Molecular analysis of a gene fromBacteroides fragilis involved in metronidazole resistance inEscherichia coli

The region ofBacteroides fragilis DNA on the recombinant plasmid pMT100 responsible for conferring metronidazole resistance inEscherichia coli strains was characterized. An open reading frame (ORF1) of 195 bp encoded a protein of 64 amino acids with a predicted M_r, of 7.3 kDa. Deletion analysis indicated that ORF1 conferred the metronidazole resistance phenotype and encoded a protein with an apparent M_r of approximately 8–10 kDa.     

Heterosigma akashiwo in central California waters

Heterosigma akashiwo (Hada) gives rise to red tides along the Atlantic and Pacific coasts and is known to produce brevetoxins. This investigation establishes baseline information showing the presence of H. akashiwo along the central California coast based on water samples collected from the Santa Cruz pier in Monterey Bay (on the open coast) and the Berkeley pier in San Francisco Bay. Light and electron microscopy as well as two species-specific DNA probe methods based on cell homogenates preparations were employed to detect H. akashiwo during the 2001–2002 field study. The DNA probe methods consisted of a sandwich hybridization assay (SHA), which targets ribosomal RNA (rRNA), and an end-point polymerase chain reaction (PCR) assay, which targets internal transcribed spacer (ITS) sequences of rRNA genes. The SHA was used to provide semi-quantitative data showing the intermittent presence of the species during a 13-month period in Monterey Bay. Samples that showed a variety of responses in the SHA (negative as well as the highest) were then subjected to the PCR assay in an attempt to confirm species identification using an independent DNA probe method that employs cell homogenates; samples included those from Monterey Bay and one from a red tide event in San Francisco Bay. SHA and PCR assays agreed on the presence or absence of H. akashiwo. Gene products from two field samples positive for H. akashiwo by PCR were cloned and sequenced and found to be identical to those of that species in GenBank. When the same samples were viewed by light microscopy, however, H. akashiwo cells were only seen in the sample with the highest abundance of that species, as evidenced by SHA. It was extremely difficult to recognize naturally occurring H. akashiwo using light microscopy in field samples that had been preserved with Lugol's iodine, including samples that gave positive results by cell homogenate methods. Results of this study indicate that H. akashiwo is present along the open California coast and could easily be missed in routine phytoplankton surveys. Despite its presence, H. akashiwo does not appear to routinely bloom with sufficient densities to cause harmful outbreaks of the frequency and severity documented in some other coastal environments. Molecular identification techniques may be the preferred approach over light microscopy when there is a need to rapidly screen many samples for fragile, harmful species and those that are otherwise problematic to identify based on their gross morphology alone.     

兴安落叶松(Larix gmelinii)火后种子萌发影响因子

林火干扰是影响兴安落叶松林结构和功能的主要因子之一。兴安落叶松种群火后更新受多种因子的影响,并决定着该群落演替轨迹。通过在大兴安岭呼中自然保护区的火烧迹地内设置原位控制实验,利用增强回归树分析方法,量化研究了落叶松火后恢复初期不同影响因子（温度、有机质层厚度、覆盖度等）对种子萌发的相对重要性。研究结果表明：落叶松火后种子萌发的最主要影响因子为温度,第二影响因子为种源,第三影响因子为草本覆盖度,分别解释了幼苗数量变异的28.51%、22.40%、20.66%;各影响因子的相对重要性在不同地形条件下有明显差异：温度在山坡顶部和阳坡底部占有重要影响,种源在阳坡中部和阴坡中部最为重要,土壤含水量在阴坡底部占有重要地位。同时,去除土壤表面有机质可以显著提高种子萌发数量,去除地面杂草则会使种子萌发数量降低。从研究结果可知,落叶松火后种子萌发在不同地形（环境条件）下的限制因子不同,人工辅助需因地制宜采取相应措施,才能更有效地促进种子萌发与森林恢复。     

八棱海棠种子超低温保存中含水量对糖代谢的影响

含水量是影响种子超低温保存效果的关键因素，而其作用机制尚不完全清楚。为探讨含水量对种子超低温保存生活力的影响途径，该研究以八棱海棠种子为材料，通过硅胶干燥法获得不同含水量的种子，测定超低温保存后种子生活力、糖含量及相关酶指标的变化并分析相关性。结果表明：(1)超低温保存15 d后，不同含水量种子生活力不同，随着种子含水量的降低，种子生活力呈现先升高后降低的趋势，含水量为9.02%的八棱海棠种子生活力最高，为53.33%;超低温保存120 d后，种子生活力随着含水量下降一直升高，含水量为6.40%生活力最高，为27.78%。这表明八棱海棠种子含水量对超低温保存后的生活力有明显影响，但受液氮保存时间影响，随着液氮保存时间的延长，最适含水量降低。(2)相关分析显示，超低温保存后种子含水量与生活力呈极显著负相关(r=-0.82);与果糖和蔗糖含量、酸性转化酶、果糖激酶呈显著负相关，而种子萌发率与这些指标呈显著正相关。这表明种子含水量通过影响酸性转化酶活性而影响蔗糖和果糖含量，进而影响蔗糖代谢，响应低温和脱水胁迫，最终导致生活力差异。种子生活力还受到介导果糖激酶的果糖代谢影响。此外，海藻糖也是种...     

水淹生境下秋华柳对镉污染土壤研究修复能力

以耐水淹和耐重金属的秋华柳（Salix variegata）作为试验材料,从土壤角度出发,探究秋华柳在水淹条件下对镉污染土壤的修复能力。设置无植物和种植秋华柳两个处理组,分别对两组设置两个水分处理组：正常供水组（CK）及土壤水淹组（FL）,4个镉浓度处理组：对照组（0 mg/kg）、低浓度（0.5 mg/kg）、中浓度（2 mg/kg）及高浓度（10 mg/kg）。分别于处理后的第30天和第60天对各处理组的土壤和水淹组水样进行取样。试验结果表明：（1）镉处理浓度越高,土壤中镉活性态浓度越高,生物毒性越强。（2）水淹显著降低土壤活性态Cd浓度（P < 0.05）,增大土壤修复难度。（3）种植秋华柳对30d土壤Cd全量及各形态镉浓度均无显著影响（P > 0.05）;对第60天正常供水和水淹组土壤中交换态、碳酸盐结合态的镉浓度以及60d水淹组土壤Cd全量均有显著影响（P < 0.05）。（4）秋华柳种植显著降低土壤Cd迁移系数,30d正常供水组、水淹组以及60d正常供水组和水淹组平均降幅分别为2.0%、4.12%、9.71%、9.32%。处理时间过短和试验用苗生物量小可能是秋华柳对土壤全量Cd影响不大的主要原因,但秋华柳均显著降低正常供水和水淹组土壤迁移系数且二组降幅差异不大。研究表明：水淹生境下,秋华柳对Cd污染的土壤仍有较好的修复能力。     

感染内生真菌的羽茅对大针茅的化感作用

以羽茅(Achnatherum sibiricum)为材料,采用室内生物测定法研究感染不同内生真菌的羽茅浸提液对大针茅(Stipa grandis)种子萌发和幼苗生长的化感作用,并采用气相色谱-质谱(GC-MS)联用技术对羽茅浸提液中的化学成分进行分析。结果表明:(1)内生真菌感染可缓解宿主羽茅对群落优势种大针茅的化感作用;(2)内生真菌感染对宿主羽茅化感作用的影响与内生真菌种类有关,具体表现为相比于未染菌羽茅,感染Epichloё gansuensis可显著缓解宿主羽茅对大针茅的抑制作用,而感染Neotyphodium sibiricum、Neotyphodium gansuensis有缓解羽茅对大针茅化感作用的趋势,但未达到显著影响;(3)羽茅浸提液中含有2,4-二叔丁基苯酚、邻苯二甲酸、邻苯二甲酸二丁酯和硬脂酸甲酯等潜在化感物质,这4种物质相对含量的差异可能是不同内生真菌感染状态的羽茅产生不同化感效应的主要原因;(4)对羽茅浸提液中4种潜在化感物质的化感作用进行验证实验,结果表明,2,4-二叔丁基苯酚对大针茅的化感作用基本表现为低浓度无影响,高浓度抑制,邻苯二甲酸二丁酯的化感作用表现为"低促高抑"的效应趋势,而邻苯二甲酸和硬脂酸甲酯对大针茅均表现出"剂量效应"的化感作用趋势,即4种潜在化感物质在高浓度时均可显著抑制大针茅的种子萌发和幼苗生长。     

研究报告: 基于核酸适配体的胶体金比色法检测鳗弧菌

目的 鳗弧菌（Vibrio anguillarum）可引起鲑鱼、鳗鲡、鲈鱼和牙鲆等多种水产养殖动物的疾病，是水产养殖中的一种重要病原菌，对其进行快速检测是确保水产养殖安全和食品安全所必需的。方法 本文利用鳗弧菌与其核酸适配体之间较强的亲和力，通过鳗弧菌夺取胶体金颗粒表面的核酸适配体，使胶体金溶液的吸光度发生变化，从而建立一种可定量检测鳗弧菌的方法。结果 该方法对鳗弧菌的吸光度值显著高于对溶藻弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌和迟钝爱德华氏菌等非目标菌的吸光度值（P<0.01），并在1~10⁵ CFU/ml的检测范围内呈现较好的线性关系。用该方法对不同盐度和鱼体组织样品进行加标回收检测，结果显示回收率和相对标准偏差等指标都符合相应检测标准。结论 该检测方法对鳗弧菌有较好的特异性，可用于水产品或食品中鳗弧菌的定量检测。     

Associations between fish health and Pfiesteria spp. in Chesapeake Bay and mid-Atlantic estuaries

In response to concerns that there may be an association between harmful algal bloom (HAB) species and fish health, including the widespread use of fish health as one indicator of a possible HAB warranting further investigation, evidence for such an association was evaluated in Chesapeake Bay and other mid-Atlantic estuaries (1999–2001). A statistical approach was used, without invoking causality, to test whether there is an association between the prevalence of externally-visible lesions in fish populations above background levels and the presence of Pfiesteria spp. in co-located water and fish samples. Externally visible anomalies (e.g. ulcers, necrosis, parasites, etc.) were recorded for Atlantic menhaden (Brevoortia tyrannus) and all other fish collected. Polymerase chain reaction (PCR) techniques were used to test for the presence of Pfiesteria spp. in water samples collected at routine and rapid response sampling events. No actively toxic Pfiesteria was found during this study. Fine-scale (within a given sample site) and broad-scale (estuary-wide sampling) comparisons showed positive associations between externally-visible fish lesions in menhaden populations and the presence of Pfiesteria spp. in co-located samples. Logistic regression modeling of Pfiesteria detection probabilities as a function of prevalence of menhaden with lesions was significant (P = 0.0096). Reductions in the false positive (tests indicating Pfiesteria presence when its absent) and false negative (tests indicating Pfiesteria is absent when it is actually present) rates occurred when the minimum sample size threshold increased from 1 to 30 fish (P = 0.003–0.001). This association served as a useful field indicator of potential HAB activity that could warrant further field investigation and testing.     

水淹条件下灰化薹草和虉草活体、枯落物分解过程的比较

为了揭示鄱阳湖湿地活体植物和自然枯落物在水淹条件下分解过程的差异,以鄱阳湖湿地优势植被物种灰化薹草(Carex cinerascens)和虉草(Phalaris arundinacea)为研究对象,采用分解袋法开展室内模拟实验。植物样品设置新鲜和风干两种处理,分别模拟活体植物和自然枯落物在水下的分解过程。研究结果表明:(1)在180 d的淹水实验过程中,两种处理下的灰化薹草和虉草的分解过程都具有阶段性,前期分解速率较快,后期较慢。(2)两种植物枯落物的分解速率与植物C/N比有显著相关性,而活体植物的分解速率与植物C/N比相关性不强。(3)活体植物和自然枯落物的水下分解速率与物种有关,虉草活体比虉草枯落物分解速率快,而薹草活体比薹草枯落物分解速率慢。(4)模拟实验结束时活体植物仍有34%—43%未分解,推测鄱阳湖丰水期退水后大量洲滩植被消失的原因并非是植被在水淹下完全分解,而是一部分植被被水生动物啃噬或被水流冲走。     

Identification of Encephalitozoon cuniculi genotype III and two novel genotypes of Enterocytozoon bieneusi in swine

Samples of intestinal content from thirty fattened pigs of six farms slaughtered at an abattoir in North-Western Germany, and faecal samples of four pigs kept as laboratory animals at the Federal Institute for Risk Assessment (BfR, Berlin, Germany) were investigated for the occurrence of microsporidia by light microscopy, PCR and sequencing. A modified Webers trichrome staining and the immunohistochemistry (the Avidin-Biotin-Peroxidase-Complex technique with a polyclonal anti-Encephalitozoon cuniculi-serum and monoclonal antibodies against Encephalitozoon intestinalis and Enterocytozoon bieneusi) was used as a screening method for the light microscopical detection of these pathogenic eukaryotes. By this light microscopically methods microsporidia suspected organisms were found in all samples (100%). By the use of PCR, microsporidia were identified in fourteen samples (41.2%). The prevalence of microsporidia infections among the farms diversifies from 0 to 80% as considered by PCR. E. bieneusi was the most prevalent species and was identified in twelve fattened pigs (40%) from five of the six tested farms (83.3%) and in two of the four laboratory animals (50%). Three of the E. bieneusi species belonged to the genotype O, one to the genotype E, and one to the genotype F. Two isolates were identified as novel genotypes and two samples showed a mixed infection of different genotypes. In three faecal samples of the pigs from two farms E. cuniculi genotype III was identified. One sample contained both microsporidia species. To our knowledge, this is the first time that the genotype III of E. cuniculi was identified in swine.     

不同土壤水盐条件下多枝柽柳(Tamarix ramosissima)对胡杨(Populus euphratica)幼苗的影响

目前胡杨更新困难,种群处于退化阶段,而与其生态位高度重叠的多枝柽柳却在扩张。为探讨多枝柽柳对胡杨生长的影响,本研究设置了3个水平的水分、盐分梯度,对胡杨进行了单种和混种的盆栽控制实验,通过测定胡杨幼苗的生长和存活状况,分析不同水、盐梯度下多枝柽柳如何影响胡杨生长。结果表明：（1）多枝柽柳伴生降低当年生胡杨幼苗的存活率,随着水分条件改善,胡杨存活率提高,而盐分对存活率没有显著的影响。（2）水分、盐分和伴生模式几个因子对胡杨的生长特征的影响存在显著的交互作用（P<0.05）。各水盐条件下,多枝柽柳会不同程度降低胡杨幼苗的株高、主根长和地上地下生物量,而根冠比增大。混种条件下,胡杨幼苗对水盐的响应更为敏感。（3）相对竞争强度对水盐环境有明显的响应（P<0.05）,随水分条件改善,地上、地下相对竞争强度降低。多枝柽柳对胡杨的生长产生不良影响的机制是通过快速消耗土壤的水分,从而导致胡杨幼苗水分亏缺。土壤水分条件是胡杨和多枝柽柳幼苗共存的关键因素,在水分供给不足的情况下,对水分偏好的差异导致胡杨无法通过实生苗进行有效的更新从而加剧胡杨种群的衰退,而多枝柽柳更为耐旱的特性使其逐步占据河岸的生境。我们的研究结果强调了两个树种对环境因子的适应性差异决定了河岸带植被发育过程中植物-土壤的相互作用导致胡杨在演替过程中表现出的衰退现象。     

柠条锦鸡儿F3H基因克隆及功能分析

柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。     

Morphological, chemical and genetic differentiation of two subspecies of Cistus creticus L. (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus)

Cistus creticus L., an aromatic species from the Mediterranean area, contains various diterpenes bearing the labdane skeleton. The production of essential oil from this species has potential economic value, but so far, it has not been optimized. In order to contribute to a better knowledge of this species and to its differentiation, the morphological characters, volatile chemical composition and genetic data of two subspecies (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus) were investigated. The leaf trichomes were studied using scanning electron microscopy. The chemical composition of Corsican essential oil (C. creticus subsp. corsicus) has been reported using GC, GC/MS and ¹³C NMR; the main constituents were oxygenated labdane diterpenes (33.9%) such as 13-epi-manoyl oxide (18.5%). Using plant material (54 samples) collected from 18 geographically distinct areas of the islands of Corsica and Sardinia, the basis of variation in the headspace solid-phase microextraction volatile fraction and an inter-simple sequence repeat genetic analysis were also examined. It was shown that the two subspecies of C. creticus differed in morphology, essential oil production, volatile fraction composition and genetic data.     

Theileria ovis discovered in China

A polymerase chain reaction (PCR) using 989/990 primers was conducted to identify a newly isolated Theileria sp. in Xinjiang Province of China. The target DNA fragments of the complete 18S rRNA gene were cloned and sequenced. The phylogenetic relationship of newly isolated Theileria spp. was inferred based on the 18S rRNA gene. The results showed that the new Theileria sp. belonged to the cluster of Theileria ovis. Moreover, the findings were confirmed by T. ovis species-specific PCR. An expected 520 bp fragment of T. ovis DNA was obtained from 25 out of 320 (8%) field blood samples, and blood of an experimental sheep infested by Hyalomma anatolicum anatolicum collected in Xinjiang. The infection rate of T. ovis was 78% (25/32) in Xinjiang province. The investigation did not find T. ovis positive samples from the field samples collected from the other twelve provinces. This study indicates that T. ovis is prevalent in Xinjiang province of China and its transmission vector is H. anatolicum anatolicum.     

CRISPR/Cas9介导靶向敲除拟南芥GGB基因突变体的鉴定

GGB是抗旱负调控基因。为了获得拟南芥ggb突变体材料,构建了以拟南芥U6启动子驱动GGB sgRNA的CRISPR/Cas9基因组编辑载体。将构建好的编辑载体利用农杆菌介导的浸花法转化野生型拟南芥。对转基因后代GGB基因的测序结果分析发现,在靶位点处有缺失4个碱基和增加1个T碱基的2种突变体产生。分别对野生型拟南芥和上述2种ggb突变体进行半定量RT PCR分析结果显示,突变体材料中几乎检测不到GGB基因表达,说明获得了GGB基因敲除突变体。对野生型和ggb突变体叶片失水率、耐旱表型及单株种子量的测定结果表明,与野生型相比,拟南芥GGB基因突变后,叶片失水率显著减少,抗旱性明显增强,而单株种子量却并没有改变。研究表明,GGB是一种理想的作物分子育种的候选靶基因,获得的突变体为今后从农作物中克隆的GGB同源基因进行功能互补验证提供了有用的遗传材料。     

Rapid detection of natural cells of Alexandrium tamarense and A. catenella (Dinophyceae) by fluorescence in situ hybridization

The marine toxic dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor that cause paralytic shellfish poisoning (PSP) are identified on the basis of morphological features in routine monitoring. Rapid and simple identification is, however, often difficult because of the morphological similarity. Fluorescent in situ hybridization (FISH) using ribosomal RNA (rRNA)-targeted probes has been studied as a method of easily identifying and enumerating species responsible for harmful algal blooms (HABs). Its application to monitoring natural populations of HAB species, however, is limited. Here, we applied the FISH method to identify and enumerate cells of A. tamarense and A. catenella in natural plankton assemblages collected from Japanese coastal waters. A. tamarense-specific (Atm1) and A. catenella-specific (Act1) probes were established based on the D2 region of the large-subunit ribosomal RNA gene (28S rDNA). With these two probes, natural cells of A. tamarense or A. catenella in field samples could easily be identified when the following three conditions were met. First, cells should be concentrated by filtration, not centrifugation, in order to avoid the loss of cells. Second, autofluorescence should be minimized; acetone was an effective decolorization reagent. Third, samples should be stored at −20 or −80 °C for long-term preservation. The results indicate that FISH is a useful tool for the rapid identification of toxic Alexandrium spp. and can facilitate the analysis of numerous natural samples.     

Imbibitional damage in conidia of the entomopathogenic fungi Beauveria bassiana, Metarhizium acridum, and Metarhizium anisopliae

When dried organisms are immersed in water, rapid imbibition may cause severe damage to plasma membranes; in unicellular organisms, such damage is usually lethal. This study investigated effects of water activity (dryness) of organisms and immersion temperature on imbibitional damage in three insect pathogenic fungi. Conidial powders of Beauveria bassiana (Bb), Metarhizium anisopliae (Ma) and Metarhizium acridum (Mac) were dried/hydrated to a broad range of water activities (a_w) (0.023–0.961) prior to immersion in water at 0.5–33 °C. Imbibitional damage in conidia of each fungus occurred rapidly, with no differences in viabilities observed following immersion for 2 vs. 60 min. Damage increased with decreasing water activity of the conidia and decreasing temperature of the immersion water. Dry (a_w  0.333) Metarhizium spp. conidia were highly susceptible to imbibitional damage, with viability declining to 5% after immersion at 0.5 °C and 63% following immersion at 15 °C. Germination of the driest Ma conidia was reduced to 66% after treatment at 25 °C. In contrast, Bb was highly tolerant to damage, with significant reductions in viability (to levels as low as 43–65%) occurring only when dry conidia were immersed at 0.5 °C. Damage was prevented when conidia were slowly rehydrated by humidification prior to immersion and immersion temperature was increased to 33–34 °C; germination of all fungi was 94% under these optimal conditions. However, immersion of the driest Bb, Ma, and Mac powders in warm water (33 °C) also resulted in high viabilities (95%, 89%, and 94%, respectively), and slow-rehydrated conidia also retained high viability (87%, 92%, and 83%, respectively) after immersion in ice-cold water (0.5 °C). Formulation of conidia in pure (non-emulsifiable) paraffinic oil provided considerable protection from imbibitional damage. This study underscores a need for establishing standard protocols for preparing aqueous suspensions of sensitive fungi for both research and commercial applications.     

贝莱斯芽孢杆菌Bv-303对水稻白叶枯病菌的拮抗活性及其应用

本研究明确了一株新型贝莱斯芽孢杆菌(Bacillus velezensis) Bv-303菌株对黄单胞杆菌水稻致病变种(Xanthomonas oryzae pv. oryzae,Xoo)的拮抗活性及其对水稻白叶枯病(bacterial-blight,BB)的生物防治效果。采用牛津杯法测定了菌株Bv-303发酵上清液(cell-free supernatant, CFS)对白叶枯病菌体外拮抗的活性及其稳定性;通过对接种白叶枯病菌的水稻叶片进行喷雾处理,在水稻体内测试了该菌株发酵液(cell-culture broth,CCB)、发酵上清液及菌悬液(cell-suspension water,CSW)对白叶枯病菌的抑制效果;并统计了该菌株对水稻种子发芽率与幼苗生长的影响。结果表明,在体外,菌株Bv-303发酵上清液对白叶枯病菌的生长抑制率可达85.7%–88.0%,对热、酸、碱、紫外线等具有较好的稳定性;在水稻叶片上,喷施该菌株的发酵液、发酵上清液及菌悬液均能提高植株对白叶枯病的抗性,其中发酵液的效果最佳,抗病性提高率高达62.7%;且发酵液对水稻种子萌发和幼苗生长均没有副作用。因此,菌...