9 (GenBank Accession No. AF263833) by phylogenetic analysis. In addition, it was shown to be safe for mammalians and sturgeons, had a wide prey range, and exhibited significant bacteriolytic effects on the pathogenic A. hydrophila. To the best of our knowledge, this is the first report on a promising gut Bdellovibrio strain against pathogenic A. hydrophila., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recent studies have revealed that the use of probiotics is an alternative to control marine aeromonas. However, few probiotics are available against Aeromonas hydrophila infections in eels. In the present study, a potential antagonistic strain G1 against the eel-pathogenic A. hydrophila was isolated from sediment underlying brackish water. Its extracellular products with antibacterial activities were shown to be stable under wide range of pH, temperature, and proteinase K. It was initially identified as Bacillus amyloliquefaciens using API identification kits and confirmed to be B. amyloliquefaciens strain (GenBank accession number DQ422953) by phylogenetic analysis. In addition, it was shown to be safe for mammalians, had a wide anti-A. hydrophila spectrum, and exhibited significant effects on inhibiting the growth of the eel-pathogenic A. hydrophila both in vitro and in vivo. To the best of our knowledge, this is the first report on a promising antagonistic Bacillus amyloliquefaciens strain from brackish water sediment against eel-pathogenic A. hydrophila.