昆虫病原线虫和共生细菌培养系统中噬菌体的检测

本文利用紫外照射、温度、pH、盐度诱导五株溶源性共生菌株Xenorhabdus和Photorhabdus同时测定斯氏和异小杆属线虫Steinernema carpocapsae A24,S.carpacapsae ALL,S.feltiae English,S.feltiae SN,Heterorhabditis bacterophora H06在体外培养过程中是否存在感染共生细菌的噬菌体。结果未发现噬菌斑，说明实验菌株在实验诱导条件下以及昆虫病原线虫固体培养系统中不可能诱发噬菌体的危害。     

连续传代培养对病原斯氏线虫的带菌率和种群结构的影响

斯氏线虫Steinernema spp.是近年来发现的颇具希望的一种昆虫致病线虫,它与细菌Xenorhabdus nematophilus互惠共生。当斯氏线虫进入昆虫体腔后,排出的共生菌引起寄主败血症而使昆虫致死(poinar 1966)。因此,线虫对害虫的致病性一定程度上取决于感染期线虫所携带的共生菌。由于不同种的斯氏线虫携带不同亚种的共生菌,同种线虫中每条线虫所携带的共生菌的数量不同,所以对害虫显示了不同的致病力。在一般情况下,病原线虫的带菌率和每条线虫的带菌数目均与其致病力(毒力)成正相关     

嗜线虫致病杆菌CB6菌株对棉铃虫的生物活性

致病杆菌属(Xenorhabdus)及近缘的发光杆菌属(Photorhabdu)是昆虫病原线虫肠道共生菌。共生菌被线虫释放到寄主血腔中后,能导致昆虫迅速死亡,且产生抗菌成分防止虫尸腐烂[1]。由于从土壤或水源中未能分离到自由存活的共生细菌,因此早期认为这类细菌只有在与线虫共生状态下才对昆虫有致死作用[2]。1990年以来,Ensign等[3]和潘映红等[4]研究发现这类细菌的胞外蛋白和脂多糖注射到大蜡螟(Galleriamellonella)体内后均有杀虫活性。1998年,Science报道了发光杆菌(P.luminescens)菌株W 14分泌的高分子量复合体蛋白对烟草天蛾(Manducasexta…     

不同昆虫病原线虫—共生细菌组合的生化特性研究

本文分别以无菌的小卷蛾斯氏线虫的A24品系线虫与其自身携带的共生细菌菌株和与格氏线虫RS92品系的共生细菌菌株,以及以无菌的嗜菌异小杆线虫的H06品系线虫与其自身携带的共生细菌菌株和与大异小杆线虫HNA品系的共生细菌菌株建立单菌组合;测定了各组合感染期线虫的致病力、干重和体内主要生化物质的含量。结果发现,共生细菌除了对感染期线虫的致病力产生显著影响外,对线虫干重、蛋白质、氨基酸、糖原和脂肪酸等生化物质的含量也有很大的影响。     

不同昆虫病原线虫一共生细菌组合的生化特性研究

本文分别以无菌的小卷蛾斯氏线虫的A24品系线虫与其自身携带的共生细菌菌株和与格氏线虫RS92品系的共生细菌菌株，以及以无菌的嗜菌异小杆线虫的H06品系线虫与其自身携带的共生细菌菌株和与大异小杆线虫HNA品系的共生细菌菌株建立单菌组合；测定了各组合感染期线虫的致病力、干重和体内主要生化物质的含量。结果发现，共生细菌除了对感染期线虫的致病力产生显著影响外，对线虫干重、蛋白质、氨基酸、糖原和脂肪酸等生化物质的含量也有很大的影响。     

三种斯氏线虫与其共生细菌之间的专化性

在自然条件下,芜菁夜蛾斯氏线虫、长尾斯氏线虫和拟双角斯氏线虫体内携带的共生细菌分别为伯氏致病杆菌、Xenorhabdus ehlersii和X.budapestensis。在一种线虫的生长培养基上分别接种3种致病杆菌,测试对线虫的恢复发育、性比、后代产量及形态大小的影响。结果表明,芜菁夜蛾斯氏线虫与致病杆菌的专化性低于长尾斯氏线虫和拟双角斯氏线虫,在3种致病杆菌上均可发育,但在伯氏致病杆菌上的发育率显著高于X.ehlersii和X.budapestensis。长尾斯氏线虫和拟双角斯氏线虫侵染期幼虫仅在其各自共生细菌X.ehlersii和X.budapestensis上发育。芜菁夜蛾斯氏线虫侵染期幼虫在伯氏致病杆菌上发育后的雌雄比为1.77,而在X.ehlersii和X.budapestensis上仅为0.77和0.74;在伯氏致病杆菌上的产量高于X.ehlersii和X.budapestensis;在伯氏致病杆菌和X.ehlersii上产生的侵染期幼虫长度显著大于X.budapesten-sis。对3种致病杆菌上培养产生的芜菁夜蛾斯氏线虫侵染期幼虫体内携带的细菌进行分离和16S rDNA序列分析,表明芜菁夜蛾斯氏线虫侵染期幼虫除可携带其共生细菌伯氏致病杆菌外,还可携带X.budapestensis,但不能携带X.ehlersii。     

昆虫病原线虫共生细菌嗜线虫致病杆菌All对小菜蛾拒食作用物性质的初步研究

采用叶碟法测定了36个昆虫病原线虫共生细菌菌株对小菜蛾Plutella xylostella二龄幼虫的拒食作用,其中10个菌株对小菜蛾幼虫的选择性和非选择性拒食率高于75%。以嗜线虫致病杆菌Xenorhabdus nematophilus All菌株为代表,研究了对小菜蛾有拒食作用的物质与细菌生长过程、型变、pH值、温度、有机溶剂之间的关系。结果表明：拒食物质产生于细菌的对数增长期和稳定期,在稳定期最高,拒食率达85%左右;次生型菌液未能分泌足够的拒食物质,其拒食率在20%以下。在-80℃-100℃的范围内,其拒食率均在81%以上。pH3-11范围内,对拒食物质稳定性有一定影响,选择性拒食率变化不大,均在96%以上;非选择性拒食率变化明显,pH值为3、5、7、11时,非选择性拒食率下降至23.1%-34.9%。拒食物质可与氯仿、石油醚、乙酸乙酯、正己烷、乙醇相溶,其提取液对小菜蛾拒食率均在80%以上。     

2种昆虫病原线虫共生菌的代谢产物对苹果病原菌真菌的抑菌作用

嗜线虫致病杆菌(Xenorhabdus nematophila HB310)和发光杆菌(Photorhabdus luminescens HB140)是分别从小卷蛾斯氏线虫和异小杆线虫中分离到的2株昆虫病原线虫共生菌。为了明确这2种共生菌的抑菌活性,分别测定了其对苹果上6种主要病原真菌的抑菌活性。结果表明,这2种共生菌对6种苹果病原真菌均有不同程度的抑制作用。嗜线虫致病杆菌HB310菌株发酵液对苹果轮纹病病原菌抑制效果最好,抑制率达76.9%,对其他病原菌的抑制作用依次为苹果树腐烂病病原菌(74.1%)>苹果斑点落叶病病原菌(60.9%)>苹果灰霉病病原菌(46.7%)>苹果青霉病病原菌(44.5%)>苹果炭疽病病原菌(23.0%);发光杆菌HB140菌株发酵液对苹果灰霉病病原菌的抑制作用最好,抑制率达到86.7%,对其他病原菌的抑制作用依次为苹果树腐烂病病原菌(76.6%)>苹果轮纹病病原菌(58.8%)>苹果斑点落叶病病原菌(46.0%)>苹果青霉病病原菌(44.2%)>苹果炭疽病病原菌(39.9%)。2种共生菌发酵液对苹果轮纹病菌均有较强的抑制作用,其EC50分别为42.28和42.92 mL/L。     

不同环境下沙冬青属植物的蛋白质氨基酸变化

对 2种沙冬青叶和种子中蛋白质结合氨基酸及种子游离氨基酸的组成成分进行测定 ,其结果显示 :9种主要结合氨基酸含量之和占总氨基酸含量的 73.71%～ 76 .0 7% ,反映了属内种遗传的一致性。不同器官中的氨基酸组成含量不同 ,并随生存环境的改变而发生变化。总的趋势是 ,栽培条件下总结合氨基酸及大部分结合氨基酸含量高于野生状态 ;蒙古沙冬青(Ammopiptanthusmongolicus) 总结合氨基酸及大部分结合氨基酸含量稍高于新疆沙冬青 (A .nanus)。在吐鲁番沙漠植物园栽培条件下 ,沙冬青种子中结合氨基酸的含量大于叶中的含量 ;两种沙冬青叶中含有一定量的Cys(半胱氨酸 ) ,而种子中几乎不含Cys。总游离氨基酸的绝对含量范围在 2 .10～ 12 .5 1mg/g·dr.wt;均含有较高的Asp(天门冬氨酸 )、Glu(谷氨酸 )、Ser(丝氨酸 )和Arg(精氨酸 ) ,Met(蛋氨酸 )、Ile (异亮氨酸 )、Leu(亮氨酸 )和Gly(甘氨酸 )含量普遍较低。种间种子游离氨基酸组分含量差异明显 ,栽培条件下种子中主要的游离氨基酸含量有明显提高 ,其中Pro(脯氨酸 )和Asp含量分别是野生状态下的 2 .4和 4 .1倍。结合引种栽培区的气候环境因子分析 ,这些游离氨基酸含量的变化与植物受到一定程度的干旱胁迫有关联。     

中国昆虫病原线虫共生细菌的研究概况

Steinernema和Heierorhabditis属昆虫病原线虫作为生物防治因子已受到世界各国的广泛重视。这类线虫感染期幼虫肠腔中专化性地携带Xenorhabdus属共生细菌。近年来,明确了Xenorhabdus共生细菌对线虫大量培养的作用,筛选了廉价、方便的线虫人工培养基,从而导致了线虫的商业化生产。自五十年代,特别是七十年代末,我国从捷克斯洛伐克、澳大利亚、美国等地引进了大批Steinernema与Heterorhabditis线虫,同时也从土壤或自然寄主中分离出许多本国线虫种或品系(李丽英 1979;戴冠群 1984;王国汉 1984;刘杰,李丽英和王国汉     

水稻抗病性的研究Ⅳ、水稻品种耐肥性与对白叶枯病抗病性的关系

 用人工接种的方法测定水稻品种在不同氮肥水平下的抗病性变化,发现最感染白叶枯病的品种,发病程度一般都随氮肥水平的增加而显著提高;比较抵抗的品种,发病程度虽然亦有所增加,但是幅度要小得多。品种叶片的蛋白质氮的含量,都随着氮肥水平的提高而增加,但是其中游离氨基酸含量则根据品种抗病性的强弱而不同,感病品种一般都随着氮肥水平的提高而增加,而此较抗病的品种则在氮肥水平过高的情况下反而降低,或者始终保持在较低的水平。因此,水稻品种对氮肥水平的生理反应是不同的,其中游离氨基酸的变化可能在一定程度上反应了品种的耐肥性。水稻对胡麻斑病的反应,除品种农垦58以外,发病程度都是随着氮肥水平的提高而降低。与植株蛋白质氮量的高低呈反此,与游离氨基酸含量的变化无一定规律。     

Increased free amino acid contents of the invasive weed Ageratina adenophora and the effects on its specialist herbivore Procecidochares utilis

In order to verify the escape‐from‐enemy hypothesis from the changes of nutrient substance and fitness of natural enemies on alien plants, contents of free amino acids in native and invasive plant populations of Ageratina adenophora and life history parameters of specialist herbivore Procecidochares utilis reared on these plants were investigated. Our results showed that the contents of glycine, valine, γ‐aminobutyric acid, proline, serine, alanine, and arginine in the invasive plants were higher than those in the native plants of A. adenophora. There was a shorter developmental duration and higher fecundity of P. utilis when fed on the invasive plants. The results indicated a possible fitness tradeoff of natural enemies between invasive and native plants arose from nutrient substance changes.     

西瓜花叶病毒2号新疆昌吉分离物外壳蛋白基因核苷酸序列分析

 利用RT-PCR从新疆昌吉地区表现花叶、疱斑、扭曲等症状的南瓜病株上检测到西瓜花叶病毒2号新疆昌吉分离物(简称WMV-2-XJ-CJ),并测定了该分离物外壳蛋白(CP)基因序列。序列分析表明,新疆昌吉分离物CP基因全长850个核苷酸,编码197个氨基酸。与国内外报道的12个WMV-2CP基因相比,其核苷酸序列同源性为92.6%~98.3%,由此推导的氨基酸序列同源性为94.7%~99.3%。新疆昌吉分离物在CP N'端可变区明显不同于国内外报道的核苷酸序列。WMV-2新疆昌吉分离物与日本和郑州分离物较其它国家和地区的分离物多出6个核苷酸,但其核苷酸及其推导的氨基酸序列差异较大。新疆昌吉分离物外壳蛋白有2个氨基酸残基明显不同于其它分离物,其中蚜传株系的特征结构域DAG突变为DAE。     

Analysis of fatty acids in lipids of Verticillium dahliae and induction of lubimin accumulation in eggplant

The unsaturated fatty acid pattern and content of the main lipid fractions of three isolates of Verticillium dahliae were investigated, Oleic (C_18:1), linoleic (C_18:2) and linolenic (C_18:3) acids were the unsaturated fatty acids detected in the polar lipids and in the free fatty acids extracted from myceha of three isolates of the fungus. The concentration of the three fatty acids in polar lipids was about double that in free fatty acids, Linoleic acid was the predominant fatty acid in both lipid fractions. The capacity of linoleic and linolenic acids to induce lubimin accumulation in eggplant fruit discs was evaluated in comparison with that of the polyunsaturated fatty acid arachidonic acid, which is a known elicitor of phytoalexin synthesis in potato tuber. The accumulation of moderate levels of the phytoalexin was induced by arachidonic and linolenic acids, A higher induction was observed when browning of the tissue was inhibited by phenylthiourea. Under these conditions, linoleic acid induced only a slight accumulation of lubimin. The possible role of linolenic acid in triggering defence reactions in eggplant is discussed.     

一株PVYNTN-NW黑龙江马铃薯分离物的检测鉴定

 马铃薯Y病毒(Potato virus Y,PVY)是马铃薯、烟草等茄科作物上的重要病毒,在与寄主共同进化过程中产生了许多株系。本文从黑龙江马铃薯样品中得到PVY分离物A12。ELISA结果表明A12被PVY^O的单克隆抗体特异性识别。A12开放阅读框为9 186个核苷酸,编码3 061个氨基酸,与SYR-II-Be1分离物的核苷酸和氨基酸序列一致率均最高,分别为98.3%和99.2%。系统发育分析发现A12与PVY^NTN-NW株系SYR-II型的分离物聚类到一起;重组分析表明,A12是N-605和O_z的重组体,重组类型与SYR-II-Be1相同。综合以上结果表明,A12属于PVY^NTN-NW株系SYR-II型。但与常见PVY^NTN-NW株系分离物在珊西烟引起叶脉坏死不同,A12产生花叶症状。A12辅助成分-蛋白酶在182位和245位的氨基酸均为精氨酸,而其它PVY^NTN-NW株系分离物为赖氨酸。本研究结果可为黑龙江马铃薯PVY的早期检测和有效防控提供理论指导。     

一株PVYNTN-NW黑龙江马铃薯分离物的检测鉴定

 马铃薯Y病毒(Potato virus Y,PVY)是马铃薯、烟草等茄科作物上的重要病毒,在与寄主共同进化过程中产生了许多株系。本文从黑龙江马铃薯样品中得到PVY分离物A12。ELISA结果表明A12被PVY^O的单克隆抗体特异性识别。A12开放阅读框为9 186个核苷酸,编码3 061个氨基酸,与SYR-II-Be1分离物的核苷酸和氨基酸序列一致率均最高,分别为98.3%和99.2%。系统发育分析发现A12与PVY^NTN-NW株系SYR-II型的分离物聚类到一起;重组分析表明,A12是N-605和O_z的重组体,重组类型与SYR-II-Be1相同。综合以上结果表明,A12属于PVY^NTN-NW株系SYR-II型。但与常见PVY^NTN-NW株系分离物在珊西烟引起叶脉坏死不同,A12产生花叶症状。A12辅助成分-蛋白酶在182位和245位的氨基酸均为精氨酸,而其它PVY^NTN-NW株系分离物为赖氨酸。本研究结果可为黑龙江马铃薯PVY的早期检测和有效防控提供理论指导。     

引起玉米粗缩病的水稻黑条矮缩病毒河南分离物的分子特征

<正>目前已报道的造成玉米粗缩病的病原有3种,分别是玉米粗缩病毒(Maize rough dwarf virus,M RDV),水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)和南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)[1]。我国于1954年在新疆和甘肃发现该病,上世纪60年代曾在中东部夏玉米区流行,至70年代以来各玉米产区已陆续发生[2],近年来在黄淮海夏玉米区特别是套播或晚春播、早夏播玉米田发生危害严重。     

Molecular characterization of the CP gene and 3'UTR of Chilli veinal mottle virus from South and Southeast Asia

Twenty-four isolates of Chilli veinal mottle virus (ChiVMV) from China, India, Indonesia, Taiwan and Thailand were analysed to determine their genetic relatedness. Pathogenicity of virus isolates was confirmed by induction of systemic mosaic and/or necrotic ringspot symptoms on Capsicum annuum after mechanical inoculation. The 3' terminal sequences of the viral genomic RNA were determined. The coat protein (CP) coding regions ranged from 858 to 864 nucleotides and the 3' untranslated regions (3'UTR) from 275 to 289 nucleotides in length. All isolates had the inverted repeat sequence GUGGNNNCCAC in the 3'UTR. The DAG motif, conserved in aphid-transmitted potyviruses, was observed in all isolates. All 24 isolates were considered as belonging to ChiVMV because of their high CP amino acid and nucleotide identity (more than 94·8 and 89·5%, respectively) with the reported ChiVMV isolates including the pepper vein banding virus (PVBV), the chilli vein-banding mottle virus (CVbMV) and the CVbMV Chiengmai isolate (CVbMV-CM1). Based on phylogenetic analysis, ChiVMV isolates including all 24 isolates tested, PVBV, CVbMV and CVbMV-CM1 can be classified into three groups. In addition, a conserved region of 204 amino acids with more than 90·2% identity was identified in the C terminal of the CP gene of ChiVMV and Pepper veinal mottle virus (PVMV), and may explain the serological cross reaction between these two viruses. The conserved region may also provide useful information for developing transgenic resistance to both ChiVMV and PVMV.     

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.     

Selection and Screening of Endorhizosphere Bacteria from Solarized Soils as Biocontrol Agents Against Verticillium dahliae of Solanaceous Hosts

Verticillium dahliae antagonistic endorhizosphere bacteria were selected from root tips of tomato plants grown in solarized soils. Fifty-three out of the 435 selected bacterial isolates were found to be antagonistic against V. dahliae and several other soilborne pathogens in dual cultures. Significant biocontrol activity against V. dahliae in glasshouse trials was demonstrated in three of 18 evaluated antagonistic isolates, provisionally identified as Bacillus sp. Although fluorescent pseudomonads were also isolated from root tips of tomato plants, none of the tested isolates exercised any significant antagonistic activity against V. dahliae in dual cultures. So these isolates were not tested in glasshouse trials in this study. Finally, two of the most effective bacterial isolates, designated as K-165 and 5-127, were shown to be rhizosphere colonizers, very efficient in inhibiting mycelial growth of V. dahliae in dual cultures and successfully controlling Verticillium wilt of solanaceous hosts. In glasshouse experiments, root dipping or soil drenching of eggplants with bacterial suspension of 10⁷cfu ml^–1 resulted in reduced disease severity expressed as percentage of diseased leaves (40–70%) compared to the untreated controls under high V. dahliae inoculum level (40 microsclerotia g^–1 soil). In heavily Verticillium infested potato fields, experiments with potato seeds dusted with a bacterial talc formulation (10⁸cfu g^–1 formulation), showed a significant reduction in symptom development expressed as percentage of diseased potato plants and a 25% increase in yield over the untreated controls. As for their effectiveness in increasing plant height, both bacterial isolates K-165 and 5-127 produced indolebutyric, indolepyruvic and indole propionic acids. Both antagonists are considered as plant growth promoting rhizobacteria bacteria since significantly increased the height of treated plants compared with the untreated controls. Chitinolytic activity test showed that both isolates were able to produce chitinase. Testing rhizospheric and endophytic activity of the antagonists it was shown that although the bacteria are rhizosphere inhabitants they also preferentially colonize the endorhizosphere of tomatoes and eggplants. Fatty acid analysis showed that isolate K-165 could belong to Paenibacillus alvei while 5-127 to Bacillus amiloliquefaciens.