低量化学除草剂对菌株QZ-97a侵染波斯婆婆纳的影响

为探索提高真菌除草剂防效的途径,研究了胶孢炭疽菌菌株QZ-97a与低剂量化学除草剂混用的互作效果。结果表明,菌株QZ-97a孢子悬浮液与低剂量的化学除草剂氯氟吡氧乙酸(Fluroxypyr,氯氟吡氧乙酸)混用可显著提高对波斯婆婆纳的防效。1.0×10~8个孢子/ml的菌株QZ-97a孢子悬浮液与常规剂量1/10的氯氟吡氧乙酸混用可在波斯婆婆纳危害时的田间条件下经保湿24～72h后使子叶期波斯婆婆纳死亡。可见,该菌与常用的防除波斯婆婆纳的化学除草剂配合性好,具有被开发为防除波斯婆婆纳的真菌除草剂的潜力。     

高粱刺盘孢Colletotrichum sublineola是四川地区高粱炭疽病的主要致病菌

 高粱是四川省主要经济作物,在高粱生产上普遍发生炭疽病。关于高粱炭疽病菌多数文献描述为禾生刺盘孢Colletotrichum graminicola。已有文献通过分子生物学和基因组学分析证明高粱炭疽病的病原菌是高粱刺盘孢C. sublineola,并非C. graminicola。为明确四川省和国内高粱主产区炭疽菌的种类,本研究从四川省泸州泸县等11个地区以及重庆等其他6个省市,总共采集了198份病样,经分离纯化共获得225个炭疽菌株。通过形态学观察,结合ITS和GAPDH基因序列及进化树分析,发现其中215个来源于17个地区的菌株为C. sublineola,且分生孢子形态均为月牙型。其余10个分别来源于两个地区的菌株为兰花刺盘孢C. cliviicola,其分生孢子形态均为圆柱型。通过另外3个基因(CHS、ACT和TUB2)的系统发育分析,进一步证明来自不同地区的17个代表性的炭疽菌菌株分类相同,且均属于C. sublineola。研究结果表明,分生孢子形态及ITS和GAPDH等基因序列可以作为高粱炭疽菌的主要分类依据。在常规高粱品种Hui1等3个材料上接种19个代表性菌株,发现17个C. sublineola菌株的致病能力存在差异,2个C. cliviicola菌株均不能致病。本文研究结果表明,C. sublineola是四川高粱主产区炭疽病的致病菌。另外,通过人工接种试验,证明高粱品种402B对强致病力菌株ZG-FS-1具有较强抗性,402B具有抗病育种应用潜力。     

Role of cutinolytic enzymes in infection of cucumber by Colletotrichum lagenarium

The role of cutinolytic enzymes in infection of cucumber by Colletotrichum lagenarium was investigated. Microscopic and visual observations of C. lagenarium penetration and colonization in the presence of organophosphate cutinase inhibitors suggested that cutinolytic enzymes do not have a primary role in penetration. Use of mutants of C. lagenarium supported these observations since only a poor correlation between cutinase production and penetration and disease expression was found, whereas a very good correlation between penetration and disease development was found.     

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.     

毁灭炭疽菌Colletotrichum destructivum诱抗蛋白对烟草抗病性的诱导

为明确毁灭炭疽菌Colletotrichum destructivum诱抗蛋白诱导烟草的抗病性及其作用,采用喷雾、摩擦接种方法及RT-PCR技术研究了诱抗蛋白的预防保护作用,以及烟草悬浮细胞经诱导后过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)活性及脯氨酸(Pro)含量和病程相关基因表达的变化。结果表明,接种3、5和7 d后,该诱抗蛋白对烟草炭疽病的诱抗效果分别为58.00%、48.99%和49.65%,对烟草白粉病的诱抗效果分别为83.26%、80.76%和78.60%,并可以抑制烟草普通花叶病毒的复制及在寄主体内的扩增;经诱抗蛋白处理后,烟草悬浮细胞POD、PPO、PAL活性及Pro含量明显提高;诱抗蛋白能够诱导烟草病程相关蛋白基因PR-1a、PR-1b以及抗病信号传导途径关键基因NPR1的表达。表明毁灭炭疽菌诱抗蛋白可诱导烟草产生抗病性,可能与烟草悬浮细胞中POD、PAL、PPO的活性及Pro的含量提高以及相关病程基因表达有关。     

谷子抵御白发病菌侵染的生理生化及基因表达分析

白发病已成为我国谷子生产上的重要病害,不仅造成产量下降,还对谷子品质构成威胁。有关谷子品种响应白发病菌侵染的生理生化机制研究尚未见报道。本研究采用病菌卵孢子和谷粒混合的接菌方法分别接种抗病品种‘G1’和高感品种‘晋谷21’(JG21),并测定抗感谷子的植株形态指标、光合参数、防御酶活性、抗氧化酶活性、可溶性糖及脯氨酸含量,同时结合基因表达分析,旨在明确谷子响应白发病菌侵染机制。结果表明,白发病菌侵染抗感品种后,植株高度及叶面积均表现出不同程度的下降,感病品种的株高降低幅度远大于抗病品种;与抗病品种相比,感病品种中除了胞间CO₂浓度显著升高外,其他光合参数显著下降,且均低于抗病品种。抗病品种中抗氧化酶和防御酶活性及脯氨酸、可溶性糖含量均升高,显著高于感病品种。抗病相关基因Seita.2G024600(PR1)、Seita.7G168700(PAL)、Seita.1G240200(PAL)和Seita.9G462500(POD)在抗病品种中上调,而在感病品种中表达受到抑制或下调。以上光合参数、酶活性、Pro和可溶性糖含量变化等生理生化指标可作为抗性鉴定和种质筛选的参考...     

谷子抵御白发病菌侵染的生理生化及基因表达分析

 白发病已成为我国谷子生产上的重要病害,不仅造成产量下降,还对谷子品质构成威胁。有关谷子品种响应白发病菌侵染的生理生化机制研究尚未见报道。本研究采用病菌卵孢子和谷粒混合的接菌方法分别接种抗病品种‘G1'和高感品种‘晋谷21'(JG21),并测定抗感谷子的植株形态指标、光合参数、防御酶活性、抗氧化酶活性、可溶性糖及脯氨酸含量,同时结合基因表达分析,旨在明确谷子响应白发病菌侵染机制。结果表明,白发病菌侵染抗感品种后,植株高度及叶面积均表现出不同程度的下降,感病品种的株高降低幅度远大于抗病品种;与抗病品种相比,感病品种中除了胞间CO₂浓度显著升高外,其他光合参数显著下降,且均低于抗病品种。抗病品种中抗氧化酶和防御酶活性及脯氨酸、可溶性糖含量均升高,显著高于感病品种。抗病相关基因Seita.2G024600(PR1)、Seita.7G168700(PAL)、Seita.1G240200(PAL)和 Seita.9G462500(POD)在抗病品种中上调,而在感病品种中表达受到抑制或下调。以上光合参数、酶活性、Pro和可溶性糖含量变化等生理生化指标可作为抗性鉴定和种质筛选的参考,这些差异表达基因可能参与了谷子对白发病菌抗病调控。研究结果可为谷子抗病分子机制及分子改良育种提供理论基础。     

苹果炭疽叶枯病菌致病相关基因CgNVF1的功能初步分析

苹果炭疽叶枯病是主要由胶孢炭疽菌(Colletotrichum gloeosporioides)引起的一种苹果重要叶部病害,严重威胁着苹果树的生长。本研究从构建的苹果炭疽叶枯病菌T-DNA突变体库中筛选获得一株致病力缺失的突变菌株A3083,采用hiTAIL-PCR方法克隆了该突变体T-DNA插入位点的右翼序列;通过与胶孢炭疽菌基因组序列比对分析,发现T-DNA插入位点位于1个预测的CGGC5_9603基因内,并将该基因命名为CgNVF1。CgNVF1基因全长2252 bp,含有2个内含子,编码709个氨基酸。CgNVF1定位于细胞质,在苹果炭疽叶枯病菌菌丝、分生孢子和附着胞中均有表达。通过构建CgNVF1敲除菌株和CgNVF1互补菌株,并结合表型分析,证实CgNVF1基因在苹果炭疽叶枯病菌附着胞形成及致病中具有重要的作用。     

Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

胶孢炭疽菌致病相关基因Plv2功能分析

胶孢炭疽菌(Colletotrichum gloeosporioides)引起的橡胶树炭疽病是橡胶树三大叶部病害之一,严重威胁着橡胶树的生长。本研究从构建的胶孢炭疽菌RC178 T-DNA突变体库中筛选获得一株致病力明显减弱的突变菌株T1103,其T-DNA为单拷贝。采用TAIL-PCR克隆了该突变体T-DNA插入位点的侧翼序列,通过比对胶孢炭疽菌全基因组序列,发现该TDNA插入位点所在的序列包含一个5 400 bp的基因,将其命名为Plv2。Plv2基因包含3个外显子,编码一个假定的甾醇C-24甲基转移酶。敲除野生型菌株RC178中的Plv2,发现敲除突变体与T1103的致病力表现基本一致,由此推测Plv2基因为致病相关基因。     

小尺度空间果生刺盘孢遗传多样性分析

 本研究将来自16个油茶品种的247个Colletotrichum fructicola的多基因序列进行遗传多样性分析,结果表明,247个C. fructicola多基因序列有21个单倍型,其中Hap2占总数的54.7%,Hap4占总数的34%,为两个主要的单倍型,来自所有16个油茶品种。基本上每一个油茶品种都存在C. fructicola独特的单倍型。遗传分化系数(Fst)表明不同品种来源菌株种群间存在一定的遗传分化。对所有品种种群进行中性检测及核苷酸不配对分析,发现小尺度空间的果生次盘孢菌经历大规模的种群扩张,群体间存在有效基因流。系统发育分析表明,来自不同品种的菌株散乱地分布在系统发育树中。     

中国南方主要梨产区果生炭疽菌对苯醚甲环唑的敏感性

为明确苯醚甲环唑对果生炭疽菌Colletotrichum fructicola不同发育阶段的毒力,并建立中国南方主要梨产区(湖北、安徽、江西、江苏、浙江和福建)果生炭疽菌对苯醚甲环唑的敏感基线,采用孢子萌发法测定了苯醚甲环唑对6个代表性菌株孢子萌发和芽管伸长的影响,采用离体叶片接种法评价了苯醚甲环唑对该病菌的防治效果,采用菌丝生长速率法测定了120株果生炭疽菌对苯醚甲环唑的敏感性。结果表明:苯醚甲环唑对果生炭疽菌菌丝生长和芽管伸长的抑制效果较强,而对分生孢子萌发的抑制效果较弱,其平均EC50值分别为(0.33±0.03)、(2.61±0.26)和(29.87±1.31)μg/mL。160μg/mL的苯醚甲环唑对梨果生炭疽病的保护作用和治疗作用防效分别为92%和79%,而1000μg/mL的多菌灵则分别为51%和23%。来源于中国南方主要梨产区的120株果生炭疽菌对苯醚甲环唑均表现为敏感,EC50值范围0.27~1.12μg/mL,敏感性频率分布呈连续单峰曲线,符合正偏态分布,其平均EC50值(0.59±0.02)μg/mL可以作为我国南方主要梨产区果生炭疽菌对苯醚甲环唑的敏感基线。本研究结果为监测梨果生炭疽菌对苯醚甲环唑的田间抗药性发展奠定了基础,并为梨产区应用该药剂防治炭疽病提供了依据。     

水稻中MPK5稳态失衡抑制拟禾本科根结线虫与水稻的亲和互作

 拟禾本科根结线虫(Meloidogyne graminicola)为水稻的重要土传病原,可严重为害水稻。本研究旨在探究水稻中丝裂原活化蛋白激酶5(MPK5)稳态失衡对拟禾本科根结线虫与水稻亲和互作的影响,并初步探讨其机制。首先,通过线虫侵染试验发现,在水稻中超表达和沉默MPK5 引发的MPK5 稳态失衡均可抑制拟禾本科根结线虫与水稻的亲和互作。qRT-PCR 分析结果显示,与水稻野生型不同,在MPK5 超表达和沉默水稻株系中MPK5 均受拟禾本科根结线虫诱导表达。同时,拟禾本科根结线虫可抑制MPK5 超表达株系中几丁质酶的表达,但诱导MPK5 沉默株系中病程相关基因PR5 与PR10 的表达。上述试验结果表明,MPK5 稳态失衡可导致MPK5 响应拟禾本科根结线虫的侵染,且在MPK5 超表达和沉默水稻株系中MPK5 介导水稻产生拟禾本科根结线虫抗性的机制不同。     

Partial characterization of the extracellular matrix released onto hydrophobic surfaces by conidia and conidial germlings ofColletotrichum graminicola

The extracellular matrix (ECM) released by conidia ofColletotrichum graminicolawhen incubated on artificial hydrophobic substrata was investigated. Time-course experiments revealed that proteins and carbohydrates were released in the ECM of both ungerminated and germinated conidia. Stains for protein and carbohydrate revealed that part of the ECM remained attached to the substratum after the fungus had been removed. The ECM isolated from substrata where ungerminated conidia had adhered and from substrata where germinated conidia had adhered exhibited the same profiles when analysed by SDS-PAGE for proteins and glycoproteins and by HPLC for carbohydrate analysis. The ECM was found to be composed of glycoproteins with a molecular weight of around 200 kDa. Deglycosylation of the isolated ECM material resulted in the release of a protein with a molecular weight of approx. 116 kDa, indicating that the original protein was extensively glycosylated. Carbohydrate analysis demonstrated that mannose was the predominant sugar. That the ECM remained attached to the surface of the hydrophobic substrata suggests that it has a role in adhesion.     

斯高威尔刺盘孢菌次生代谢产物分离纯化及其对大斑凸脐蠕孢菌的抑制作用

采用硅胶柱层析、凝胶柱层析、薄层层析和高效液相色谱等技术对一株斯高威尔刺盘孢菌Colletotrichum scovillei次生代谢产物进行分离纯化,得到两个化合物1和2,结构经波谱综合解析并与文献数据比对,确认化合物1为未见文献报道的神经酰胺类化合物6-甲基-12-羟基-十四烷基-N-(2’,3’,4’-三羟基丁基)-2E,4Z-二烯酰胺,化合物2为已知化合物sambutoxin。抑菌活性测定结果表明,两个化合物均对大斑凸脐蠕孢菌Exserohilum turcicum具有较强的抑制作用,EC₅₀值分别为11.43μg/mL和18.42μg/mL。离体叶片法测定结果表明,化合物1在800μg/mL质量浓度下对玉米大斑病的相对防治效果可达84.8%,与对照药剂百菌清相当。     

引起油茶炭疽病胶孢炭疽菌种群遗传结构研究

 胶孢炭疽菌是引起油茶炭疽病的主要病原之一。目前对于该病原菌种群的遗传结构尚未见报道。研究病菌种群的遗传结构对制定科学的防治方法具有重要意义。本研究比较中国6个省12个不同地区的126个胶孢炭疽菌的ITS序列的差异,以期研究各不同群体的单倍体型多样性,不同地区群体之间的遗传关系,以及遗传距离与地理距离的线性关系。遗传分析发现,得到的126个胶孢炭疽菌样品ITS序列可定义为27种单倍体型。27种单倍体型中,一个主要的单倍体型(Haplotype12)含有78个样品,且基本上每个样品采集地都有分布。遗传分化指数(Fst)表明病菌不同地理种群间的遗传分化较大。AMOVA分析显示,种群间的遗传变异占总变异的6%,种群内部变异占总变异的94%。Mantel测试显示地理距离与遗传距离没有显著的线性关系。对所有地理种群病原菌ITS序列的核苷酸不配对进行分析,发现油茶炭疽病菌未经历过大规模的种群扩张过程。系统发育分析表明,来自不同地区的油茶炭疽病菌散乱的分布在系统树中。研究结果表明油茶炭疽病菌种群具有丰富的遗传多样性。     

谷子种子白发病菌带菌检测及种群多态性分析

为检测不同地区谷子种子携带白发病菌Sclerospora graminicola的情况以及种群多态性,以我国谷子不同主产区收集的95份代表性谷子品种种子为材料,提取种子基因组DNA并作模板,利用白发病菌28S rRNA序列设计特异性引物进行PCR扩增,将所获得的PCR产物测序后与白发病菌28S rRNA序列进行比对。结果表明,特异性引物Sg-28S-403-F/Sg-28S-1221-R对白发病菌具有高度的特异性,扩增出819 bp的特异性目标片段,有效检测灵敏度为0.125 ng/μL。在95份谷子种子中,26份种子检测出特异性扩增条带,所有扩增条带对应序列与白发病菌28S rRNA序列的相似性达98%以上,并且带菌种子全部来自春谷区。分析26份阳性样品的28S rRNA序列,得到58个变异位点和34种单倍型,其中单倍型SG2出现频率最高,内蒙古、陕西、山西、河北和辽宁省区的白发病菌都具有该单倍型,表明不同地理来源的白发病菌28S rRNA序列存在差异,SG2为优势单倍型。     

8种杀菌剂对核桃炭疽病病原菌胶孢炭疽菌的室内毒力

由胶孢炭疽菌Colletotrichum gloeosporioides引起的核桃炭疽病是目前危害山东省核桃生产的主要病害。为筛选防治该病害的高效药剂,在确定了最适菌丝生长和分生孢子萌发温度的基础上,采用菌丝生长速率法和孢子萌发法,测定了8种常用杀菌剂对胶孢炭疽菌的毒力,评估了温度对咪鲜胺和戊唑醇毒力的影响,检测了17个胶孢炭疽菌菌株对咪鲜胺和戊唑醇的敏感性,同时评价了这2种杀菌剂的预防和治疗作用。结果显示:在胶孢炭疽菌菌丝生长和孢子萌发的最适温度28℃下,供试8种杀菌剂中,咪鲜胺、戊唑醇和三唑酮对供试菌株菌丝生长的抑制作用最强,平均EC50值分别为0.07、1.45和3.04 mg/L;异菌脲、代森锰锌和咪鲜胺对分生孢子萌发的抑制作用最强,平均EC50值分别为21.07、25.14和25.18 mg/L。咪鲜胺对菌丝生长的抑制作用受温度影响较小,而戊唑醇对菌丝生长的抑制作用随温度降低而增强;供试17个菌株对咪鲜胺和戊唑醇均有较高敏感性,平均EC50值分别为0.08 和 1.03 mg/L。建议在核桃炭疽病发病的不同时期,轮换使用咪鲜胺、戊唑醇、三唑酮、异菌脲和代森锰锌等杀菌剂,以有效控制该病害。