一种新的棉花黄萎病快速接种技术及其在病原菌致病力和寄主抗病性鉴定上的应用

 棉花品种抗黄萎病鉴定一般在田间病圃中进行,其结果受病圃中病原菌分布均匀程度、气象等因素影响极大,往往导致鉴定结果不准确。为了使鉴定方法简单、科学、可靠,我们在温室条件下比较了3种苗期接种棉花黄萎病的方法,即切根蘸孢子法(接种浓度为10⁶分生孢子/mL);菌培养物土壤接种法(0.5%、1%、2%,w/w);微菌核土壤接种法(10³个微菌核/g土)。结果表明,切根蘸孢子法导致棉苗发病均匀、严重、迅速,播种35~45 d后即可得到均匀一致的发病结果。而其它2种接种方法在播种75 d后才得到相对稳定的发病结果。同时,研究还表明接种浓度为10⁴分生孢子/mL所导致的黄萎病显著比10⁵或10⁶的轻。利用切根蘸孢子法在室内鉴定12个棉花品种或品系的抗黄萎病能力,证明该方法是抗黄萎病快速鉴定的有效方法。此外,该鉴定方法还可快速鉴定黄萎病菌不同菌株的致病性,并可应用于作物对其它土传病害的抗病性鉴定上。     

分离土壤棉花黄萎病菌选择性培养基的筛选

 采用土壤检测稀释法、水筛法和不同土样接种量,对5种土壤浸提液改良培养基进行了比较研究。结果表明,土壤水筛法对土壤棉花黄萎病菌的检测效果优于稀释法,其菌落检测数增加4~10倍。培养基每皿0.25 g土样接种量的检测效果高于0.5 g和1g。5种测试培养基中,培养基D对分离土壤棉花黄萎病菌的选择性表现最好,其土壤接种体检测回收率达63.5%~83.3%.培养基D的组份为:土壤浸提液25 ml,KH₂PO₄ 1.5 g,K₂HPO₄ 4 g,尿酸钠0.2 g,半乳糖醛酸钠(果胶)1g,山梨糖1g,Dox盐2 ml,Tergitol NP-101ml,PCNB0.1g,琼脂17g,蒸馏水1000 ml,pH6.4~6.7。每1000ml融化培养基倒皿前加抗菌素液50 ml (含氯霉素0.05 g,链霉素0.05 g,青霉素-G盐0.05 g)。     

Specific Detection of Tomato Pathotype of Verticillium dahliae by PCR Assays

Verticillium dahliae Klebahn is the causal agent of tomato wilt disease. Isolates of V. dahliae can be classified based on pathogenicity to tomato, but the pathotypes are indistinguishable in morphology. We designed PCR primers for specific detection of isolates pathogenic to tomato (tomato pathotype) from the sequences of a pathotype-specific gene, vdt1. With the primer pair Tg5/Tc3, a PCR product (approximately 3.2 kb) specific to tomato pathotype was amplified from the genomic DNA of isolates. Using the primer pair, a tomato pathotype isolate was specifically detected from hypocotyls of inoculated tomato and eggplant. On the other hand, no amplification was observed from non-tomato pathotype isolates of V. dahliae, some other wilt pathogens of tomato and a healthy host plant. Therefore, the primer pair can be useful for pathotype-specific detection of V. dahliae as well as for diagnosis of wilt disease of tomato plant. Received 7 September 2001/ Accepted in revised form 3 December 2001     

Effects of granular nematicides on growth and microbial antagonism to Rhizoctonia solani

Effects of nematicides on growth and microbial antagonism toRhizoctonia solani were investigated as part of a study on the mechanisms involved in the increased incidence of this pathogen in nematicide-treated potato crops.Ethoprophos inhibited mycelial growth ofR. solani on potato dextrose agar (PDA), Czapek Dox agar (CDA) and on water agar (WA). Aldicarb stimulated its growth on PDA up to 14% but not on CDA and WA. Oxamyl inhibited mycelial growth on CDA and WA, but not on PDA.Ethoprophos and aldicarb stimulated development of the mycoparasiteVerticillium biguttatum on cultures ofR. solani. The effect was dependent on the medium on which the host fungus was grown. ForRhizoctonia cultures on PDA, growth of the mycoparasite was highly promoted by aldicarb and to a lesser extent by ethoprophos. WhenR. solani was grown on CDA, the development of the mycoparasite was not affected by aldicarb, slightly stimulated by ethoprophos and slightly inhibited by oxamyl. On water agar, its development on the host mycelium was not affected.In field trials on sandy soil, nematicides encouragedV. biguttatum probably by increased availability of substrate (i.e.Rhizoctonia mycelium) perhaps through reduced activity of the mycophagous fauna.Soil fungistasis was increased by ethoprophos and to a lesser extent by aldicarb at very high doses. At normal field rates, no effects can be expected on fungistasis. So the increased stem and stolon infection of potatoes in nematicide-treated fields was not caused by a direct effect of the nematicides on growth ofR. solani or by suppressing the microbial antagonism.Samenvatting De invloed van granulaire nematiciden op de groei vanRhizoctonia solani en op het microbiële antagonisme tegen deze schimmel werd onderzocht in het kader van een studie over de mechanismen die een rol spelen bij de toename van de aantasting in een met nematiciden behandeld aardappelgewas.Ethoprofos remde de myceliumgroei vanR. solani op aardappeldextrose agar (PDA), Czapek Dox agar (CDA) en op wateragar (WA). Aldicarb stimuleerde op PDA de groei met maximaal 14%. Op CDA en WA werd geen effect van aldicarb waargenomen. Oxamyl veroorzaakte groeiremming op CDA en WA, maar niet op PDA.Ethoprofos en aldicarb stimuleerden de ontwikkeling van de mycoparasietVerticillium biguttatum op cultures vanR. solani. De mate van groeistimulering was afhankelijk van de voedingsbodem waarop de waard,R. solani, werd gekweekt. De groei vanV. biguttatum werd sterk gestimuleerd door aldicarb en in geringere mate door ethoprofos, wanneer de waard gekweekt werd op PDA. Aldicarb had geen effect op de mycoparasiet wanneerR. solani op CDA gekweekt werd, terwijl ethoprofos de groei wel iets stimuleerde en oxamyl een gering remmend effect had. Op WA werd geen effect van de nematiciden op het mycoparasitisme vastgesteld.In veldproeven op zandgrond stimuleerden de nematiciden het voorkomen vanV. biguttatum op de stolonen. Het effect werd waarschijnlijk veroorzaakt door een verhoogde substraat beschikbaarheid (d.w.z. mycelium vanR. solani). De verhoogde beschikbaarheid van dit mycelium kan samenhangen met een door nematiciden gereduceerde activiteit van de fungivore bodemfauna.De bodemfungistase werd verhoogd door ethoprofos en, in geringere mate, door aldicarb bij hogere doseringen. Bij de in de praktijk aanbevolen doseringen kan echter geen effect op de fungistase verwacht worden. De toename in stengel- en stolonaantasting van aardappelen, geteeld in met granulaire nematiciden behandelde percelen, kon niet worden toegeschreven aan een direct effect van de nematiciden op de groei vanR. solani of aan een vermindering van het microbiële antagonisme.     

Effect of crop rotation on the incidence of soil-borne fungal diseases of potato

Effects of crop rotation on the incidence of soil-borne pathogens and on the performance of potato were investigated in five field experiments. Rotations differed in cropping frequency of potato and in crop sequence.Incidence of stem canker caused byRhizoctonia solani was strongly influenced by the cropping frequency of potato and not by crops with which the potato was alternated in the rotation. Cropping frequency of potato also affected the occurrence of black scurf, but less pronounced than for stem canker. The antagonistVerticillium bigutatum slightly reducedR. solani (black scurf) in plots on sandy soil continuously cropped with potato. Incidence of stem canker was also strongly affected by granular nematicides applied to the soil, nitrogen level and the cultivar grown.     

土传病害茄子大丽轮枝菌致病类型的研究

将天津市５个地区采集的２０个茄子黄萎病致病菌——大丽轮枝菌菌株，在６个茄子品种上做致病类型的测定。结果表明，各菌株的致病力可分为强、中、弱３个类型；明确了天津市３个茄子主栽品种的抗感反应型：快圆茄为感病型，大苠茄为中感型，二苠茄为耐病型     

大丽轮枝菌分泌蛋白提取方法比较

【目的】采用超滤法（UF）、三氯乙酸沉淀法（TCA）、离子交换富集法（IEX）分别从大丽轮枝菌（Verticillium dahliae）高致病性菌株VdG1培养上清液中提取分泌蛋白,对提取样品进行质谱鉴定。寻找适合大丽轮枝菌分泌蛋白的提取方法,为大丽轮枝菌分泌蛋白组深入研究奠定基础。挖掘大丽轮枝菌分泌蛋白中潜在的致病相关蛋白,为其深入功能验证及致病机理研究提供平台。【方法】3种方法提取大丽轮枝菌VdG1分泌蛋白,shot-gun方法进行质谱鉴定。利用生物信息学软件WoLFPSORT、SignalP、TMHMM、PHOBIUS对其中经典分泌蛋白进行预测。通过CAZy和PHI数据库进行经典分泌蛋白中碳水化合物水解酶（CAZy）和病原菌寄主互作因子（PHI）注释,BLASTp软件进行RxLx、LysM、Cys Pattern等模体蛋白比对分析。提取的蛋白样品接种感病棉种（军棉1号）进行样品生物学活性及致病性检测。【结果】TCA法、IEX法和UF法制备大丽轮枝菌分泌蛋白,其提取效率分别为1.18、0.96和0.56 mg?L-1。SDS-PAGE电泳检测显示UF法提取的蛋白样品条带少而模糊。而TCA法和IEX法提取的蛋白样品条带较多,样品的纯度也较高;经生物信息学软件预测分析,提取的蛋白样品中含有经典分泌蛋白分别为124、112和75个;质谱鉴定的分泌蛋白中含有潜在致病因子分别为98、85和57个;另外,IEX法制备大丽轮枝菌分泌蛋白效果较好,具有小分子偏好性,且能够保持样品的生物学活性,该蛋白样品接种棉花叶片能够引起萎蔫、坏死的症状。【结论】3种方法提取大丽轮枝菌分泌蛋白,UF法提取效率最低,纯度最差,蛋白数量最少,蛋白样品中含有潜在的毒素蛋白数量也最少,而且对于大体积处理相当耗时。TCA法和IEX法都适合大丽轮枝菌分泌蛋白的制备,但是TCA法制备过程中不能保持样品的生物学活性,后续适合于2-D电泳检测等试验。IEX法首次应用于病原菌分泌蛋白的制备,操作简单、方便,全部过程均由仪器系统完成。制备的大丽轮枝菌分泌蛋白具有生物学活性,能够引起棉花叶片的萎蔫、坏死病症。另外,数据库及软件比对分析发现大丽轮枝菌分泌蛋白组中含有CAZy酶类61个、PHI相关蛋白38个、RxLx模体蛋白13个、LysM模体蛋白1个、Cys Pattern的模体蛋白15个、VdNEP蛋白家族2个。该结果将为致病相关蛋白的筛选和深入功能验证提供重要依据。     

环介导等温扩增技术检测大丽轮枝菌

 本研究基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),通过比对分析大丽轮枝菌(Verticillium dahliae)与其相近种不同靶标序列间的差异,选取Gpd(glyceraldehyde-3-phosphate dehydrogenase,甘油醛-3-磷酸脱氢酶)基因作为靶标基因,设计并筛选了四条特异性强、灵敏度高的LAMP引物和两条环引物,建立了一种基于颜色判定的简单、快速和灵敏的大丽轮枝菌的检测方法,并进行了特异性、灵敏度实验及田间发病组织的检测。该方法在62 ℃等温条件下进行核酸扩增反应70 min,扩增前加入染料HNB(羟基萘酚蓝),反应后根据染料颜色变化判定扩增结果。特异性试验中,仅含有大丽轮枝菌菌株DNA的反应管扩增后呈天蓝色的阳性反应,而其他供试菌株均呈紫色的阴性反应。该方法的最低检测限为100 pg·μL^-1,在土壤中检测的灵敏度为10个孢子/0.25g土壤。该技术能够检测出棉花发病组织中的目标菌,对采自江苏和山东的24份疑似病害样本进行检测,11份为阳性。该方法的建立为大丽轮枝菌的检测及其所致病害的诊断提供了新的技术。     

海岛棉单萜合酶基因克隆及其受黄萎病诱导的表达分析

[目的]克隆海岛棉品种新海15中单萜合成酶基因(GbTPS),研究其受黄萎病诱导的表达情况,为进一步研究该基因在棉花抗黄萎病中的功能打下基础.[方法]克隆Gb TPS基因的开放阅读框(ORF)全长序列,对其蛋白序列进行生物信息学分析,并采用实时荧光定量PCR及病毒诱导的基因沉默对其抗病性进行分析.[结果]通过PCR扩增得到一个全长为1761 bp的ORF序列,命名为Gb TPS(GenBank登录号:KP247548).生物信息学分析发现,该基因编码586个氨基酸,预测分子量为67.7 kD,等电点为5.79.系统进化树分析结果表明,GbTPS属于Tps-g亚家族.GbTPS基因经黄萎病菌诱导后,表达量在4~24h内显著(P<0.05)或极显著(P<0.01)高于空白对照组.病毒诱导的基因沉默受黄萎病诱导后其病情指数为52.38,与对照组相比并无明显差异.[结论]GbTPS基因仅在棉花抗黄萎病的早期发挥积极作用.     

4株拮抗细菌对棉花黄萎病的防治效果及机制

棉花黄萎病严重制约新疆棉花持续高产和稳产,拮抗细菌在棉花黄萎病生物防治中潜力巨大。本研究旨在明确4株拮抗细菌对棉花黄萎病的防治效果及其机制,为棉花黄萎病的生物防治提供理论依据。本文采用共培养法,研究4株拮抗细菌对黄萎病菌分生孢子浓度、菌丝和孢子形态、膜透性和产毒素能力的影响,采用滤液接种法进行盆栽试验验证其对棉花黄萎病的防治效果,采用愈创木酚法和氮蓝四唑光化还原法测定棉株体内防御酶系的活性。结果表明,4株拮抗细菌能够减少黄萎病菌分生孢子浓度,破坏其细胞形态,导致其细胞膜破裂,降低其毒蛋白浓度。接种4株拮抗细菌,能够诱导棉株体内POD酶和SOD酶等防御酶系的积累,提高了棉花的抗病能力,由菌株SHZ-24、SHT-15、SMT-24和BHZ-29处理的棉花,其对棉花黄萎病的最高防治效果分别为73.82%、80.48%、84.91%和84.18%。4株拮抗细菌通过抑制黄萎病菌的生长和诱导植物产生防御反应来提高对棉花黄萎病的抗性,其对棉花黄萎病具有良好的防治效果。