贪婪倔海绵中抗菌活性细菌的筛选及初步鉴定*

采用平板涂布法从我国南海三亚周边海域贪婪倔海绵(Dysidea avara)中分离海绵共附生细菌,采用金黄色葡萄球菌、大肠埃希氏菌、荧光假单胞菌、枯草芽孢杆菌、白假丝酵母、宛氏拟青霉、黑曲霉7种指标菌进行抑菌试验筛选抗菌活性菌,同时对于得到的活性菌进行生理生化鉴定。共分离获得个149个细菌菌株,发现20株具有抑制真菌和革兰氏阳性细菌的活性,占细菌总数的13.4%。经过细菌形态观察和生理生化试验,发现此20株活性菌属于革兰氏阳性芽孢杆菌属(Bacillussp.)。     

红树内生细菌CⅢ-1菌株鉴定及其胞外抗菌蛋白性质研究

用对峙生长法从422株红树内生细菌中筛选到1株对青枯雷尔氏菌(Ralstonia solanacearum)、金黄色葡萄球菌(Staphylococcus aureus)、溶藻弧菌(Vibrio alginolyticus)、辣椒疫霉菌(Phytophthora capsici)、香蕉枯萎病菌(Fusarium oxysporum f. sp. cubense)等动植物病原细菌和真菌均具有较强的拮抗作用的海洋细菌CⅢ-1菌株, 经形态和生理生化特性及16S rRNA序列分析, 鉴定该菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。用33%硫酸铵从菌株的培养滤液中盐析获得对供试病原菌均具有较强拮抗作用的抗菌活性蛋白, 性质测定发现, 该抗菌活性蛋白对热不稳定, 60?C和100?C下处理10 min, 其抗菌活性分别下降了62.5%和完全丧失, 在pH 5.0?10.0范围内均具有抑菌作用, 以pH 7.0时抗菌活性最强。     

红树内生细菌CⅢ-1菌株鉴定及其胞外抗菌蛋白性质

用对峙生长法从422株红树内生细菌中筛选到1株对青枯雷尔氏菌(Ralstonia solanacea-rum)、金黄色葡萄球菌(Staphylococcus aureus)、溶藻弧菌(Vibrio alginolyticus)、辣椒疫霉菌(Phytophthora capsici)、香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)等动植物病原细菌和真菌均具有较强的拮抗作用的海洋细菌CⅢ-1菌株,经形态和生理生化特性及16S rRNA序列分析,鉴定该菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。用33%硫酸铵从菌株的培养滤液中盐析获得对供试病原菌均具有较强拮抗作用的抗菌活性蛋白,性质测定发现,该抗菌活性蛋白对热不稳定,60°C和100°C下处理10min,其抗菌活性分别下降了62.5%和完全丧失,在pH5.0?10.0范围内均具有抑菌作用,以pH7.0时抗菌活性最强。     

恩诺沙星对4种水产致病弧菌的抑杀菌效应

采用二倍稀释法测定恩诺沙星对溶藻弧菌、最小弧菌、哈维氏弧菌、创伤弧菌4种12株菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),结果表明:恩诺沙星对12株弧菌的MIC为0.1?0.8 mg/L,MBC为0.4?3.2 mg/L。在此基础上,从每种菌中选取一株对恩诺沙星较为敏感的菌株,研究恩诺沙星不同药物浓度(1 MIC、2 MIC、4 MIC)对4种弧菌的杀菌动力学和抗菌后效应(PAE),结果显示:各浓度恩诺沙星对溶藻弧菌X040625-R菌株均有较强的杀菌作用,而对哈维氏弧菌M071202-H、创伤弧菌Q050723-C、最小弧菌H010911-Z等3菌株却表现出了低浓度抑菌、高浓度缓慢杀菌的作用。恩诺沙星的抗菌后效应PAE与药物浓度及细菌与药物的接触时间成正相关,恩诺沙星对溶藻弧菌X040625-R的PAE最长,对哈维氏弧菌M071202-H的PAE最短。     

南极沉积物来源抗菌细菌的筛选及抑菌物质的鉴定

【背景】科学研究表明,由于南极环境条件特殊,微生物资源丰富,有望筛选出功效显著的抗菌微生物。【目的】以黄瓜枯萎病致病菌木贼镰刀菌(Fusarium equiseti)为指示菌,从南极沉积物中分离筛选具有拮抗作用的细菌菌株并对其抑菌物质进行初步鉴定。【方法】应用平板对峙法和琼脂扩散法分别对样品和发酵液进行初筛和复筛,筛选出对F. equiseti抑菌效果最强的菌株,基于形态学、生理生化、分子生物学分析,对该菌株进行鉴定。之后,对目标菌株发酵上清中的抑菌物质进行抑菌谱研究,并对其抑菌成分进行温度和pH的稳定性检测,通过硫酸铵沉淀的方法初步鉴定发酵液中的抑菌物质。【结果】从南极沉积物样品中共分离纯化出62株细菌,有5株具有较好的抑菌效果,其中抑菌效果最强的菌株鉴定为枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp.spizizenii),命名为JYM35。抑菌谱检测结果显示,菌株JYM35对丝瓜枯萎病致病菌层生镰刀菌(Fusarium proliferatum)和辣椒枯萎病致病菌F. equiseti具有较强的抑菌效果,对长豆褐腐病致病菌笄霉属(Choanephora)有较明显的拮抗作用,对水产致病菌副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V.alginolyticus)也有一定的拮抗作用。菌株JYM35发酵上清中所含的抑菌物质热稳定性强且耐碱不耐酸,硫酸铵沉淀后可初步确定其抑菌物质隶属蛋白类。【结论】菌株JYM35是一株产蛋白类活性物质的广谱型抑菌菌株,对枯萎病致病菌的拮抗作用最强。因此具有较好的开发利用价值。     

抗耐药细菌药用植物内生菌的筛选与鉴定

耐甲氧西林金黄色葡萄球菌(MRSA)、耐头孢霉素大肠杆菌和耐亚胺培南铜绿假单胞菌在医院临床治疗中引起的严重感染,极大地危害着人类身体健康。旨在从植物组织中分离和筛选高活性的拮抗上述耐药细菌的内生菌株。从分离自22种药用植物组织的197株内生菌中,经过两步筛选获得18株对MRSA有拮抗作用的菌株,未筛选到对耐头孢霉素大肠杆菌和耐亚胺培南铜绿假单胞菌有拮抗作用的菌株;经16SrRNA序列分析,其中8株为链霉菌属,6株为芽孢杆菌属,4株为假单胞菌属;对抑菌效果较强的5株菌进行发酵培养,检测发酵液对MRSA的抑菌效果,其中QN1和CF2的发酵液对MRSA有很好的抑制效果。     

一株抗水稻纹枯病菌的解淀粉芽胞杆菌分离与鉴定

【目的】筛选对水稻纹枯病菌(Rhizoctonia solani)具有强拮抗作用的细菌菌株。【方法】用指示菌法筛选拮抗菌株;通过形态观察、生理生化实验、Biolog及16S rDNA序列分析鉴定目标菌株;利用平板双向培养法和滤纸片扩散法测定抑菌谱及拮抗性质。【结果】分离到一株高活力的水稻纹枯病菌拮抗菌株YB-3,该菌株属于解淀粉芽胞杆菌(Bacillus amyloliquefaciens);菌株YB-3对常见的14株病原真菌和7株细菌具有较强的拮抗作用,并发现其对亲缘关系较近的芽孢菌属有较强的拮抗作用;该菌株的抑制活性具有温度稳定、耐酸、但对蛋白酶敏感的特点。【结论】通过指示菌法筛选到一株对水稻纹枯病菌有强拮抗作用的解淀粉芽胞杆菌(B.amyloliquefaciens)YB-3,它具有广谱、高效的植物病原菌拮抗活性。     

斑节对虾(非洲群体)肠道中哈维氏弧菌拮抗菌的筛选、鉴定及生理特性分析

基于斑节对虾(非洲群体)(Penaeus monodon)肠道菌的生理生化特性, 为解决斑节对虾急性肝胰腺坏死综合征(AHPND)提供新思路, 文章分离纯化健康斑节对虾肠道中的优势菌株; 以致病性哈维氏弧菌为指示菌, 用牛津杯法从纯化菌株中筛选拮抗菌; 通过生理生化特征、Biolog系统鉴定及16S rDNA技术综合方法对菌株进行鉴定; 通过药敏实验, 评价拮抗菌株的安全性; 绘制高敏拮抗菌株的生长曲线, 得出其生长特性; 通过测定其产酶活性, 形成对其益生机制的初步推断。从斑节对虾肠道中共分离得到14株的优势土著菌, 分别标记为P.m-1、P.m-2······P.m-14, 经过拮抗实验得到了3株拮抗菌(P.m-1、P.m-9和P.m-13)。经鉴定P.m-1、P.m-9和P.m-13分别为枯草芽孢杆菌(Bacillus subtilis)、屎肠球菌(Enterococcus faecium)和溶藻弧菌(Vibrio alginolyticus)。药敏实验结果表明, 3株拮抗菌均不属于耐药菌, P.m-1对药物高度敏感。生长特征实验表明: P.m-1在2h进入对数期, 10h达到生长高峰, 菌体密度可达1.14×109 cfu/mL, 表现出了强劲的生命力。经过蛋白酶活性测定, P.m-1具有较强的产酶能力。优势菌P.m-1具有成为益生菌的潜力, 后续将作为功能性饲料添加剂或水质调节剂进行应用, 为AHPND进行生物防治提供新思路。     

一株解淀粉芽孢杆菌的分离鉴定及其抑菌条件研究

旨在从海洋环境中分离筛选抑制鲈鱼病原鳗弧菌的拮抗菌,进行菌种鉴定,并对其抑菌条件及药物敏感性进行研究。采用十字交叉划线法和平板点种法筛选鲈鱼病原鳗弧菌的拮抗菌,采用形态学、生理生化反应及16S r DNA基因序列的系统发育分析方法对分离拮抗菌进行鉴定,采用单因子变量法对拮抗菌WTD的最佳抑菌条件进行研究,采用平板药敏纸片法对其药物敏感性进行研究。结果显示,从海洋环境样品中分离出126株细菌,筛选出19株拮抗菌。其中菌株WTD的拮抗效果最强,细菌鉴定结果表明,该菌为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。最佳抑菌条件研究表明,该菌在温度35℃、p H7、盐度为1%Na Cl的时候,拮抗效果最好。药敏实验结果表明,该菌对麦迪霉素等29种抗生素敏感,而对多粘菌素具有抗性。拮抗菌解淀粉芽孢杆菌WTD对鲈鱼病原菌鳗弧菌有较强的抑制作用,具有潜在的应用价值。     

南极抗细菌活性菌株的筛选及系统发育分析

分别以大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、青枯假单胞菌、绿脓假单胞菌和苏云金芽孢杆菌为指示菌,采用琼脂扩散法对实验室保存的580株极地细菌进行了抗菌活性菌株的筛选与活性验证,从中筛选出4株对上述指示菌株具有明显抗菌效果的活性菌株,其编号分别为97、Z11、Z18及Z19,并对其生长曲线、抗菌活性曲线和系统发育地位进行研究。结果表明,4株菌均在培养24 h后进入指数生长期,菌株97在培养48 h后达到稳定期,而菌株Z11、Z18及Z19在培养60 h后达到稳定期。抗菌活性分别在培养84、96、72和72 h时达到最高。系统发育分析表明,该4株菌分别属于伦黑墨氏菌属(Rheinheimera)、嗜冷杆菌属(Psychrobacter)、假单胞菌属(Pseudomonas)和嗜冷杆菌属(Psychrobacter)。     

利用形态学及mtDNA基因分子鉴定长须鲸和大村鲸

大型鲸类死亡个体,往往因腐烂程度较高或外部形态损坏,很难鉴定物种。2018年11月和2019年1月在江苏南通和浙江宁波各发现一头死亡须鲸。本文仔细辩别了两头须鲸的形态学特征,又应用分子生物学技术,分别得到903 bp和972 bp mtDNA控制区序列,以及467 bp和438 bp Cyt b序列。经GeneBank 序列比对,南通样本与长须鲸相似度最高,达99%~100%;宁波样本与大村鲸相似度最高,达97%~99%。通过MEGA 7.0软件的最大似然法（ML）构建的系统发育树与Blast结果一致。综上,南通死亡须鲸鉴定为长须鲸,宁波死亡须鲸鉴定为大村鲸。同时,由于这两个物种在中国的分布信息比较缺乏,本研究证实了长须鲸在江苏省南通市及大村鲸在浙江省宁波市象山县的新分布。     

中国沿海浒苔属rbcL和ITS序列比较及系统进化分析

为阐明中国沿海浒苔的亲缘关系及地理分布特点,采集青岛栈桥、盐城弶港、宁波象山、温州平阳四地浒苔样本,克隆测序得到ITS1、5.8SrDNA和ITS23种不同长度序列片段。四个地区的rbcL目的片段,长度均为1201bp。分析核苷酸差异和遗传距离,采用邻接法建立系统发生树。结果显示,ITS序列进化速率较快,rbcL序列相当保守。ITS区较短,GC含量均在65%以上,5.8SrDNA的CG含量在50%左右,ITS1区的序列差异大于ITS2区。四个地区的浒苔存在一定的地理差异,盐城和青岛的样本间的亲缘关系较近;宁波和温州的样本间的亲缘关系较近。石莼属(Ulva)和浒苔属(Enteromorpha)的物种没有聚成各自独立的分枝,而是相互混合在一起,应是两个亲缘关系相近的属。引起青岛绿潮的海藻很可能是来自盐城海域的Enteromorpha linza或Enteromorpha prolifera。     

First palynological data from the Jurassic South Xiangshan Formation (Nanjing area,China)

The outcrops of the Xiangshan Group have been studied since the beginning of the twentieth century, yielding a rich macroflora (the Xiangshan Flora), mainly from the lower part of the Xiangshan Group (South Xiangshan Formation). Nevertheless, no palynological data have been published so far from the South Xiangshan Formation. The present study provides the first palynostratigraphic data of the South Xiangshan Formation. More than 50 fossil taxa from 30 fossil genera have been identified, allowing for a more accurate dating. The most characteristic taxa are Polycingulatisporites triangularis, Quadraeculina anellaeformis, Manumia delcourtii, Ischyosporites variegatus, Callialasporites turbatus, C. trilobatus, C. minus, and Sestrosporites pseudoalveolatus, suggesting a late Toarcian-late Aalenian age for the South Xiangshan Formation. These results are consistent with previous studies suggesting a middle-late Early Jurassic age for this formation; consequently, the most probable age for the South Xiangshan Formation is late Toarcian (late Early Jurassic).     

DNA Base Composition and Esterase Patterns of Bacteria Isolated from Deep Sea Sediments

Electrophoretic patterns of esterase isozymes and DNA base compositions were determined for 38 marine bacteria, 31 strains of which were from sediment samples collected at depths of 9400–10,400 in in the Philippine and Marianas Trenches of the Pacific Ocean, and 7 from seawater samples collected at various depths of surface–4000 m and from several locations in the Pacific Ocean. Previous numerical taxonomic analysis indicated 5 distinct phenetic clusters; 4 identified as Pseudomonas and 1 as aerogenic Aeromonas. In this study, the DNA base composition range for the entire sample set was found to be 41–59 moles % of GC. However, 32 of the 38 strains possessed a GC of c. 57%. In general, strains belonging to the same phenon possessed nearly identical DNA base composition and demonstrated similar esterase patterns.     

黄精种子萌发及组培技术研究

研究了不同的激素对多花黄精种子的萌发及组培的影响。研究结果表明:多花黄精种子萌发的最佳激素条件为6-BA 180.0 mg/L+GA_3 60.0 mg/L,两种激素可能有交互作用;多花黄精块茎外植体的最佳灭菌时间为12 min;最佳诱导外植体愈伤组织培养基为:6-BA 2.0 mg/L+2,4-D 0.2 mg/L+MS,低浓度的6-BA利于多花黄精块茎愈伤诱导;最佳诱芽培养基为:6-BA 6.0 mg/L+NAA 0.2 mg/L+MS和6-BA 4.0 mg/L+TDZ 0.2 mg/L+NAA 0.2 mg/L+MS,低浓度的TDZ对不定芽的诱导和生长有促进作用;最佳生根培养基为:IBA 1.0 mg/L+1/2MS,但是过高浓度的IBA会抑制根的诱导。通过多花黄精的繁殖技术的研究,以期为多花黄精的快繁提供技术支持。     

凡纳滨对虾健康状态分化前后养殖水体浮游细菌群落的比较

养殖水体浮游细菌群落与对虾健康状态息息相关.本研究基于16S rRNA高通量测序技术手段比较了凡纳滨对虾病害发生前后病害趋向塘与健康塘的浮游细菌群落多样性、组成及关键类群互作关系的动态变化.结果表明:对虾发病前,病害趋向养殖水体浮游细菌群落Shannon指数显著低于健康养殖水体,且呈现持续下降的趋势;伴随疾病爆发,病害趋向养殖水体浮游细菌群落结构离散程度显著增大,群落稳定性较差;发病前,病害趋向塘养殖水体中绿弯菌门、δ-变形菌纲和Saccharibacteria平均相对丰度均显著高于健康水体,而拟杆菌门显著低于健康水体;部分病害指示OTU隶属于弧菌科及黄杆菌科,隶属于红杆菌科的OTU1是发病前健康塘的健康指示OTU.网络分析表明,大多数与OTU1存在相互作用的指示OTUs属于黄杆菌科,说明黄杆菌科在对虾养殖水体浮游细菌群落中扮演重要角色.鉴于以上结果,对虾发病可能与多种浮游细菌群落相关,当出现“生态位机会”时,条件致病菌快速繁殖进而导致对虾发病,而保持和恢复细菌群落平衡可以成为预防对虾发病的有效策略.     

Properties of Bacteria Isolated from Deep-Sea Sediments

Thirty-eight isolates were subjected to taxonomic analysis by computer. Of the 38 isolates, 31 were from sediment samples collected at depths from 9,400 to 10,400 meters in the Philippine and Marianas Trenches of the Pacific Ocean, and 7 cultures were from seawater samples collected at various depths from surface to 4,000 meters and from several locations in the Pacific Ocean. A total of 116 characteristics were determined for each isolate, coded, and transferred to punch cards. Similarity values were obtained by computer analysis, with the use of two recently developed computer programs. Five distinct phenetic clusters were observed from the numerical analyses. Four of the clusters were identified as species of the genus Pseudomonas, and one, as an aerogenic species of Aeromonas. Group IV was identified as pigmented Pseudomonas fluorescens, and the major cluster, consisting of groups I and II, which merged at a species level of similarity, was treated as a new species of Pseudomonas. The 38 strain data were compared with data for 132 marine and nonmarine strains previously subjected to computer taxonomic analysis. The barotolerant deep-sea strains, with the exception of the deep-sea P. fluorescens isolates, clustered separately from all other marine strains.     

Quantitative assessment of fish assemblages on artificial reefs using acoustic and conventional netting methods,in Xiangshan Bay,Zhejiang Province,China

Artificial reefs are often deployed by many countries for the purpose of enhancing fishing after a period of several years. The objective of this study was to analyze the effect of a pilot artificial reef system deployment in enhancing the demersal fish assemblages, investigated using both acoustic methods and bottom trammel nets, during four years between 2011 and 2017, in Xiangshan Bay, Zhejiang Province, China. Comparisons of community indices, including fish biomass, species richness and species diversity, indicated relatively consistent trends between the control sites and the artificial reefs following their deployment in 2012. Fish density, represented by the value of the nautical area scattering coefficient, and fish biomass were significantly higher on the artificial reefs than at the control sites in 2015 and 2017, and species richness and diversity were significantly greater at the reefs from 2013 to 2017. Blackhead seabream (Acanthopagrus schlegelii) and false kelpfish (Sebastiscus marmoratus) were the dominant fishes on the artificial reefs, and the average body lengths of these two species were significantly greater 40 and 64 months after deployment. Thus, construction of the artificial reefs appears to have achieved their primary purpose as fish-attraction devices, thereby contributing to ecological restoration in Xiangshan Bay.     

宁波港压载水的细菌多样性研究

为揭示宁波港压载水的微生物群落结构在人类活动影响下的分布差异及对环境因子变化的响应趋势。该课题从压载水中分离细菌,通过微生物自动鉴定系统(Sherlock MIS)、分子生物学16S rRNA基因的PCR扩增及RFLP技术,对宁波港压载水的细菌结构特征进行比对和分析,并通过16S rRNA基因文库解析研究压载水中微生物群落多样性。结果表明印度洋水样中存在假交替单胞菌属(Pseudoalteromonas sp.)、交替单胞菌属(Alteromonas sp.)、海杆菌属(Marinobacter sp.)和玫瑰杆菌属(Roseobacter sp.)、肠杆菌属(Enterobacter sp.);新加坡水样中存在人苍白杆菌属(Ochrobactrum sp.)、甲基杆菌属(Methylobacterium sp.)、鞘鞍醇单胞菌(Sphingomonas sp.)、嗜冷杆菌属(Psychrobacter sp.)、假单胞菌(Pseudomonas sp.)、伯克霍尔德氏菌(Burkholderia sp.)和葡萄球菌属(Staphylococcus sp.)假单胞菌属(Pseudomonas sp.);而美洲水样中存在弧菌属(Vibrio sp.)、军团菌属(Legionella sp.)、莫拉氏菌属(Moraxella sp.)和不动杆菌属(Acinetobacter sp.)。压载水中含有大量的潜在致病菌。     

旅游干扰对香山黄栌林的影响研究

 旅游生态学是生态学应用的一个新领域。香山公园是北京近郊重要的风景游览区,游客在时间上和空间上具有集中性。本文从种群、群落和土壤特性几个方面出发,根据道路状况确定旅游干扰程度,探讨了旅游干扰对香山最重要的旅游资源——黄栌林的影响,其结果可以为香山公园的旅游生态规划提供科学依据。