多堆柄锈菌侵染不同抗性玉米的组织病理学研究

［目的］ 了解不同抗感玉米自交系对南方锈病的组织病理学反应,为今后筛选不同类型抗病自交系提供参考。［方法］ 采用曲利苯蓝透明染色法,以4个玉米自交系为材料,研究了玉米南方锈病致病菌—多堆柄锈菌在不同抗性材料上侵染过程的组织学特征。［结果］ 多堆柄锈菌侵入和定殖可以分为5个阶段：孢子萌发与芽管形成、附着胞形成、侵入细胞、胞内吸器产生、菌丝在细胞间扩展。在不同抗性的玉米材料上,病菌孢子萌发和芽管形成差别不明显,但侵入后病菌在不同抗性材料内的发育进程和发育程度具有显著差异。在抗病玉米材料上,病菌初生菌丝、吸器母细胞、次生菌丝的形成时间推迟,胞内吸器少,菌丝分枝少,菌丝生长缓慢。［结论］ 这些抗性特征与田间表现出的细胞过敏性坏死、叶片上夏孢子堆少的特征具有一致性。     

玉米感染肿囊腐霉后寄主-病原物互作的超微结构研究

 本文利用透射电镜,首次对玉米不同抗性寄主与肿囊腐霉(Pythium inflatum)相互作用中的寄主反应及菌丝在寄主内的发展进行了系统研究。结果表明:玉米苗期根部接种后,孢子迅速萌发成菌丝在根表蔓延,随即穿透根表皮,进入表皮细胞、皮层甚至感病寄主的维管束组织。与此同时,寄主相应的反应迅速,寄主反应采取如下方式:细胞壁的沉积物及乳突在真菌的入侵处形成,各种无定形物质或纤丝构成的质网包围入侵菌丝。沉积在寄主细胞壁上并在受侵染细胞内聚集的化合物可能是酚类物质,这些嗜锇酸的电子致密物是用来机械地加强细胞壁的强度及产生抗真菌的环境。寄主反应的早晚及程度的强弱决定着菌丝在寄主体内发展繁殖的程度及寄主抗性的强弱。抗病玉米自交系的反应程度及速度显著强于感病玉米。     

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。     

菜豆锈菌侵染过程的显微和超微观察

 本文报道了通过微分干涉衬显微镜、荧光显微镜及扫描电镜和透射电镜所观察到的菜豆锈菌的侵入和扩展过程。菜豆锈菌夏孢子萌发多产生1个芽管,偶尔也产生双芽管。芽管以气孔侵入为主,也可从表皮直接侵入。侵入前形成或不形成明显的附着胞。气孔侵入的芽管首先在气孔腔内形成气孔下囊,再进一步分化出圆形的膨大体,由膨大体产生1~2支初生菌丝。初生菌丝与叶肉细胞壁接触后分化出吸器母细胞,吸器母细胞进入叶肉细胞内部形成吸器。初生侵染菌丝在产生吸器母细胞的部位的后部产生分枝,形成次生侵染菌丝在叶肉细胞间蔓延。     

柿树炭疽菌侵染柿树叶柄的超微结构观察

 利用透射电镜技术研究了柿树炭疽菌侵染柿树叶柄的超微结构。结果表明:病原菌侵入寄主细胞后,产生细胞内的初生菌丝,其表面沉积凹凸不平的电子不透明物质。一层界面基质(interfacial matrix)把表初生菌丝细胞壁和凹陷的寄主原生质膜分开。随着初生菌丝定殖下一个细胞,原先细胞中的细胞膜消失,形成许多泡囊,随后叶绿体消失,内质网和高尔基体也逐渐降解,最后细胞内物质全部被降解成电子不透明的颗粒,降解的物质沿着初生菌丝和细胞壁表面沉积。初生菌丝穿透细胞壁的过程中,菌丝顶端接触细胞壁后膨大,并在中部产生一个隔膜,然后顶端细胞产生一个较细的穿透菌丝,穿透寄主细胞壁。穿透菌丝在寄主细胞壁中的狭窄处产生一个隔膜,一旦穿透寄主细胞壁后,迅速膨大。次生菌丝在细胞间和细胞内扩展,通过菌丝体对细胞壁施加的机械压力引起寄主细胞壁破裂,或同初生菌丝一起使细胞壁解体。侵染90 h后,形成垫形分生孢子盘。在分生孢子盘周围的表皮细胞中,次生菌丝不断形成子座组织,使原来的子座扩大,子座不断分化形成产梗细胞,产梗细胞产生分生孢子梗,分生孢子梗生长和发育对角质层和表皮细胞壁组织折叠处施加机械压力,使角质层和表皮细胞壁组织进一步折叠,分生孢子盘也相应扩大。     

玉米丝黑穗病原菌侵染的一些细胞学研究

 在玉米生育早期,以幼芽鞘为材料,对11个抗病性不同的自交系进行了研究。玉米丝黑穗病原菌[Sphacelotheca reiliana (Kühn) Clint var.zeae Pass]可在玉米幼芽鞘内表皮上生长和繁殖;其侵染菌丝可直接侵入表皮细胞或通过气孔进入寄主体内。菌丝的侵入诱导了芽鞘内表皮细胞的细胞壁、细胞质和细胞膜特性的异常变化;抗性不同的自交系对病原菌侵入的反应不同,揭示了在生育早期玉米芽鞘细胞就表现了其固有的抗病性。     

玉米叶片受新月弯孢菌侵染后的细胞病理学变化

 本文利用透射电子显微镜技术与细胞化学技术研究了玉米叶片受弯孢菌侵染后的超微结构和细胞壁的组成成份变化。透射电镜观察发现,病菌侵入后,菌丝先在寄主细胞间扩展,随着寄主细胞病变、坏死,菌丝可进入寄主细胞形成胞内菌丝。随病菌侵入和在寄主体内扩展,寄主细胞先后发生了一系列的超微结构变化,叶绿体、液泡等细胞器解体,出现质壁分离现象,并最终解体、坏死、变形。细胞化学标记定位发现,受侵寄主细胞壁中纤维素、木聚糖和果胶质的标记密度明显低于未接种的健康组织,表明细胞壁降解酶(如纤维素酶、木聚糖酶和果胶酶)的产生与病菌侵染和致病过程密切相关。     

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。     

大豆疫霉菌对大豆下胚轴侵染过程的细胞学研究

 接种后1.5~24h,用光镜和电镜研究了2个大豆品种与大豆疫霉菌Ps411的亲和性和非亲和性互作。观察结果表明,大豆疫霉菌对大豆下胚轴的侵染过程可分为侵入前、侵入、皮层组织中的扩展和进入维管束组织4个连续阶段。大豆下胚轴接种后在25℃保湿培养,1.5h后游动孢子即形成休止孢并萌发产生附着孢,3h后侵入表皮细胞,6h后进入皮层组织,24h后进入维管束组织。病原菌主要以侵染菌丝直接侵入表皮,表皮细胞间隙是主要侵入部位。皮层细胞是病原菌定殖和发展的主要场所,胞间菌丝侵入皮层细胞并形成吸器。在菌丝与寄主细胞接触部位的寄主细胞壁与质膜之间常有胞壁沉积物的形成。在抗病品种上病菌的侵染事件与感病品种基本一致,但不能形成正常的吸器,胞壁沉积物明显多于感病品种,菌丝在寄主组织内的扩展明显受到抑制。利用β-1,3-葡聚糖免疫金标记单克隆抗体进行的免疫细胞化学的研究表明,胞壁沉积物内含有大量的β-1,3-葡聚糖,在大豆疫霉菌菌丝壁中也存在β-1,3-葡聚糖。以上结果表明,病原菌的侵染可诱导抗病寄主细胞内β-1,3-葡聚糖迅速的合成与积累、并形成胞壁沉积物,以抵御病菌的侵染与扩展。     

如何识别与防治大蒜锈病

大蒜锈病对大蒜品质和产量均有较大的影响。要有效地控制该病,必须正确掌握识别与防治方法。大蒜锈病由葱柄锈菌(属担子菌亚门真菌)侵染所致。除侵染大蒜外,还侵染洋葱、韭菜等。病菌主要侵染叶片和假茎。病部初为梭形褪绿斑,后在表皮下出现圆形或圆形稍凸起的夏孢子堆,表皮破裂后散出橙黄色粉状物,即夏孢子。病斑四周有黄色晕圈,后病斑连片致全叶黄枯,植株提前枯死。生长后期,在未破裂的夏孢子堆上产出表皮不破裂的黑色冬孢子堆。病菌多以夏孢子在留种葱和越冬青葱及大蒜病组织上越冬。翌年入夏形成多次再侵染,这时正值蒜头形成或膨大期,为…     

Ultrastructural study on interaction between a sterile,white endophytic fungus and eggplant roots

Eggplant roots colonized by a sterile, white mycelial endophyte (SWM) were previously found to become highly resistant to Verticillium wilt. SWM alone, however, caused no visible, disease symptoms, such as wilting or necrosis. The mechanism of the symptomless infection by SWM was investigated in this study. Electron microscopy revealed that hyphae of SWM were abundant on and inside the root epidermal cells 2 weeks after inoculation. Many terminal appressoria formed from apical tips of hyphae, and heavy degradation of the host cell walls was evident where hyphae accumulated. By 4 weeks following inoculation, penetration pegs easily breached epidermal cells, and the infection hyphae penetrated outer cortical cells. In response to the hyphal ingress, numerous tubule-like vesicles and membrane-bound, multivesicular bodies accumulated in cortical cytoplasm near the infection sites of the outer cortical cells, but no visible signs of the host reactions were seen in the epidermal cells. Papillae developed at the spaces between cell walls and plasma membranes at the infection sites. The penetration hyphae often grew out of the papillae, but further hyphal ingress was halted in the middle cortical cell layer. By 8 weeks following inoculation, papillae that developed in these cells contained larger amounts of highly electron-dense material and were reinforced by multilamellate, fibrous elements. Hyphae that entered such papillae were confined to them, and the hyphal cytoplasm degenerated. As the result of the activated resistance reactions, root vascular cylinders remained intact, and the host plants did not wilt.     

栗疫病菌侵染板栗枝条的显微观察

栗疫病是一种严重危害栗属植物的病害。为了明确栗疫病菌侵染板栗枝条的过程及侵染的关键时间点,本研究利用病理组织切片技术、显微镜和扫描电镜技术对栗疫病菌侵染板栗枝条的过程进行了观察。结果表明:接种栗疫病菌后0～5 h,菌丝先降解枝条表皮,进行横向营养生长的同时沿着伤口纵向侵染,为进入皮层做准备;接种后6 h病菌开始在表皮定殖,并侵入皮层;接种后9 h在皮层可观察到侵染性菌丝沿着细胞间隙向相邻细胞延伸;接种后12 h栗疫病菌侵入韧皮部,在皮层的侵染面积扩大。随着侵染程度加深,皮层、韧皮部等处细胞被菌丝降解,最终在形成层附近聚集。接菌后9 h为栗疫病菌侵染板栗枝条的关键时间点。     

Histopathology of compatibility and incompatibility between oilseed rape and Albugo Candida

Cotyledons of one resistant and three susceptible rape lines/cultivars were inoculated with zoospores of Albugo Candida race 7. Samples of whole cotyledons were examined by differential interference contrast microscopy. The time course of the infection process was followed histologically. Germination of zoospore cysts occurred 2-3 h after inoculation. Infection was initiated with germ-tubes penetrating through stomata. Haustorium formation was first observed in the palisade mesophyll cells adjacent to the substomatal chambers 8 h after inoculation.
Only after the establishment of the first haustorium did compatible and incompatible interactions begin to differentiate. In the resistant cultivar, most primary hyphae produced single haustoria. Necrosis of the invaded host cell was first observed 12 h after inoculation followed by cessation of fungal growth. The death of host cells was largely restricted to the penetration site; the adjacent non-penetrated cells remained apparently unaffected. In the susceptible hosts, necrosis of infected cells occurred only infrequently, and hyphal growth continued unabated, resulting in mycelial ramification into the mesophyll. Numerous haustoria were produced.
Histological studies showed that the earliest event distinguishing a compatible from an incompatible interaction occurred after formation of the first haustorium and that resistance was not manifested until the host mesophyll cell had come into contact with the first haustorium. The distinction between compatibility and incompatibility was substantiated by quantitative analysis of white rust development on both resistant and susceptible lines/cultivars.     

Infection of wheat spikes by <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> and alterations of cell wall components in the infected tissue

The infection process of Fusarium avenaceum on wheat spikes and the alteration of cell wall components in the infected host tissue were examined by means of electron microscopy and cytochemical labelling techniques following spray inoculation at growth stage (GS) 65 (mid-flowering). Macroconidia of the pathogen germinated with one to several germ-tubes 6–12 h after inoculation (hai) on host surfaces. The germ-tubes did not penetrate host tissues immediately, but extended and branched on the host surfaces. Hyphal growth on abaxial surfaces of the glume, lemma and palea was scanty 3–4 days after inoculation (dai) and no direct penetration of the outer surfaces of the spikelet was observed. Dense mycelial networks formed on the inner surfaces of the glume, lemma, palea and ovary 36–48 hai. Penetration of the host tissue occurred 36 hai by infection hyphae only on the adaxial surfaces of the glume, lemma, palea and upper part of ovary. The fungus penetrated the cuticle and hyphae extended subcuticularly or between the epidermal wall layers. The subcuticular growth phase was followed by penetration of the epidermal wall, and hyphae spread rapidly inter- and intracellularly in the glume, lemma, palea and ovary. During this necrotrophic colonization phase of the wheat spike, a series of alterations occurred in the host tissues, such as degeneration of cytoplasm and cell organelles, collapse of host cells and disintegration of host cell walls. Immunogold labelling techniques showed that cell walls of spike tissues contained reduced amounts of cellulose, xylan and pectin near intercellular hyphae or infection pegs compared to walls of healthy host tissues. These studies suggest that cell wall degrading enzymes produced by F. avenaceum facilitated rapid colonization of wheat spikes. The different penetration properties of abaxial and adaxial surfaces of the spikelet tissues as well as the two distinct colonization strategies of host tissues by F. avenaceum are discussed. The penetration and colonization behaviour of F. avenaceum in wheat spikelets resembled that of F. culmorum and F. graminearum, although mycotoxins produced by F. avenaceum differed from those of the latter two Fusarium species.     

Infection and colonization of strawberry by <Emphasis Type="Italic">Gnomonia fragariae</Emphasis> strain expressing green fluorescent protein

Gnomonia fragariae is a poorly studied ascomycete, which was recently demonstrated to be a cause of severe root rot and petiole blight of strawberry. The pathogen was genetically transformed with the GFP as a vital marker and hygromycin resistance gene. Several stable transformants were obtained, which did not differ in their phenotype from the wild type isolate. Using one of the GFP-tagged isolates the infection process and colonization of roots and petioles of host plant by the pathogen were studied. Fluorescence microscopy examinations of the inoculated plants at different time points showed that plant infection occurs 24 h after inoculation and intensively continues during first 3 days. The specific penetration sites on epidermal cells and preferences in colonization for certain root and petiole tissues were observed. The pathogen intensively colonized and destroyed cortex of roots and petioles and spread rapidly longitudinally within intercellular spaces. The petioles were colonized by the hyphae, which grew mostly in the intracellular spaces of the cortical cells while in the roots the intracellular growth of hyphae occurred only in the later stages of infection. The fungus was also capable to infect the vascular tissues of petioles although these were not the primary tissues colonized by the pathogen. The mature ascomata were formed on the infected petiole bases several weeks after the inoculation. This study presents a genetic transformation method for Gnomonia fragariae and it demonstrates details on infection process and colonization of root, crown and petiole tissues of strawberry by the pathogen.     

Histological characterization of the early-stage infection events of Setosphaeria turcica in maize

Northern corn leaf blight (NCLB) caused by Setosphaeria turcica is a major foliar disease of maize. The early-stage infection events of this pathogen on maize leaves are unclear. We investigated the optimum temperature for conidial germination and appressorium formation, and characterized penetration and growth of S. turcica in maize leaf sheath and onion epidermis cells, including use of histological staining to assess plant cell viability. The results showed that the optimum temperature for conidial germination and appressorium formation was 20°C. On the maize leaf sheath, the appressoria were formed by germinated conidia, and penetration on the epidermal cells occurred at 8 h postinoculation (hpi). Round vesicles developed beneath the appressoria. Between 16 and 24 hpi, the branched invasive hyphae invaded three to five adjacent cells at most infection sites. The invasive hyphae tended to move along the cell wall and crossed from one cell to another. In the onion epidermis cells, the appressoria formed at 8 hpi, and in most cases the epidermal cells were penetrated through the juncture of the cell walls. At 16–24 hpi, the primary hyphal terminus swelled to a vesicle. The maize leaf sheath cells died at 8 hpi, whereas the onion cells did not. Our findings documented in detail the penetration and invasive hyphal growth in maize leaf sheath and onion epidermis, as well as viability of plant cells, at the early stages of infection, and provide a foundation for elucidating the underlying mechanism of S. turcica–maize interactions.     

禾谷镰刀菌在小麦穗部侵染过程的细胞学研究

 采用扫描和透射电镜技术系统地观察了禾谷镰刀菌(Fusarium graminearum)在小麦穗部的侵染过程。接种后6~12 h,分生孢子在小麦穗部的任何部位均可萌发,每个孢子可产生1至多个芽管,新产生的芽管并不立即入侵寄主组织,而是在寄主体表生长扩展;接种后36~48 h观察,小穗颖片、外稃、内稃的内侧和子房的表面形成了密集的菌丝网,然而在小麦穗轴表面、颖片和内稃的外表面,菌丝生长缓慢、分布稀疏,但颖片外表边缘的菌丝可跨越边缘扩展到颖片的内表皮上;接种后36 h,寄主体表菌丝产生入侵菌丝,以直接入侵方式由颖片、外稃、内稃的内侧及子房的顶部侵入寄主组织体内,随后,菌丝以胞间和胞内生长的方式向下扩展;接种后4~5 d,菌丝由上述组织扩展到达穗轴后,在穗轴内沿微管束组织和皮层组织向上和向下扩展,延伸到相邻小花,随菌丝在小麦穗部组织内不断地生长扩展,使得寄主细胞坏死、解体,并最终导致整个麦穗的枯死。     

小麦穗组织中脱氧镰刀菌烯醇毒素的免疫细胞化学定位

 采用免疫细胞化学技术对禾谷镰刀菌(Fusarium graminearum)在侵染小麦穗部过程中产生的脱氧镰刀菌烯醇毒素(deoxynivalenol,DON)进行了定位分析。在接种后24h,当菌丝在外稃、内稃的内侧表面扩展而尚未侵入寄主细胞前,病菌已分泌DON,并且DON已扩散到寄主组织内。在菌丝细胞内,DON主要被定位于细胞质、线粒体及细胞壁上;在寄主细胞中DON主要分布于细胞壁、叶绿体、细胞质和内质网上。在侵染初期(接种后2 d),菌丝仅能在寄主细胞间隙扩展,随寄主组织中DON浓度的升高,寄主细胞相应发生了一系列病理变化。随寄主细胞坏死(接种后3~4d),病菌进入坏死的寄主细胞。上述结果表明,DON在禾谷镰刀菌的侵染、致病和定殖过程中起着重要的作用。毒素标记结果表明病菌产生的毒素可通过穗轴微管束组织从侵染部位向上、向下转输,毒素向上的转输量明显高于向下转输     

Histological studies of the expression of the Lr9, Lr20 and Lr28 alleles for resistance to leaf rust in wheat

The development of the leaf rust fungus ( Puccinia recondita f.sp. tritici ) in a susceptible cultivar and three other cultivars possessing the Lr9, Lr20 and Lr28 alleles for resistance was studied by light and fluorescence microscopy. Formation of the substomatal vesicle, intercellular hypha and the first haustorial mother cell was unaffected by resistance. Lr9 and Lr28 expression was rapid, first seen as early initiation of hyphal branching at 16 h after inoculation, then reduced haustorial diameters at 19 h. Limited host cell necrosis was seen immediately afterwards. Elongation of intercellular hyphae was reduced between 20 and 24 h, and virtually ceased by about 30 h. Numbers of infection sites with a second haustorial mother cell were briefly higher at 24 h. Reduced hyphal branching and haustorial mother cell numbers were seen at 20–24 h and 36 h respectively. Lr20 expression was not seen until 36 h when reduced hyphal branching was observed, accompanied by extensive host cell necrosis. Reduced haustorial mother cell numbers were detected at 48 h. Findings suggested a secondary role for host cell necrosis in the expression of the Lr9 and Lr28 alleles. Host necrosis may play a determinant role in Lr20- based expression.