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.     

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。     

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。     

Ultrastructure of the Penetration and Infection of Pansy Roots by Thielaviopsis basicola

ABSTRACT Transmission electron microscopy was used to study the penetration and infection of pansy roots by Thielaviopsis basicola. Events observed in 7- to 10-day-old roots produced on moist filter paper differed slightly from those in roots from 4-week-old plants washed free of potting media prior to inoculation. By 3 h postinoculation (PI), epidermal cells of roots produced on filter paper exhibited aggregated cytoplasm and papilla formation in response to germ tube tips. The presence of callose in papillae was demonstrated using immunogold labeling. Papilla formation was not effective in preventing host cell penetration. A slender infection hypha emerged from a germ tube tip and grew through a papilla. Its tip then expanded to form a globose infection vesicle. By 6 h PI, infection hyphae emerged from infection vesicles, and invaded host cells showed signs of necrosis. By 8 h PI, infection hyphae had grown into cortical cells in spite of papilla formation in these cells. By 24 h PI, distinctive intracellular hyphae were present in necrotic cortical cells. In washed roots, most epidermal cells failed to respond to invasion. Hyphae simply grew through these cells and contacted cortical cells that exhibited aggregated cytoplasm and papillae formation. Infection structures similar to those produced in epidermal cells from roots grown on filter paper then formed in cortical cells of washed roots. The fact that T. basicola formed infection structures only in cells that responded to invasion suggests that T. basicola has a more complex relationship with its host than would be expected in a nectrotrophic pathogen. We believe that T. basicola is best described as a necrotrophic hemibiotroph.     

球毛壳ND35菌株在宿主植物上的侵染定殖

为了解球毛壳Chaetomium globosum ND35菌株在宿主植物上的侵染定殖方式和途径,以毛白杨组培苗为宿主植物,借助光学显微镜、扫描电镜、透射电镜,结合免疫荧光标记技术,研究了球毛壳ND35菌株子囊孢子萌发后在毛白杨上的侵染行为及其菌丝在组培苗根部的定殖。结果显示,子囊孢子萌发后形成的菌丝,能从杨树苗根、茎部表面细胞间的缝隙侵入或在根表面形成附着胞,进而形成侵染钉直接从表皮细胞侵入,在叶部主要从气孔侵入叶片内部。侵入根部的菌丝主要定殖于表皮细胞、外皮层细胞和细胞间隙,未进入内皮层和维管束组织。     

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.     

Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare

Colletotrichum gloeosporioides f. sp. malvae, isolate Biomal®, ATCC 20767, was originally isolated from round-leaved mallow (Malva pusilla) and developed as a weed biocontrol agent. Ribosomal DNA sequence analysis was recently used to re-classify this fungus as C. orbiculare, which is an aggregate species with a number of formae speciales. Several morphological features of ATCC 20767 were examined that were consistent with those described for C. orbiculare, and inoculation of a number of Nicotiana species and several cultivars of N. tabacum showed that this fungus was pathogenic to many of these previously undescribed hosts. Spore germination and appressorium formation were higher on tobacco than previously observed on round-leaved mallow. The pathogen produced melanized appressoria on N. tabacum leaves that formed preferentially at the anticlinal epidermal cell wall. A symptomless phase of infection persisted for 72–96 h postinoculation, during which time the fungus first produced a spherical infection vesicle from an infection peg, and then large primary hyphae which grew through the epidermal cells. The large primary hyphae were highly constricted at the points of penetration of the host cell walls. Thin secondary hyphae appeared at 96–120 h postinoculation coinciding with the appearance of light green, water-soaked spots and the formation of acervuli. The infection of tobacco by C. orbiculare ATCC 20767 is not a non-specific interaction but appears to follow an intracellular hemibiotrophic infection process that is very similar to that established for the C. orbiculare infection of round-leaved mallow, cucurbits and beans.     

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。     

Ultrastructure of the Infection of Sorghum bicolor by Colletotrichum sublineolum

ABSTRACT Ultrastructural studies of the infection of susceptible and resistant cultivars of Sorghum bicolor by Colletotrichum sublineolum were conducted. Initial penetration events were the same on both susceptible and resistant cultivars. Germ tubes originating from germinated conidia formed globose, melanized appressoria, that penetrated host epidermal cells directly. Appressoria did not produce appressorial cones, but each penetration pore was surrounded by an annular wall thickening. Inward deformation of the cuticle and localized changes in staining properties of the host cell wall around the infection peg suggests that penetration involves both mechanical force and enzymic dissolution. In compatible interactions, penetration was followed by formation of biotrophic globular infection vesicles in epidermal cells. Filamentous primary hyphae developed from the vesicles and went on to colonize many other host cells as an intracellular mycelium. Host cells initially survived penetration. The host plasma membrane invaginated around infection vesicles and primary hyphae and was appressed tightly to the fungal cell wall, with no detectable matrix layer at the interface. Necrotrophic secondary hyphae appeared after 66 h and ramified through host tissue both intercellularly and intracellularly, forming hypostromatic acervuli by 114 h. Production of secondary hyphae was accompanied by the appearance of electron-opaque material within infected cells. This was thought to represent the host phytoalexin response. In incompatible interactions, infection vesicles and primary hyphae were formed in epidermal cells by 42 h. However, they were encrusted with electron-opaque material and appeared dead. These observations are discussed in relation to the infection processes of other Colletotrichum spp. and the host phytoalexin response.     

利用绿色荧光蛋白GFP研究稻瘟病菌与水稻的互作

稻瘟病菌Magnaporthe oryzae严重威胁水稻的产量与质量,明确稻瘟病菌与水稻互作过程及机理,对防治稻瘟病具有重要意义。本研究利用稻瘟病菌常用致病菌株GUY11和ZB25,构建了绿色荧光蛋白GFP的过量表达菌株,并通过荧光显微观察菌株侵染寄主水稻过程中侵染结构的形成与发育,包括孢子萌发、附着胞形成、侵染钉形成、侵染菌丝增殖、坏死斑形成及产孢。另外,通过比较过量表达菌株对稻瘟病高抗水稻和易感水稻的侵染过程,发现侵染过程的差异主要集中于侵染钉的穿透和侵染菌丝的定殖。本研究为分析稻瘟病菌对寄主水稻的定殖规律提供了一种有效工具。     

The development of different pathotype groups of Mycosphaerella pinocles in susceptible and partially resistant pea leaves

The progress of infection by high- and low-virulence isolates of Mycosphaerella pinodes was examined in susceptible and partially resistant pea leaves. Conidia germinated with one or more germ tubes which frequently branched and formed appressorium-like structures on the leaf surface. Penetration occurred through the epidermal walls. A structure similar to an infection vesicle was formed, lying partly in the epidermal wail and partly in the cell lumen. From this structure, a penetration hypha was derived which initiated the development of the intra- and intercellularly-growing fungal colony. Infections led to rapid tissue collapse in both susceptible and resistant interactions. In resistant interactions, the formation of infection vesicles and penetration hyphae was reduced, and the development and spread of lesions was retarded.     

小麦雪霉叶枯病菌的侵染过程

 本文报道了小麦雪霉叶枯病菌Gerlachia nivalis的叶面侵染过程。该菌分生孢子发芽后相互结合,形成网状复合体,产生粗壮的叶面菌丝,经由气孔保卫细胞间隙侵入,不产生特化的侵染机构。侵染菌丝在叶肉细胞间和细胞内扩展,也可由气孔逸出在叶面蔓延。分生孢子梗由分生孢子座上产生,由气孔抽出。产孢细胞顶端有环痕。叶面菌丝亦能产孢。寄主组织病变特点表明该菌产生活性很强的胞外酶。     

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。     

Histological investigation of stripe rust (Puccinia striiformis f.sp. tritici) development in resistant and susceptible wheat cultivars

The wheat cultivar Kariega expresses complete adult plant resistance against stripe rust, whereas cv. Avocet S is susceptible. Using confocal laser scanning microscopy, initial fungal penetration into flag leaves was identical in both cultivars, with directional germ-tube growth towards stomata that were penetrated without the formation of an appressorium, followed by differentiation of a substomatal vesicle, infection hyphae, haustorial mother cells and haustoria. During the following 4 days, further fungal development occurred more quickly in the resistant than in the susceptible cultivar. However, by 7 days postinoculation (dpi) the situation changed, with exponential growth of the pathogen occurring only in the susceptible line. Induced cellular lignification, a typical defence reaction of cereals, was observed at 4 dpi in the resistant cultivar, and 2 days later lignified tissue completely surrounded the fungal colonies. In the susceptible cultivar, isolated lignified host cells occurred at 6 dpi, and long, unbranched fungal hyphae outgrowing the resistance reaction were observed.     

Hemibiotrophic infection of Pisum sativum by Colletotrichum truncatum

The infection of pea ( Pisum sativum ) by Colletotrichum truncatum was studied by light and electron microscopy. These investigations were facilitated by use of an Argenteum pea mutant, which has a readily detachable epidermis. Infection pegs emerging from appressoria penetrated epidermal cells directly. Large intracellular primary hyphae formed a dense stromatic mycelium confined within a single epidermal cell. Primary mycelia gave rise to thinner secondary hyphae which radiated into surrounding cells and caused extensive wall dissolution. Melanized sclerotia developed in the centre of chlorotic water-soaked lesions. Acervuli were not observed. Epidermal cells survived initial penetration by primary hyphae, as shown by their ability to plasmolyse and accumulate Neutral red, but all infected cells were dead when the secondary hyphae had formed. Six cultivars of pea were susceptible, but seven other legumes were resistant. A single isoform of polygalacturonase with a pI of 8·3 and apparent M _r of 40000 was purified from culture filtrates and the TV-terminal amino acid sequence determined. The relevance of the results to the taxonomy of C. truncatum and the relationships between infection process and host range are discussed.     

Histopathology of the initial stages of the interaction between rose flowers and Botrytis cinerea

The early stages of the interaction between flowers of the cut rose cv. Sonia andBotrytis cinerea was investigated by scanning electron microscopy and light microscopy. Infection of petals by conidial germ tubes evoked a susceptible reaction. In contrast to general findings nutrient addition to the inoculum was not a prerequisite for this phenomenon. At the lower side of germ tube tips the cuticle was penetrated by infection pegs. Already at this early stage of the infection process, the infection sites were macroscopically visible as scattered white spots. After penetration, pegs enlarged to form infection hyphae, which invaded the periclinal wall of outer epidermal cells. At those sites, the petals formed outgrowths of variable appearance at their abaxial side. Thee outgrowths consisted of remanants of collapsed epidermal cells and of infection hyphae. Subsequent intra- and intercellular growth of hyphae led to a collapse of epidermal and mesophyll cells. The symptoms described generally developed within 24 h. After subsequent incubation the lesions became necrotic. Eventually, the necrosis would spread leading to the death of whole petals.     

Cytology of the host-pathogen interactions between Lolium perenne and Drechslera dictyoides

The interactions between Drechslera dictyoides and Lolium perenne resulting in the development of the net blotch lesion were examined using light and electron microscopy. Following appressorium formation a reorganization of the network of cytoplasmic strands in underlying epidermal cells resulted in the accumulation of cytoplasm and the migration of the nucleus towards the appressorium. Successful penetration was followed by extensive colonization of the invaded epidermal cell. Abortive penetration attempts often resulted in the formation of hyphae within the epidermal cell wall. During lesion development, advancing hyphae came into contact with unmodified host cells which responded with the production of wall appositions. Hyphae were sparsely distributed within the developing lesions and their sparingly branched, predominantly linear pattern of growth resulted in the formation of discrete net lesions. Prior to the commencement of sporulation, the density of hyphae increased and the invasion of moribund cells of the senescent host tissue was observed.     

核盘菌侵入油菜超微结构及侵染机制的研究

 通过电子显微镜观察核盘菌在油菜叶片上侵染过程,发现该菌首先在叶片上形成复合附着器。每个分枝末端一般生出一个侵染钉。侵染钉侵入叶表面腊质、角质层和表皮细胞壁时.不仅靠附着器产生的压力,而且供助于酶对寄主表面的软化、消解作用。该菌通过角质层和表皮细胞壁侵入油菜叶片,尚未发现通过气孔侵入的现象。侵入叶片后,该菌的继续生长,导致了油菜组织的溃烂。然后菌丝在腐烂的叶片上集结形成菌核。     

The early development of Erysiphe pisi on Pisum sativum L.

Erysiphe pisi , the powdery mildew pathogen of Pisum sativum , followed a developmental sequence that allowed the identification of 10 distinct growth stages (GS) over 30 h following inoculation. The growth stages were ungerminated conidia (GS1), germinated conidia, having produced a germ tube (GS2), germlings where the germ tube had forked (GS3), germlings with a multi-lobed germ tube (GS4), germlings with a single hypha (GS5), germlings with two (GS6 and 7) or three (GS8 and 9) hyphae, one of which may have formed from the appressorium (GS7 and 9), and germlings with abnormally long germ tubes (GS10), which did not develop hyphae. Conidia germinated rapidly, with a quarter of conidia producing germ tubes by 2 h after inoculation (hai). Most germlings produced multi-lobed appressoria, which showed considerable variation in structure. Haustoria, although often difficult to visualize, were first seen 4 hai, and the first hyphae 14 hai, growing from the body of the conidium. Subsequent hyphae developed from both the body of the conidium and from the appressorium.     

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.