紫菜腐霉拮抗菌的筛选和鉴定

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。     

Comparison of electrophoretic patterns of soluble proteins and isozymes of the red rot disease fungus Pythium sp. isolated from Porphyra yezoensis from Korea and Japan

SUMMARY: Pythium sp., the causative organism of red rot disease, isolated from Porphyra yezoensis from Wando in Korea was compared with those from Miyagi, Aichi, and Fukuoka Prefectures by means of electrophoretic patterns of soluble proteins and isozymes. Native and sodium dodecylsulfate–polyacrylamide gel electrophoretic patterns of soluble proteins revealed small but significant differences among the four isolates. Among the 14 detected enzymes, catalase (CAT), carboxylesterase (CE), glucose-6-phosphate dehydrogenase (G6PD), glucosephosphate isomerase (GPI), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), superoxide dismutase (SOD), and xanthine dehydrogenase (XDH) possessed isozymes. The dissimilarity value of isozyme banding patterns for four isolates by unweighted pair-group method using arithmetic averages (UPGMA) cluster analysis was highest (0.51) between the isolate from Fukuoka and those from Miyagi and Aichi, and lowest (0.19) between the isolate from Miyagi and that from Aichi. Cluster analysis of data from isozyme banding patterns grouped the isolates from Miyagi and Aichi. These results indicate that the isolates from Miyagi and Aichi are similar, but each isolate from Wando and Fukuoka differed slightly from those of Miyagi and Aichi.     

Polysaccharolytic activities of bacterial enzymes that degrade the cell walls of Pythium porphyrae, a causative fungus of red rot disease in Porphyra yezoensis

ABSTRACT: Four bacteria with degrading activity against the cell walls of Pythium porphyrae were successfully isolated from Porphyra -culturing environments. The crude enzymes from these bacterial isolates, Bacillus sp. BE1, Bacillus sp. FE1, Pseudomonas sp. PE1, and Pseudomonas sp. PE2, degraded the mycelial cell walls of Pythium sp. N -Acetylglucosamine and glucose were detected in the supernatants of Pythium cell walls treated with the enzymes from Bacillus sp. BE1, Bacillus sp. FE1, and Pseudomonas sp. PE2 as the final degrading products by high-performance liquid chromatography, whereas only glucose was detected in the supernatant of the cell walls treated with the enzyme from Pseudomonas sp. PE1. Moreover, the activities of β-1,3-glucanase, β-1,3-1,4-glucanase, and chitinase were observed by polysaccharolytic analysis using the enzymes from Bacillus sp. BE1, Bacillus sp. FE1, and Pseudomonas sp. PE2, whereas only the activities of β-1,3-glucanase and β-1,3-1,4-glucanase were found during analysis using Pseudomonas sp. PE1. β-1,4-Glucanase, β-1,6-glucanase, and mannanase activities were not detected in any of the crude enzymes obtained from these isolates. From the four isolates, the molecular weights (MW) of β-1,3-glucanase and chitinase were estimated to be approximately 50 000–100 000 by the ultrafiltration method. Two Pseudomonas spp. were also suggested to have β-1,3-1,4-glucanases with MW of 50 000–100 000. However, the MW of β-1,3-1,4-glucanases from the two Bacillus spp. might be close to 50 000 or they produce at least two β-1,3-1,4-glucanases with MW of 30 000–50 000 and 50 000–100 000, respectively.     

Physicochemical responses of Pythium porphyrae (Oomycota), the causative organism of red rot disease in Porphyra to acidification

Physicochemical responses to acidification in Pythium porphyrae , the causative organism of red rot disease in Porphyra , were investigated. The acid tolerance of P. porphyrae mycelia under pH 4 (acidic condition) condition increased significantly compared with that of the mycelia under pH 8 (condition equivalent to seawater) condition. Free amino acid levels in the mycelia decreased 1.3–8.8-fold under pH 4 condition. However, some free amino acids such as the d -cysteinolic acid-like component, phosphoethanolamine, glutamic acid, aminoadipic acid and methionine increased 2.6–21.7-fold under the same condition. Proton flux on the mycelia exposed to pH 8 increased significantly compared with the mycelia exposed to pH 4. The patterns of proteins present (examined by two-dimensional polyacrylamide gel electrophoresis) differed among the pH conditions. These results suggest that P. porphyrae acquires acid tolerance and is able to adapt to the changing pH conditions. This has significant implications for the use of acidic fungicide treatment for prevention of red rot disease on Porphyra farms.     

Genetic variation detected with random amplified polymorphicDNA markers among isolates of the red rot disease fungus Pythiumporphyrae isolated from Porphyra yezoensis from Koreaand Japan

ABSTRACT:   Genetic variation of the fungal parasite Pythium porphyrae ,causative organism of red rot disease of Porphyra , isolatedfrom Asan, Mokpo, Pusan and Wando in Korea, and from Aichi, Fukuokaand Miyagi in Japan was investigated by random amplified polymorphicDNA (RAPD) cluster analysis. The total 67 RAPD markers were generatedfrom 38 isolates by RAPD-polymerase chain reaction (PCR) using arbitraryprimers consisting of 10 nucleotide sequences and 33 of them indicatedpolymorphisms. The dissimilarity coefficients calculated from theRAPD banding patterns ranged from 0.0010 to 0.6983. The dendrogramgenerated by the unweighted pair-group method using arithmetic averagesshowed that the 38 isolates were classified into three clusters(Groups 1, 2 and 3). Group 1 consisting of two isolates from Miyagiwas separated from all other isolates by a genetic distance of 0.6983.Groups 2 and 3 containing the majority of the isolates were branchedon genetic distance of 0.3957. These two clusters subdivided intofour and three subclusters, respectively, which were apparentlyassociated with geographic origins of the isolates. Interestingly,the isolates from Asan of Korea were close to Japanese isolatesrather than Korean isolates on genetic diversity. In addition, thegenetic distances of intra-isolates from Japan were higher thanthose from Korea.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven,Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg^-1) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10^-5M) glycogen breakdown and insulin-mediated (10^-8M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10^-7M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver. Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation. Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.     

Simulation and quantification of enrichment and retention processes in the southern Benguela upwelling ecosystem

Important environmental processes for the survival and recruitment of early life stages of pelagic fishes have been synthesized through Bakun's fundamental triad as enrichment, concentration and retention processes (A. Bakun, 1996, Patterns in the Ocean. Ocean Processes and Marine Population Dynamics. San Diego, CA, USA: University of California Sea Grant). This conceptual framework states that from favourable spawning habitats, eggs and larvae would be transported to and/or retained in places where food originating from enrichment areas would be concentrated. We propose a method for quantifying two of the triad processes, enrichment and retention, based on the Lagrangian tracking of particles transported within water velocity fields generated by a three‐dimensional hydrodynamic model. We apply this method to the southern Benguela upwelling ecosystem, constructing putative maps of enrichment and retention. We comment on these maps regarding main features of the circulation in the region, and investigate seasonal variability of the processes. We finally discuss the results in relation to available knowledge on the reproductive strategies of two pelagic clupeoid species abundant in the southern Benguela, anchovy (Engraulis encrasicolus) and sardine (Sardinops sagax). Our approach is intended to be sufficiently generic so as to allow its application to other upwelling systems.     

Status of aquatic and riparian biodiversity in artificial lake ecosystems with and without management for recreational fisheries: Implications for conservation

1. Humanity is facing a biodiversity crisis, with freshwater-associated biodiversity in a particularly dire state. Novel ecosystems created through human use of mineral resources, such as gravel pit lakes, can provide substitute habitats for the conservation of freshwater and riparian biodiversity. Many of these artificial ecosystems are subject to a high intensity of recreational use, however, which may limit their biodiversity potential.
2. The species richness of several taxa (plants, amphibians, dragonflies, damselflies, waterfowl, and songbirds) was assessed and a range of taxonomic biodiversity metrics were compared between gravel pit lakes managed for recreational fisheries (n = 16) and unmanaged reference lakes (n = 10), controlling for non-fishing-related environmental variation.
3. The average species richness of all the taxa examined was similar among lakes in both lake types and no substantial differences in species composition were found when examining the pooled species inventory. Similarly, there were no differences between lake types in the presence of rare species and in the Simpson diversity index across all of the taxa assessed.
4. Variation in species richness among lakes was correlated with woody habitat, lake morphology (surface area and steepness), and land use, but was not correlated with the presence of recreational fisheries. Thus, non-fishing-related environmental variables had stronger effects on local species presence than recreational fisheries management or the presence of recreational anglers.
5. Collectively, no evidence was found that anglers and recreational fisheries management constrain the development of aquatic and riparian biodiversity in gravel pit lakes in the study region; however, the conservation of species diversity in gravel pit lakes could benefit from an increasing reliance on habitat enhancement activities.