黄颡鱼卵致病性绵霉的分离鉴定与药敏特性

【目的】对黄颡鱼水霉病病原进行分离鉴定,并对其药敏特性进行研究。【方法】参照传统方法对患水霉病的黄颡鱼卵上的丝状真菌进行分离,通过人工感染实验证实分离菌株的致病性,然后根据形态学特征和ITS rDNA序列分析对致病菌株进行鉴定,并进一步采用倍比稀释法研究其药敏特性。【结果】从患水霉病的黄颡鱼卵上分离到8株丝状真菌,经人工感染试验证实菌株YC对黄颡鱼卵具有致病性,并进一步研究了其形态与药敏特性,开展了ITS rDNA序列分析。结果表明,菌株YC为透明管状结构,中间无横隔;游动孢子囊多呈圆筒形、棍棒形或穗状,游动孢子发育成熟后不断从孢子囊顶端释放出来,成团聚集在游动孢子囊口,并经过一个时期的静休后,成团脱落或直接分散在水中游动;次生孢子囊具有典型的侧生现象;藏卵器呈球形或梨形,大多与雄器异枝,少数与雄器同枝,含1?15个卵孢子;成熟卵孢子中生或亚中生,偏生一个大油球。其ITS rDNA序列与GenBank基因库中绵霉属菌株自然聚类,同源性高达99%,与异丝绵霉(Achlya klebsiana)菌株CBS101.49(GenBank登录号AF119579)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株YC为异丝绵霉(Achlya klebsiana)。此外,在实验选用的中草药和消毒剂中,黄连和异噻唑啉酮分别对菌株YC的抑菌效果最好,其对菌株YC的最小抑菌浓度分别为256 mg/L和2 mg/L。【结论】首次分离了黄颡鱼卵致病性异丝绵霉菌株YC,并确定了其药敏特性,可以作为该病防治用药的依据。     

彭泽鲫卵源致病性水霉的鉴定及其生物学特性

从患病的彭泽鲫卵上分离3株丝状真菌,经人工感染试验证实其中1株丝状真菌JL1对彭泽鲫卵具有致病性,并进一步研究了其形态与生长特性,开展了ITS rDNA序列分析。实验结果表明,菌株JL1菌丝为透明管状结构,中间无横隔,分枝较少;游动孢子囊多数呈棒状,游动孢子呈多排排列,发育成熟后从孢子囊中释放出来,并迅速游离;藏卵器呈球形,与雄器同枝或异枝。菌株JL1的ITS rDNA序列与GenBank基因库中水霉属菌株自然聚类,同源性高达99%,与Saprolegnia sp.H(登录号:EF460351)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株JL1为水霉菌(Saprolegnia sp.)。此外,菌株JL1在5°C-30°C、pH 4-11范围内均能生长,最适生长温度和pH范围分别为25°C-30°C和6-9。同时菌株JL1对NaCl敏感,质量分数为2%的NaCl即可抑制其生长,可以作为该病防治的依据。     

水霉菌的形态及ITS区分子鉴定

研究利用形态学和分子生物学相结合的方法, 对从患病黄颡鱼病灶处分离的水霉菌株(HSY)的分类地位进行了研究。结果显示, 该菌在18—23℃均能形成游动孢子囊, 但只在20℃才大量释放游动孢子; 在15℃培养约3 周出现大量的藏卵器, 多数顶生, 形态与Saprolegnia litoralis 和Saprolegnia ferax 的均非常相似。序列比对分析显示, HSY 菌株与S. litoralis、S. bulbosa、S. oliviae、S. longicaulis、S. ferax、S. mixta 和S.anomalies 的ITS 区(包括5.8S rDNA)序列相似性均高于99%。系统发育分析显示, 所选的真菌分为3 个群,HSY 落于第一个群, MP 树显示HSY 菌株与S. litoralis、S. bulbosa、S. oliviae、S. longicaulis、S. ferax 以及S. mixta 枝系均互相平行, 而NJ 树显示其与S. ferax 和S. mixta 更近。综合形态学、ITS 区序列及系统发育分析, 将HSY 菌株暂定为多子水霉(Saprolegnia ferax)。       

rDNA ITS序列在ACCC真菌鉴定中的应用

【目的】真菌无处不在,并与人类生活紧密相关。真菌的鉴定是科学研究和资源开发利用的基础。本研究从中国农业微生物菌种保藏管理中心(ACCC)随机选取已经定名的112株库藏真菌菌株作为实验材料,通过DNA测序,评估核糖体DNA内转录间隔区(rDNA ITS)序列在真菌属级鉴定上的应用。【方法】采用真菌DNA条形码rDNA ITS的序列测定和在GenBank中查寻比对的方法,对这些菌株进行属水平的鉴定复核。【结果】通过研究,供试菌株中有80株的属名与原鉴定相符,同时表明序列中套峰的情况降低与Gen Bank中序列比对的相似性。【结论】rDNA ITS序列测定法可以用于真菌菌株进行属水平的鉴定复核,国家菌种保藏机构应对已入库保藏和即将入库的所有真菌菌株都进行属水平的鉴定复核,并对菌株鉴定结果作审慎处理,原定名的名称及其变更历史应以"曾用名"在数据库中保留。     

一株表面活性剂产生菌的分离及抑菌活性

【目的】研究分离得到的表面活性剂产生菌的产表面活性剂能力、分类地位和抑菌活性。【方法】采用血平板、油平板进行表面活性剂产生菌的分离,以排油圈法进行表面活性的测定;通过生理生化特性和16S rDNA序列相似性分析对BS1菌株进行初步鉴定;利用对峙培养法和菌丝生长、孢子囊形成、孢子萌发的抑制率测定研究其抑菌活性。【结果】从石油污染土壤中分离到的BS1菌株可产生表面活性剂,在分类学地位上属于假单胞菌属(Pseudomonas sp.)。BS1菌体、发酵上清液、挥发性物质对12种供试病原真菌均表现出一定的抑制作用。BS1菌体、发酵上清液对大豆疫霉菌(Phytophthora sojae)的抑制率最大,分别达到65.31%和95.93%。发酵上清液通过影响大豆疫霉菌菌丝生长、孢子囊形成、孢子萌发等方式抑制病原菌的正常生长,稀释20倍的发酵上清液依然具有明显的抑制作用。BS1菌株产生的挥发性物质对大豆菌核菌(Sclerotinia sclerotiorum)的抑菌效果最好,抑制率达到84.25%。【结论】BS1菌株在产生表面活性剂的同时,还具有生物防治作用潜力。     

黄颡鱼暴发性出血病病原菌的分离鉴定及其生物膜形成特性

【背景】安徽省当涂县某池塘养殖黄颡鱼发生暴发性出血病,而当前对该病的病原存在争议。【目的】确定引起黄颡鱼暴发性出血病的病原菌,并明确分离菌株的生物膜形成特性,为从抗生物膜形成角度防治病原菌感染提供参考。【方法】取濒死期黄颡鱼病变脏器分别接种EPC细胞与培养基(TSB琼脂平板和血琼脂平板)分离病原,并通过人工感染回归试验确定其致病性;采用表型鉴定与16S rRNA基因序列分析相结合的方法鉴定分离菌株,并对其生物膜形成最佳条件、成膜能力及携带的生物膜形成相关基因进行研究。【结果】从病变脏器中分离纯化到一株优势菌株(HSY-2),对黄颡鱼的半数致死量为1.05×106 CFU/mL。经形态学、生化特性和细菌16S rRNA基因测序等分析确定分离株HSY-2为简达气单胞菌。其形成生物膜的最佳条件是将细菌接种TSB培养基于30 °C培养静置96 h,可形成中等强度的生物膜。同时,分离菌株携带气单胞菌甘油-3-磷酸脱氢酶D编码基因glpD、S-核糖同型半胱氨酸裂解酶基因luxS和LuxI家族蛋白同系物编码基因ahyI三种生物膜形成相关基因,但未检测到甘露糖敏感型血凝素菌毛合成蛋白Q编码基因。【结论】本实验为进一步研究简达气单胞菌生物膜形成的调控机制打下基础,并且从抗生物膜形成角度防治简达气单胞菌感染提供了参考。     

一株引起马来甜龙竹组培污染内生菌的分离与鉴定

【目的】对一株引起马来甜龙竹组培污染内生菌的分离与鉴定。【方法】采用改良的NA培养基分离纯化菌株,并通过菌体的形态结构观察、生理生化试验及其16SrDNA序列同源性分析对其进行鉴定。【结果】菌株SWFU01的形态特征及生理生化试验结果与解淀粉芽孢杆菌[Bacillus amyloliquefaciens(Fukumoto)Priest et al.]的描述基本相同;16S rDNA序列分析表明,该菌株与解淀粉芽孢杆菌JS在同一系统发育分支,其同源性为99.28%。【结论】综合形态学特征、生理生化特征以及16S rDNA序列分析的研究结果,菌株SWFU01被鉴定为解淀粉芽孢杆菌。     

水质净化酵母菌的分离筛选及鉴定

摘要:【目的】针对目前水产养殖专用优良菌种资源缺乏的现状,从养殖环境和养殖生物体中分离筛选具有水质净化功能的酵母菌,并对优良菌株进行鉴定。【方法】在低温和常温条件下从皮皮虾、南美白对虾肠道及养殖池底质活性污泥中分离具有水质净化功能的酵母菌,在模拟水体中对分离菌株的水质净化能力进行筛选,并对优良菌株采用形态、生理生化实验及5.8S rDNA ITS序列分析进行鉴定。【结果】从3种介质中共分离到酵母菌37株,其中常温分离16株,低温分离21株。水质净化实验结果表明,常温分离的16株酵母菌中有5株,低温分离的21株酵母菌中有6株对模拟水体中亚硝态氮和氨氮有显著的去除效果;其中低温分离的DN9和常温分离的CN6 48h能将10.64mg/L的亚硝态氮彻底转化,96h对630mg/L CODcr的去除率分别达52%和67%。DN9和CN6均产红色色素,经形态特征、生理生化特性及5.8S rDNA基因序列分析,鉴定菌株CN6为沼泽生红冬胞酵母(Rhodosporidium paludigenum),DN9为胶红酵母(Rhodotorula mucilaginosa)。【结论】红酵母DN9和CN6能有效去除养殖水体中的有机污染物和亚硝态氮,有望开发成 水产养殖水质净化高效微生态制剂。     

醉马草内生菌的分离、鉴定及杀虫研究

摘要：【目的】探明醉马草内生菌的种类,筛选对农作物虫害有毒杀作用的菌株。【方法】采用研磨法从健康醉马草植物的根、茎、叶和种子中进行菌种分离;通过对其形态、培养特征、生理生化及其他生物学特性的研究;16S rDNA及ITS序列的系统发育学分析进行鉴定;用玻片浸渍法和喷雾法筛选产杀虫活性物质菌株。【结果】获得细菌89株,分别属于枯草芽孢杆菌属（Bacillus）、链霉菌属(Streptomyces)、棒状杆菌属（Corynebacterium）、叶杆菌属（Phyllobacterium）、鞘脂单胞菌属（Sph     

蛇足石杉中产石杉碱甲内生真菌的分离和鉴定

【目的】从野生蛇足石杉(Huperzia serrata)中分离筛选产石杉碱甲的内生真菌。【方法】采用薄层层析及高效液相色谱法对内生真菌代谢产物进行测定和分析以期分离获得产石杉碱甲菌株,运用形态及ITS序列分析方法对产石杉碱甲菌株进行鉴定,并利用连续传代方法考察菌株遗传稳定性。【结果】经筛选获得一株产石杉碱甲内生真菌NSH-5,经形态学鉴定及ITS序列分析鉴定为轮枝镰孢菌(Fusarium verticillioides),其石杉碱甲产量为11.76 mg/100 m L,菌株经20次连续传代后遗传稳定。【结论】NSH-5菌株为一株具有产石杉碱甲能力的轮枝镰孢菌,该菌株的发现为生物合成石杉碱甲提供了新的菌种资源。     

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.     

Re-evaluation of the enigmatic species complex Saprolegnia diclina-Saprolegnia parasitica based on morphological, physiological and molecular data

The phylogenetic relationships among isolates of the Saprolegnia diclina-Saprolegnia parasitica complex were investigated based on ITS rDNA sequences, and correlated with morphological and physiological characters. The isolates studied belong to five phylogenetically separate clades. The majority of presumed parasitic isolates, mostly isolated from fish lesions, fell within a clade that comprises isolates which has been variously named as S. diclina Type 1, S. parasitica, Saprolegnia salmonis or just as unnamed Saprolegnia sp. Presence of bundles of long-hooked hairs on secondary cysts, high frequency of retracted germination, and oogonia production at 7 degrees C (when occurring) were characteristic of this clade. A single isolate identified as S. diclina Type 2 clustered in a clade along with Saprolegnia ferax isolates. The isolates identified as S. diclina s. str. (S. diclina Type 3) distributed in two clades and appeared closely related to Saprolegnia multispora and to a number of Chilean isolates identified as Saprolegnia australis. The ITS sequences of clade I were almost identical even though the isolates were of diverse geographical origins and showed physiological and morphological differences and variations in their pathogenicity. This suggest these species reproduces clonally even in apparently sexually competent isolates. Adaptation to parasitism in Saprolegnia might have occurred at spore level by the development of long-hooked hairs to facilitate host attachment and selection of a retracting germination. The use of the name S. parasitica should be assigned to isolates of clade I that contained isolates forming cysts with bundles of long-hooked hairs.     

Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.     

Molecular identification of a bronopol tolerant strain of Saprolegnia australis causing egg and fry mortality in farmed brown trout, Salmo trutta

Some species of the genus Saprolegnia, such as Saprolegnia diclina and Saprolegnia ferax are responsible for devastating infections on salmonid eggs. Members of this group cause saprolegniasis, a disease resulting in considerable economic losses in aquaculture. Although both S. diclina and S. ferax have received much attention, the role of other Saprolegnia species in infecting fish eggs is less known. For this purpose, we have investigated the aetiology of chronic egg mortality events occurring in farmed brown trout, Salmo trutta. A total of 48 isolates were obtained from eggs with signs of infection as well as from water samples. A molecular analysis based on nrDNA internal transcribed spacer (ITS) operational taxonomic units indicated that the majority of the isolates correspond to Saprolegnia australis. All isolates of S. australis exhibited the same random amplified polymorphic DNA (RAPD) band patterns suggesting that a single strain is implicated in egg infections. The isolates followed Koch postulates using trout eggs and fry. Under standard concentrations of bronopol commonly used in farms, these isolates could grow, but not sporulate. However, both growth and sporulation were recovered when treatment was removed. This study shows that S. australis can infect and kill salmon eggs, and helps in defining oomycetes core pathogens.     

Fungi isolated from cultured eggs, alevins and broodfish of brown trout in a hatchery affected by saprolegniosis

The aquatic fungi cultured from eggs, alevins and broodfish of brown trout Salmo trutta belonged to the genus Saprolegnia and were identified as S. diclina , S. australis , S. ferax , S. furcata , S. hypogyna , S. unispora and S. parasitica . The species obtained from infected eggs and alevins were different to those from infected fish. Several Saprolegnia species were isolated from eggs and alevins, whereas all the isolates obtained from broodfish were the pathogenic S. parasitica .     

雄性普通黄颡鱼与所保护受精卵间的亲缘关系分析

父母通过保护自己的子代来确保其繁殖的成功率.在普通黄颡鱼(Pelteobagrus fulvidraco)中,雄性普通黄颡鱼具有筑巢产卵保护后代的习性,其所保护的子代与其是否有亲缘关系是有待探讨的问题.本文利用10对微卫星分子标记鉴定12窝普通黄颡鱼受精卵与护卵鱼之间的亲缘关系,并对子代的遗传多样性进行分析.在单亲鉴定中,累积非父排除概率为0.9986,平均父权相对机会(RCP)在99.989%-99.999%之间,每个子代在10个微卫星位点上的累积PI值在2006.73-604464.07之间.同时在亲权鉴定分析中,发现3窝卵子的等位基因来自2个母亲,说明雄性黄颡鱼可以和2条雌性黄颡鱼发生交配;在遗传多样性分析中,黄颡鱼子代的平均等位基因数为11.7, 无偏观测杂合度值(Ho)在0.2473-0.9866之间,多态信息含量(PIC)值0.7096-0.8993之间.通过亲权鉴定分析,可以确认看护受精卵的雄性普通黄颡鱼与受精卵间的亲子关系.     

Saprolegnia australis R. F. Elliott 1968 infection in Prussian carp Carassius gibelio (Bloch, 1782) eggs and its control with herb extracts

In order to control saprolegniosis in Prussian carp (Carassius gibelio (Bloch, 1782) eggs, it is important to screen herb extracts as potential anti‐Saprolegnia drugs in Prussian carp hatcheries. For this purpose, an oomycete water mould (strain SC) isolated from Prussian carp [Carassius gibelio (Bloch, 1782)] eggs suffering from saprolegniosis was characterised morphologically as well as from ITS rDNA sequence data. Initially identified as a Saprolegnia sp. based on its morphological features, the constructed phylogenetic tree using the neighbour joining method further indicated that the SC strain was closely related to Saprolegnia australis R. F. Elliott 1968 strain VI05733 (GenBank accession no. HE798564 ), and which could form biofilm communities as virulence factors. In addition, aqueous extracts from forty Chinese herbs were screened as possible anti‐Saprolegnia agents. Among them, a 1 g ml^?1 extract from Radix sanguisorbae was the most efficacious anti‐Saprolegnia agent, indicated by the minimum inhibitory concentration that was as low as 256 mg L^?1. Relative survival of 73 and 88% was obtained against the SC strain in fish eggs at concentrations of 256 and 1280 mg L^?1, respectively. This is the first known report of Saprolegnia australis R. F. Elliott 1968 infection in C. gibelio (Bloch, 1782) eggs involving the screening of R. sanguisorbae extracts as potential anti‐Saprolegnia agents.