龙须菜体表附生细菌的几种分离方法比较

采用超声波粉碎法、涡旋振荡法、超声波清洗法、研磨匀浆法等4种不同的方法处理龙须菜,分离其体表的附生细菌,并对分离细菌的数量、种类、形态结构、细胞壁特性等进行了观察和分析。通过对不同方法和相间方法的不同处理所得结果比较显示,超声波清洗法和研磨匀浆法对分离细菌的数量和种类效果都较差;超声波粉碎法和涡旋振荡法效果较好,尤以超声波粉碎法的30W30s处理效果最好,该方法分离到本项目4个方法13个处理获得的16个菌株中的12个菌株,龙须菜的细菌数1．75×10~6cells/g。     

番茄灰霉病内生拮抗细菌的分离筛选初报

采用常规法对番茄植株进行内生细菌的分离 ,结果表明 ,不同品种不同生长时期 ,内生细菌的数量有所不同 ;同一品种不同生长时期不同部位 ,内生细菌的数量也存在变化。在所分离的 96个菌株中 ,有 7个菌株对番茄灰霉病有拮抗性 ,其中菌株x 9和x 1 5的拮抗效果较为明显 ,对番茄灰霉病防病效果达到 40 %和2 5 %。此 7个菌株对番茄植株无致病性 ,且对番茄苗无明显促生长作用。     

超声波处理土样分离放线菌

【目的】探索超声波处理土壤悬液,增加稀有放线菌的类群。【方法】将西双版纳热带雨林的混合土样,制成土壤悬液,用超声波分别处理0-120s,用平板稀释法分离放线菌,得到纯菌落后测定其16S rRNA基因序列,进行系统发育分析,将分离菌株鉴定到属;用超声波处理已经鉴定到种且常见的10种链霉菌0-5min,后进行培养,测定其存活率。【结果】土壤悬液经超声波处理不同时间,放线菌的数量和种类逐渐增加。超声波处理已知链霉菌1-5min,对链霉菌的数量没有明显影响。【结论】用超声波处理土壤悬液40s,可以大大增加放线菌的出菌总数,明显增加稀有放线菌的种类,是一种经济且简便易行的方法。     

药用植物青蒿不同种类的内生菌抑菌活性分析

为了研究青蒿不同种类的内生菌抑制细菌和抑制真菌的活性,该研究采用组织块法和研磨法从青蒿的根、茎、叶中分离内生细菌、放线菌和真菌,以大肠埃希菌(Escherichia coli)(CICC 23657)、枯草芽孢杆菌(Bacillus subtilis)(CICC 10275)、金黄色葡萄球菌(Staphylococcus aureus)(CICC 10384)、黑曲霉(Aspergillus niger)(CICC 2487)、酿酒酵母(Saccharomyces cerevisiae)(CICC 33032)为指示菌,采用琼脂块法和双层平板法检测内生菌的抑菌活性。结果表明:(1)从青蒿植株中共分离到76株内生菌,其中内生细菌19株、内生放线菌34株、内生真菌23株。从分离部位来看,56株来自于茎段、17株来自于根段、3株来自叶片。(2)内生细菌中抑菌活性菌株占总菌株的比例最高,为95%,内生放线菌和内生真菌中抑菌活性菌株的比例分别为41%、35%。(3)内生细菌的抗菌谱较广;虽然内生放线菌的抗菌谱较窄,但其中高抗菌株较多,尤其对酿酒酵母的抑菌效果好。综上结果显示,药用植物青蒿中存在着丰富的有抑菌活性的内生菌,且不同种类的内生菌抑菌活性不同。     

白肋烟内生细菌的分离鉴定及降低N-亚硝胺含量研究

以表面消毒法分离白肋烟TN90和TRM品种 30个样品的内生细菌 ,共分离到 33株内生细菌。对这些菌株进行了初步分类鉴定 ,它们属于假单胞菌属、黄单胞菌属、节杆菌属、葡萄球菌属、棒杆菌属、黄杆菌属、气球菌属和利斯特氏菌属。筛选出TEB1 1、TEB1 7、TEB2 3、TEB2 6、TEB30、TEB34等 6株还原硝酸盐和亚硝酸盐能力较强的菌株 ,以粉碎烟叶接种、叶柄浸泡接种和叶面喷雾接种 3种方式处理调制后 ,化验分析结果表明 ,接种内生细菌能降低2 7 5 6 %～ 99 88%白肋烟TSNA含量 ,降低百分率以粉碎烟叶接种最高 ,其次是叶柄浸泡接种 ,叶面喷雾接种最低     

拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定

红树内生细菌分离及拮抗辣椒疫霉(Phytophthora capsici)筛选结果表明：各红树体内均有大量的内生细菌, 不同红树种类及部位内生细菌的数量均不同, 被测定的红树内生细菌中约有27.97%的可培养菌株对辣椒疫霉具有拮抗作用; 其中18株拮抗作用较强的细菌在辣椒果上对辣椒疫病菌均有一定的抑制效果, 以来自红海榄叶片内的RS261菌株效果最好; 经形态、生理生化特征和分子生物学等测定分析, 将RS261菌株初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。     

稻草秸秆预处理方法对烟曲霉产纤维素酶的影响

采用机械粉碎、高温、酸碱处理等方法对稻草秸秆进行预处理,以烟曲霉为实验菌株,研究预处理方法对菌株产纤维素酶的影响。结果表明,取机械粉碎后的稻草（30~120目）进行121℃高压蒸汽处理20min（即灭菌处理）,有利于菌株的生长与纤维素酶的产生;与未粉碎的稻草秸秆相比,烟曲霉羧甲基纤维素钠（CMC）酶、微晶纤维素酶、β-葡萄糖苷酶和滤纸（FPA）酶的活力分别提高了63.2%、164.0%、10.2%和14.1%。而采用不同种类、不同浓度的酸碱常温处理稻草秸秆4d或100℃高温处理30min,纤维素酶活力均出现了不同程度的下降。     

酸溶辅助超声波法提取类球红细菌类胡萝卜素条件优化

通过单因素和正交试验,对类球红细菌3757产类胡萝卜素的提取条件进行了研究。先采用超声波法、酸溶法、研磨法、冻融法、酸溶辅助超声波法和冻融辅助超声波法优化了从类球红细菌3757菌株中提取类胡萝卜素的方法,然后开展了酸溶辅助超声波法的单因素和正交实验,最后进行了重复性实验。结果表明,酸溶辅助超声波法是较优的提取方法,丙酮是较好的提取溶剂。最佳提取条件为料液比110、超声波处理总时间20 min、酸浓度3 mol/L、酸溶时间25 min、超声波振幅40%、超声工作/间隔时间2 min/1 min、酸溶温度27℃,实验重现较好。优化后类胡萝卜素的提取率较优化前提高了74.8%,为其产业化创造了条件。     

红树林内生细菌的分离及拮抗菌筛选*

红树林内生细菌及拮抗菌分离筛选结果表明:各品种红树体内均有大量的内生细菌,不同红树品种及部位内生细菌的数量不同,其中以红海榄体内的含量最高,达4．225×10⁴cfu／g(fw),其它依次为木榄、桐花树、秋茄和白骨壤等;不同部位以茎组织体内内生细菌的含量最多,达1．649×10⁴cfu/g(fw),其次为根和叶。获得的内生细菌中约有43．53％的内生细菌菌株对枯萎病菌(Fusarium oxysporum)、炭疽病菌(Colletotrich     

贵州兴义喀斯特洞穴土可培养细菌多样性及其产蛋白酶、淀粉酶活性筛选

【目的】进一步了解贵州喀斯特洞穴土可培养细菌的物种多样性组成及其产蛋白酶、淀粉酶生物活性能力。【方法】选取11种分离培养基,利用稀释直接涂布平板法对贵州黔西南兴义市喀斯特地区白碗窑镇魔家大溶洞洞内土壤进行可培养细菌分离;利用两种鉴定培养基对相关细菌进行生物活性判定。【结果】根据16S rRNA基因序列的系统进化分析,将分离得到的217株细菌分别归类到24个属的63个不同种类,其中红球菌属(Rhodococcus)和链霉菌属(Streptomyces)为该洞内土壤可培养细菌的优势菌群,分别占24.42%和21.66%。大多数菌株与已知典型菌株的16S rRNA基因序列相似性为97.90%-99.99%,其中至少有4株菌株(D3T01、D911、D961和D502)为潜在的新分类单元。对217株细菌进行蛋白酶和淀粉酶活性筛选,其中具有蛋白酶或淀粉酶活性的99株,占分离菌株的45.62%,分别属于18个属的38个不同种;同时具有蛋白酶和淀粉酶活性的36株,占具有酶活性菌株的36.36%,占分离菌株的16.59%。【结论】贵州兴义喀斯特洞穴土中存在丰富多样的细菌类群,且蕴藏着一定数量的潜在新物种资源;此外功能酶菌株在喀斯特洞穴土壤中大量存在,为工业应用奠定了资源基础,极具进一步发掘和研究的价值。     

Comparative study of soil bacterial flora as influenced by the application of a pesticide,pentachlorophenol (PCP)

Summary The effects of pentachlorophenol (PCP) applications on the taxonomic composition of bacterial microflora were studied in water-logged soil (WS) and in shake cultures of suspended soil (SS). PCP applications resulted in a predominancy of Gram-negative bacteria over Gram-positive species. Members of theAcinetobacter group were the most common in PCP-treated soil although a small portion of the flora were in thePseudomonas-Alcaligenes group or belonged to theEnterobacteriaceae. Coryneform bacteria and species of theBacillus were the dominant forms in untreated WS; however, WS cultures treated with PCP at recommended rates (2.67 gm/m²) evidenced species ofPseudomonas, Alcaligenes, Acinetobacter, and members of theEnterobacteriaceae as the predominant bacterial species. The dominance of Gram-negative bacteria in PCP-treated soil was evidenced for 3 months after application of the compound but was not evident after 17 months when PCP had dissipated. Gram-negative bacteria found in PCP-treated soil were highly tolerant of the phenol. In WS cultures coryneform bacteria were the most common although PCP tolerance was heterogenous in nature.     

不同生境黑果枸杞根际与非根际土壤微生物群落多样性

研究典型生境黑果枸杞根际与非根际土壤微生物群落多样性及其与土壤理化性质间的关系,为进一步研究黑果枸杞抗逆性提供理论数据。采集新疆精河县艾比湖地区(EB)盐碱地、乌苏市(WS)路旁荒地、五家渠市(WQ)人工林带的黑果枸杞根际与非根际土壤,利用Illumina-MiSeq高通量测序技术分析细菌和真菌群落组成和多样性。结果表明:根际土壤细菌多样性高于非根际土壤(WQ除外),而根际真菌多样性低于非根际土壤。WQ非根际土壤细菌和真菌多样性均高于EB和WS;根际细菌多样性排序为EBWSWQ,根际真菌多样性排序为WSEBWQ。根际土壤优势细菌门依次是变形菌门、拟杆菌门、放线菌门、酸杆菌门,真菌优势门为子囊菌门、担子菌门。根际土壤细菌变形菌门、拟杆菌门、酸杆菌门的相对丰度高于非根际土壤,而厚壁菌在根际土壤中的丰度显著降低,真菌优势门丰度在根际土和非根际土中的变化趋势因地区而异; Haliea、Gp10、Pelagibius、Microbulbifer、假单胞菌属、Thioprofundum、Deferrisoma是根际土壤细菌优势属;多孢子菌属、支顶孢属、Corollospora、Cochlonema是根际真菌优势属。细菌、真菌优势类群(门、属)的组成以及丰富度存在地区间差异,厚壁菌门在EB地区的丰富度显著高于含盐量较低的WS、WQ;盐碱生境EB中根际土壤嗜盐细菌的丰度高于非盐碱生境(WQ、WS),如盐单胞菌属、动性球菌属、Geminicoccu、Pelagibius、Gracilimonas、Salinimicrobium等。小囊菌属是EB根际真菌的最优势属,Melanoleuca是WQ和WS的最优势属,地孔菌属、Xenobotrytis、Brachyconidiellopsis、多孢子菌属等在EB根际土壤中的丰度显著高于WQ和WS。非盐碱生境(WS和WQ)的微生物群落之间的相似性较高,并且高于与盐碱环境(EB)之间的相似性,表明土壤含盐量对微生物群落组成丰度具有重要的影响。     

Molecular identification of intestinal microflora in Takifugu niphobles

We investigated the intestinal microflora of coastal fish including Takifugu niphobles using both culture techniques and library cloning. As a result, the numbers of bacteria appeared on agar media were 1.0 × 10⁴ to 1.4 × 10⁹ CFU/g (colony forming units/gram), whereas those of total bacteria stained with 4′,6-diamidino-2-phenylindole were 4.7 × 10¹⁰ to 1.9 × 10¹¹ cells/gram, irrespective of different fish species. In addition, the culture technique showed that the intestinal microflora in all specimens was mainly composed of the genus Vibrio. In contrast, the direct count method showed that spirochaetes with length of 2.5-4.5 μm were present in the intestinal contents of T. niphobles at high densities, whereas such bacteria could not be detected in those of other fish species. Library cloning yielded the sequences of 16S rRNA genes that were divided into seven taxonomic categories of bacteria including Actinobacteria, Bacilli, Clostridia, Gammaproteobacteria, Mollicutes, Spirochaetes and an unclassified bacterial group. These results demonstrate that the molecular diversity of the intestinal bacteria in T. niphobles based on the clone library method reflects the direct observation by fluorescence microscopy to some extent.     

Indicator bacteria in freshwater and marine molluscs

The freshwater mussel Anodonta cygnea and four marine shellfish (mussels, Mytilus edulis; cockles, Cerastoderma edule; clams, Mya arenaria; Scrobicularia plana) from a total of six sites were surveyed for Escherichia coli, Clostridium perfringens, faecal streptococci, 25 and 37 °C coliforms, 25 °C and 37 °C total viable numbers and fluorescent pseudomonads. The A. cygnea from an urban lake contained greater numbers of the faecal indicator bacteria than animals from a rural lake. There were also differences in the other bacterial counts and these were discussed with respect to bacterial parameter and animal characteristics. When freshwater mussels were transferred from the city site to the rural site for 24 h the load of faecal indicator bacteria was eliminated or significantly reduced. Other bacterial types took longer to become stabilised. Loss of indicator bacteria from Anodonta was also demonstrated using cleansing in the laboratory. Very high bacterial numbers were found in some marine molluscs notably Scrobicularia plana and most shellfish contained significant numbers of the three faecal indicator bacteria at every sampling occasion. The relationship between bacterial types was discussed and it was concluded that in both freshwater and marine animals the bacterial numbers were determined more by sampling site than by species of shellfish.     

Isolation,partial characterization,and the effect of plant growth-promoting bacteria (PGPB) on micro-propagated sugarcane in vitro

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 10⁶ bacteria per gram dry weight of stem and 10⁷ bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the¹⁵N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.     

Incidence of Legionella and heterotrophic bacteria in household rainwater tanks in Azumino,Nagano prefecture,Japan

Many administrative agencies in Japan are encouraging installation of household rainwater‐storage tanks for more effective use of natural rainwater. Water samples were collected periodically from 43 rainwater tanks from 40 households and tested for the presence of Legionella species and the extent of heterotrophic bacteria in Azumino city, Nagano prefecture, Japan. PCR assays indicated the presence of Legionella spp. in 12 (30%) of the 43 tank water samples. Attempts were made to identify correlations between PCR positive samples, topography, pH, chemical oxygen demand (COD), atmospheric temperature and the numbers of heterotrophic bacteria. Between June and October, 2012, the numbers of heterotrophic bacteria in rainwater tanks and the values of COD positively correlated with the presence of Legionella species. In most of the Legionella‐positive cases, heterotrophic bacterial cell counts were >10⁴ CFU/mL. Moreover, Legionella species were less frequently detected when the COD value was >5 mg KMnO₄/L. Therefore, at least in Azumino, Japan between June and October 2012, both heterotrophic bacterial counts and COD values may be considered index parameters for the presence of Legionella cells in rainwater tanks. Much more accumulation of such data is needed to verify the accuracy of these findings.     

Occurrence of bacterivory in Cryptomonas,a common freshwater phytoplankter

Summary Bacterivory was detected by incorporation of 0.57 m diameter, fluorescent polystyrene beads and fluorescently labeled bacteria (FLB) in two cultured species of Cryptomonas (C. ovata and C. erosa), and a population of Cryptomonas sp in a humic, mesotrophic lake. Rates of ingestion and clearance were very low, and similar for the cultures and the in situ population. The in situ population incorporated 0.7–1.7 bacteria cell^-1 h^-1, thereby ingesting 0.3%–2.0% of the total bacterial numbers present in the water per day, and receiving less than 2% of its carbon content per day through bacterivory. Thus, bacterivory by Cryptomonas was quantitatively important neither as a sink for bacterial biomass, nor as a carbon source for the algal cells. Possibly, it served in the uptake of essential nutrients.     

Genotypic differences in the uptake and distribution of radiocaesium in plants

The ability of endophytic bacteria to influence Erwinia carotovora var. atroseptica (Eca) growth and disease development was examined in potatoes. Bacterial populations isolated from within the tubers of five potato (Solanum tuberosum L.) cultivars (Kennebec, Butte, Green Mountain, Russet Burbank and Sebago) showed antibiosis toward Eca in an in vitro assay. Sebago was host to the highest percentage of bacterial isolates inhibiting Eca growth in vitro (49.5%), followed by Green Mountain (33.3%), Kennebec (29.3%), Russet Burbank (12.9%) and Butte (1.8%). Of these, Curtobacterium luteum was the most common species. Few endophytic bacteria from Butte were inhibitory to Erwinia; all were from Pantoea agglomerans. Significantly higher populations of Erwinia-inhibiting bacteria were recovered from Kennebec (1.89 × 10⁶ cfu fresh weight tuber tissue) as compared to the other cultivars; the lowest populations were recovered from Butte (0.01 × 10⁶ cfu per g fresh weight tuber tissue). Published levels of cultivar disease resistance to blackleg did not correspond to actual bacterial soft rot development (induced by Eca) in an in vivo (tuber) assay. However, bacterial soft rot development was negatively correlated with the density of tuber populations of endophytic bacteria found able to inhibit Eca growth in vitro (R=−0.879, p=0.05).     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.