Ultrastructural characterisation of the host–pathogen interface in white blister-infected Arabidopsis leaves

In this study transmission electron microscopy (TEM) was used to examine details of the host–pathogen interface in Arabidopsis thaliana cotyledons infected by Albugo candida, causal agent of white blister. After successful entry through stomatal pores, the pathogen developed a substomatal vesicle and subsequently produced intercellular hyphae. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were spherical and 4.5 μm in diameter. Each haustorium was connected to intercellular hyphae by a narrow, slender haustorium neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. No obvious response was observed in host cells following formation of haustoria. Most of the mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cells suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotrophic Oomycete differs considerably from responses to other pathogens such as necrotrophs. Modification of the host plasma membrane (PM) along the cell wall and around the haustoria, was detected by applying the periodic acid-chromic acid-phosphotungstic acid (PACP) staining technique. After staining with PACP, the host PM was found to be intensely electron dense where it was adjacent to the host cell wall and the distal region of the haustorial neck. By contrast, the extrahaustorial membrane, where the host PM surrounded the haustorium, was consistently very lightly stained.     

The infection biology of Fusarium graminearum: defining the pathways of spikelet to spikelet colonisation in wheat ears

Fusarium graminearum is one of the main causal agents of Fusarium Ear Blight on wheat. How the pathogen colonises the entire ear is not known. There is controversy over whether this mycotoxin producing pathogenic fungus invades wheat floral tissue using a necrotrophic or another mode of nutrition. A detailed microscopic investigation has revealed how wild-type fungal hyphae, of the sequenced strain PH-1, colonised susceptible wheat ears and spread from spikelet to spikelet. At the advancing infection front, colonisation of the host cortex occurred ahead of any vascular colonisation and the hyphae adapted to the available intercellular space between host cells. Intercellular hyphae then became abundant and host cells lost their entire cellular contents just prior to intracellular colonisation. No host cells died ahead of the infection. However, while these deep cortex infections progressed, just below the surface the highly photosynthetic chlorenchyma cells were observed to have died prior to colonisation. Behind the infection front, hyphae were abundant in the vasculature and the cortex, often growing through the pit fields of thick walled cells. This high level of inter- and intracellular fungal colonisation resulted in the collapse of the non-lignified cell-types. In this middle zone of infection, hyphal diameters were considerably enlarged. Far behind the infection front inter- and intracellular hyphae were devoid of contents and had often collapsed. At later stages of infection, the pathogen switched from predominately vertical to lateral growth and accumulated below the surface of the rachis. Here the lignified host cell walls became heavily degraded and hyphae ruptured the epidermis and produced an aerial mycelium.     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

Herpobasidium filicinum (Eocronartiaceae,Platygloeales) occurs on Dennstaedtia wilfordii (Dennstaedtiaceae) in Japan

A fungus parasitic on a fern, Dennstaedtia wilfordii (Dennstaedtiaceae), was found at the foothill of Mt. Fuji, Yamanashi Prefecture, Japan. Its hyphae spread within host mesophyll cells and through intercellular spaces, forming coiled haustoria in the epidermal and mesophyll cells. The hyphae emerged either through stomata or by disrupting epidermal cell junctions. The hyphae spreading over the abaxial leaf surface generated one-septate, thin-walled basidia. All the morphological features observed were characteristic of the genus Herpobasidium. The species identification of the fungus as H. filicinum by morphology was supported by molecular phylogenetic analyses of the D1/D2 region of the large subunit rRNA gene.     

The responses of guard and mesophyll cell photosynthesis to CO2, O2, light,and water stress in a range of species are similar

High resolution chlorophyll a fluorescence imaging was used to compare the photosynthetic efficiency of PSII electron transport (estimated by Fq'/Fm') in guard cell chloroplasts and the underlying mesophyll in intact leaves of six different species: Commelina communis, Vicia faba, Amaranthus caudatus, Polypodium vulgare, Nicotiana tabacum, and Tradescantia albifora. While photosynthetic efficiency varied between the species, the efficiencies of guard cells and mesophyll cells were always closely matched. As measurement light intensity was increased, guard cells from the lower leaf surfaces of C. communis and V. faba showed larger reductions in photosynthetic efficiency than those from the upper surfaces. In these two species, guard cell photosynthetic efficiency responded similarly to that of the mesophyll when either light intensity or CO2 concentration during either measurement or growth was changed. In all six species, reducing the O2 concentration from 21% to 2% reduced guard cell photosynthetic efficiency, even for the C4 species A. caudatus, although the mesophyll of the C4 species did not show any O2 modulation of photosynthetic efficiency. This suggests that Rubisco activity is significant in the guard cells of these six species. When C. communis plants were water-stressed, the guard cell photosynthetic efficiency declined in parallel with that of the mesophyll. It was concluded that the photosynthetic efficiency in guard cells is determined by the same factors that determine it in the mesophyll.     

Infection of Subterranean Clover (Trifolium subterraneum) by Kabatiella caulivora

Kabatiella caulivora is a serious pathogen of clover ( Trifolium ) spp. Subterranean clover ( T. subterraneum ) cv. Woogenellup was inoculated with K. caulivora , to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10–11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15–18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease.     

Compared Anatomy of Young Leaves of Prunus persica (L.) Batsch with Different Degrees of Susceptibility to Taphrina deformans (Berk.) Tul

Anatomical observations of leaves infected by Taphrina deformans were studied in tolerant peach trees (TPT) and in very susceptible (VSPT) ones. Leaves from the first sampling (2nd April) showed hyphae penetrating through the stomata or into the cuticle of the host tissue; anatomical structures of leaf sections were similar for both TPT and VSPT. The ultrastructure of the leaves of TPT showed seemingly normal mesophyll cells. In contrast, mesophyll cells of the VSPT showed important signs of degradation. Cells were organelle‐free and the middle lamella was expanded and invaded by hyphae of T. deformans. In some samples, the leaves of TPT showed deformed epidermal cells, loss of some spongy cells and increase of the intercellular spaces and division of the palisade cells. The pathogen proliferation in the leaves of the VSPT was considerably superior. In this case, stimulation of cell division occurred in the abaxial epidermis. Cells showed periclinal and oblique divisions, with an increased number of plasmodesmata; palisade or spongy cells were not differentiable. Leaves from TPT collected on 26th April showed hyphae with a non‐cylindrical section and with a squashed aspect. The hyphae were very evident in the intercellular spaces, showing abundant endoplasmic reticulum of rough type (RER) in the cytoplasm. On the other hand, epidermis of the leaves of the VSPT had numerous hyphae under the cuticle, which were growing in a thick pectin matrix. Leaves from TPT and VSPT collected on 6th May showed relevant differences. The leaves of TPT had a palisade mesophyll with fewer cells but with active chloroplasts. In contrast, the leaves from VSPT showed empty mesophyll cells, the cytoplasm was collapsed and the adaxial epidermis was covered with the fungus fructification. The observed anatomical and ultrastructural differences of leaves from TPT and VSPT confirm a different behaviour in plant‐host reaction at early stages of infection.     

冠盘二胞Marssonina coronaria侵染不同抗性苹果叶片的组织病理学研究

利用光学和电子显微镜,从组织细胞学水平系统研究了冠盘二胞Marssonina coronaria在苹果抗、感病品种叶片上的侵染过程及侵染后寄主细胞的超微结构特征。结果表明：冠盘二胞的侵入和定殖过程可以分为6个阶段：孢子萌发与芽管形成、附着胞形成、侵入细胞角质层、在叶肉细胞内产生吸器、菌丝在叶肉细胞间和细胞内扩展、分生孢子盘形成。随着菌丝扩展,受侵寄主细胞出现细胞壁加厚,细胞壁降解,质壁分离,叶绿体内淀粉粒、嗜饿颗粒积累,叶绿体基粒片层瓦解,线粒体空泡化等现象。在不同抗性的苹果品种上,分生孢子萌发率差别不明     

Cytological Study of Wheat Spike Infection by Bipolaris sorokiniana

The infection of wheat spikelets by Bipolaris sorokiniana , the causal agent of black point on grains and grain shrivelling, was examined by light and electron microscopy. Conidia of the pathogen germinated 6–12 h after inoculation on the surfaces of the different spike tissues. Extracellular sheaths were observed on germ tubes and appressoria attached to the surfaces of lemma, palea and seeds, but were only scarcely detected on the surface of conidia. Appressoria, frequently found over grooves, formed penetration hyphae invading the epidermal cell walls. Infection process was similar on the surface of the lemma, palea and glume. Growth of the fungus in the epidermal and parenchyma cells was found predominantly in the cell walls, and hyphae also extended intercellularly and intracellularly. Infection of seeds appeared to occur via two ways: (i) direct infection of the outer layers of the cell walls of the pericarp and (ii) through entering the stigma into the pericarp cells. Secretion of host cell wall hydrolytic enzymes at the apex of the penetrating hyphae may facilitate the spread of the fungus. In addition, toxins secreted by the fungus might explain the rapid death of host cells in contact with or distant to fungal cells. A host response to fungal infection involved the development of appositions between cell wall and plasma membrane in cells adjacent to fungal cells. Fungal hyphae were sometimes also surrounded by electron dense material.     

The histopathology of Phaeocryptopus gaeumannii on Douglas-fir needles

Germinating ascospores of Phaeocryptopus gaeumannii produce suprastomatal appressoria from which penetration pegs enter needles. Initial infection occurs between late May and early Jul and coincides with budbreak and shoot elongation. Colonization within needles is exclusively intercellular and increases continuously during Jul-May. No intracellular hyphae or haustoria were observed, but hyphae closely appressed to mesophyll and palisade cell walls are abundant by 3-5 mo after initial infection. Pseudothecial primordia begin to form in epistomatal chambers Oct-Apr, 4-9 mo after initial infection. Pseudothecial primordia developing in the epistomatal chamber are connected to the endophytic thallus by specialized cells in the substomatal chamber that have thickened apical walls and resemble phialides but are not involved in asexual reproduction. The apical wall thickenings instead appear to function as reinforcement against the turgor pressure of the guard cells, allowing cytoplasmic continuity to be maintained between the developing pseudothecium and vegetative hyphae within the needle. Concurrent with the formation of pseudothecial primorida, epiphytic hyphae emerge from the periphery of developing pseudothecia, grow across the needle surface, form numerous anastomoses and reenter the needle by producing appressoria above unoccupied stomata. Epiphytic hyphae and their associated appressoria gradually become more abundant during Oct-Jan.     

Induced Systemic Resistance in Tomato Plants against Phytophthora infestans

Infection of lower leaves of tomato plants with Phytophthora infestans followed by a period unfavourable to disease development increased the general resistance of the plants against the pathogen. Induced resistance to a second infection of upper leaves was expressed in the development of necrotic lesions that were sharply defined and reduced in size. Sporulation of the pathogen was suppressed. Lesions on unprotected plants expanded with a sporulating zone passing gradually to the healthy tissue under the same conditions. Induced resistance delayed and reduced penetration of the pathogen into the epidermis and subsequent colonisation of the mesophyll by formation of papillae in epidermal cells and hypersensitive-like reactions of penetrated, mesophyll cells.     

柿树炭疽菌侵染不同柿树种、品种和部位的细胞学特征

用柿树炭疽病菌Colletotrichumgloeosporioides的分生孢子制备孢子悬浮液,接种无核柿、野柿、冬柿和浙江柿的新梢、叶柄和叶片,并观察致病性、附着胞形成和侵染特性。柿树炭疽菌可以侵染无核柿枝条和叶柄以及野柿枝条,但不侵染无核柿叶片、野柿叶柄和叶片,也不侵染冬柿和浙江柿枝条、叶柄和叶片。室内接种试验与田间自然发病结果一致。柿树炭疽菌在不同柿树表面均能形成附着胞,附着胞产生在寄主表皮背斜细胞壁间结合处(JACWs)或近结合处的百分率达81%~93%。接种12h后,不同柿树表面都有附着胞形成;36h后,无核柿枝条、叶柄中有侵染菌丝存在;48h后,无核柿枝条、叶柄中观察到膨大初生菌丝和较细次生菌丝,初生菌丝可扩展到相邻细胞中,而野柿枝条中仅观察到侵染菌丝;60h后,野柿枝条中也观察到膨大的初生菌丝和较细的次生菌丝,但初生菌丝仅局限在最初侵染的细胞中,无核柿枝条和叶柄以及野柿枝条中都有分枝的次生菌丝在细胞内、细胞间或相邻的细胞中扩展;直到接种90h后,在冬柿和浙江柿上都未观察到侵染菌丝的形成。结果表明,柿树炭疽菌在不同柿树种和品种上侵染菌丝的形成和扩展方式可能是其寄主专化性(或致病性)差异的重要机制之一。     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

Ultrastructural and cytochemical studies on the infection of wheat spikes by<Emphasis Type="Italic">Fusarium culmorum</Emphasis> as well as on degradation of cell wall components and localization of mycotoxins in the host tissue

The infection process and pathway of spreading ofFusarium culmorum in wheat spikes was examined by means of light, scanning and transmission electron microscopy after spray inoculation and single spikelet inoculation. Macroconidia of the pathogen germinated on the host surfaces, however, hyphal development and penetration of host tissues normally occurred on the inner surfaces of the lemma, glume and palea as well as on the ovary. The pathogen spread downward to the rachilla and rachis node by inter- and intracellular growth from the glume, lemma, palea and ovary. The pathogen extended in the rachis in upward and downward direction by inter- and intracellular growth inside and outside of the vascular bundles of the rachis. The spreading of the hyphae in the host tissues was associated with pronounced alterations including disintegration and digestion of host cell walls, suggesting production of cell wall degrading enzymes during infection and spreading in the host tissues. Immunogold labelling studies revealed that accumulation ofFusarium toxins in infected wheat spike tissue showed a close relationship to pathological changes in the host cells, symptom appearance and pathogen colonisation of the host tissue.Fusarium toxins may play an important role in wheat head blight development.     

Two immunological approaches to the detection of ribulose-1,5-bisphosphate carboxylase in guard cell chloroplasts

Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.     

Fungal endophytes in Juncus imbricatus var chamissonis: identification of colonization patterns.

Colonization patterns corresponding to individual endophytic species were observed in asymptomatic leaves and culms of Juncus imbricatus var chamissonis, a perennial plant. The presence of the fungi within the host was demonstrated by light microscopy after the cleaning and staining of tissues. The main endophytic species, Pleospora aff togwotiensis, was found colonizing the substomatal chamber and growing intercellularly between mesophyll cells. In a second colonization pattern, an unknown fungus was restricted to a single host epidermal cell, showing similar characteristics to other fungal taxa observed on Juncus effusus var pacificus, Pseudotsuga menziessii and Fagus sylvatica. A third colonization pattern, where the mycelium is confined to the substomatal chamber, was also described.     

Identification of the determinants of host resistantce and pathogenicity in interactions between Alternaria linicola Groves & Skolko and Linum Usitatissiumum L. accessions using multivariate analyses

Multivariate analysis of variance (MANOVA) and canonical variates analysis (CVA) were used to examine differences in host plant resistance and pathogen behaviour in interactions between Altemaria linicola and three genotypes of Linum usitatissimum, previously identified as susceptible, moderately resistant and resistant to the pathogen. Significant differences in pathogen development were found among the Linum accessions at 18, 24, and 40 h after inoculation. At 18 h after inoculation attempted penetration by the pathogen was relatively rare on all three accessions and canonical variates analysis revealed that overall differences among accessions resulted from large differences with respect to a small number of variables associated with successful penetration on the most susceptible accession. At later times after inoculation, when attempted penetration was more common, overall differences among accessions were found to result from smaller absolute differences among a group of variables which characterised the early colonisation of the host tissue. The results from these investigations are discussed in relation to recent research on the ecology of the pathogen and the importance of the timing of host responses to infection in determining host plant resistance.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

The binding of isolated mesophyll cells from barley leaves to hyphae of Pyrenophora teres

Live mesophyll cells from barley leaves were isolated and used to investigate contact phenomena between these cells and hyphae of the barley pathogen Pyrenophora teres. When incubated with colonies of P. teres, the isolated cells became attached to the hyphae. The binding of the cells may have been mediated by an affinity between cellulose in the mesophyll cell wall and extracellular material produced by the fungal hyphae. Different fungi exhibited a wide range of affinities for the mesophyll cells. It is suggested that isolated mesophyll cells may be a useful tool in the study of host-pathogen interfaces.     

Microtubules and coated vesicles in guard-cell protoplasts ofAllium cepa L.

Protoplasts were prepared from the guard cells ofA. cepa. Epidermal peels taken from expanding green leaves and largely free of mesophyll were treated with Cellulysin, and protoplasts were harvested after 18 h of digestion. That the protoplasts were derived from guard cells was ascertained from their characteristic vacuolar autofluorescence and from observations showing that all other epidermal cells are killed in the peeling procedure. The protoplasts proved to be a good system with which to view the cell cortex and inner surface of the plasmalemma. The lysis of cells adhering to polylysine-treated, Formvar-coated grids, followed by negative staining in uranyl acetate, showed that many microtubules normally present in ordered arrays in situ remain closely applied to the inner surface of the plasmalemma in protoplasts. In addition, numerous vesiculate elements including coated vesicles and/or pits are present amongst the microtubules. Similar vesicles are evident in thin sections of fixed, embedded guard cells and protoplasts. The significance of these structures in the cell cortex is discussed.