褐藻酸降解菌感染下海带活性氧产生的研究

以海带为材料，选用5株感染能力较强的褐藻酸降解菌作为致病菌，研究了活性氧（ROS）在海带抗褐藻酸降解菌感染中的产生及其作用。结果表明：（1）海带在褐藻酸降解菌感染下活性氧的产生具有普遍性。（2）活性氧的大量产生只发生在褐藻酸降解菌感染的早期阶段，随着感染时间的不断延长，活性氧的产生速率逐渐降低。说明活性氧的产生是海带在褐藻酸降解感染早期启动的反应特征之一。     

褐藻酸降解菌的筛选及其生长条件

采用安藤芳明选择性培养基从自然发病明显的海带幼苗上分离出8株褐藻酸降解菌。研究表明，8株菌能够不同程度地降解褐藻酸钠，菌株A1和A2表现出较强的降解能力，接种1d后液体培养基即变清。温度是该菌大量繁殖的决定性因子，其生长的最适温度为20℃，最适pH7.5。褐藻酸钠质量分数为0.5％-0.6％时，不同氮源和碳源对菌体生长的影响不同，其中有机氮和铵态氮有利于菌体生长，尿素和硝酸钠抑制菌体生长；只有在培养基中含有褐藻酸钠时，菌体才能正常生长。     

海带对褐藻酸降解菌的抗性与其抗氧化能力的相关性分析

采用生物化学的方法测试 2个品系的海带 (Laminariajaponica)对褐藻酸降解菌抗性的差异性 ,并对其抗氧化能力进行研究 ,旨为海带烂病的防治提供生物学基础依据。结果表明 ,1)海带荣城 1号对褐藻酸降解菌的抗性远高于海带 90 1;2 )海带荣城 1号细胞内 2种抗氧化酶 -超氧化物歧化酶 (SOD)和过氧化物酶 (POD)的活性明显高于海带 90 1;而另一种抗氧化酶—过氧化氢酶 (CAT)的活性与海带 90 1相比没有明显的差别 ;3 )海带荣城 1号的脂溶性抗氧化能力同样明显高于海带 90 1。说明海带的抗氧化能力与其对褐藻酸降解菌的抗性有一定的相关性。     

活性氧在海带抗褐藻酸降解菌感染中不同时期的抗氧化能力

以海带为材料,研究了活性氧(ROS)在褐藻酸降解菌感染中的产生及其作用.结果表明①褐藻酸降解菌感染引起海带活性氧的大量产生,且海带No.1活性氧的产生速率始终明显大于海带901(P<0.05).②在褐藻酸降解菌感染的早期阶段,海带维持着较高的抗氧化能力和对褐藻酸降解菌感染较高的抗性,而且海带的抗感染能力与活性氧产生速率呈一定的正相关性;2个品系相比,海带No.1的抗性又明显大于海带901(P<0.01).③在褐藻酸降解菌感染的后期,海带的抗氧化能力显著降低,膜脂过氧化和脱酯化伤害作用加剧,同时对褐藻酸降解菌感染的抗性显著降低,海带的抗感染能力与活性氧产生速率呈一定的负相关性,其中海带 No.1活性氧的产生速率、膜脂过氧化作用和脱酯化作用都远远高于海带901;相反,海带No.1对褐藻酸降解菌的抗性明显低于海带901(P<0.05).指示在感染的早期阶段,活性氧的产生在海带抵抗褐藻酸降解菌的感染中起着重要作用;而在后期阶段,活性氧的产生又大大降低了海带对褐藻酸降解菌感染的抗性.海带在感染的早期维持较高水平的抗氧化能力,而在感染的后期抗氧化能力显著下降是活性氧表现出双重作用的重要原因之一.     

对虾养殖水体中一株降解氨氮芽孢杆菌的分离鉴定及其发酵配方的优化

本实验在(28±1)℃下从对虾养殖池塘中分离筛选具有氨氮(包括游离氨NH_3、铵盐NH~+_4)降解效果的芽孢杆菌(Bacillus),并采用单因素实验对筛选出的芽孢杆菌进行发酵配方优化,以此挑选出低廉高产的发酵培养基配方。菌株分离筛选及氨氮降解实验结果显示:经初步分离,得到5株芽孢杆菌(DS1、DS2、DS3、DS4、DS5),再通过氨氮降解实验得到一株能够显著降解水体氨氮的芽孢杆菌DS5,5 d内菌株DS5对水体氨氮降解迅速,在第4天达到最大降解率(36.41±0.07)%,且最后3 d水体氨氮持续稳定在36%左右;通过菌株16S rDNA序列分析及生理生化鉴定,将菌株DS5(GenBank登录号:MK629979)初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。菌株DS5发酵配方实验结果表明:在28℃下,当碳源为5‰葡萄糖、氮源为10‰豆粕、无机盐为5‰氯化钠时,菌株生长良好,培养基含菌数较高,综合考虑成本、菌含量等因素,选择5‰葡萄糖-10‰豆粕-5‰氯化钠作为该菌的最优发酵培养基配方。     

凡纳滨对虾养殖体系中群体感应淬灭菌的筛选、安全性评估及发酵条件优化

从凡纳滨对虾(Litopenaeus vannamai)养殖体系中筛选得到具有群体感应淬灭(Quorum quenching, QQ)活性的潜在功能菌，并对菌株进行16S rDNA鉴定、安全性评估及发酵条件优化。采用根癌农杆菌(Agrobacterium tumefaciens) A136液体X-gal法进行活性菌株筛选，发现2株细菌(BDZ5和W1B)的发酵液对信号分子己酰基高丝氨酸内酯(C6-HSL)具有显著的降解作用，但经沸水浴处理后均丧失降解活性。经16S rDNA基因序列分析，菌株BDZ5和W1B分别被鉴定为芽孢杆菌属(Bacillus)和科贝特氏菌属(Cobetia)。安全性评估结果显示，菌株BDZ5和W1B均无溶血性，且对多数抗生素具有敏感性，注射2株活性菌未提高凡纳滨对虾死亡率，且对凡纳滨对虾血清免疫酶活性无显著影响。单因素条件优化结果显示，在起始pH为7.0、盐度为20、0.0005%的MnCl2条件下培养48 h后，菌株BDZ5达到最大生长量；在起始pH为6.0、盐度为40、0.05%的CaCl2条件下培养48 h，菌株W1B达到最大生长量。2株菌的最适信号分子C6-HSL降解条件与其生长条件有所不同，在起始pH为7.5、盐度为30、0.0005%的MnCl2条件下培养48 h，2株活性菌对信号分子C6-HSL的降解率达到最大值。研究表明，菌株BDZ5和W1B可作为益生菌应用于水产养殖，为基于QQ角度防治对虾细菌性病害提供优良菌种。     

褐藻酸降解菌对海带配子体克隆的作用及其药物防治研究

通过回染实验，确认褐藻酸降解菌是海带配子体克隆病害的主要致病菌；观察了对其海带配子体克隆细胞侵染的现象，并研究了抗生素对海带配子体克隆细胞和褐藻酸降解菌的作用，发现0．5－100mg/L青霉素对配子体克隆细胞的生长有一定促进作用，实验范围内各浓度青霉素对褐藻酸降解菌均有较好的抑菌效果，且抑菌效果随浓度的提高而加强。其中100mg/L青霉素10d内可将褐藻酸降解菌浓度降低到初始浓度的1／20以下，可作为海带配子体克隆保存中有效的抑菌药物。     

孔雀石绿降解菌Enterobacter sp.B-20的分离、鉴定和降解特性研究

从受孔雀石绿污染的淡水鱼养殖池塘底泥中,筛选到一株孔雀石绿(C23H25Cl N2,malachite green,MG)高效降解菌,经16S r RNA基因序列及系统进化树分析,初步鉴定为Enterobacter sp.B-20。采用硼氢化钾(KBH4)还原-高效液相色谱法对Enterobacter sp.B-20的孔雀石绿降解特性进行研究。结果显示,孔雀石绿对菌株的生长有一定抑制作用,24 h内菌株对5~20 mg·L~(-1)孔雀石绿降解率超过97%,12 h内对1~40 mg·L~(-1)孔雀石绿降解率超过82%。菌株的孔雀石绿降解能力随着p H的增加而显著提高,最适降解p H为9。Enterobacter sp.B-20具有较强的耐盐性,在10~40 g·L~(-1)的氯化钠(Na Cl)质量浓度下具有稳定的孔雀石绿降解特性。在高浓度金属离子条件下,Enterobacter sp.B-20表现出较强的耐性及稳定的孔雀石绿降解能力,0.1~1 mmol·L~(-1)铁离子(Fe3+)、0.1~0.5 mmol·L~(-1)铜离子(Cu2+)、0.1 mmol·L~(-1)锰离子(Mn2+)、0.1~1 mmol·L~(-1)铅离子(Pb2+)均能显著提高菌株的孔雀石绿降解率。结果表明,Enterobacter sp.B-20具有在复杂养殖水环境下降解孔雀石绿的应用潜力。     

褐藻酸降解菌诱发海带病烂的组织细胞学观察

用褐藻酸降解菌对海带进行人为感染，经培养使其产生烂斑，通过组织显微切片观察表明：1、褐藻酸降解菌首先侵入内皮层，然后再逐渐侵入到外皮层、髓部和表皮，最终导致海带病烂。2．感染后内皮层伤害最重，其次是外皮层和髓部，表皮的伤害最轻。3、侵染海皮层的细胞中，有大量颗粒状储藏物质，其直径范围在1．5－4．0μm之间，平均直径为2．7μm。形状为椭圆或圆形。     

褐藻酸降解菌引起海带内皮层细胞储藏颗粒变化的初步研究

实验对正常海带用褐藻酸降解菌进行了人工感染，经培养使其产生烂斑，通过组织显微切片进行比较观察，发现褐藻酸降解菌首先侵入内皮层，然后再逐渐侵入到外皮层、髓部，最终导致海带病烂。切片中发现在侵染海带切片内皮层的细胞中，有大量储藏颗粒，形成椭圆或圆形。其直径范围在1．5－4．0μm之间，平均直径为2．7μm。该颗粒主要存在内皮层，从其产生到消亡的过程分析，与褐藻酸降解菌的侵入有关，并作为营养起到促进菌增殖的作用。     

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.     

Does chromic oxide (Cr2O3) affect faecal lipid and intestinal bacterial flora in Arctic charr. Salvelinus alpinus (L.)?

Abstract. Arctic charr. Salvelinus alpinus (L.), were fed a commercial diet with or without a supplement of 1% chromic oxide (Cr₂O₃) in fresh water at 8°C for 70 days. Supplementation of the diet with 1% chromic oxide did not affect the lipid composition of stomach contents, but did affect that of the faeces.
Highest total lipid content in faeces was observed when fish were fed on the diet supplemented with chromic oxide. The proportion of triacylglycerols and free fatty acids increased in the neutral lipid fraction. The level of the dietary fatty acids 16:0, 16:1 and 18:1 increased, while 20:1 (n-9), 22:1 (n-11) and 18:2 (n-6) decreased significantly (P < 0·05) compared to the unsupplemented diet.
Total viable counts (TVC) of aerobic microorganisms were somewhat higher in stomach contents when the charr were fed on the unsupplemented diet. In faeces from charr fed the unsupplemented diet, TVC were higher, by a factor of 10 compared to fish fed diet supplemented with chromic oxide. Bacterial composition in stomach content of both rearing groups were dominated by Pseudomonas sp. and Agrobacterium sp., but the number of Lactobacillus sp. and Streptococcus sp. enhanced in stomach content when the fish were fed on the chromic oxide enriched diet. The faecal bacterial flora were also different between the rearing groups. The predominant bacterial species present in the faeces of fish fed unsupplemented diet were identified as Pseudomonas sp. and Aeromonas sp. Other bacterial species isolated included Lactobacillus sp., Streptococcus sp., Agrobacterium sp., Flavobacterium sp. and Vibrio sp. In contrast, the bacteria flora in faeces of fish fed the chromic oxide diet was dominated by the Gram-positive bacteria Streptococcus and Lactobacillus.
It is suggested that the use of chromic oxide as a marker in digestibility studies in Arctic charr may not be ideal.     

Inhibitory effects of Bacillus licheniformis (DAB1) and Pseudomonas aeruginosa (DAP1) against Vibrio parahaemolyticus isolated from Fenneropenaeus indicus

In aquaculture industries, there is an urgent need to develop microbial control strategies, to control disease outbreaks. In recent years, probiotics are considered as a valid alternative for the use of antibiotics in aquaculture to prevent high mortality and promote growth. In the present study, seven strains of bacteria such as Bacillus licheniformis (DAB1), Bacillus pumilus (DAB2), Bacillus sp. (DAB3), Pseudomonas aeruginosa (DAP1), Pseudomonas sp. (DAP2), Pseudomonas aeruginosa (DAP3), Pseudomonas aeruginosa (DAP4), and three pathogenic Vibrio parahaemolyticus (DAV1, DAV2, DAV3) were isolated from healthy and diseased Fenneropenaeus indicus collected from the east coast of Tamilnadu, India. The strains were identified by biochemical analysis and 16S rRNA sequence methods. Among the seven probiotic strains tested, the cell-free extract from DAB1 and DAP1 exhibited higher inhibitory activity of V. parahaemolyticus than other isolates under in vitro conditions. The LC₅₀ of DAV1, DAV2, and DAV3 was found to be ～10³ CFU mL^?1. Pathogenicity of three V. parahaemolyticus DAV1, DAV2, and DAV3 showed significant mortalities (40 %) in Artemia nauplii at inoculation densities of 10³ CFU mL^?1 when compared to the controls (unchallenged nauplii). A significant reduction in mortality (P < 0.001) was found by addition of 10⁶ CFU mL^?1 of DAB1 and DAP1 strains in nauplii against the pathogens. In conclusion, the present study result reveals that DAB1 and DAP1 have potential applications for controlling pathogenic V. parahaemolyticus in Artemia culture systems and aquaculture practices.     

根瘤菌N_1对扁藻生长影响的研究

研究了由海藻培养体系中分离获得的10株细菌对扁藻生长影响。试验结果表明,菌株N1对自然带菌扁藻及除菌扁藻的生长均有明显的促进作用;经16SrDNA分析表明,菌株N1为根瘤菌属细菌。     

不同食物组合与投喂方法对蒙古裸腹溞生长和生殖的影响

蒙古裸腹(Moina mongolica Daday)是旧大陆上分布的一种盐水裸腹溞[1],1988年在我国被首次报道[2].有关该溞的生物学和培养技术已被较为系统地研究[3].20世纪90年代以来,该溞在我国沿海地区逐渐被用做海水和半咸水鱼、虾、蟹人工育苗生产中的活饵料.     

虾夷扇贝面盘幼虫细菌性疾病的病原研究

从发生下沉死亡的虾夷扇贝面盘幼虫中分离到3株细菌X1,X2,X3,经过人工感染试验证实,3株细菌均对虾夷扇贝面盘幼虫具有致病性。应用PCR技术扩增16S rRNA基因的部分序列,并对其序列进行测定,同源性分析结果表明,这3株细菌分别与交替单胞菌属、冰居壶属和假交替单胞菌属的菌株具有较高的相似性,相似性均超过98%。10种抗生素的药敏试验结果表明,3株细菌对菌必治均敏感。     

A Lactobacillus sp. from diseased female rainbow trout, Salmo gairdneri Richardson, in Newfoundland, Canada

Abstract. Post-spawning mortalities of 2- to 3-year old female rainbow trout, Salmo gairdneri Richardson, at a Newfoundland hatchery were studied. The fish were found to have a mixed bacterial infection involving primarily a Lactobacillus sp. (a Gram-positive, chain-forming coccobacillus) and to a lesser degree Enterobacteriaceae, Aeromonas hydrophila and Pseudomonas fluorescens. The bacteria were isolated from the visceral organs, kidney, and ascitic fluid present in the coelomic cavity. The fish had extensive degeneration and necrosis of the liver, spleen and kidney, and sloughing of intestinal epithelium, but whether these were caused directly by the bacterial infection was not established because the fish appeared to have received substantial stress from the mechanical stripping and from the retention of dead, unshed eggs in the coelomic cavity. Tissue sections suggested that substantial growth of the Lactobacillus sp. was occurring within these unshed eggs. The present study describes this unusual observation and compares the Lactobacillus sp. to two other Lactobacillus isolates from cultured trout.     

海链藻对氮磷需求量的研究

本实验初步研究了海链藻对氮磷的需求，研究结果表明：以等摩尔氮的NH4Cl、NaNO，和NFLNO3作为氮源时，选择NH4NO3为氮源可以获得较快的生长率并维持较长时间的指数生长期；氮源浓度的最适范围在0．54-1．08mmol／L之间。而2．16mmol／L以上的高浓度NH4NO3则对海链藻的生长有一定的抑制作用；在不同磷浓度(0．015-0．12mmol／L)的条件下，海链藻保持大致相同的生长速率。     

美国青蛙膨气病病原菌的分离鉴定及药敏试验

经对膨气病病原菌的分离鉴定，嗜水气单胞菌与假单胞菌属细菌是致病菌。药敏试验结果：庆大霉素，氯霉素对致病菌有较强的杀灭作用，中草药黄连，黄岑也有一定的抑制作用，该病发生与环境条件密切相关，保持良好的养殖水质环境是预防该病的最好方法。     

利玛原甲藻PL11共附生菌多样性研究

为揭示产腹泻性贝类毒素(diarrheic shellfish poisoning,DSP)典型赤潮甲藻-利玛原甲藻(Prorocentrum lima)PL11的共附生菌群多样性信息,通过Illumina MiSeq高通量测序分析了PL11的共附生菌群种类及相对丰度,并且对其可培养共附生菌株进行了选择性分离及其16S rRNA基因序列扩增与系统发育的分析。利玛原甲藻PL11高通量测序结果显示:样品共附生细菌包括5门,14纲,26目,38科及54属。其中优势门(>5%)为3个,包括变形菌门(Proteobacteria,75.5%)、拟杆菌门(Bacteriodetes,11.5%)以及浮霉菌门(Planctomycetex,8.5%);优势纲(>5%)为4个,包括α-变形菌纲(Alphaproteobacteria,51.7%)、δ-变形菌纲(Deltaproteobacteria,31.8%)、鞘脂杆菌纲(Sphingobacteria,9.9%)以及OM190纲(8.9%);优势属(>5%)为6个,侏囊菌科下未知属(norank Nannocystaceae,21.8%)、Pyruvatibacte属(11.6%)、Phaeodactylibacter属(9.4%)、生丝单胞菌科下未知属(norank Hyphomonadaceae,8.5%)、OM190纲下未知属(8.1%)以及Roseovarius属(7.9%)。从利玛原甲藻PL11培养物中分离获得可培养微生物菌株9株,分属于8个属,包括Ochrobactrum sp.(2株)及Microbacterium sp.、Algoriphagus sp.、Ponticoccus sp.、Hoeflea sp.、Labrenzia sp.、Sphingopyxis sp.、Erythrobacter sp.各1株。其中PL11-1为Ponticoccus属潜在新种。