一株牙鲆源黏质沙雷氏菌YP1的分离鉴定及致病性分析

黏质沙雷氏菌(Serratia marcescens)是引起人类、动物及植物感染的重要条件致病菌,但其作为鱼类致病菌却鲜有报道。【目的】本研究以从患病牙鲆(Paralichthys olivaceus)病灶处分离的一株黏质沙雷氏菌YP1为研究对象,分析黏质沙雷氏菌对鱼类的致病性及对疾控的影响。【方法】利用形态学、分子生物学及生理生化实验综合鉴定菌株YP1;利用菌株YP1进行人工感染实验、组织病理实验及药敏试验,研究其感染症状、组织病理学、毒力和药物敏感性。【结果】分离自患病牙鲆体表溃疡病灶处的菌株YP1鉴定为黏质沙雷氏菌。感染实验结果显示,牙鲆和斑马鱼的半数致死量(LD₅₀)分别为3.44×10⁷CFU/g和6.28×10⁵CFU/g,除牙鲆外菌株YP1对其他鱼类也具有高致病性;菌株YP1主要导致牙鲆腹水,同时伴有呼吸急促、摄食减弱、脱肛、白便、鳃缺血及多脏器膨大出血等症状,并随着感染时间的延长对脏器损伤呈加重趋势。病理组织切片结果显示,菌株YP1对牙鲆鳃、肠、肝、脾、肾、心均造成损伤。药敏试验结果表明,YP1对左氧氟沙星、诺氟沙星等14种药物敏感;但对氨苄西林、头孢拉定等19种药物具有耐药性。【结论】本研究结果证实了黏质沙雷氏菌是能导致牙鲆腹水病的一种病原菌,同时对其他鱼类也具高致病性,为该菌感染鱼类导致疾病的检测、鉴别和防治提供科学依据。     

灵杆菌合成灵菌红素的转录调控

灵菌红素是一种具有多种生物活性的红色素,具有巨大的经济价值和广阔的应用前景。灵杆菌是灵菌红素的生产菌株,同时也是研究灵菌红素合成的模式菌株。本文综述了转录水平上调控灵杆菌合成灵菌红素的研究进展,总结了双(多)组分调控系统、群体感应系统、σ因子和转录因子在调控灵杆菌合成灵菌红素过程中发挥的作用,并对未来的研究方向进行了展望。     

粘质沙雷氏菌几丁质酶基因ChiA克隆及生物信息学分析

通过生物信息学技术对Chi A基因序列进行分析预测,了解Chi A的基因结构及蛋白质性质。从自有菌株(粘质沙雷氏菌Serratia mareescens S68)中克隆到几丁质酶基因Chi A,利用相关软件对Chi A基因序列进行分析预测。Chi A基因全长1 714 bp,开放阅读框编码563个氨基酸,推测其编码的蛋白质分子量为60 983.8Da,等电点为6.35,是一种稳定的亲水性蛋白质。预测Chi A可能存在信号肽,切割位点在第23~24位氨基酸之间,1~23位氨基酸为其跨膜结构,其余肽链位于细胞外。Chi A主要存在3种二级结构元件,在二级、三级结构中都有体现。该Chi A是一种水溶性蛋白质,结构稳定且可以分泌到胞外。     

水稻内生成团泛菌YS19与菠萝泛菌YJ76共培养提高环境胁迫生存适应性

成团泛菌(Pantoea agglomerans)YS19和菠萝泛菌(Pantoea ananatis)YJ76是从水稻(Oryza sativa)“越富”品种中分离得到的2株优势内生固氮细菌。利用YS19和YJ76共培养方式模拟菌体栖息微环境,探索潜在的种间生物效应,发现适量的YJ76能够促进YS19共质体的形成并且YJ76细胞可以参与到YS19的共质体结构中,YS19?YJ76接种量比例为10?3时促进效果最为显著。相比之下,当使用吲哚产量显著下降的YJ76突变株Δzwf与YS19共培养时,上述促进效应均显著减弱。与单独培养相比,共培养显著提高了两个菌株对重金属Zn²⁺、四环素、紫外线以及干燥的耐受能力,YS19分别提高了63.9%、56.0%、57.4%、72.0%,YJ76分别提高了147.9%、172.7%、266.3%、348.6%。结果表明水稻的两种主要内生菌YS19和YJ76在共栖息时表现为强烈的协同作用,而YJ76产生的吲哚信号分子在这个过程中起到重要调节作用。     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70 kb, pRK10 is only 4241 bp long with 53% G + C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5α. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.     

黏质沙雷氏菌AHPC29对松树萎蔫病的防治效果及其增菌条件

【目的】从松材线虫的媒介天牛蛹室及其气管中获得的黏质沙雷氏菌Serratia marcescens AHPC29对松材线虫具有致病能力,本研究旨在探究黏质沙雷氏菌AHPC29对松材线虫引起的松树萎蔫病的防治效果以及该菌株在实验室的增菌条件。【方法】通过对温室内人工感染松材线虫的松树灌溉菌剂,分析黏质沙雷氏菌AHPC29对松树萎蔫病的作用效果;通过单组分筛选和正交实验确定培养基组分和培养条件对其生长的影响,探究黏质沙雷氏菌AHPC29的最佳增菌条件。【结果】对于感染松材线虫的松树,灌溉黏质沙雷氏菌AHPC29菌液的处理组生长状态优于对照组,并且树内松材线虫含量显著降低;黏质沙雷氏菌AHPC29增菌的最佳培养基配比为0.1%乳糖、0.5%复合氨基酸、0.5% KNO₃、1.5% MgSO₄,其中影响最大的组分为氮源;培养时其菌液最佳接种量为7%,最佳装液量为40%,最佳转速为180 r·min^-1,最佳温度为30 ℃,最适培养时长为36 h。【结论】本研究获得了黏质沙雷氏菌AHPC29的最佳增菌条件,并证实该菌具有良好的松树萎蔫病防治应用潜力,为其作为生防菌的应用提供了理论基础。     

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。     

巴西橡胶树HbSUT3和HbSUT5 mRNA在嫩茎和中脉中的原位杂交分析

为研究巴西橡胶树(Hevea brasiliensis)中HbSUT3和HbSUT5基因的功能,采用地高辛标记的RNA探针与橡胶树嫩茎和中脉两种组织切片分别进行RNA原位杂交,对这2种SUT基因在组织中的表达区域与表达特点进行了分析。结果表明,在橡胶树嫩茎中,两个SUT基因主要在树皮的韧皮部和皮层细胞中表达;在中脉中,两个SUT基因在除木质部导管系统外的其它部位均有表达;HbSUT3基因在嫩茎和中脉中的表达量相近,而HbSUT5基因在嫩茎中的表达量远高于中脉。这些揭示HbSUT3和HbSUT5基因可能广泛参与韧皮部装载、蔗糖运输与库细胞供给等活动,同时两个SUT基因也存在功能分化。     

药用野生稻拮抗稻瘟病内生细菌的筛选、鉴定及发酵条件优化

【背景】稻瘟病是水稻的三大重要病害之一,每年对水稻生产都会造成较大的损失,生物防治稻瘟病已成防治该病害的重要手段,而目前鲜少有报道从药用野生稻中分离内生菌用以防治稻瘟病。【目的】从药用野生稻内生菌中挖掘对稻瘟病具有拮抗作用的微生物资源。【方法】采用分离法从勐遮药用野生稻中先分离出内生菌,再通过平板对峙法筛选对稻瘟病菌具有拮抗作用的菌株,通过形态学和16S rRNA基因序列分析对菌株进行鉴定,同时筛选该菌株的最适培养基、pH、培养温度和摇床转速。【结果】筛选得到拮抗菌株Z5,在LB培养基上对稻瘟病菌菌丝生长的抑制率达到96.43%,经形态学和16S rRNA基因序列分析,初步鉴定为短小芽孢杆菌(Bacillus pumilus)。在NYBD、CM、LB、NA和YSP这5种培养基中,菌株Z5的最适培养基为NYBD培养基、最适pH值为8.0-9.0、最适培养温度为35℃、最适摇床转速为250 r/min。【结论】短小芽孢杆菌Z5对稻瘟病菌的生长具有较好的抑制作用,且该菌株耐高温、较耐酸碱,作为稻瘟病生防制剂研发具有一定的价值。     

Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a new Chrysoporthe species from Colombia

The pathogen Chrysoporthe cubensis (formerly Cryphonectria cubensis) is best known for the important canker disease that it causes on Eucalyptus species. This fungus is also a pathogen of Syzygium aromaticum (clove), which is native to Indonesia, and like Eucalyptus, is a member of Myrtaceae. Furthermore, C. cubensis has been found on Miconia spp. native to South America and residing in Melastomataceae. Recent surveys have yielded C. cubensis isolates from new hosts, characterized in this study based on DNA sequences for the ITS and β-tubulin gene regions. These hosts include native Clidemia sericea and Rhynchanthera mexicana (Melastomataceae) in Mexico, and non-native Lagerstroemia indica (Pride of India, Lythraceae) in Cuba. Isolates from these hosts and areas group in the sub-clade of C. cubensis accommodating the South American collections of the fungus. This sub-clade also includes isolates recently collected from Eucalyptus in Cuba, which are used to epitypify C. cubensis. New host records from Southeast Asia include exotic Tibouchina urvilleana from Singapore and Thailand and native Melastoma malabathricum (Melastomataceae) in Sumatra, Indonesia. Consistent with their areas of occurrence isolates from the latter collections group in the Asian sub-clade of C. cubensis. DNA sequence comparisons of isolates from Tibouchina lepidota in Colombia revealed that they represent a new sub-clade within the greater Chrysoporthe clade. Isolates in this clade are described as Chrysoporthe inopina sp. nov., based on distinctive morphological differences.     

转鞭毛蛋白基因水稻细菌性条斑病抗性研究

该研究从生防菌枯草芽胞杆菌Bs-916中克隆了鞭毛蛋白基因,利用转基因载体pCAMBIA1300转入水稻,筛选得到98株阳性转基因植株。分子检测结果表明,有12个转基因株系可检测到目的基因的表达。随后抗病性鉴定表明,有3个转基因株系对水稻细菌性条斑病具有较高的抗性。该研究为目前水稻抗细菌性条斑病转基因研究拓宽了可应用基因资源的范围。     

Biological Control of Rice Blast byPseudomonas fluorescensStrainPf7–14: Evaluation of a Marker Gene and Formulations

Pseudomonas fluorescensstrainPf7–14 was evaluated for biological control of rice blast in field experiments. StrainPf7–14 was formulated in methylcellulose:talc (1:4) and applied to IR50 rice (Oryza sativa) seeds as a seed treatment and as foliar sprays in seedbed and field experiments. When applied as a seed treatment followed by three foliar applications, the strain provided a 68.5% suppression of rice blast in the seedbed experiment and a 59.6% suppression in the field experiment. The persistence and migration ofPf7–14 on the rice plant was studied with the aid oflacZYgenes inserted into the bacterium. In greenhouse experiments,Pf7–14gal was detected on rice roots at 10⁶to 10⁵cfu/g of root tissue for 110 days, the duration of the rice crop. Migration of the strain from the seeds to the leaves occurred only until the seedlings were 16 days old. WhenPf7–14 was applied to the rice plants by foliar sprays, 10⁴cfu of the bacterium per gram of leaf tissue was detected for the next 40 days. The limited migration of the bacterial biocontrol agent emphasizes the need for multiple foliar applications of the bacterium to sustain the bacterial population for effective suppression of rice blast.     

异角毛藻和平滑角毛藻的分类学修订

为了澄清异角毛藻和平滑角毛藻的分类学疑问,在广东南澳岛海域建立了单克隆培养株系,利用光学显微镜和电子显微镜技术,开展了基于生活史的连续形态学观察,发现粗大角毛的延伸方向易变,是不稳定特征,不宜继续赋予分类学价值。对核糖体大亚基编码基因的D1～D3区序列进行扩增和分析,结果异角毛藻和平滑角毛藻的目标基因序列基本一致,仅有1个平滑角毛藻株系存在2个差异碱基。因此,两者具有一致的系统学位置,属于同一物种,平滑角毛藻应该是异角毛藻的同种异名。     

Cloning of the gene coding for chitobiase ofSerratia marcescens

Summary A λ phage DNA library ofSerratia marcescens was constructed and a clone carrying the gene coding for chitobiase (E.C.3.2.1.29) was isolated and characterized. Deletion analysis limited the cloned region to 4.5 kb that is capable of efficient expression of chitobiase.Escherichia coli cells harboring a plasmid carrying the cloned gene express chitobiase constitutively. The molecular weight of the protein is about 95000 daltons. In exponentially growingE. coli cells the chitobiase enzyme was found to be secreted into the periplasm.     

水稻内生成团泛菌YS19共质体形成差异表达蛋白MalE及其兼职功能

成团泛菌YS19是从水稻“越富”品种中分离的一种优势内生细菌,与宿主水稻互作时具有多种促生作用,其形成的共质体(symplasmata)结构与菌体抗逆及与宿主互作有重要意义．研究发现了一种在YS19共质体形成阶段高表达的差异蛋白,对其用肽指纹图谱进行鉴定,发现其属于周质空间麦芽糖结合蛋白家族．克隆了该蛋白质的基因,重组表达并分离纯化了该蛋白质,发现它是一种兼职功能蛋白,其不仅参与麦芽糖的ABC运输系统,而且在强酸环境下不易发生变性沉淀,并可通过疏水面的显著暴露结合底物蛋白来发挥分子伴侣活性,这些兼职功能构成了菌体抗逆生存适应性的重要分子基础．     

珍稀药用石斛内生菌研究进展

药用石斛是我国传统名贵中药材,具有重要的药用价值,由于人为和环境等因素,药用石斛资源已变得极其匮乏,难以满足市场的需求。药用石斛内生菌资源丰富,在与宿主石斛长期共生的过程中,对药用石斛的生长发育、环境的适应、活性代谢产物的积累等方面具有很强的促进作用。深入研究药用石斛内生菌及其与宿主的关系,对药用石斛生产、资源保护与利用有着重要意义。本文简要概述了药用石斛内生菌资源及其生物学功能,并探讨了存在的问题和未来发展方向,以期为药用石斛内生菌的开发与利用提供参考。     

水稻OsWRKY89启动子紫外线-B反应元件区的鉴定

植物需要利用太阳光能进行光合作用,因而不可避免地受到紫外线-B(UV-B) 辐射的影响．为了鉴定水稻WRKY转 录因子OsWRKY89基因启动子中的UV-B反应相关元件,分析了转启动子不同缺失片段与gus融合基因的水稻幼苗,发现在该启动子中存在UV-B反应元件,位于基因翻译起始位点上游-1 213～-1 188之间的25 bp区域,碱基序列为AAGATCTACCATTGCTCTATAGCTT．结合OsWRKY89和UV-B诱导上调表达基因启动子序列分析发现,该元件区在水稻UV-B反应基因启动子上具有高度的保守性,而且与已知保守的光反应元件位置邻近,表明该区域在水稻UV-B反应的转录调控中可能具有重要功能．