不同致病力松材线虫和拟松材线虫种群繁殖对温度的响应

为探讨松材线虫Bursaphelenchus xylophilus和拟松材线虫Bursaphelenchus mucronatus在菌食和植食阶段的种群繁殖力对环境温度的响应及其与种群致病力的关系,本研究对不同致病力的松材线虫和拟松材线虫,通过单异活体培养（菌食）和接种寄主黑松（植食）两种方式,探讨了不同温度条件下线虫种群孵化率、繁殖量和后代性比等繁殖参数的变化情况。结果表明,在单异活体培养时,供试的松材线虫强毒和弱毒虫株以及拟松材线虫有毒和无毒虫株在25℃下均具有最高的卵孵化率;不同致病力的松材线虫和拟松材线虫种群具有不同的最适生长温度,松材线虫强致病力虫株ZJ在35℃出现最大繁殖量,弱致病力虫株YW4在30℃下繁殖最多,而拟松材线虫无致病力虫株JNL10和有致病力虫株15#则在25℃下繁殖量较大。但总体来看,在25~35℃下,松材线虫ZJ虫株和YW4虫株繁殖量无显著性差异,而拟松材线虫JNL10虫株和15#虫株的繁殖量具有显著差异。在寄主体内,15~20℃时松材线虫ZJ虫株和YW4虫株繁殖量无显著性差异,25~35℃时,松材线虫ZJ虫株的繁殖量明显高于YW4虫株;而在各个温度下,拟松材线虫繁殖量基本低于松材线虫弱毒虫株YW4且远远低于强毒虫株ZJ。在单异活体培养下大部分经不同温度处理线虫种群的雌雄性比都大于2.0;而在寄主黑松体内各个虫株的种群性比均低于1.5,其中松材线虫种群的雌雄性比多数低于1.0,且随着温度的升高（15~30℃）出现先上升后下降的趋势;拟松材线虫各处理的种群雌雄性比在1.0~1.5之间,且随着温度的上升而上升。表明在不同温度下,单异活体培养阶段的松材线虫和拟松材线虫的种群增长与其致病力不呈相关性;而在寄主体内,不同温度条件下的松材线虫和拟松材线虫的繁殖力与其致病性存在正相关关系,致病力越强的虫株,其繁殖力越大。     

松材线虫和拟松材线虫体内细菌的透射电镜观察及分离鉴定

【目的】松材线虫是松树萎蔫病的病原,拟松材线虫在形态等方面与松材线虫极其相似。关于两种线虫与细菌的研究多集中于体表伴生细菌。本文要揭示松材线虫和拟松材线虫体内是否存在细菌。【方法】对松材线虫和拟松材线虫进行透射电镜观察;并采用1%升汞和抗菌素混合液对两种线虫虫体进行体表消毒后研磨,制备悬浮液涂布NA平板;通过生理生化测定和16S rDNA序列分析鉴定细菌种类。【结果】松材线虫和拟松材线虫透射电镜照片显示在两种线虫肠道内均发现细菌;体表无菌的松材线虫和拟松材线虫共分离到3株体内细菌;这3株细菌分别属于寡养单胞菌属(Stenotrophomonas)和爱文氏菌属(Ewingella)。【结论】松材线虫和拟松材线虫体内均存在细菌;这些细菌对这两种线虫可能具有一定的生理生态作用。本文是松材线虫和拟松材线虫体内存在细菌的首次报导。     

松材线虫与伴生微生物的生态关系

松材线虫是重要的外来有害生物,造成松树大量死亡,带来严重的经济损失和生态破坏.松材线虫与伴生微生物存在密切的生态关系.本文综述了松材线虫伴生微生物的种类及其在松材线虫繁殖和致病过程中的重要生态作用,从微生态系统角度对松材线虫病进行探讨.伴生真菌能为松材线虫提供食物,维持松材线虫次生侵染循环,提高分散型第三龄幼虫在种群中的比例,利于松材线虫侵染扩散;伴生细菌能够提高松材线虫致病性,促进其繁殖,并菌有助于松材线虫降解松萜类物质,从而提高松材线虫的适应性.     

松材线虫入侵种群形成与扩张机制——国家重点基础研究发展计划“农林危险生物入侵机理与控制基础研究”进展

从种群遗传学和生态学两个不同角度分析了松材线虫成功入侵的机制,种群遗传多样性研究表明,松材线虫入侵过程中,无明显的奠基者效应和由遗传漂变引起的种群遗传瓶颈,由于不同入侵来源的种群多次大量入侵,导致我国入侵种群具有丰富的遗传多样性,入侵过程中保持有高的种群遗传多样性,是松材线虫成功入侵的遗传学机制之一,种间竞争实验结果表明,松材线虫具有高的繁殖潜能和强的竞争能力,在入侵过程中能对本地近缘种产生种间竞争和生态替代,竞争性替代是松材线虫成功入侵的生态学机制之一,种间杂交和基因渗入,有利于松材线虫的入侵,同时,从功能基因组的角度,研究了与松材线虫适应性相关的一些重要基因的结构、功能及其进化,探讨松材线虫成功入侵的生态适应性分子机制,并从生态系统的抵御性和恢复性角度,探讨了生态系统对松材线虫入侵和扩散的影响,此外,对尚需继续深入研究的科学问题进行了讨论,研究结果为进一步揭示松材线虫成功入侵机理的研究奠定了基础,并为松材线虫病的有效管理和控制提供了科学依据。     

植物根际益生细菌代表性菌株贝莱斯芽孢杆菌FZB42对松材线虫的抑杀性

【目的】由松材线虫导致的松树萎蔫病是松树的毁灭性病害,也是我国最主要的林业病害之一。本研究测评了在农业上广泛使用的、我国微生物肥料行业主要菌种资源之一——贝莱斯芽孢杆菌,对松材线虫的潜在抑杀性能。【方法】选用贝莱斯芽孢杆菌的代表性菌株FZB42为材料,测定对不同条件下的菌液上清、不同菌株的菌液上清、细菌素plantazolicin的提取物以及菌体直接接触等方式,对松材线虫死亡率/存活率的影响,并构建FZB42生物膜合成缺损菌株,测定比较其对松材线虫存活率的影响。【结果】相比于Landy培养基,使用LB培养基得到的菌液上清,对松材线虫具有明显抑杀性,培养48h的菌液上清比培养24h的菌液上清抑杀性更强,同时,菌液上清的抑杀效能随浓度升高及处理时间增长而有所增加。其中,使用培养48h后收集的LB菌液上清处理48h,松材线虫死亡率可高达约50%。之前报道对秀丽隐杆线虫有杀线虫活性的细菌素plantazolicin,经不同突变株上清以及plantazolicin提取物测试,被证实对松材线虫无抑制作用。直接接触实验表明,尽管FZB42的生物膜形成作用会刺激松材线虫提高抗胁迫能力,但整体而言,菌体接触对松材线虫亦有明显抑杀效果。【结论】本研究通过严格细致的测试证明,贝莱斯芽孢杆菌FZB42对松材线虫具有抑杀性,该抑杀作用的分子基础和作用机理有待深入挖掘,但其与plantazolicin无关。作为安全性好、研究开发程度高的一类生防菌株,贝莱斯芽孢杆菌在防治松材线虫病方面的潜在价值值得关注。     

携带与非携带松材线虫的松墨天牛miRNA表达谱比较分析

【目的】松墨天牛是松树的重要蛀干害虫,也是林业重大外来入侵种松材线虫的媒介昆虫。虽然松墨天牛和松材线虫互作的化学生态和分子进化机制受到人们的广泛关注,但miRNA等表观遗传因子在天牛—线虫互作中的作用未见报道。【方法】使用illumina HiSeq 2000平台进行miRNA高通量测序,得到4个携带线虫的天牛miRNA库和4个未携带线虫的天牛miRNA库。此外,对鉴定出的miRNA进行了差异表达分析,并对这些miRNA的靶基因进行了GO注释和KEGG通路富集分析。【结果】在携带线虫的天牛表皮、脂肪体、中肠和气管样本中分别鉴定出780、802、617和762个miRNA;在未携带松材线虫的天牛的不同组织样本中分别鉴定出784、723、713和837个miRNA。在携带松材线虫的松墨天牛中,某些已知miRNA表达量会显著升高,如miR-14、miR279和miR-312等。差异表达miRNA的功能大多指向代谢、免疫等方面。【结论】miRNA在松墨天牛和松材线虫互作中起着重要的调控作用。     

扩散型与繁殖型松材线虫数字基因表达谱对比分析

【目的】松材线虫作为林业重大外来入侵种,扩散型幼虫的形成对其传播扩散起着非常重要的作用,但扩散虫态的形成与维持机制尚未阐明。【方法】通过构建松材线虫数字基因表达谱(DGE),从滞育状态的维持、化学感受、代谢途径等方面分析松材线虫不同虫态的基因表达差异。【结果】参考松材线虫基因组数据,鉴定出2种扩散型幼虫(LⅢ,LⅣ)和繁殖型幼虫(Ln)各有11184、8533和10781个基因。相对于繁殖型虫态,大多数基因在LⅣ中下调表达,该虫态中特异上调表达的基因有化感受体基因、核受体基因以及一些代谢相关基因。推测这可能与扩散型线虫滞育状态的维持相关,并在其生理功能如化学感受和媒介/寄主互作中发挥作用。GO和Pathway富集分析显示,多数代谢相关通路在LⅣ中下调表达,而在LⅢ中的表达均活跃。【结论】以上结果与LⅣ处于不进食、总体代谢水平较低等生理状态的表型相一致。     

松材线虫伴生细菌多样性的宏基因组分析

【目的】松材线虫是松材线虫病的病原,且与其伴生细菌之间存在互作关系,它们构成一个微生态系统。本研究旨在揭示松材线虫-伴生细菌群落细菌多样性。【方法】采用16S rRNA基因文库和454测序对伴生细菌群落的宏基因组进行初步分析。【结果】依据97%序列相似性划分OTU(Operational Taxonomic Unit),构建的16S rRNA文库包含25个OTU,分别属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Bacteroidetes,其中优势菌群为Gammaproteobacteria,特别是Stenotrophomonas maltophilia为优势细菌。在伴生细菌优势种群上,454测序结果与16SrRNA基因文库结果基本一致。【结论】松材线虫的伴生细菌多样性较高,这些细菌可能对松材线虫具有一定的生态意义。     

松材线虫伴生细菌多样性的宏基组分析

摘要：【目的】松材线虫是松材线虫病的病原,且与其伴生细菌之间存在互作关系,它们构成一个微生态系统。本研究旨在揭示松材线虫-伴生细菌群落细菌多样性。【方法】采用16S rRNA基因文库和454测序对伴生细菌群落的宏基因组进行初步分析。【结果】依据97％序列相似性划分OTU（Operational Taxonomic Unit）,构建的16S rRNA文库包含25个OTU,分别属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Bacter     

松材线虫galectin-1基因的克隆、原核表达和表达模式分析

【目的】galectin-1是凝集素的一种,广泛存在于各种生物体内,在生长发育、免疫调节方面起重要作用。本研究克隆和表达了松材线虫的galectin-1蛋白,并分析了各个龄期的表达量。【方法】设计引物,扩增松材线虫的galectin-1基因,使用双酶切的方法连接p ET-28a载体和目的基因,转化到大肠杆菌DH5α感受态细胞,筛选阳性克隆;在不同温度下,用不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,Western blot检验蛋白表达情况;采用RT-PCR技术检验松材线虫各个龄期galectin-1基因的表达情况。【结果】由SMART和Predict Protein软件分析可知,该蛋白有2个结构域,并且主要由无规卷曲和β折叠构成;生物学信息分析显示,松材线虫的galectin-1与小卷蛾斯氏线虫的相似性更高;与日本血吸虫相比,线虫能很好地聚集在一个分支上。Western blot检测纯化蛋白大小与查询所得蛋白分子质量一致。RT-q PCR结果显示,以繁殖型2龄松材线虫(L_2)为对照,galectin-1基因在繁殖型3龄(L_3)、繁殖型4龄(L_4)、扩散型3龄(L_(Ⅲ))和扩散型4龄(L_(Ⅳ))松材线虫中的表达量高,尤其是在LⅢ中的表达量最高;雌雄成虫没有显著性差异。【结论】松材线虫的galectin-1基因在p ET-28a原核表达系统中呈可溶性表达,在不同龄期的表达量有差异。本研究为进一步研究松材线虫的galectin-1基因奠定了基础,为松材线虫的防治提供了新的方向。     

Occurrence of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) and the microhabitat distribution of fungi in declining pine trees in a locale in Korea

A total of 33 pine trees with symptoms of decline were collected in Jeonnam Province, South Korea, and were examined for the presence of nematodes. About 20% of the trees sampled were positive with Bursaphelenchus species. All Bursaphelenchus species were found in recently dead or dying trees. Based on morphological observations, the nematode extracted from the declining pine trees was identified as B. mucronatus. The highly pathogenic pine wood nematode B. xylophilus was not found in any pine trees sampled. B. mucronatus was easily reared on fungus Botrytis cinerea. Twenty one fungal isolates were isolated from dead trees, fallen twigs, and healthy pine trees. The fungal isolates belonged to Trichoderma genus and were dominant in the wood of partially declining pines. The blue‐stain fungi transmitted by the Monochamus beetle were not detected. The B. mucronatus population decreased markedly on Auxarthron reticulatum DY‐2 isolated from soils. The number of nematodes also reduced on Verticillium saksenae A‐1, a nematophagous fungus, and Beauveria bassiana, an entomopathogenic fungus. This observation suggested the fungal production of nematicidal activity against B. mucronatus. When the fungal culture filtrates were also used for nematicidal activity on B. mucronatus, the culture filtrates of A‐1, DY‐2 and B. bassiana showed over 50% mortality within 48 h exposure. The fungi BC4, BC5 and BC6 isolated from declining pine trees inhibited the reproduction of B. mucronatus, and their culture filtrates also expressed nematicidal activity, indicating a possible interaction between the fungi in pine trees and nematodes at microhabitat level.     

里氏木霉翻转酶DRS2对木质纤维素降解酶基因表达及分泌的影响

[目的]本研究以工业生产菌株里氏木霉为研究对象,鉴定翻转酶基因drs2对其纤维素酶表达及分泌的影响.[方法]首先通过BLAST序列比对,从里氏木霉中鉴定出翻转酶基因drs2,并通过同源重组的方法在里氏木霉中构建了drs2基因的敲除菌株△drs2.对△drs2菌株及其对照株在不同碳源上的生长发育、蛋白分泌、纤维素酶及半纤...     

Immunohistochemical, in situ hybridization and biochemical studies on endogenous cellulase of Corbicula japonica

We previously reported on the endogenous cellulase gene of Corbicula japonica, CjCel9A. In this study, the tissue localization of the mRNA and translated products of CjCel9A was investigated in order to understand how this gene is physiologically involved in cellulose decomposition by C. japonica. Antiserum against recombinant CjCel9A protein was prepared. Multiple bands were observed mainly on western blot analysis of the crystalline style, and the band sizes partially corresponded to the active bands detected using zymographic analysis. In situ hybridization and immunohistochemical analyses clarified the exclusive production and secretion of this cellulase by the secretory cells localized in the epithelium of the digestive tubules in the digestive gland. These data strongly support our previous assumption that the endogenous cellulase of C. japonica is produced in the digestive gland and transported to the crystalline style to act as a component of its cellulolytic activity.     

狐、貉源大肠杆菌分离鉴定及毒力基因检测

[背景]狐、貉肺炎频发导致养殖户遭受巨大经济损失.[目的]调查黑龙江省、吉林省、河北省、山东省等地狐、貉肺炎原因,对病原菌进行分离、鉴定及部分生物学特性研究.[方法]无菌采集患病狐、貉肺组织进行细菌分离培养;对分离菌进行革兰氏染色、生化试验、特异性基因PCR鉴定、药物纸片扩散法敏感性试验、毒力基因检测及小鼠致病性试验....     

New Insights into the Phylogeny and Worldwide Dispersion of Two Closely Related Nematode Species,Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to coniferous forests worldwide, causing severe ecological damage and economic loss. The biology of B. xylophilus is similar to that of its closest relative, B. mucronatus, as both species share food resources and insect vectors, and have very similar morphological characteristics, although little pathogenicity to conifers has been associated with B. mucronatus. Using both nuclear and mitochondrial DNA markers, we show that B. xylophilus and B. mucronatus form distinct phylogenetic groups with contrasting phylogeographic patterns. B. xylophilus presents lower levels of intraspecific diversity than B. mucronatus, as expected for a species that evolved relatively recently through geographical or reproductive isolation. Genetic diversity was particularly low in recently colonised areas, such as in southwestern Europe. By contrast, B. mucronatus displays high levels of genetic diversity and two well-differentiated clades in both mitochondrial and nuclear DNA phylogenies. The lack of correlation between genetic and geographic distances in B. mucronatus suggests intense gene flow among distant regions, a phenomenon that may have remained unnoticed due to the reduced pathogenicity of the species. Overall, our findings suggest that B. xylophilus and B. mucronatus have different demographic histories despite their morphological resemblance and ecological overlap. These results suggest that Bursaphelenchus species are a valuable model for understanding the dispersion of invasive species and the risks posed to native biodiversity and ecosystems.     

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- -glucosidase [β- -glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater.     

23种中草药的体外抗菌活性筛选研究

为考察中草药抗菌物质基础筛选出活性提取物,该研究通过80%乙醇冷浸和95%乙醇回流提取制备23种中草药的提取物,采用琼脂扩散法测量抑菌圈直径,用微量液体培养基倍比稀释法测定最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal/fungicidal concentration,MBC/MFC),并测定了提取物对临床4种常见病原菌体外抗菌活性。结果表明:紫珠草、千斤拔、黄龙尾等9种中草药对金黄色葡萄球菌有较强的抑菌活性,其MIC/MBC值除个别菌是12.5 mg·mL~(-1)外,其他都在0.09～3.12 mg·mL~(-1)之间;千斤拔、大红袍、过江龙等5种中草药对铜绿假单胞菌有较强抑菌活性,其MIC/MBC值在3.12～12.5 mg·mL~(-1)之间;紫珠草、千里光、石楠等13种中草药对大肠埃希菌有较强的抑菌活性,其MIC/MBC值在0.09～6.25 mg·mL~(-1)之间;八角对白色念珠菌有较强抑菌活性,其MIC/MFC值在0.78～12.5 mg·mL~(-1)之间。23种中草药的抗细菌活性较好,尤其是千斤拔、大红袍、过江龙、八角、黄药子对金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌都具有较好的抑菌活性,具有广谱抗菌活性;但对真菌抑菌效果不明显,仅有八角对白色念珠菌有抑菌活性。此外,提取溶剂浓度、提取温度和提取时间对中草药的提取率和活性均有影响,冷提稍优于热提。     

我国口岸瓜实蝇监测样本的SSR分子标记分析

[目的] 瓜实蝇是世界性检疫害虫,被列入我国口岸常年监测计划中。本研究以检疫性有害生物瓜实蝇为研究对象,利用不同地理种群瓜实蝇的转录组发现特异性的微卫星（simple sequence repeats,SSR）序列,分析我国口岸监测的瓜实蝇样本的遗传多样性。[方法] 利用生物信息学方法,对4个不同地理来源的瓜实蝇转录组进行分析,发现了特异性的SSR序列,并设计特异性引物,选用我国11个省市的49个瓜实蝇监测样本进行测试及验证,使用NTsys和Popgene 32软件进行遗传多样性分析。[结果] 筛选出15对多态性较好的引物,UPGMA聚类分析显示,新疆、四川和广西种群归为一支,浙江、广东、江苏种群归为一支,上海、云南、海南、天津种群归为一支,北京种群单独聚为一支。[结论] 聚类分析结果显示,11个不同省市的瓜实蝇监测样本间基因分化系数为0.6712,表明不同口岸的瓜实蝇监测样本间的遗传分化较大,可能具有不同的来源地,可为进一步开发监测样本溯源技术提供理论依据。     

牛源致病性蜡样芽孢杆菌溶血素BL亚基的原核表达及其生物学活性分析

蜡样芽孢杆菌属于革兰氏阳性菌,分布广泛,具有一定的致病性。不同的蜡样芽孢杆菌携带有不同的毒力因子,这直接决定了蜡样芽孢杆菌株致病性的差异。研究和探讨毒力因子分布以及具体毒素的生物活性有助于对蜡样芽孢杆菌病采取更科学的防控。本研究通过原核表达系统将溶血素BL三亚基重组表达,并对表达蛋白进行了纯化和部分生物学活性的检测。研究结果表明,牛源致病性蜡样芽孢杆菌溶血素BL可以在原核表达系统中成功表达和纯化,牛源致病性蜡样芽孢杆菌溶血素BL拥有溶血性、细胞毒性、很好的免疫原性以及对小鼠具有一定的免疫保护性。本研究表达了溶血素BL三亚基,并探究了牛源致病性蜡样芽孢杆溶血素BL的生物学活性。本研究为进一步揭示蜡样芽孢杆菌溶血素BL的致病作用机制和建立针对牛源致病性蜡样芽孢杆菌病的检测方法奠定了理论基础。     

Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.