Intercellular Hyphae Research Articles

Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae)

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts ( Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.

Cercosporella and Ramularia

Cercosporella and Ramularia (hyphomycetous anamorphs with relationship to Mycosphaerellaceae, Ascomycota) are difficult to distinguish based on light microscopic characteristics of conidiophores and conidia. Although both genera have been known for more than 120 y, new morphological characteristics were found by “simple” light and scanning electron microscopy now allowing an unambiguous differentiation between both genera. Newly discovered morphological characteristics of interaction structures and ultrastructure of conidiogenous loci are congruent with LSU rDNA sequence analysis of C. virgaureae and Ramularia species. DNA sequences of the type species of both genera, C. virgaureae and R. pusilla, were generated. For the first time a complex cup-shaped appressorium is reported for a member of plant pathogenic cercosporoid hyphomycetes, based on light, scanning and transmission electron microscopy. The appressorium of C. virgaureae is formed by strongly branched hyphae radiating from a supporting intercellular hypha and remaining closely attached to each other. With its concave side the appressorium adheres to a cell of the leaf mesophyll. This structure is significant in comparison with morphologically unspecific intercellular hyphae known in other cercosporoid hyphomycetes. Scanning electron microscopy of conidiogenous loci also provide additional characteristics for distinguishing Cercosporella and Ramularia. Conidiogenous loci are smooth in Cercosporella but similar to the Cladosporium-type (consisting of a circular rim and a central dome) in Ramularia.

A novelPhytophthora infestanshaustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.

Ultrastructural observations on Puccinia menthae infections.

Haustoria and associated regions of the hostpathogen interfaces of the autoecious rust Puccinia menthae, during mono- and dikariotic phases of development on its host Mentha spicata, differed in ultrastructural and cytochemical features. By application of the PA-TCH-SP procedure, polysaccharides were shown not only to be major constituents of the walls of intercellular hyphae, haustorial mother cells (HMC) and haustoria of both the monokaryon and dikaryon (m- and dhaustoria, respectively) of P. menthae, but were also present within the extra-haustorial matrix (EM) of both types of haustoria. In the neck region of d-haustoria, which is structurally more differentiated than in m-haustoria, there was evidence for the presence of lipopolysaccharides and glycoprotein. However, the characteristic neck-band, which stains densely in conventional staining of the d-haustorium, did not react with PA-TCH-SP. The outermost layer of the dikaryotic HMC wall was completely digested by protease, whereas treatment with protease and cellulase did not affect either mono- or dikariotic intercellular hyphae or walls of m-haustoria, although these treatments indicated the presence of protein and cellulose in the EM. In dikaryotic infections, protein was found to be a major constituent of the EM. Cellulase treatment resulted in almost complete digestion of the host wall, except for a thin outer layer, whereas hyphal and haustorial walls were unaffected. The morphology and structural differences observed between the mono- and dikaryotic infection structures of P. menthae, and their interactions with host cells were clarified, and confirmed the possibility of their functional differences. Such information should facilitate further comparative studies on host-parasite interfaces at different stages of the rust life cycle and other biotrophic fungi.

Occurrence and ultrastructure of Albugo candida on a new host, Arabis alpina in Saudi Arabia

A population of Arabis alpina (Brassicaceae) growing in Saudi Arabia was observed to be infected for the first time by the Oomycete, Albugo candida. Both conventional chemical fixation and high pressure freezing followed by freeze substitution (HPF/FS) were used to prepare zoosporangia, intercellular hyphae, haustoria, invading host cells and host–parasite interface of A. candida for study with both scanning and transmission electron microscopy. Both fixations gave good preservation of ultrastructural details and data from the two sample types were highly complementary. Scanning electron microscopic observation revealed that mature zoosporangia of A. candida are spherical or ellipsoidal in shape and characterized by a smooth surface and faint terminal secession scar at each end. Transmission electron microscopic observation indicated that coenocytic intercellular hyphae are located in intercellular spaces of host leaf tissue forming haustoria in host mesophyll cell. Each haustorium is connected to intercellular hyphae by a narrow, slender neck which enclosed by a collar as a response of host cell to infection. The cytoplasm of the haustorium contains different organelles characteristic of the Oomycetes. No obvious responses are observed in host cell organelles following infection which may be due to the presence of a compatibility between the host and the Oomycete. Modifications of the host plasma membrane around the haustorium are detected. Many tubular elements were found to be continuous with the extrahaustorial membrane. This appears to be the first report of the presence of these tubular elements in case of A. candida haustoria. These tubular elements may increase membrane surface area and consequently increase the efficacy of nutrients uptake by haustoria from host cell.

Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest

Fungi were isolated from the roots of 17 plant species from the families Apiaceae, Cunoniaceae, Cyperaceae, Droseraceae, Fabaceae-Mimosoideae, Lomandraceae, Myrtaceae, Pittosporaceae, Proteaceae and Stylidiaceae at a sclerophyll forest site in New South Wales, Australia. Internal transcribed spacer (ITS) restriction fragment length polymorphism (RFLP) and sequence comparisons indicated that the isolated fungi had affinities to a range of ascomycetes, basidiomycetes and zygomycetes. Four RFLP types had closest affinities to previously identified Helotiales ericoid mycorrhizal (ERM) or Oidiodendron spp. Isolates representing six RFLP types, which were variously isolated from all 17 plant species, formed ERM coils in hair root epidermal cells of Woollsia pungens (Ericaceae) under gnotobiotic conditions. Three of these isolates formed intercellular hyphae, intracellular hyphae and/or microsclerotia, which are typical of dark septate endophyte infection, in roots of Stylidium productum (Stylidiaceae), indicating an ability to form different types of association with roots of different hosts. Overall the data indicate that a broad range of plant taxa may act as repositories for ERM fungi in sclerophyll forest soil.

Interaction between an isolate of dark-septate fungi and its host plant Saussurea involucrata

A dark-septate endophytic (DSE) fungus EF-M was isolated from the roots of an alpine plant Saussurea involucrata Kar. et Kir. ex Maxim. The fungus was identified by sequencing the PCR-amplified rDNA 5.8S gene and ITS regions. The sequence was compared with similar sequences in the GenBank, and results showed that EF-M was congeneric to Leptodontidium. Resynthesis study was conducted to clarify the relationship between the root endophyte EF-M and the host plant S. involucrata using the material grown in sterile culture bottle. In roots recovered 6 weeks after inoculation, epidermal cells were colonized by intercellular and intracellular hyphae and "microsclerotia" formed within individual cells in the epidermis layers. However, hyphae did not invade the cortex and the stele. There were no profound effects of endophyte EF-M on plant root development, but significant differences were detected in plant height and shoot dry weight between the treatments. The present study is the first report hitherto on DSE fungi in S. involucrata.

Root infection and systemic colonization of maize by Colletotrichum graminicola.

Colletotrichum graminicola is a filamentous ascomycete that causes anthracnose disease of maize. While the fungus can cause devastating foliar leaf blight and stalk rot diseases, little is known about its ability to infect roots. Previously published reports suggest that C. graminicola may infect maize roots and that root infections may contribute to the colonization of aboveground plant tissues, leading to disease. To determine whether C. graminicola can infect maize roots and whether root infections can result in the colonization of aboveground plant tissues, we developed a green fluorescent protein-tagged strain and used it to study the plant root colonization and infection process in vivo. We observed structures produced by other root pathogenic fungi, including runner hyphae, hyphopodia, and microsclerotia. A mosaic pattern of infection resulted from specific epidermal and cortical cells becoming infected by intercellular hyphae while surrounding cells were uninfected, a pattern that is distinctly different from that described for leaves. Interestingly, falcate conidia, normally restricted to acervuli, were also found filling epidermal cells and root hairs. Twenty-eight percent of plants challenged with soilborne inoculum became infected in aboveground plant parts (stem and/or leaves), indicating that root infection can lead to asymptomatic systemic colonization of the plants. Many of the traits observed for C. graminicola have been previously reported for other root-pathogenic fungi, suggesting that these traits are evolutionally conserved in multiple fungal lineages. These observations suggest that root infection may be an important component of the maize anthracnose disease cycle.

Comparative Ultrastructure of Cucumbers Pretreated with Plant Growth‐promoting Rhizobacteria, DL‐3‐aminobutyric Acid or Amino Salicylic Acid after Inoculation with Colletotrichum orbiculare

AbstractThe infection behaviour of Colletotrichum orbiculare was observed using a transmission electron microscope (TEM) in the leaves of cucumber plants preinoculated with plant growth‐promoting rhizobacteria (PGPR) Serratia marcescens (90‐166) or Pseudomonas fluorescens (89B61) after inoculation with the anthracnose pathogen. Moreover, the structural defence mechanisms mediated by pretreating dl‐3‐amino butyric acid or amino salicylic acid was compared with those mediated by these two PGPR. In untreated plants broadly spread intercellular/intracellular hyphae were found in leaf tissues. The cytoplasm of fungal hyphae appeared intact and the electron densities of intercellular and intracellular hyphae were not high even at 5 days after fungal inoculation, indicating lack of defence responses. However, in the leaves of plants preinoculated with both PGPR active defence responses were observed, i.e. sheath formation at penetration sites, and accumulations of endoplasmic reticula or of numerous vesicles around intracellular hyphae. Furthermore, the electron densities of most intracellular and intercellular hyphae were higher than those of untreated control plants. In the leaves of plants pretreated with both chemicals, the hyphae of the pathogen were rarely observed at 5 days after challenge inoculation. Similar to the case of PGPR‐pretreated plants, the most intercellular and intracellular hyphae in plant tissues were electron dense. However, no active defence responses were observed in leaf tissues. Moreover, in some cases, hyphal growth was restricted to the epidermal cell layer, and in particular, was not observed to develop in mesophyll cells of the plants. These results suggest that the mechanisms of systemically induced resistance are differently expressed by preinoculating with PGPR or by pretreating with chemicals.

Microscopic and Macroscopic Studies of the Development of Puccinia hemerocallidis in Resistant and Susceptible Daylily Cultivars.

Infection and colonization of eight daylily cultivars, which varied in resistance to daylily rust, by Puccinia hemerocallidis was studied macroscopically and microscopically. After germination of urediniospores, appressoria formed at the tip of germ tubes and the fungus penetrated the host through stomatal openings 2 days after inoculation (DAI). Under the infection sites, intercellular hyphae aggregated and formed uredia, which released urediniospores 8 DAI. Resistant cultivars, characterized by the development of rapid death of host cells, were separated into three qualitative categories based on absence and presence of necrotic lesions without or with sporulation. In highly resistant cvs. Prairie Blue Eyes and Bertie Ferris, no macroscopic disease symptoms were observed on leaf surfaces although a few collapsed cells were detected microscopically. Both resistant and moderately resistant reactions were characterized by necrotic lesions with many collapsed cells under infection sites. The difference between these two reactions was that uredia and urediniospores were observed in the moderately resistant cv. Chicago Apache, but not in resistant cvs. Buttered Popcorn and Stella De Oro. Susceptible cultivars, characterized by the absence of a hypersensitive response, were separated into two qualitative categories based on restriction of intercellular hyphal growth that delayed development of uredia and formation of urediniospores. Compared to the susceptible cv. Pardon Me, moderately susceptible cvs. Mary Todd and Chorus Line had a delayed latent period and reduced amount of sporulation. The results indicate that hypersensitive cell death is one of the resistance responses to daylily rust. Necrotic lesions on leaf surfaces are associated with the number of collapsed host cells. Delayed latent period and reduced sporulation that resulted from restriction of intercellular hyphal growth could represent another type of resistance response in the daylily-rust pathosystem.

Effects of thymol fumigation on survival and ultrastracture of Monilinia fructicola

Postharvest treatment of stone fruit with thymol vapours controls brown rot, caused by Monilinia fructicola. While the mechanism of the fungicidal action is not known, the gross effect of the thymol vapours appeared to be on the fungal spores and surface mycelia. Harvested plums were inoculated with conidia of M. fructicola and incubated for 4–6 days at 21 °C to allow for sporulation. Sporulating fruit was treated with thymol vapours from 0 to 8 μg mL −1. The viability of the conidia obtained from thymol treated and untreated brown rot lesions was tested with four vital stains. M. fructicola conidia were immediately affected with a 50% reduction in viability after treatment with 2 μg mL −1 thymol as detected by the vital stains fluorescein diacetate, propidium iodide, fluorescent dye DiOC 6(3) and the commercial product FungoLight™. Conidia treated with 8 μg mL −1 thymol exhibited 17–23% survival. One millimeter square sections of the infected fruit skin with the associated surface mycelial mass were prepared for transmission electron microscopy. The electron micrographs display the intra and intercellular ultrastructure of the fungal conidia. Thymol crystallized on the outer surface of the fungal cell wall. The cytoplasm of the thick walled spores did not appear changed from the untreated spores. In contrast, all other fungal fine structures on the surface of the lesion were severely affected by thymol vapours. Sections of germ tubes, appressoria and surface hyphae exposed to thymol vapours were characterised by disrupted and disorganised cytoplasmic organelles. The fine structures of the fungal intra- and intercellular hyphae inside the plum tissue were unaffected by thymol treatments.

Wood‐decay fungi in fine living roots of conifer seedlings

The mycorrhizal basidiomycetes are known to have multiple, independent evolutionary origins from saprotrophic ancestors. To date, a number of studies have revealed functional resemblance of mycorrhizal fungi to free-living saprotrophs, but information on the ability of saprotrophic fungi to perform as mycorrhizal symbionts is scarce. Here, the objective was to investigate the ability of three wood-decay fungi, Phlebiopsis gigantea, Phlebia centrifuga and Hypholoma fasciculare, to colonize fine roots of conifer seedlings. For each fungus, mycorrhizal syntheses were attempted with Picea abies and Pinus sylvestris. After 24 wk, isolation of fungi and direct sequencing of fungal internal transcribed spacer (ITS) rDNA were carried out from healthy-looking surface-sterilized root tips that yielded both pure cultures and ITS sequences of each inoculated strain. Mycelial mantle of P. gigantea was frequently formed on root tips of P. abies, and microscopical examination has shown the presence of intercellular hyphae inside the roots. The results provide evidence of the ability of certain wood-decay fungi to colonise fine roots of tree seedlings.

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.

Light and electron microscopic observations of the infection of Camellia sasanqua by the fungus Exobasidum camelliae var. gracilis

Only a very small percentage of the shoots of the numerous Camellia sasanqua Thunb. plants examined in this study were infected by Exobasidium camelliae var. gracilis Shirai, and prior to the onset of new leaf formation in the spring, it was impossible to distinguish these shoots from healthy shoots with the naked eye. However, beginning in early April, an infected shoot gave rise to a group of tightly clustered, pale-green leaves that were significantly larger and thicker than the dark-green healthy leaves. Light and transmission electron microscopic observations revealed the presence of intercellular hyphae of E. camelliae var. gracilis in the apical meristems of shoots that gave rise to enlarged infected leaves. Very slender intercellular hyphae were observed between the tightly packed cells of the apical meristem as well as between the cells of the tunica that covered the meristem. Hyphae also filled many of the intercellular spaces in the stem immediately below the apical meristem. Some hyphae formed distinctive haustoria that protruded for short distances into the invaded host cells. Hyphae also were present in the leaf primordia and proliferated in the intercellular spaces while the primordium differentiated into a leaf. Eventually a distinct pseudoparenchymatous layer of intercellular hyphae developed some four to six cell layers above the lower epidermis. This layer then gave rise to the basidia that made up the hymenium of E. camelliae var. gracilis. As basidia developed, the underlying cell layers of the leaf separated from the overlying hymenium and eventually sloughed away to expose the basidia. Localized infections of random leaves also were observed. In many cases only a small part of a leaf was infected. Localized infections also culminated in successful hymenium development.

Mycorrhizal associations in Ailanthus altissima (Simaroubaceae) from forested and non-forested sites1

Ailanthus altissima tree seedlings were excavated from each of two habitats: (1) a forest adjacent to a trail and stream and (2) a non-forested steep, barren slope adjacent to a major highway. Each seedling root system was examined for colonization by mycorrhizal structures using light microscopy and transmission electron microscopy. The roots were colonized by one or more endomycorrhizal fungi with Arum-type colonization. Endomycorrhizal colonization of the seedlings from the non-forested site (65.2%) was significantly greater than that of the seedlings from the forested site (37.9%). Colonization by intercellular hyphae and vesicles was significantly greater in the non-forested habitat than the forested habitat. This exotic invasive species may benefit from the rapid colonization of endomycorrhizae in more extreme open environments.

Binucleate Rhizoctonia (Ceratorhiza spp.) as non-mycorrhizal endophytes alter Pinus sylvestris L. seedling root architecture and affect growth of rooted cuttings

The main aim of this study was to assess critically the effects of binucleate Rhizoctonia (BnR) inoculation on Scots pine (Pinus sylvestris L.) seedling and hypocotyl root architecture and early plant growth in tree seedling nursery soil, Sphagnum peat and forest humus. The BnR isolates (251, 266, 268, 269) stimulated early seedling growth in the nitrogen-limited nursery soil 86 days postinoculation (p.i.). These seedlings exhibited significantly higher root length and reduced root width, although percentage root infection levels were <6%. At a harvest 240 days p.i., no significant plant and root growth differences were identified, although short root numbers were significantly increased. BnR infection detected in roots was characterized by the presence of intercellular fungal hyphae and subtending intracellular monilioid fungal cells located in outer cortical cells of long roots. Similar endophytic infection morphology was detected in adventitious roots generated in prerooted hypocotyl cuttings [Kaparakis, G. & Sen, R. (2006). Scandinavian Journal of Forest Research, 21] exposed to all BnR isolates in nursery peat and forest humus, although isolate-specific cutting growth was detected. In conclusion, these BnR isolates, known to be genetically related to orchid mycorrhizal Ceratorhiza spp., are non-pathogenic and have the capacity to stimulate significantly early Scots pine root development in nursery soils and forest humus.

Ultrastructure of the fungus Ophiodothella vaccinii in infected leaves of Vaccinium arboreum

Light and electron microscopy were used to study the fungus Ophiodothella vaccinii E.S. Boyd in infected leaves of Vaccinium arboreum Marsh. This pathogen produces large lesions on leaves in which host cells are surrounded by intercellular hyphae and penetrated by intracellular hyphae. In this study, we examined details of acervuli that are produced on these lesions, the process of conidiogenesis that took place within acervuli, and the relationships between hyphae and host cells within lesions. Most acervuli developed on the adaxial leaf surface just beneath the epidermis, which also was permeated by fungal hyphae. The epidermis covering an acervulus separated from the underlying host cells as numerous conidia embedded in extracellular material accumulated within an acervulus. The raised portion of the epidermis formed a shield-like clypeus over the acervulus. Mature conidia coated with extracellular material were released through a small pore in the clypeus. A columnar mass of sterile hyphae was present in each acervulus near the pore region. This mass was surrounded by conidia embedded in extracellular material. Conidia arose from a layer of phialides present at the base of an acervulus. Phialides arose from both intercellular hyphae and intracellular hyphae that emerged from host cells and also were surrounded by extracellular material. In older lesions, O. vaccinii clearly functioned as a necrotroph. However, in young lesions, we found evidence indicating that some cells were alive when penetrated by hyphae. Hyphae were highly constricted at host cell wall penetration sites and intracellular hyphae commonly grew completely through infected host cells and directly into adjacent cells. Hyphae in older lesions became extremely thick walled and were packed with large lipid bodies.

(44) Microscopic and Macroscopic Studies on the Development of Puccinia hemerocallidis in Resistant and Susceptible Daylily Cultivars

Infection process of Puccinia hemerocallidis, the causal agent of daylily rust, and resistance responses in eight daylily cultivars, were studied macroscopically and microscopically. After germination of urediniospores, appressoria were formed at the tip of germ tubes and penetrated through stomatal openings. Intercellular hyphae aggregated and formed uredia under the infection sites, and released urediniospores after rupturing the epidermis. In highly resistant cultivars `Prairie Blue Eyes' and `Bertie Ferris', intercellular hyphal growth was restricted and uredia were not formed. No macroscopic symptoms of the disease were present on the leaf surface, although a few collapsed cells were observed microscopically. Both resistant and moderately resistant reactions were characterized by necrotic lesions with many collapsed cells under infection sites. The difference between these two reactions was that uredia and urediniospores were observed on the moderately resistant cultivar `Chicago Apache', but not on resistant cultivars, `Buttered Popcorn' and `Stella De Oro'. Sporulation was observed on both moderately susceptible and susceptible cultivars, but latent periods were delayed and the amount of urediniospore production was reduced on moderately susceptible cultivars, `Mary Todd' and `Chorus Line', compared to the susceptible cultivar `Pardon Me'. The results indicate that the hypersensitive cell death is one of the resistance responses to daylily rust, but necrotic lesions on leaf surfaces are associated with the amount of collapsed host cells. The delayed latent periods and reduced sporulation that resulted from restricted intercellular hyphal growth could represent another resistance mechanism in the daylily rust pathosystem.

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development ofColletotrichum acutatumon Almond

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.

Growth, occurrence and development of septa in Plasmopara viticola and other members of the Peronosporaceae using light- and epifluorescence-microscopy

Although Plasmopara viticola causes grape downy mildew in most, if not all, wine producing countries, many basic biological or chemical aspects are still unknown and thus the histopathological changes during development of this oomycete pathogen were studied. The fluorochromes Aniline blue and Uvitex 2B were successfully used in whole leaf stainings to visualise intercellular hyphae and to investigate septal-development in sporangiophores. The occurrence and transfer of cytoplasm in sporangiophores was studied with the aid of Chlorazole Black E and Phloxin-B. Application of Chlorazole Black E was the most reliable method to differentiate between cytoplasm and septa in sporangiophores due to a high contrast between the dark cytoplasm and the pale septa. In P. viticola, septa were found in the stem and branches of the sporangiophores, but not in the intercellular hyphae, which was in contrast to other oomycetes, such as P. tabacina, Pseudoperonospora cubensis and P. humuli, where septa were frequently found in the mycelium. In order to verify chemical composition of septa, sporangiophores were digested using chitinase and beta-1,3-glucanase. After staining with Aniline blue and Uvitex 2B, the intense fluorescence of septa was conserved after the application of chitinase but not after beta-1,3-glucanase, indicating that septa are mainly composed of beta-1,3-glucans. Pretreatments of infected vine leaves with 2-deoxy-D-glucose (2-DOG) at low concentrations (1-5 mM) led to a disorganisation of the sporangiophore structure of P. viticola, whereas the production of septa was unaffected. Application of 2-DOG at higher concentrations resulted in a reduced length of intercellular hyphae (10 mM) or total inhibition (50 mM) of the intercellular mycelium. Thus, 2 DOG or its analogues interferes with morphogenesis in Plasmopara viticola and may have a deleterious effect on the spread of this downy mildew between plants.