内吸杀菌剂烯唑醇对小麦条锈菌和白粉菌发育影响的研究

 小麦幼苗分别用小麦条锈菌和白粉菌接种3天后,用内吸杀菌剂烯唑醇喷雾施药。电镜观察烯唑醇对条锈菌和白粉菌在寄主上发育的影响,结果表明,烯唑醇引起两种病菌和寄主细胞发生一系列变化。小麦条锈菌和白粉菌菌丝细胞壁的内层普遍不规则地加厚,菌丝细胞的隔膜发育受阻而成为畸形;两病菌的吸器外间质变宽,并沉积有电子致密度较高的物质。小麦条锈菌的部分吸器母细胞产生的畸形入侵栓,不能穿透寄主细胞壁,部分吸器不能正常发育,吸器体呈分枝状,不能完全扩张、膨大。此外,被侵小麦细胞所分泌的物质可将条锈菌吸器完全包围起来。小麦条锈菌和白粉菌以上的细胞学变化可能导致了它们进一步发育受阻。     

小麦叶锈菌与近等基因系TcLr41互作的组织病理学变化

为探讨小麦抗叶锈病机制,以小麦抗叶锈病近等基因系TcLr41和感病对照Thatcher为材料,利用荧光显微技术研究小麦叶锈菌与寄主互作的组织病理学变化.结果表明,在接菌24 h内亲和与非亲和反应过程之间差异不大,但在接种后36h仅在非亲和反应中出现早期的过敏性坏死反应,在接种后48h非亲和反应中进一步出现大量过敏性坏死反应,抑制了菌丝的生长,而在亲和互作的过程中始终未发现有过敏性坏死反应产生.     

小麦新抗源一粒葡抗条锈病的组织学和超微结构研究

 采用荧光显微镜、微分干涉显微镜和电子显微镜技术,系统研究了小麦新抗源一粒葡抗小麦条锈病的组织学和超微结构特征。结果表明:相对于感病品种铭贤169,一粒葡对条锈菌的侵染,在组织学和超微结构上均表现出明显的抗性特征。在组织学水平,表现为菌丝生长受抑,菌落发育延迟或败育,吸器母细胞和吸器数目明显减少;同时,侵染点的寄主细胞表现出不同程度的过敏性坏死症状。电镜观察发现,在一粒葡和感病品种中,条锈菌均可由芽管顶端直接进入或通过形成附着胞进入小麦气孔。其后,在一粒葡上,病菌胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,表现为原生质染色逐渐加深,液泡增多变大,逐渐消解原生质;胞间菌丝、吸器母细胞细胞壁不规则增厚;胞间菌丝线粒体肿胀,数目增多,逐渐解体;吸器母细胞细胞质逐渐空泡化后丧失其生理功能;吸器外质膜皱褶;吸器外间质加宽并有丝状或颗粒状物质形成,吸器体壁逐渐消解出现孔洞,吸器体最终畸形坏死。同时,寄主细胞产生一系列显著的结构防卫反应:形成胞壁沉积物、乳突、吸器鞘等结构,以及发生坏死,阻碍并抑制病菌的发育及扩展。     

小麦-条锈菌互作过程中活性氧及保护酶系的变化研究

 对条锈菌与小麦不同互作体系中组织学特征、活性氧产生及其相关酶系的变化进行了研究。组织学观察表明:非亲和组合相对于亲和组合存在明显差异,表现为菌丝生长受抑,吸器母细胞和吸器形成减少,寄主细胞在接种后18h左右出现过敏性坏死。生化测定结果表明:在非亲和组合中,O₂^-的产生速率和H₂O₂的含量均高于亲和组合,且O₂^-产生速率在接种后12h达到一个峰值,H₂O₂的含量在接种后20和72h出现2个高峰。而在亲和组合中,O₂^-产生速率低于对照或与对照相似,H₂O₂的含量虽然高于对照,但却普遍低于非亲和组合;SOD在亲和组合中的活性总体上要高于非亲和组合;接种24h后,CAT在2种组合中的活性均高于对照,在接种后36和48h时,亲和组合中的CAT活性高于非亲和组合,而在接种60h后又开始低于非亲和组合;POD活性在接种24h后均明显升高,但亲和组合中POD活性增幅大;MDA在非亲和组合中于接种后72h含量明显上升。结果表明,亲和与非亲和组合中条锈菌扩展、活性氧的产生及相关酶活变化都存在明显差异,这些差异与小麦抗锈性的表达可能有密切联系。     

种衣剂17号包衣对小麦条锈菌影响的组织病理学研究

种衣剂１７号内含三唑醇等杀菌剂和其它化学成分，小麦种子用种衣剂包衣，出苗后一叶期接咱小麦条锈菌毒性小种，采用荧光染色技术和整叶透明技术，研究了种衣剂１７号对条锈菌在寄主内发育的影响。观察结果表明，该种衣剂对对叶表的夏孢子萌发和芽管入侵无明显影响，但可使菌丝扩展和菌丝分枝严重受抑，吸器母细胞和吸器形成的数目减少，每一侵染点的吸器一般不超过２个，并且对吸器具有致畸作用。在多数侵染点内可观察到寄主细胞坏     

三唑酮拌种对小麦条锈病菌叶部侵染过程的影响

 供试小麦种子用干拌法施药,在一叶期接种小麦条锈菌(Puccinia striiformis West)毒性小种,用整叶透明染色方法研究三唑酮拌种对条锈菌叶部侵染过程的影响.结果表明,由初生吸器形成开始,三唑酮对吸器产生和菌落扩展表现出多方面的影响.拌种植株叶片中条锈菌侵染菌丝的生长和分枝严重受阻,吸器母细胞和吸器形成显著减少,菌落亦不再进一步分化,多数侵染点还出现寄主细胞坏死。这些组织病理学变化与抗病品种的过敏性坏死反应很相似.     

小麦凝集素类受体激酶TaLecRLK1的鉴定及抗锈病功能分析

凝集素类受体激酶(lectin receptor-like kinase, LecRLKs)是一类在植物应答多种生物/非生物胁迫中发挥重要作用的类受体激酶。本研究在小麦与条锈菌互作的转录组中筛选到1个在小麦与条锈菌非亲和互作中显著上调表达的LecRLKs基因TaLecRLK1。该基因全长2 292 bp,编码蛋白含有1个胞外信号肽、B-lectin结构域、PAN＿AP结构域、跨膜域和胞内酪氨酸激酶域。qRT-PCR分析表明：TaLecRLK1在小麦与条锈菌非亲和互作早期诱导表达,在烟草及小麦原生质体中瞬时表达,TaLecRLK1-GFP定位在细胞膜上。利用大麦条纹花叶病毒介导的基因沉默技术(BSMV-VIGS)沉默TaLecRLK1,接种无毒性条锈菌小种CYR23后,沉默叶片表面产生少量夏孢子堆,组织学观察发现沉默植株中条锈菌菌丝长度增长、侵染点附近活性氧积累面积减少;TaPR1、TaPR2、TaPR5的表达受到抑制,TaCAT和TaSOD则被迅速诱导表达。综上所述,TaLecRLK1对小麦抗条锈病起到正调控作用。     

不同亲和性的小麦品种和叶锈菌小种相互作用的组织病理学研究

用整叶透明染色法系统地观察了不同亲和性的小麦品种和叶锈菌小种相互作用的组织病理学表现。结果表明,(1)从气孔下泡囊形成迟早和初生侵染丝的生长开始,各组合间呈现明显的差别,暗示着品种——小种相互识别和抗性表达从这个阶段开始;(2)除了不亲和组合和慢锈组合出现过敏性反应外,在亲和组合的感病品种5389与叶锈菌小种互作中也观察到了少数侵染点有坏死细胞出现,但出现时间较晚,坏死细胞数目很少,并不影响菌丝的扩展。根据上述现象,本文讨论了过敏性坏死细胞形成的专化性和利用生理和组织学某些特征作为品种抗性鉴定的指标的意义。     

小麦抗条锈反应中活性氧及细胞质钙离子的作用

 为探讨活性氧和细胞质钙离子在小麦抗条锈病反应中的作用,以小麦品种洛夫林13与具有不同致病力的单孢锈菌CY29、CY25的互作体系为平台,对锈菌侵染后小麦叶片中活性氧(ROS)积累、保护酶系(SOD、CAT和APX)活性变化动态、细胞质膜的透性改变及细胞质钙离子浓度变化做了研究。结果表明,不亲和锈菌CY25侵染可引起小麦叶片内2次ROS的爆发,第1次出现在接种后前期(接种后第2天),强度较小,第2次出现在接种后期(接种后第5天),强度较大;亲和锈菌CY29侵染只引起1次ROS爆发,出现在接种后期(接种后第5和第6天之间),但强度极大。过敏性坏死反应HR只出现在不亲和互作小麦叶片上前期ROS爆发之后,表明前期ROS爆发与HR的产生有关。伴随着后期小麦叶片中强度极高的ROS的爆发,叶片细胞原生质膜遭到了破坏,细胞内物质外渗,细胞不久便死亡,表明高强度的ROS爆发会导致细胞死亡。根据不同互作体系ROS爆发时期SOD、CAT和APX等保护酶的活性变化分析,不亲和互作体系前期强度较小的ROS爆发主要成分是H₂O₂,后期强度较高的ROS爆发主要成分是O₂^-_·和H₂O₂;亲和互作体系强度极高的ROS爆发主要成分是O₂^-_·和H₂O₂。由此说明H₂O₂是引起小麦抗病反应HR发生的因素,而O₂^-_·则是引起细胞死亡的因素。细胞质钙离子浓度变化研究表明,HR的发生与细胞质钙离子浓度增加相关。细胞质钙离子浓度的降低推迟了HR的发生,这说明小麦叶片细胞内细胞质钙离子浓度的增加是HR的必要条件,同时也说明Ca²⁺是植物HR的胞内第二信使参与植物抗病防卫反应。     

寡聚糖诱导黄瓜对白粉病抗病反应的超微结构研究

 中科6号(2%氨基寡糖素)处理黄瓜植株叶片,5 d后接种白粉菌Sphaerotheca fuliginea(Schlecht.) Poll.,可诱导黄瓜Cucumis sativus产生对白粉病的抗病性,寄主细胞对病原菌的侵入产生了防卫反应结构和物质以及过敏性坏死反应。表现为寄主细胞壁加厚,染色加深,寄主细胞壁下产生多层次结构的乳突,在寄主细胞壁与质膜之间有黑色物质沉积;吸器外质膜皱褶,染色加深,吸器外基质中出现染色加深的颗粒状电子沉积物;寄主细胞质紊乱,细胞器解体,整个寄主细胞解体、坏死。     

Histological and ultrastructural comparisons of compatible, incompatible and -β-amino- n -butyric acid-induced resistance responses of pepper stems to Phytophthora capsici

The compatible interaction of pepper stems with Phytophthora capsici showed more rapid and severe disease development than did the incompatible interaction, although pathogen penetration styles of host cells in compatible and incompatible interactions were similar to each other. Treatment with -β-amino- n -butyric acid (BABA) protected the pepper plants against P. capsici infection. Reduced hyphal growth and sporangial formation were found after P. capsici infection in BABA-induced resistant and incompatible reactions. One of the most noticeable ultrastructural features of the BABA-induced resistant reaction was the formation of electron-dense wall appositions. The thick and dense wall appositions that encased the haustoria restricted haustorial development, thus leading to limitation of further pathogen penetration into inner plant tissues. A main host response in the incompatible interaction was the occlusion of cortical cells with an amorphous material. Plugging of the intercellular spaces in the cortical cells with electron opaque material was frequently observed in the incompatible interaction, but not in the compatible interaction. Another common feature of the BABA-induced resistant and incompatible reactions was degeneration of mitochondrial structure within penetrating hyphal cytoplasm. The mitochondrial structure in the BABA-induced resistant or incompatible reactions had no distinct double membrane layer and well-shaped cristae.     

The haustorial interface in a resistant interaction of Erysiphe pisi with Pisum sativum

The interface between Erysiphe pisi and pea cv. JI 1049 was studied at the ultrastructural and cytochemical levels and compared with those in two susceptible cultivars. Haustorial efficiencies, as indexed by the length of mycelium associated with each haustorium, and growth rates on the resistant and one susceptible cultivar were also compared.The interaction in the resistant cultivar differed from those in the susceptible cultivars in the following ways: (i) there was no contact between the host plasmalemma and the A neckband region; (ii) papillae were contiguous with the surface of the neck and thus were probably formed before haustoria and (iii) there appeared to be less polysaccharide in the extrahaustorial membrane. The extrahaustorial membrane in the resistant cultivar lacked ATPase activity, whereas the rest of the host plasmamembrane had normal activity.Each haustorium supported a significantly greater total hyphal length in the resistant than in the susceptible cultivars. Growth rates of superficial hyphae were very similar on the susceptible and resistant cultivars but there was a delay in the onset on hyphal growth on the resistant cultivar which correlated with the previously reported delay in formation of the first haustorium. In contrast to hyphal growth rates, the rate of haustorium production was significantly less in the resistant cultivar. It is proposed that resistance in cv. JI 1049 operates at the stages prior to haustorium formation, similarly to that in some non-host and partial resistance systems. Once formed, however, the function of the haustorium seems to be unimpaired, despite the observed interfacial differences.     

小麦叶锈菌在感病寄主上发育的组织病理学和超微结构研究

 应用荧光显微技术、微分干涉技术和生物电镜技术,系统地研究了小麦叶锈菌在感病寄主上的发育过程及其超微结构特征。小麦叶锈菌在感病品种上的发育过程可分为几个明显的阶段,即孢子的萌发、附着胞的形成、气孔下囊的分化、初生菌丝和次生菌丝的形成和生长、吸器母细胞和吸器的形成、夏孢子床和夏孢子堆的产生以及夏孢子的形成。小麦叶锈菌的胞间菌丝呈丝状,生长和分枝通常沿寄主细胞壁进行。胞间菌丝与寄主细胞的接触诱导了吸器母细胞的分化,吸器母细胞在与寄主细胞壁的接触部位发育形成入侵栓,穿透寄主细胞壁后于细胞内形成吸器。胞间菌丝和吸器母细胞均含有双核,而成熟吸器则含有单核。经常规染色后,胞间菌丝和吸器母细胞的壁与隔膜均可分辨出由多层构成。     

Encasement of Erysiphe graminis haustoria after treatment with bromuconazole

Preventive application of bromuconazole caused reduction in size and increased encasement rate of haustoria of Erysiphe graminis DC. For example, seven days after inoculation, 60 and 70% of haustoria had been encased in leaves treated with 8 mg litre⁻¹ and 16 mg litre⁻¹ respectively; the average length of the digitations was 8–10 μm in treated cells compared to 24 μm in untreated cells. The encasement process extended from the neck region to the whole haustorium. Haustorial bodies from treated plants had electron-dense cytoplasm and their organelles were more difficult to identify than in control plants. Extrahaustorial matrix was reduced to an unusually thin, osmiophilic pellicle, surrounded by abundant heterogeneous encasement material. Curative treatment induced similar changes, especially in the margin of the colonies. In the centre of the colony, haustoria were less affected by the fungicide; deposition of collar-like material, modification of extrahaustorial matrix and membrane and accumulation of plant cytoplasm around the digitations resulted in an intermediate, ‘swollen’ state of digitate haustoria. The possible pathway of encasement events is discussed.     

青杨叶锈病菌(Melampsora larici-populina Kleb.)侵染过程的超微结构研究

 采用电子显微镜技术对青杨叶锈病菌(Melampsora larici-populina Kleb.)的侵染过程进行了研究。发现该菌夏孢子萌发产生1~3个芽管,且具较多的树杈状分枝。芽管由气孔侵入,侵入前不形成明显的附着胞或仅个别芽管形成附着胞。芽管侵入气孔后在气孔腔内形成气孔下囊,再分化出圆形的膨大体而产生1~2支初生菌丝。初生菌丝在寄主细胞间扩展,与叶肉细胞壁接触后分化出吸器母细胞,吸器母细胞中的细胞器与胞间菌丝相同,双核。吸器母细胞产生侵入钉侵入叶肉细胞内部形成吸器,成熟吸器由细长具颈环的管状颈部和膨大的吸器体组成,此时胞间菌丝在吸器母细胞处分化出次生菌丝,在叶肉细胞间扩展形成次生菌落,产生孢子堆。病菌在寄主细胞间隙或沿寄主细胞壁延伸时,寄主细胞仍保持正常状态。     

Cytological Evidence of an Active Role of Silicon in Wheat Resistance to Powdery Mildew (Blumeria graminis f. sp. tritici)

ABSTRACT Silicon (Si) amendments in the form of exogenously supplied nutrient solution or calcium silicate slag protect wheat plants from powdery mildew disease caused by the fungus Blumeria graminis f. sp. tritici. The most striking difference between Si- and Si+ plants challenged with B. graminis f. sp. tritici was the extent of epidermal cell infection and colonization by B. graminis f. sp. tritici. Histological and ultrastructural analyses revealed that epidermal cells of Si+ plants reacted to B. graminis f. sp. tritici attack with specific defense reactions including papilla formation, production of callose, and release of electron-dense osmiophilic material identified by cytochemical labeling as glycosilated phenolics. Phenolic material not only accumulated along the cell wall but also was associated with altered integrity of haustoria in a manner similar to localized phytoalexins as reported from other pathosystems. These results strongly suggest that Si mediates active localized cell defenses against B. graminis f. sp. tritici attack.     

The haustorial host-parasite interface in rust-infected bean leaves after high-pressure freezing

The haustorial fine structure of the bean rust fungus, Uromyces appendiculatus var. appendiculatus, was studied within the cells of its host, Phaseolus vulgaris. Results were obtained after high-pressure freezing and subsequent freeze-substitution or freeze-fracturing. Good preservation of leaf tissue after freeze-substitution needed cryoprotection with 8% methanol. For freeze-fracturing, no chemical treatment was applied. In addition to the organelles which are generally found in fungi after cryo-fixation, tubular-vesicular complexes were found in the cytoplasm. Both techniques revealed an extrahaustorial matrix of even width, surrounding the haustorial body. The extrahaustorial membrane was not undulated, and the side facing the plant cytoplasm was lined with a delicate fringe of well-stained material. The extrahaustorial membrane was nearly devoid of intramembrane particles. The host plasma membrane in infected tissue, especially the protoplasmic face, had fewer intramembrane particles than those in uninfected tissue. The haustorial plasma membrane contained many intramembrane particles.     

Permeability of the haustorium-host interface in powdery mildews

The extent to which uranyl ions and peroxidase permeated haustorial complexes isolated from Pisum sativum infected with Erysiphe pisi was determined by transmission electron microscopy (TEM).Freshly isolated complexes were treated with uranyl acetate, either directly, or after treatment with cell wall degrading enzymes, or with Triton X-100 and the uranyl ion, precipitated with phosphate. The complexes were then fixed with glutaraldehyde and examined by TEM to determine the distribution of uranyl phosphate crystals.Two independent experiments, each involving the examination of 80 complexes, gave similar results. After direct uranyl treatment, crystals were excluded from 38% of the complexes, were present in the extrahaustorial matrix only in 19% and occurred throughout in 43%. Breaks were observed in the extrahaustorial membrane of the last two categories. Uranyl crystals bound to the outer (host cytoplasmic) surface of the extrahaustorial membrane and also to the matrical surface, where this was accessible. In addition, the few necrotic haustorial complexes observed were completely permeated.Following pre-treatments with pectinase and cellulase, or Triton X-100, uranyl crystals were present throughout the complexes in all or 96%, of the complexes respectively.In similar experiments involving treatment with horseradish peroxidase (instead of uranyl acetate) followed by the addition of hydrogen peroxide and 3,3-diaminobenzidene, electron opaque deposit occurred around the complexes but never internally in any part of them. Even where breaks in the extrahaustorial membrane were visible the extrahaustorial matrix was not permeated.It is concluded that the extrahaustorial membrane and haustorial plasma membrane are semipermeable in many of the isolated haustorial complexes but that some are damaged during isolation and purification procedures; that a barrier to diffusion exists where the extrahaustorial membrane is joined to the wall of the haustorial neck and in other parts of the neck wall so that the whole boundary of the extrahaustorial matrix restricts diffusion of solutes; and that the extrahaustorial matrix is a gel which precludes the passage of large molecules through it. The physiological implications of the results are discussed.