节丛孢属丝孢菌对松材线虫和拟松材线虫的捕食*

利用节丛孢属捕食线虫真菌的 8个种 ,1 1个菌株 ,对松材线虫 (Bx)和拟松材线虫(Bm)进行了捕食力的测定。结果显示节丛孢属真菌对这两种线虫的捕食能力既存在种间差异 ,也存在同种不同菌株间的差异。指状节丛孢的 3个菌株Ad 1 ,Ad 2 ,Ad 3菌株对两种线虫均表现出较高的捕食率 ,接种 7d后对Bx和Bm的捕食率分别达到了 98.08%,91.16 %,86.3 %和 96.28%,90.45 %,85.38%;同一菌株对松材线虫和拟松材线虫的捕食没有选择性。此外 ,通过对松材线虫和拟松材线虫     

节丛孢属丝孢菌对松材线虫和拟松材线虫的捕食

利用节丛孢属捕食线虫真菌的8个种,11个菌株,对松材线虫（Bx）和拟松材线虫（Bm）进行了捕食力的测定。结果显示节丛孢属真菌对这两种线虫的捕食能力既存在种间差异,也存在同种不同菌株间的差异。指状节丛孢的3个菌株Ad－1,Ad－2,Ad－3菌株对两种线虫均表现出较高的捕食率,接川7d后对Bx和Bm的捕食率分别达到了98．08％,91．16％,86．3％和96．28％,90．45％,85．38％;同一菌株对松材线虫和拟松材线虫的捕食没有选择性。此外,通过对松材线虫和拟松材线虫繁殖真菌的筛选实验,肯定了利用拟松材线虫代替松材线虫进行生防菌株筛选的可行性。     

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...     

拟松材线虫纤维素酶活性与其致病性关系

【目的】研究拟松材线虫是否分泌纤维素酶以及纤维素酶与其致病力的关系。【方法】对拟松材线虫的不同致病性种群以及松材线虫的虫体蛋白提取液、分泌液的纤维素酶活性进行定量测定,比较群体间纤维素同工酶谱型差异。【结果】拟松材线虫也含有纤维素酶,并向体外分泌;且不同致病性种群的纤维素酶活性与其致病性有一定相关性,致病性越强纤维素酶活性越强。【结论】纤维素酶活性大小是导致拟松材线虫不同种群间致病性差异的重要原因,这对于全面认识松树线虫萎蔫病的致病机理有重要意义。     

携带松材线虫对松墨天牛成虫肠道和气管细菌的影响

【目的】探究被松墨天牛Monochamus alternatus携带的松材线虫Bursaphelenchus xylophilus对松墨天牛肠道和气管细菌的影响。【方法】野外采集携带松材线虫的松墨天牛成虫后,取出完整肠道和气管进行细菌总DNA抽提后进行16S rDNA基因测序并拼接,并利用生物信息学方法分析松墨天牛成虫肠道和气管细菌组成、结构、丰度和多样性。【结果】携带松材线虫的松墨天牛成虫肠道和气管细菌菌群共检测到15门26纲66目110科201属296种,可操作分类单元(operational taxonomic unit, OTU)数目为444。携带松材线虫的松墨天牛成虫比未携带松材线虫的松墨天牛成虫肠道优势细菌菌群变化不显著,均为变形菌门(Proteobacteria)肠杆菌目(Enterobacterales);携带松材线虫的松墨天牛成虫气管优势细菌菌群为变形菌门肠杆菌目,未携带松材线虫的松墨天牛成虫气管优势细菌菌群为厚壁菌门(Firmicutes)乳杆菌目(Lactobacillales)。携带松材线虫的松墨天牛成虫较未携带松材线虫的松墨天牛成虫气管细菌多样性和丰度升高,细...     

一株北里孢菌株的分离鉴定及其对松材线虫的致病性

【目的】筛选、鉴定出对松材线虫杀灭活性较高的放线菌菌株,并确定生防菌株的毒力因子。【方法】采用平板活性测试及代谢杀虫活性检测方法进行筛选,采用形态学及16S rDNA序列分析等进行鉴定。对发酵液中的活性物质稳定性分析后,利用醇沉、萃取、层析、气相色谱/质谱分析等方法分离纯化出杀虫毒力因子。【结果】从河南南阳宝天曼的腐木及枯枝落叶样品中共分离获得了79株放线菌,从中筛选出对松材线虫有灭活作用的放线菌6株,其中分离株C620菌株对松材线虫的灭活性最高:该菌株的发酵液处理松材线虫48、60 h后线虫的死亡率分别达到60.0%、81.5%。结合该菌株的形态学、生理学特征及16S rDNA序列分析等结果将其归为北里孢菌属中的一个种,菌株编号Kitasatospora sp.strain C620。该菌株的发酵液中杀线虫活性物质的热稳定性、光稳定性及耐储藏性均较强,在中性偏碱性环境较稳定;经pH纸电泳层析初步确定该物质属于碱性水溶性物质。对菌株C620发酵液分离纯化,得到活性化合物为1-苯基-3-(2-吡啶)-5-吡唑啉酮。【结论】获得一株松材线虫高效生防菌Kitasatospora sp.strain C620,其活性物质为1-苯基-3-(2-吡啶)-5-吡唑啉酮。     

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     

甲酸乙酯对松材线虫的熏蒸效果

【背景】在溴甲烷面临禁用的情形下,探讨新熏蒸剂甲酸乙酯对松材线虫的处理效果,可以科学地评估甲酸乙酯的使用前景。【方法】采用松材线虫分离液和带疫松木段,设置甲酸乙酯5个剂量梯度、5个处理温度和5个处理时间,测定其对松材线虫的毒力及CT值。【结果】在25℃下处理3、6、12、24、48h,甲酸乙酯对松材线虫的Lc50分别为2．63、1．60、0．99、0．41、0．20mg·L-1。温度对毒力有显著影响,在10～29℃,随温度升高,甲酸乙酯对松材线虫的毒力降低,19和29℃下,1．85mg·L-1甲酸乙酯处理松材线虫的死亡率分别为63％和100％。甲酸乙酯熏蒸12h内,能完全杀灭木段中的松材线虫。在23℃下处理6和12h,松材线虫死亡率达到99％时,甲酸乙酯的cr值分别为453．94和424．14mg·h-1·L-1。【结论与意义】甲酸乙酯可用于松材线虫的检疫处理。     

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。     

三株芽胞杆菌菌株对松材线虫低温存活与繁殖的影响

为明确细菌在松材线虫生态适应性中的作用,本研究选择与致病相关的松材线虫伴生细菌GD1、马尾松内生细菌GD2以及具有杀松材线虫活性的湿地松内生细菌NJSZ-13为试验对象,测定经这3株芽胞杆菌菌株3个浓度低温驯化10、15和20 d后,在冷冻条件下松材线虫强毒虫株AMA3、中毒虫株AA3和弱毒虫株YW4的存活率和繁殖量。结果表明：低温驯化15 d和20 d后3株菌株对不同毒力线虫的活力影响较驯化10 d后的更显著。在低温驯化15 d、-20℃冷冻处理1 h后,5×10⁶ CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的存活率分别为77.22%、83.68%和84.26%,与对照差异显著;5×10⁵ CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的存活率分别为75.76%、80.67%和81.50%,与对照差异显著。5×10⁶ CFU/mL和5×10⁵ CFU/mL浓度菌株GD2处理下,与GD1处理组结果相似,菌株NJSZ-13处理组则与菌株GD1和GD2的结果相反。低温驯化15 d、-20℃冷冻处理1 h后,5×10⁶ CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的繁殖量分别为7 530、9 317和12 793条/皿,与对照（3 192、3 840和5 823条/皿）差异显著;5×10⁵ CFU/mL浓度菌株GD1处理时,3个虫株的繁殖量均与对照差异显著。而菌株GD2和NJSZ-13处理后,3个虫株的繁殖量均无显著变化。表明不同芽胞杆菌对松材线虫的低温适应性影响存在差异,松材线虫伴生细菌GD1和马尾松内生细菌GD2能增强其低温适应性,而湿地松内生细菌NJSZ-13菌株则相反。     

Pathogenicity and repulsion for toxin‐producing bacteria of dominant bacteria on the surface of American pine wood nematodes

Bacteria were isolated from the surface of two samples of American pine wood nematodes to identify methods of controlling pine wilt disease. The dominant bacterial strains were identified, and their toxicity and pathogenicity, in addition to their competitiveness with other pathogenic bacteria, were measured to ascertain how bacteria on the surface of American pine wood nematodes might be used to prevent and control pine wilt disease. The bacterial isolates show that the dominant bacteria carried by the two samples of pine wood nematodes are US4, US5, Smal‐007 and Rrad‐006. Based on routine staining, morphological observation and 16S rDNA sequence analysis, the four strains were identified as Delftia lacustris, Pseudomonas putida, Stenotrophomonas maltophilia and Rhizobium nepotum. The incubation of four dominant bacterial strains and Chinese dominant bacterial strains on the surface of aseptic nematodes and in nutrient broth showed that Smal‐007 and Rrad‐006 have strong competitiveness on the surface of pine wood nematodes. Using a bacterial culture medium to measure the propensity of pine seedlings to wilt, all the American dominant bacterial strains were shown to be less toxic than the Chinese dominant strains. If pine seedlings are inoculated with both bacterial and aseptic pine wood nematodes, American dominant bacterial strains present less pathogenicity than the Chinese dominant bacterial strains. In particular, Smal‐007 and Rrad‐006 show the lowest pathogenicity. If pine seedlings are inoculated with both bacterial and wild pine wood nematodes, American dominant bacterial strains significantly reduce the pathogenicity of wild pine wood nematodes isolated from Zhejiang Province, China. The effects of Smal‐007 and Rrad‐006 are confirmed as the most prominent. The American dominant strains Smal‐007 and Rrad‐006 satisfy two main requirements: excellent repulsion performance and low pathogenicity. Therefore, they can be used as candidate strains for biocontrol bacteria.     

季也蒙假丝酵母对松材线虫病的抑制作用

松材线虫病是重要的森林病害,该病与松材线虫携带的病原菌和松树的内生病原菌密切相关。在室内条件下,初步研究了从人工培养的松材线虫上分离到的菌株C对松材线虫病的抑制作用。在健康的水培马尾松枝上分别接种松材线虫接种液、菌株C接种液、松材线虫与菌株C的混合接种液。处理后松枝的相对重量与相对蒸腾强度均为:接种菌株C的松枝>混合接种的松枝>接种线虫的松枝。处理后15d时,接种线虫的松枝与混合接种的松枝的相对重量有显著性差异(p<0.05)。接种线虫松枝的存活期显著短于其它处理松枝的存活期(p<0.05)。接种菌株C的针叶褐变株数少于接种线虫的松枝,两者有显著性差异(p<0.05)。从接种线虫和混合接种的所有松枝中都分离到松材线虫,且分离出的线虫量没有显著性差异。将8个月生的断根马尾松苗插入菌株C的查彼培养液的滤液中培养,6d后松苗的平均感病指数和感病株率均显著少于对照(p<0.05)。这表明,菌株C对松材线虫病有抑制作用,菌株C培养液中产生的某些代谢物质有利于松苗的抗病和存活。菌株C可能抑制了松树上的内生病原菌和松材线虫携带的病原微生物,或提高了松树的生长力和抗逆能力。经电子显微镜观察并参照AP I 20 C AUX鉴定系统鉴定,菌株C为季也蒙假丝酵母C and id a gu ilierm ond ii。     

松墨天牛成虫体内的松材线虫分布和速检研究

为了探索快速测定松墨天牛成虫(下称成虫)的带疫性,用解剖方法和自制便携式显微镜,对成虫携带的松材线虫部位和速检(下称线虫)作了研究,得到下列结果：87．9％的线虫分布在成虫的节间、腹腔和生殖器内;雄成虫阳茎是松材线虫集中分布区,两头平均携带线虫498．5条;交尾时雄成虫把阳茎上的线虫传到雌成虫外生殖器,在雌成虫产卵时再把线虫带入松树形成层组织;在野外检测成虫带疫性平均需时1．57min。     

Response of Plant Parasitic and Free Living Soil Nematodes to Composted Animal Manure Soil Amendments

In an outside pot experiment, dry pig manure processed on pine sawdust litter and fermented for seven days by house fly larvae (fermented manure), and pine sawdust applied alone, and in combination with a spring application of inorganic nitrogen fertilizer were used to determine their effects on plant parasitic and free-living soil nematodes on sugar beets (cv. Antek). Non amended soil was used as a control. All treatments with fermented pig manure and sawdust with nitrogen fertilizer decreased number of plant parasitic nematodes and also root-fungal feeding nematodes compared to the untreated control. Sawdust applied alone had no effect on plant parasitic and root-fungal feeding nematode suppression. Free-living nematodes which were mainly bacteriovores and fungivores were significantly more abundant in soil amended with fermented pig manure, while the sawdust had no effect on these nematodes. The effect of all tested treatments on omnivores-predators was rather random, and in general, the number of these nematodes decreased after soil amendment applications compared to the untreated control.     

Pathogenicity of Bursaphelenchus xylophilus on Pines in Minnesota and Wisconsin

The pinewood nematode, Bursaphelenchus xylophilus, was inoculated into established native jack and red pines (Pinus banksiana and P. resinosa) and exotic Austrian pine (P. nigra) in Minnesota and Wisconsin forests during summer 1981. The nematode isolates did not kill established nonstressed pine trees growing in the forest. However, the same nematode isolates killed pine seedlings under greenhouse conditions. Girdling the main stem of some trees to induce stress resulted in the death of the majority of inoculated and noninoculated branches of Austrian and jack pines, but no branch death was observed on red pine. Greater numbers of nematodes were extracted from branches of inoculated, girdled trees than from nongirdled trees. The mean number of nematodes extracted from branches of inoculated, nongirdled trees was 0.3 - 14 nematodes per gram of wood.     

Transmission of Bursaphelenchus xylophilus through Oviposition Wounds of Monochamm carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode through Monochamus carolinensis oviposition wounds was documented. Nematode transmission was measured as the average number of nematodes isolated per oviposition wound excavated and also as the percentage of oviposition wounds from which nematodes were isolated. The influence of three factors that might affect nematode transmission was investigated: age of the beetle vector, number of nematodes carried per beetle, and egg deposition in the oviposition wound. Only the number of nematodes carried by the beetle was found to have a significant effect on transmission. Nematodes were transmitted more frequently and in slightly greater numbers by beetles carrying more nematodes. The influence of pinewood on nematode exit from beetles were investigated by comparing nematode exit from beetles placed over pine chips with those placed over distilled water. Nematodes exited in greater numbers and at a higher frequency from beetles over pine chips than from beetles over distilled water. Apparently, the nematodes are able to detect a factor from the pine chips that promotes their exit from the beetles.     

Horizontal Transmission of Bursaphelenchus xylophilus between Sexes of Monochamus alternatus

Four experiments were conducted using nematode-infested and nematode-free adults of the cerambycid beetle, Monochamus alternatus, to determine horizontal transmission pathways of Bursaphelenchus xylophilus. When nematode-infested beetles of one sex and nematode-free beetles of the opposite sex were paired in containers for 48 or 72 hours, the number of nematodes carried by nematode-free beetles tended to increase with increased number of nematodes carried by nematode-infested beetles. The nematodes acquired by "nematode-free" beetles could be transmitted to pine. A female beetle that received 13 nematodes from a male transmitted one nematode to a Pinus densiflora bolt via an oviposition wound. When the nematode-infested and nematode-free beetles were observed continuously, it was observed that the number of nematodes carried by nematode-free beetles at the end of the first sexual mounting increased as the number of nematodes carried by nematode-infested beetles just before mounting increased. The number of nematodes transferred to nematode-free beetles was positively related to duration time of mounting. There was no difference in transmission efficacy between male-to-female transmission and female-to-male transmission. The horizontal transmission pathways are discussed relative to the persistence of B. xylophilus in resistant pine forests and the control of pine wilt disease.     

Maturation feeding and transmission of Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) by Monochamus alternatus (Coleoptera: Cerambycidae) inoculated with Beauveria bassiana (Deuteromycotina: Hyphomycetes)

We examined the amount of maturation feeding and transmission of pinewood nematodes, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Nematoda: Parasitaphelenchidae), to healthy pine (Pinus spp.) trees by pine sawyer Monochamus alternatus Hope (Coleoptera: Cerambycidae) adults infected with Beauveria bassiana (Balsamo) Vuill. (Deuteromycotina: Hyphomycetes). Inoculated beetles fed less than noninoculated beetles, probably because feeding by inoculated beetles began to decrease at about 4 d postinoculation and inoculated beetles ceased to feed for several days before their death. In inoculated beetles carrying >1,000 nematodes, some beetles died before nematode departure. The remaining heavily nematode-infested beetles lived until the beginning of nematode departure, but they had stopped feeding, preventing the nematodes from entering pine twigs. We suggest that microbial control of pine sawyer adults by B. bassiana may be effective in preventing transmission of pine wilt disease to healthy pine trees.     

Precision and Selection of Extraction Methods of Aphelenchid Nematodes from Maritime Pine Wood,Pinus pinaster L.

Four extraction methods for Bursaphelenchus xylophilus and other aphelenchid nematodes were compared on the number of nematodes per gram recovered, and on the precision of the mean number of nematodes per gram of pine wood. The number of nematodes per gram recovered by each method, in addition to its inherent shortcomings when the actual number of nematodes is unknown, failed to provide clear rankings among the extraction methods. The precision of the mean number of nematodes per gram did provide clear guidelines for selection. Selection of the method may be based on prior knowledge about the range of nematodes to be expected or the independence of precision from the mean number of nematodes.