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。     

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。     

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。     

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。     

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。     

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。     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

稻叶黑粉病菌侵染水稻叶片的细胞学研究

稻叶黑粉病是由担子菌Entyloma oryzae侵染水稻叶片或叶鞘引起的真菌病害，但对病原菌与寄主互作的细胞学机制一直缺少了解。本文对采自田间自然发病叶片上的病斑进行了初步的细胞学分析，结果发现，病原菌侵染后，寄主病斑部位的表皮细胞外部形态基本保持完整；病原菌主要在寄主叶肉细胞部位产生大量的厚垣孢子并逐渐取代叶肉细胞；病原菌菌丝在寄主胞外扩展，未见其穿透寄主细胞壁进入细胞内，也没有产生典型的真菌吸器。靠近病原菌菌丝的寄主各种细胞内的细胞器均发生降解，降解产生的脂类物质凝聚成了体积较大的脂质球。寄主维管束组织的细胞壁一直保持完整，未发现病原菌菌丝进入维管束，病原菌菌丝和孢子被限制在寄主相邻两个维管束之间。在发病后期，由于寄主叶片表皮结构整体性破坏导致大量细菌进入，加速了叶片的衰老死亡。本研究结果表明，稻叶黑粉病菌的侵染模式为胞外侵染，类似于活体营养真菌；但病原菌的侵染导致附近寄主细胞降解死亡，类似于腐生营养真菌。     

禾顶囊壳小麦变种对小麦种子根的侵染过程

 光镜和电镜观察表明,禾顶囊壳小麦变种(Gaeumannomyces graminis var.tritici,小麦全蚀病菌)对小麦种子根的侵染过程可分为侵入前、侵入表皮层、进入皮层和进入中柱等4个连续阶段。麦根接菌后在15℃下培养,48 h后侵入表皮层细胞,60 h后进入皮层,120 h后进入中柱。病原菌主要以侵染菌丝直接侵入表皮层,表皮细胞间隙和根毛基细胞是主要侵入部位,少数由附着枝侵入。菌丝穿透细胞壁有明显的酶解作用特征,菌丝先端前方胞壁上还产生电子密物质。皮层细胞是病原菌定殖和发展的主要场所,病原菌还能离解胞间层,形成胞外空间,特别有利于菌丝和菌丝束的扩展。在侵入位点的寄主细胞壁和质膜之间,形成多种形状的木质管,其数量与侵入菌丝的数目相对应,但木质管不能阻止菌丝进入细胞。菌丝进入中柱后,可阻塞导管和筛管。小麦细胞发生退行性病变,尤以细胞壁膨大崩坏和早期质壁分离最明显,细胞间隙还产生性质不明的黄色物质。     

小麦条锈菌夏孢子阶段核相状况的研究

 本文应用电镜和荧光染色技术对小麦条锈菌夏孢子阶段的细胞核相状况进行了系统研究。观察发现夏孢子通常为双核体,夏孢子萌发的芽管可为双核、三核或四核,但仍以双核芽管为主。尽管在胞间菌丝和吸器母细胞中可观察到典型的双核细胞,但胞间菌丝和吸器母细胞中的多核现象极为普遍。经连续切片观察证实,吸器的细胞核可为双核、三核、四核、五核和六核,一般以四核为主。夏孢子堆中的不同类型的细胞均为双核。上述研究结果表明,小麦条锈菌的细胞核相状况较为复杂,并且显然不向于其它锈菌。关于小麦条锈菌多核现象的生物学意义值得进一步研究。     

荸荠茎点霉秆枯病菌侵染过程的超微观察

<正>荸荠(Eleocharis dulcis),又称马蹄,为莎草科多年生草本植物,是一种具有食用和药用价值的水生蔬菜。近年来,随着荸荠在我国种植面积的不断扩大,病害发生也呈逐年上升趋势。荸荠茎点霉秆枯病是2009年在湖北省荸荠产区发现的一种新病害,由Phoma bellidis侵染引起,该病在湖北省团风地区发生尤为严重,对荸荠的产量和品质造成严重影响;病害一般在8~12月发生,发病初期在荸荠茎秆上产生圆形或梭形红褐色小斑,随后病斑沿茎     

Scanning electron microscopy study of infection structure formation of Puccinia graminis f.sp. tritici in host and non-host cereal species

The development of uredospore-derived infection structures of Puccinia graminis f.sp. tritici in wheat, barley, sorghum and maize was examined by scanning electron microscopy (SEM). Germ tubes grew over the leaf surface until a stoma was located. An appressorium formed over the stoma and the leaf was penetrated by an infection peg. Within the substomatal chamber of all species the infection peg developed a substomatal vesicle by 6 h post-inoculation (hpi). from which a primary infection hypha developed parallel to the long axis of the leaf. In wheat, barley and maize, when a primary infection hypha abutted onto a host cell, a septum was laid down between the tip of the hypha and the substomatal vesicle, delimiting a haustorial mother cell by 12 hpi; haustorial mother cells did not form in sorghum. Secondary infection hyphae arose on the substomatal vesicle side of the septum; infection did not progress further in maize, but in wheat and barley secondary infection hyphae branched, and proliferated intercellularly forming the fungal thallus. A haustorial mother cell was delimited when an intercellular hypha abutted onto a host cell. Infection sites with haustorial mother cells were observed at 12 hpi in barley and 24 hpi in wheat. In all four plant species, some atypical substomatal vesicle initials, substomatal vesicles and primary infection hyphae were observed.     

Cytology of infection of apple leaves by Diplocarpon mali

Diplocarpon mali, the causal agent of Marssonina leaf blotch of apple, causes severe defoliation during the growing season. Little information is available on the mode of infection and the infection process. In this study, the infection strategies of D. mali in apple leaves were investigated using fluorescence and electron microscopy. Conidia attached to leaf surface apparently by mucilage and germinated on both sides of leaves 6 h post-inoculation (hpi). The pathogen penetrated the cuticle by infection pegs formed either in germ tubes or appressoria in 6 hpi, and then formed haustoria in host epidermal and mesophyll cells accompanied by extension of subcuticular and intercellular hyphae. Five days post-inoculation (dpi), the intracellular hyphae were observed. At the same time, the subcuticular hyphal strands (SHS) were produced as a means for fast expansion and reproduction. About 7 dpi, acervuli formed on inoculated leaves. This was the first observation that D. mali formed haustoria and SHS as infection strategies. Our results suggest that D. mali may behave like a hemibiotroph, which can use both biotrophic and necrotrophic strategies to establish infections on apple leaves.     

Initial Infection and Colonization of Leaves and Stems of Cling Peach by Tranzschelia discolor

ABSTRACT Initial infection processes and the subsequent colonization of leaves and young stems of peach by Tranzschelia discolor were studied. On leaves where multiple disease cycles of peach rust occur during the growing season, urediniospores germinated after 4 h of wetness. Germ tubes became septate and formed appressoria only over leaf stomata beginning 18 h after inoculation. No appressoria, however, formed over stomata of positive replicas of leaf surfaces indicating nonthigmotropic responses of germ tubes. On young, primary-growth stems (ca. 8 weeks old), stomata were mostly closed, less frequent than on leaves, and recessed from the surface of the cuticle of the epidermis. Although appressoria formation was not observed on inoculated stems, germ tube growth of urediniospores was directional toward stomata. Penetration of stem tissue is apparently a less common event that was reflected by a lower occurrence of stem lesions compared with that of leaf lesions in our potted plant inoculation studies and previous field observations. Still, stem lesions are important as sources of primary inoculum each spring and were reproduced in this study for the first time. Fungal colonization of leaves and stems was subepidermal-intercellular and haustoria were commonly found within mesophyll or cortical cells, respectively. No fungal colonization was observed in cambial stem tissue. Vascular tissue was also not colonized and delimited lesions in leaves and stems. Morphological host responses were not observed in infections on either leaves or young stems. In older stems (>32 weeks old), however, the infection was delimited by a wound periderm after uredinial formation. Furthermore, with continued secondary growth, stems recovered and fungal lesions became part of the bark tissue of woody branches. Thus, the fungus must infect primary-growth branches each year to establish stem lesions.     

Nonhost versus host resistance to the grapevine downy mildew, Plasmopara viticola, studied at the tissue level

Following inoculation of host and nonhost plants with Plasmopara viticola, the grapevine downy mildew, a histological survey was undertaken to identify the stage where its development is contained in nonhosts and in resistant host plants. Three herbaceous nonhost species, Beta vulgaris, Lactuca sativa, and Capsicum annuum, and three grapevine species displaying different level of resistance (Vitis vinifera [susceptible], Vitis riparia [partially resistant] and Muscadinia rotundifolia [totally resistant]) where inoculated by P. viticola using a controlled leaf disk inoculation bioassay. During the early steps of infection, defined as encystment of zoospores on stomata, penetration of the germ tube, and production of the vesicle with the primary hypha, there was no evidence of a clear-cut preference to grapevine tissues that could attest to host specificity. The main difference between host grapevine species and nonhosts was observed during the haustorium formation stage. In nonhost tissues, the infection was stopped by cell wall-associated defense responses before any mature haustorium could appear. Defense responses in resistant grapevines were triggered when haustoria were fully visible and corresponded to hypersensitive responses. These observations illustrate that, for P. viticola, haustorium formation is not only a key stage for the establishment of biotrophy but also for the host specificity and the recognition by grapevine resistance factors.     

Leaf wax layer may prevent appressorium differentiation but does not influence orientation of the leaf rust fungus Puccinia hordei on Hordeum chilense leaves

The infection process of most rust fungi start with spore germination, directional growth of the germ tube towards a stoma, differentiation of an appressorium over the stoma, and penetration into the substomatal cavity. In the South American wild barley Hordeum chilense Roem. & Schult., wide variation occurs in the degree to which several rust fungal species are able to form appressoria over the stomata. Apparently, features of the plant may hamper early stages of the infection process. Such an early defence is called avoidance. In order to find out how germ tube growth is directed towards stomata, and whether the cuticular wax layer plays a role in this orientated growth and in appressorium differentiation, several orientation and differentiation parameters of Puccinia hordei germ tubes were measured on H. chilense leaves with and without the wax layer. Orientated growth of the germ tubes started upon contact with the epidermal cell junctions. The growth of lateral branches of the germ tube over the first epidermal cell junction that it meets, may help the germ tube to grow along the transverse axis of the leaf. No evidence was found of attraction of the germ tube to stomata. Removal of the cuticular wax layer did not result in loss of germ tube orientation. This suggests that the leaf wax layer has no role in the guidance of germ tubes. On high avoidance accessions, removal of the wax layer allowed appressoria to develop over stomata that would otherwise be overgrown. No effect of the cell widths in stomatal complexes was found on the chance that stomata were overgrown. This suggests that the overgrowth of stomata on H. chilense leaves by P. hordei germ tubes is mainly caused by the wax covering of the stomatal apparatus.