荧光假单胞菌CZ菌株定殖及抗病毒活性研究

荧光假单胞菌Pseudomonas fluorescens CZ能分泌包括碱性磷酸酶在内的抗病毒蛋白抑制烟草花叶病毒Tobacco mosaic virus(TMV)侵染?本文利用抗生素利福平逐级诱导获得抗药性标记菌株CZ-rif, 生测试验显示:野生型和抗药型菌株的发酵上清和粗蛋白液对TMV的体外钝化效果均大于90%, 无显著差异?灭菌和非灭菌的根际土拌CZ-rif菌液后盆栽烟苗, 取根际土在含抗生素的平板上检测定殖菌量, 结果显示, CZ-rif能够在烟株根际土壤中有效定殖, 第31天在灭菌土中的定殖菌量为4.3×104 cfu/g, 显著大于在非灭菌土中的定殖菌量6×103 cfu/g, 在叶面上的定殖菌量显著低于根际土壤?叶面喷施CZ-rif菌液后24 h接种病毒, 对TMV-GFP的预防效果为45.85%?田间试验中喷淋菌株发酵稀释液, 对烟草花叶病毒病的防效为42.02%?此外, 水培试验显示生防菌发酵液对烟草幼苗具有促生作用?总之, 荧光假单胞菌CZ能在烟草根际定殖和促进植株生长, 叶面喷施能钝化TMV并抑制其初侵染, 可以研发防治病毒病害的生物制剂?     

施用外源有机酸对早期烟草青枯病菌的影响

在常年青枯病发生较为严重的彭水县润溪乡,于烟草移栽期、小培土期、团棵期施外源有机酸,通过平板稀释计数法,测定根际土壤青枯菌数量,并调查烟草青枯病的发病情况和烟株前期的生长情况。试验结果表明,在烟草早期的生长过程中,有机酸能够显著刺激青枯菌的定殖与侵染,并且对烟草生长的作用影响不明显,且能加重烟草青枯病的发生。因此,在实际应用中,应注重田间有机酸的调控,要采取相应的措施避免有机酸的积累。     

健康与感染青枯病烟株根际土壤与茎秆真菌群落结构与多样性

为研究感染青枯病后烟株根际土壤与茎秆真菌群落结构与多样性的变化,对健康和感染青枯病烟株的根际土壤、病株茎秆发病组织和健株茎秆健康组织等样品中真菌ITS区的rDNA进行了PCR扩增、用Illumina MiSeq测序技术对扩增DNA片段进行高通量测序,并分析不同样品的真菌群落组成与多样性。结果表明,所有烟株根际土壤中优势门为子囊菌门Ascomycota和接合菌门Zygomycota;所有茎秆样品中优势门为担子菌门Basidiomycota和子囊菌门。在属水平,被孢霉属Mortierella、镰刀菌属Fusarium和隐球菌属Cryptococcus为所有土壤中的主要菌属,Boeremia主要存在于发病烟株根际土壤中,而木霉属Trichoderma主要存在于健康烟株根际土壤。发病茎秆病害组织中优势属为小画线壳属Monographella、隐球菌属、鬼伞属Coprinopsis和赤霉属Gibberella;发病茎秆病健交界处组织中优势属为隐球菌属、红酵母属Rhodotorula和小画线壳属。健康烟株茎秆组织中优势属为隐球菌属、链格孢属Alternaria和红酵母属;健康烟株中与发病茎秆病健交界处组织等高茎秆中优势属为镰刀菌属、隐球菌属、链格孢属和Gibellulopsis。青枯菌侵染烟株后根际土壤、发病茎秆病害组织和发病茎秆病健交界处组织的真菌群落中物种丰富度与多样性均显著提高,且发病茎秆病害组织与发病茎秆病健交界处组织真菌群落的变化大于根际土壤。研究结果为烟草青枯病的生物防治提供了参考。     

生防菌EN5的定殖能力及其对根际土壤微生物类群的影响

［目的］ 明确生防菌EN5菌株在番茄根、茎及根际土壤中和在黄瓜、烟草体内的定殖情况,及对根际土壤微生态的影响,为细菌性青枯病的生态治理提供理论依据。［方法］ 采用抗生素抗性标记法测定生防芽胞杆菌EN5的定殖能力;以平板培养及ERIC PCR法分析菌株EN5对番茄根际土壤微生物种群的影响。［结果］ 菌株EN5在番茄根、茎及根际土壤中均能稳定定殖,当菌株EN5处理植株15 d时,其在番茄茎内的定殖量可达5.6×104 cfu/g;此外,菌株EN5在烟草和黄瓜体内亦可定殖,其在烟草体内的定殖量较大。菌株EN5处理使根际土壤中细菌、真菌和放线菌的数量明显高于对照土壤,对氨化细菌、固氮菌、纤维素降解菌等微生物群体有促进作用,对反硫化细菌群体有抑制作用。同时,菌株EN5处理还改善了根际土壤中细菌群体的多样性。［结论］ 菌株EN5可以在其自然寄主番茄的根、茎及根际土壤中稳定定殖。同时,菌株EN5处理有助于改善根际土壤微生态环境,抑制病菌繁殖。     

育苗基质中添加不同微生物菌剂对烟草抗病性及产质量的影响

土传病害给烟草的产质量造成严重损失,而在大田施用生物菌剂进行生物防治往往因生防菌剂在土壤中适应增殖困难而效果不理想.本研究采用基质拌菌的方法,选取4种常用拮抗微生物菌剂(枯草芽孢杆菌菌剂、荧光假单胞杆菌菌剂、解淀粉芽孢杆菌菌剂和多粘类芽孢杆菌菌剂),探究不同微生物菌剂早期根际定殖对烟草青枯病的防控效果及对烟株农艺性状和产量的影响.结果表明:在育苗基质中加入微生物菌剂可以优化烟草苗期和团棵期农艺性状,提高烟草对青枯病的抗病性,其中以培育1 000株烟苗的育苗基质中加入多粘类芽孢杆菌500g(有效芽孢量5×10~3亿cfu)效果最好.本试验结果为育苗基质拌菌技术在烟草促生和抗病方面提供理论支撑.     

拮抗青枯劳尔氏菌的荧光假单胞菌SN15-2分离鉴定及其生防能力分析

为了筛选出对番茄青枯病具有较好防效的生防菌,采用皿内测定法从上海地区的番茄青枯病自然衰退土壤中,分离到一株对番茄青枯病有很强抑制作用的菌株SN15-2,并进行了分子鉴定和对荧光假单胞菌SN15-2产抗生素能力和定殖能力测定。结果表明,菌株SN15-2为荧光假单胞菌(Pseudomonas fluorescens)。其菌株能产生2,4-二乙酰基间苯三酚(2,4-DAPG)、硝吡咯菌素(PRN)、藤黄绿脓菌素(PLT)。同时,可以产生HCN、噬铁素,能够形成生物膜。SN15-2在施入番茄根际后的前20 d定殖数量减少,20 d后基本稳定,60 d时,在干土中的定殖数量可达3.67×105cfu/g。盆栽防效分析表明,荧光假单胞菌SN15-2对番茄青枯病防效达到46.58%。     

荧光素酶标记无致病力青枯雷尔氏菌的生物学和定殖特性

为示踪青枯雷尔氏菌Ralstonia solanacearum无致病力菌株FJAT1458在番茄植株及根际土壤中的定殖特性,采用电击法对菌株FJAT1458进行荧光素酶(luciferase,LUC)基因标记,并于室内采用生测法测定标记菌株的生物学特性、遗传稳定性、致病力及在番茄植株和根际土壤中的定殖能力。结果表明,成功将luc基因整合至无致病力菌株FJAT1458染色体上,标记菌株FJAT1458-LUC发出强烈的荧光,且PCR扩增出1 612 bp的luc基因片段;与野生型菌株FJAT1458相比,标记菌株FJAT1458-LUC的生长明显滞后,培养8h之后标记菌株FJAT1458-LUC的OD_600nm值均小于野生型菌株FJAT1458;且标记菌株FJAT1458-LUC连续传代20次后菌体浓度显著增加,发光菌体比例显著降低,LUC活性和luc基因表达量均随着传代数增加而显著降低;标记前、后菌株FJAT1458的弱化指数分别为0.90和0.89,且接种番茄植株30 d均未引起植株发病。标记菌株FJAT1458-LUC能在番茄根际土壤、根及茎中定殖,定殖数量呈...     

假茄科雷尔氏菌引起向日葵青枯病在中国的首次报道

茄科雷尔氏菌复合种侵染引起的青枯病是众多作物上的毁灭性病害。2020年笔者首次在广东省东莞市发现向日葵青枯病,并对其病原菌进行了鉴定。室内人工接种试验、16S rDNA序列比对和演化型鉴定结果表明,引起向日葵青枯病的病原菌为假茄科雷尔氏菌Ralstonia pseudosolanacearum。生理生化特性和致病性鉴定结果表明,分离自向日葵的15株假茄科雷尔氏菌为1号生理小种和生化变种3。egl基因部分序列系统进化分析表明,15株假茄科雷尔氏菌分属4个序列变种,其中8株菌株为序列变种17,5株菌株为序列变种13,其余2株菌株分别为序列变种14和序列变种54。本文是我国首次报道假茄科雷尔氏菌侵染引起向日葵青枯病。     

五种药剂对烟草青枯病菌的抑制活性及碳代谢的影响

由茄科劳尔氏菌Ralstonia solanacearum引起的烟草青枯病是烟叶生产上的重要病害之一，严重影响烟叶的产量和品质。为筛选高效防治烟草青枯病的药剂，本研究采用平板菌落计数法测定了中生菌素、土霉素、噻霉酮、春雷霉素和氯溴异氰尿酸对茄科劳尔氏菌的抑制活性，并通过Biolog GEN Ⅲ微平板法分析了上述5种药剂胁迫下茄科劳尔氏菌的碳代谢情况和对化学物质的敏感性差异。结果表明：5种药剂均能抑制茄科劳尔氏菌的生长，抑制活性从强到弱依次为中生菌素>土霉素>噻霉酮>春雷霉素>氯溴异氰尿酸，对应的EC50值分别为0.24、0.74、2.84、7.95和273.99 mg/L。Biolog GEN Ⅲ碳代谢测定结果显示：茄科劳尔氏菌能利用Biolog GEN Ⅲ微平板中糖类及氨基酸类等71种碳源，但在药剂胁迫下，该菌对碳源的代谢受到了不同程度的抑制，其中抑制程度最显著的是氨基酸、己糖酸、羧酸、酯和脂肪酸类碳水化合物；随着5种药剂质量浓度增加，在6 mg/L中生菌素、8 mg/L土霉素、30 mg/L春雷霉素和5738 mg/L氯溴异氰尿酸胁迫下，茄科劳尔氏菌对Bio...     

用gfp基因标记法研究金龟子绿僵菌在玉米根际的定殖动态

利用草胺磷抗性基因bar为筛选标记,将绿色荧光蛋白基因（gfp）转入载体pbarGPE1,得到了组成性表达载体pGPE-GFP,并转入金龟子绿僵菌RCEF3377,获成功表达。用含有gfp基因的金龟子绿僵菌孢子粉配制种衣剂,对玉米种子包衣。于玉米苗期对根际定殖情况进行定期取样观测。结果表明,与非根际土壤相比,在根际土壤中被标记的绿僵菌带菌量不仅最大,而且在播种后10d数量达最大,20～30d内数量即不再显著变动。相比之下,非根际土壤中的带菌量不仅极显著少于根际土壤,且随时间的延长明显降低;在表层土中的带菌量很低,亦随时间的延长显著降低。表明应用该种衣剂剂型,金龟子绿僵菌孢子在玉米根际有较高活力和定殖效果。     

Tachinid parasitoids (Diptera: Tachinidae) of<Emphasis Type="Italic">Spodoptera exigua</Emphasis> in cotton fields in Diyarbakır,Turkey

Tachinid parasitoidsExorista xanthaspis (Wiedemann),Nemorilla maculosa (Meigen),Palesisa maculosa (Villeneuve) andDrino imberbis (Wiedemann) were obtained fromSpodoptera exigua (Hübner) (Noctuidae) larvae collected from cotton fields in Turkey.S. exigua is a new host record forN. maculosa, andP. maculosa is recorded from Turkey for the first time. http://www.phytoparasitica.org posting July 14, 2004.     

感染马铃薯Y病毒烟株对烟蚜取食行为及烟蚜茧蜂适应性的影响

为探明烟蚜茧蜂Aphidius gifuensis Ashmead对烟蚜Myzus persicae(Sulzer)取食感染马铃薯Y病毒(Potato virus Y,PVY)烟株的适应性,利用刺探电位图谱(EPG)技术记录了烟蚜在感病烟株与健康烟株上的取食行为,并测定了烟蚜茧蜂对取食2种烟株烟蚜的寄生率以及烟蚜茧蜂的羽化率、发育时间及性比等指标。结果表明,与健康烟株相比,烟蚜在感染PVY烟株上的第1次刺探取食持续时间显著延长,且口针遇到阻力的次数(F波)和总持续时间均显著减少。烟蚜在感病烟株木质部的吸食时间(G波)显著长于健康烟株。感病烟株上烟蚜在韧皮部阶段的分泌唾液时间(E1波)较在健康烟株上显著缩短,而被动吸食汁液时间(E2波)显著延长。烟蚜茧蜂寄生取食感染PVY烟株的烟蚜,虽然能成功完成其生活史,但适应性与寄生取食健康烟株烟蚜的蚜茧蜂存在差异。在感染PVY烟株上,烟蚜茧蜂对烟蚜的寄生率为33.67%,显著低于对照的64.67%;且僵蚜体重明显下降,羽化的成蜂个体较小;成蜂存活时间1.48 d也极显著短于对照组的2.25 d。表明烟蚜茧蜂对取食感染PVY烟株烟蚜的适应性较低,PVY可通过烟蚜为介体间接降低烟蚜茧蜂的适应力。     

油菜菌核病抗(耐)病性筛选方法的研究

本文评价了不同接种鉴定方法在油菜菌核病抗(耐)病性筛选上的应用。结果表明:花期离体或活体叶片菌丝体接种和限期接种法能显著区分不同品种(系)的抗感水平;苗期菌丝体悬浮液接种和试管苗接种法适于早期育种世代进行大规模筛选。对83份品种(系)鉴定结果,821、083等呈轻度感病,大部分属中感至高感范围。     

Complementary host–pathogen genetic analyses of the role of fumonisins in the Zea mays–Gibberella moniliformis interaction

Zea mays often is colonized with the fungus Gibberella moniliformis, which produces fumonisin toxins. The role of fumonisins in seedling colonization and blight was studied using complementary genetic analyses of host and pathogen. Only one of two fumonisin B1 (FB1)-insensitive maize backcross lines was more resistant than the FB1-sensitive parent to seedling blight, indicating that the increase in FB1-insensitivity was not associated with an increase in resistance. FB1-producing and nonproducing isogenic fungal strains did not differ in ability to cause seedling blight, but the FB1-producing strain was more effective in systemic colonization of seedlings in reciprocal strain challenge tests. Together, these and previous results indicate that the role of fumonisins depends on complex environmental and genetic contexts in this host–pathogen interaction.     

Contribution to the taxonomy and pathogenicity of fungicolous Verticillium species. I. Taxonomy

The genusVerticillium is divided into four sections (two new) and a residual group. The new sectionNigrescentia comprises the well-known plant-pathogenic and some other saprophytic species with dark resting structures. The new sectionAlbo-erecta is characterized by white (or yellowish) colonies and erect conidiophores and contains mainly fungicolous species. Seven (two new) species and two new varieties are described and keyed out. Three other species have known teleomorphs ofNectriopsis (one new and two new combinations). This genus had been merged withNectria by Samuels, but its retention is justified, as the original and the here described fungicolous (and myxomyceticolous) species are more suitably accomodated inNectriopsis than inHypomyces.The common causal agent of dry bubble inAgaricus bisporus, Verticillium fungicola var.fungicola, is redescribed and defined more narrowly than by Gams (1971) by its maximum growth temperature below 27°C. A similar fungus with a maximum growth temperature near 33°C, causing brown spots inA. bitorquis, is described asV. fungicola var.aleophilum. Isolates from wild agarics with a strongly reduced growth at 24°C and a maximum below 27°C, a yellowish mycelium and inconspicuous sclerotia, are described asV. fungicola var.flavidum. V. biguttatum W. Gams,sp. nov., with cylindrical biguttulate conidia, is a common soil fungus and hyperparasite ofRhizoctonia solani. Gliocladium microspermum (Sacc.) W. Gams,comb. nov., the anamorph ofNectriopsis broomeana (Tul.) W. Gams,comb. nov., which is intermediate betweenGliocladium andVerticillium, is included because of its fungicolous habit.Samenvatting Het geslachtVerticillium wordt onderverdeeld in vier secties (waaronder twee nieuwe) en een restgroep. De nieuwe sectieNigrescentia bevat onder meer de bekende ziekteverwekkende soorten met donkere ruststructuren. De nieuwe sectieAlbo-erecta wordt gekarakteriseerd door witte (of geelachtige) kolonies en rechtopstaande conidioforen en bevat hoofdzakelijk op andere fungi groeiende soorten. Zeven soorten (waaronder twee nieuwe) en twee nieuwe variëteiten worden beschreven en in een sleutel verwerkt. Drie andere soorten bezitten perfecte vormen vanNectriopsis (waaronder één nieuwe en twee nieuwe combinaties). Dit geslacht werd door Samuels metNectria samengevoegd, maar kan gehandhaafd worden en lijkt beter geschikt om de hier beschreven ascomyceten onder te brengen danHypomyces. Verticillium fungicola var.fungicola, de algemene veroorzaker van droge mollen bij de champignon (Agaricus bisporus), wordt beschreven en met zijn temperatuur-maximum beneden 27°C nauwkeuriger gedefiniëerd dan in een vroegere studie (Gams, 1971). Een vergelijkbare schimmel met een temperatuurmaximum van ongeveer 33°C, die bijA. bitorquis bruine vlekken veroorzaakt, wordt beschreven alsV. fungicola var.aleophilum. Isolaten van wilde paddestoelen met een optimum duidelijk beneden 24°C en een maximum beneden 27°C, geelachtig mycelium en onopvallende sclerotiën, worden beschreven alsV. fungicola var.flavidum. V. biguttatum W. Gams, sp. nov., met cilindrische, biguttate conidiën, is een algemene grondschimmel en hyperparasiet vanRhizoctonia solani. Gliocladium microspermum (Sacc.) W. Gams, comb. nov., de imperfecte vorm vanNectriopsis broomeana (Tul.) W. Gams, comb. nov., een tussenvorm tussenGliocladium enVerticillium, wordt opgenomen wegens zijn voorkomen op vruchtlichamen van de dennemoorder.     

Epigallocatechin gallate,a major tea catechin,induces biofilm formation of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Plants constitutively produce a variety of secondary metabolites that have antimicrobial activities against phytopathogens; however, interactions between these performed antimicrobial compounds and phytopathogens were poorly understood. In this study, interactions between epigallocatechin gallate (EGCg), which was a major tea catechin that had antimicrobial activities against varieties of bacteria, and Pseudomonas syringae pv. theae (P.s. theae), the causal of bacterial shoot blight of tea, were investigated. EGCg had less antimicrobial activity against P.s. theae; however, subinhibitory concentrations of EGCg induced biofilm formation. Because biofilms are induced in the presence of sucrose in the culture medium but not by P.s. theae strains deficient in exopolysaccharide levan production, biofilm induction by EGCg and levan production are closely related. EGCg increased survival of P.s. theae under dry conditions on nonwounded leaf surfaces in the presence of sucrose. These data indicate the possibility that tea catechins affect the survival of P.s. theae on the phyllosphere. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bacterial diseases of potatoes: from classical phytobacteriology to molecular pathogenicity

Various bacterial pathogens attack potato plants and tubers. These pathogens includeErwinia, Corynebacterium (Clavibacter), Psuedomonas andStreptomyces. Over the past few years there have been significant advances in the molecular biological analysis of several of these pathogens and this is now helping us to understand the major aspects of virulence mechanisms. However, to date, such information is not sufficiently useful to allow us to intervene rationally in these potato diseases, except by the standard practises of good husbandry.     

黄瓜花叶病毒和马铃薯Y病毒混合侵染烟株对烟蚜取食行为的影响

为探究黄瓜花叶病毒(Cucumber mosaic virus,CMV)、马铃薯Y病毒(Potato virus Y,PVY)混合侵染烟株对烟蚜取食行为的影响,利用刺探电位图谱(electrical penetration graph,EPG)技术记录了烟蚜在健康烟株与CMV、PVY混合侵染后不同发病级别烟株上的取食波形。结果显示:烟蚜在健康烟株上的刺探次数最少,在感病烟株上的C波总持续时间均显著长于健康烟株;第1次到达韧皮部前的刺探次数,健康植株上仅为4.00次,3级感病烟株上的为健康烟株上的2倍;在健康烟株上E2波总持续时间为120.65 min,极显著大于2级和3级感病烟株;刺探过程中,感病烟株上的pd波出现次数均高于健康烟株,且pd波II-1和II-3亚波的持续时间也显著高于健康烟株。研究表明,CMV、PVY混合侵染烟株可降低寄主对烟蚜的适合度,且能促进烟蚜对病毒的传播。     

Biofilm formation and resistance to bactericides of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">theae</Emphasis>

Biofilm-grown cells of Pseudomonas syringae pv. theae (P.s.theae) wild-type strain K9301 on abiotic surface had remarkable resistance to kasugamycin in comparison to planktonically grown cells; however, the biofilm-grown cells of K9301 had the same sensitivity to copper sulfate. Because both the lesser biofilm-forming strain K9301S3 and enhanced biofilm-forming strain K9301-6 also had remarkable biofilm resistance to kasugamycin just as K9301 did and because epigallocatechin gallate, which enhanced biofilm formation of P.s.theae, had no effect on biofilm resistance to kasugamaycin, the degree of biofilm formation was not correlated with the antibiotic susceptibilities. In addition, K9301 and K9301S3 had less sensitivity to kasugamycin but had high sensitivity to copper sulfate on nonwounded leaf surfaces. These results indicate a possibility that the mechanism of P.s.theae biofilm resistance to bactericide functions on both abiotic and nonwounded leaf surfaces.     

Grapevine crown gall caused by <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> (Ti) in Japan

In 2005, characteristic symptoms of crown gall on grapevines (Vitis vinifera L. cv. Muscat of Alexandria, and cv. Seto Giants) were observed in a commercial greenhouse-orchard in Okayama Prefecture, Japan. Isolations from diseased tissues consistently yielded bacterial colonies that were white, glistening, and produced abundant polysaccharide on potato semi-synthetic agar (PSA) medium. Ten representative isolates were chosen for further characterization. A multiplex polymerase chain reaction (PCR) assay showed these strains were not Rhizobium vitis but did possess a Ti plasmid. The bacteriological characteristics of the isolates corresponded well with R. radiobacter. The almost complete 16S ribosomal DNA sequences of isolates AT06-1 and AT06-2, selected from 10 grapevine isolates, were determined and corresponded to sequences of R. radiobacter. The pathogenicity of the isolates was tested on young grapevine and tomato (Lycopersicon esculentum Mill.) plants. Gall symptoms developed on both plant species after inoculation, and bacteria with the same colony morphology as those inoculated were reisolated. Based on these results, the isolates were identified as R. radiobacter (Ti). This report is the first of the occurrence of R. radiobacter (Ti) on grapevine in Japan. Phylogenetic analyses using the partial nucleotide sequences of virC operon located on a Ti plasmid showed that the isolate of R. radiobacter (Ti) isolated from grapevine and some strains of R. vitis (Ti) belonged to the same monophyletic group, which differed from the groups of R. radiobacter (Ti) isolated from plants other than grapevine and of the majority of R. vitis (Ti) strains isolated from grapevine. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accessions AB306890, AB306891, and AB465432–AB465459.