Host Wall Research Articles

Histopathology of Fusarium wilt of staghorn sumac (Rhus typhina) caused by Fusarium oxysporum f. sp. callistephi race 3. II. Characteristics of opaque matter associated with extensive host cell and cell wall alterations

Light and transmission electron microscopy observations of staghorn sumac plants inoculated or naturally infected with Fusarium oxysporum f. sp. callistephi are reported. One aspect of infection was the presence of large intercellular masses of opaque matter (OM) in middle lamellae between ray cells and/or fibres, often bypassing several intercellular areas; similar OM confluent with the intercellular OM also occurred in secondary walls and in the periphery of numerous cells. A gradual increase in the abundance of the OM in host tissues vertically from the inoculation point and then radially was noted over infection time and was related to host wall and cell alterations. In the region of recently deposited tissue, the OM was associated with pronounced cell hyperplasia and hypertrophy. The OM was delimited by thin, compact bands, and when it was less compact, displayed opaque particles and other fine structures. No indications were obtained that it contained or had contained intact or altered organelles. The DNA probe bound to OM in middle lamellae and in cell periplasmic areas, and to material of a similar texture lining vessel walls. Samples from Fusarium-infected plants, incubated on an agar medium before fixing to determine from which elements the pathogens could develop, displayed bodies as the sole elements present in mature xylem cells and in intercellular areas. These bodies were delimited by membranous structures and profiles of a wall layer and contained opaque particles and areas of fine structures. Certain inter- or intracellular fungal cells in the same tissue frequently had similar content. In the light of these observations it is proposed that the OM is primarily of a pathogen rather than of a host origin.

Diversity, phenomenology and epidemiology of epiphytism in farmed Gracilaria chilensis (Rhodophyta) in northern Chile

This study identified the most common epiphytes infecting the algal host Gracilaria chilensis on a farm in northern Chile. Simultaneously, the types of host–epiphyte interfaces were characterized and their relative abundance and temporal variability were monitored. Five types of anatomical relationships were detected. Infection type I included the epiphytes weakly attached to the surface of the host and not associated with damage of host tissues (i.e. Hincksia mitchelliae, H. granulosa and Ectocarpus acutus). Infection type II included those epiphytes strongly attached to the surface of the host but not associated with any host tissue damage (i.e. Acrochaetium sp., Antithamnionella sp. and Colpomenia sinuosa). Infection type III included all the epiphytes that penetrated the outer layer of the host wall without damaging its cortical cells (i.e. Xenococcus sp. and Sahlingia subintegra). Infection type IV included epiphytes penetrating deep into the host cell wall, disorganizing the cortical tissue (i.e. Ulva lactuca and Acrosorium corallinarum). Infection type V included epiphytes that penetrated deeply into the cortex, reached the medullary tissue and caused destruction of the host's cells in the area around the infection (i.e. Ceramium rubrum and Polysiphonia harveyi). Prevalence varied with time and with infection type, with types II and III reaching up to 80% and 90% of the thalli respectively. Severity of epiphyte infection was similar to the distribution of infection prevalence, with crustose epiphytes colonizing up to 80% of the host surface.

A comparative cytological analysis of fungal endophytes in the sporophyte rhizomes and vascularized gametophytes of Tmesipteris and Psilotum

This article describes the results of a light and electron microscopic study of the fungal endophytes and vascular anatomy in the rhizomes and gametophytes of Tmesipteris and Psilotum. The parenchymatous cortical cells of the rhizomes and subterranean gametophytes of Tmesipteris and Psilotum contain intracellular aseptate glomeromycotean fungi resembling the “Paris-type” of arbuscular mycorrhizas found in seed plants. The fungi differentiate into multinucleate vesicles and hyphal coils, both containing bacteria-like structures and accumulating lipid masses and crystals as they age. After several cycles of infection in the same cell, degenerate hyphae form amorphous masses encased by host wall material. Nearly identical host–fungus cytology between the autotrophic sporophytes and the heterotrophic gametophytes suggests that these psilophyte associations are exploitative of the fungus in both generations. Following the description of tracheids nearly 60 years ago in the gametophytes of Psilotum, vascular elements are described for the first time in the haploid generation of Tmesipteris. Close similarities between the water- and food-conducting elements in both generations, viz. vessel elements with scalariform perforation plates and sieve cells with refractive spherules and lacking callose at all stages in their develoment, add support to the homologous theory of the alternations of generations. Mitochondrial aggregations, cross-linked by small electron-opaque rods, are common in the stelar cells of both generations and appear to be a unique feature of the psilophyte clade.

Interactions between the mycoparasite Pythium oligandrum and two types of sclerotia of plant-pathogenic fungi

The interactions between Pythium oligandrum hyphae and two types of sclerotia, i.e. the plano-convexoid sclerotium of Botrytis cinerea and the tuberoid sclerotium of Sclerotinia minor, were investigated by ultrastructural and cytochemical experiments. In the mycoparasitism of P. oligandrum, some differences in relation to sclerotium anatomy and the role of the rind layer in preventing invasion are documented. Both types of sclerotia showed neither alterations of the heavily melanised rind walls, nor direct rind wall penetration by P. oligandrum. This oomycete successfully entered B. cinerea sclerotia only through breaches at the junction of rind cells and corresponding to gaps in melanin deposits. On the other hand, none of these breaches was observed in the small sclerotia of S. minor, and P. oligandrum ingress in the sclerotia stopped at the inner rind layer. After the penetration of B. cinerea sclerotia by the mycoparasite, it extensively colonised the cortical and medulla areas by intercellular growth. The invaded tissues displayed pronounced alterations as well as some disorganisation of tissue in places. Colonisation was associated with severe chitin degradations of all host walls, which occurred even at some distance from P. oligandrum hyphae. The observation of wall thickenings in some P. oligandrum-hyphae suggests that the sclerotial cells constitute a harsh environment unsuitable for survival of the mycoparasite. These wall thickenings could be interpreted as P. oligandrum defence-like reactions.

Occurrence of paracrystalloids and their particles in resistant and susceptible carnation plants infected with Fusarium oxysporum f.sp. dianthi race 2

Uncommon, opaque particles (of approximately 20-22 nm, referred to as OP), aggregating into paracrystalloids occurred only next to colonized cells in carnation plants of either a susceptible or resistant cultivar (cv.) infected with Fusarium oxysporum f.sp. dianthi. In the susceptible plant, those structures occurred in vessel lumina and host walls, apparently associated with their alterations, but not in parenchyma cells, a situation which was the exact opposite of that observed in resistant plants. In comparison with apparently similar structures reported in other systems, paracrystalloids and their OPs did not seem to have exact counterparts in plants infected with viruses or fungi, although similar paracrystalloids were observed in nematode-infected plants. The OPs were associated in both cvs. with fine opaque matter, often displaying fine filamentous structures, and were in addition connected to fungal cells in the susceptible cv. Similar structures also extended through host walls into adjoining cells; these relations with parenchyma cells in resistant plants were interpreted as if the particles therein were akin to, if not exactly of the same nature as those in susceptible plants. As the opaque matter, the filamentous structures and the OPs were interrelated and associated with pathogen cells, it seemed warranted to assume that the OPs were issued from the pathogen.

Peculiar ultrastructural characteristics of fungal cells and of other elements apposed to and in vessel walls in plants of a susceptible carnation cultivar, infected with Fusarium oxysporum f.sp. dianthi race 2

Ultrastructural characteristics and cytochemical reactions of unusual, irregular elements (IE) in vessel elements in susceptible carnation plants infected with Fusarium oxysporum are reported. As revealed by labelling for chitin, fungal cells in contact with host cell walls or content had altered or defective lucent layers, and labelling was frequently associated with their outer, opaque layer or matter located outside the cells. Coating matter on vessel walls occurred at all stages of infection, and IEs only in later stages. IEs were delineated by opaque, often folded bands, some contouring pit borders, and contained membranous and vesicular structures mixed with other fine components. Only then, IEs were strongly but not uniformly labelled for chitin. Coating, IE-delineating bands, and the opaque outer layer of typical fungal cells were texturally similar, not labelled for chitin or cellulose, except where they impinged upon host walls. Both probes for chitin and cellulose strongly attached to vessel secondary walls. IEs were often confluent with coating, and with fungal cells connected to them by means of microfilamentous structures. Similar microfilamentous structures and opaque bands connected to IEs, the coating, and the microhyphae, or protruding from fungal cells reached into host walls, associated with alterations of these walls. The possible malleable IEs might be a counterpart of the coating, and although they do not occur in the initial stages of the disease, they could play an important role in the final stages of tissue degradation.

Ultrastructure of the early stages of Colletotrichum acutatum infection of strawberry tissues

The early stages of the infection of attached leaves and petioles of strawberry (Fragaria ×ananassa Duch. 'Camarosa') by Colletotrichum acutatum Simmonds were studied using scanning and transmission electron microscopy. Pre-penetration events of these tissues were similar, but the production of secondary conidia (microcyclic conidiation) was detected only on leaves. At the ultrastructural level, different stages of maturation of appressoria were observed and described. In young appressoria, the cell wall was composed of two layers and the plasma membrane displayed a wavy appearance. In the following stage, the appressorium developed a third electron-transparent layer between the cell wall and the plasma membrane. This new electron-transparent material was especially visible in the region of the appressorium near the cuticle. The plasma membrane of this appressorium showed a smooth appearance. Afterwards, a penetration peg emerged through the pore penetrating the cuticle and reached the epidermal wall where it enlarged to form an intramural infection vesicle. Both structures of infection, the penetration peg and the intramural infection vesicle, produced during the early phases of infection of strawbery tissues by C. acutatum, have not been previously reported and confirm that its invasion strategy is that of a subcuticular intramural pathogen. Once the infection was well established, abundant subcuticular and intramural hyphae were produced on petioles, causing severe degradation of the host cell walls. Occasionally, the cuticle appeared disrupted in those regions where the host walls were very degraded and dilated. Differences between colonization of petioles and leaves were observed.Key words: Colletotrichum acutatum, Fragaria ×ananassa, infection vesicle, invasion strategy, penetration peg.

Extracellular sheath formation by Sphaeropsis hypodermia and association with its infection in elm trees

Sphaeropsis hypodermia, isolated from a cankered American elm branch, was grown on agar medium (PDA), on autoclaved wiping paper (Kimwipes), and American elm (Ulmus americana) wood chips, or inoculated into greenhouse-grown American elm saplings. Samples from each treatment were double-fixed with glutaraldehyde and osmium tetroxide and examined with the light and the transmission electron microscopes. Ultrastructurally, the hyphae on PDA and inert substrates appeared surrounded by large extracellular sheaths which were delimited by rigid opaque bands of various thicknesses. The sheaths extended appreciable distances from the fungal cells, as evidenced by their adherence to rigid substrates. Individual or aggregated opaque bodies, even as large masses on elm wood chips, were the main components of the sheath. This opaque material was often associated with penetration and ruptures of the wood cells. Inoculated into elm trees, the fungus rapidly caused pronounced alterations of cambial tissues and colonized the adjoining bark and xylem cells. The prominent penetration and breakdown of the inner and outer bark cells by the fungus were associated with opaque material, particularly in cortical fibres. The material was structurally similar to the sheath formed on the rigid sterilized substrates. In the xylem, only the walls of the recently deposited cells were visibly altered, and although mature fibres were generally colonized, the passage of the fungus from one fibre to another was rarely observed, contrary to the passage from vessel and ray cells to adjoining cells. In that instance, only bands of opaque material present in the walls of fibres were connected with fungal cells in their lumen. In the inner bark and cambial regions, cell hypertrophy and hyperplasia occurred next to host walls that were altered and contained similar opaque material. The extracellular sheath of S. hypodermia under in vitro conditions and the opaque material associated with host wall alterations in vivo are considered to be analogous.

A Cucurbit[6]uril Analogue: Host Properties Monitored by Fluorescence Spectroscopy

This paper describes the host properties of a new cucurbit[6]uril analogue, studied by fluorescence and 1H NMR spectroscopy. This host has an elongated cavity with oval-shaped portals. It is intrinsically fluorescent, and more importantly, this fluorescence is sensitive to guest encapsulation, allowing for the study of the inclusion of nonfluorescent guests by fluorescence spectroscopy. In the case of benzene as guest, significant enhancement of the cucurbit[6]uril analogue host fluorescence was observed upon addition of benzene; this allowed for the determination of the binding constant for 1:1 host-guest complexation, yielding a value of K = 6900 +/- 1100 M(-1). This complexation was also studied by 1H NMR, yielding a similar value of K = 8980 +/- 500 M(-1). The binding of a much larger guest, the dye Nile Red, was also studied, but in this case using guest fluorescence. Significant suppression of the Nile Red fluorescence was observed upon 1:1 complexation with the cucurbit[6]uril analogue, with an extremely large binding constant of 8.2 +/- 0.5 x 10(6) M(-1), indicating a very strong host-guest interaction and an excellent size and shape match. In both cases, binding was much stronger than in the case of the same guests with cucurbit[6]uril itself, and in the case of Nile Red, binding was also much stronger than with modified beta- or gamma-cyclodextrins. This is partly a result of the partial aromatic nature of the host walls, which allow for pi-pi interactions not possible in cucurbiturils or cyclodextrins. The ability to study its inclusion complexes using either host or guest fluorescence, and the very high binding constants observed, illustrates the versatility and potential usefulness of this new host compound.

Interactions between parasitic fungi and mosses: pegged and swollen-tipped rhizoids in Funaria and Bryum

Two kinds of parasitic fungal infections are described in the rhizoidal systems of both wild and cultured materials of Funaria hygrometrica, Bryum capillare and B. pseudotriquetrum. Protonemal and rhizoidal cells of Funaria produce pegs of host wall materialaroundsites of hyphal penetration. Similar structures appear to be much more widespread in hepatics and in Monoclea have been misinterpreted as rudimentary pegs, the characteristic feature ofpeggedrhizoids.Swollen side branch initials and the tip cells of major rhizoidal axescontaina single rozellopsidalean (Oomycota formerly Chytridiomycota) zoosporangium in Bryum pseudotriquetrum and up to three similar structures in B. capillare. Each zoosporangium produces a single exit tube. Whereas the infected cells die the subapical cells remain alive and,afterzoospore discharge, produce side branches whose tip cells also become infected. Restriction of the parasites to sidebranchinitials and rhizoid tip cells is perhaps because these are the only cells that the zoospores are able to penetrate. Nutrients translocated along rhizoids to sustaintipgrowth are diverted to nurture the fungi in infected plants. Differences between the number of zoosporangia per host cell and the number of exit tubes between different mosses suggest that more than one species of rozellopsidalean fungus may form rhizoid galls in mosses and not just Pleotracheluswildemanidescribedpreviously.Rhizoid galls appear to be genuinely uncommon in mosses but, following clarification of the differences between these and rhizoidal tubers, further discoveries may be anticipated.

The intercellular biotrophic leaf pathogen Cymadothea trifolii locally degrades pectins, but not cellulose or xyloglucan in cell walls of Trifolium repens

The intercellular ascomycetous pathogen Cymadothea trifolii, causing sooty blotch of clover, proliferates within leaves of Trifolium spp. and produces a complex structure called interaction apparatus (IA) in its own hyphae. Opposite the IA the plant plasmalemma invaginates to form a bubble. Both structures are connected by a tube with an electron-dense sheath. Using immunocytochemistry on high-pressure frozen and freeze-substituted samples, we examined several plant and fungal cell wall components, including those in new host wall appositions at the interaction site, as well as a fungal polygalacturonase. Within the tube linking IA and host bubble, labelling was obtained for cellulose and xyloglucan but not for rhamnogalacturonan-I and homogalacturonans. The IA labelled for chitin and beta-1,3-glucans, and for a fungal polygalacturonase. Plant wall appositions reacted with antibodies against callose, xyloglucans and rhamnogalacturonan-I. Cymadothea trifolii partly degrades the host cell wall. Structural elements remain intact, but the pectin matrix is dissolved. A fungal polygalacturonase detected in the IA is probably a key factor in this process. Owing to the presence of chitin and beta-1,3-glucans, the IA itself is considered an apoplastic compartment.

Characterisation of the early events in atypical tomato root colonisation by a biocontrol agent, Pythium oligandrum

The specific oomycete–plant relationship established between a biological agent, Pythium oligandrum, and tomato ( Lycopersicon esculentum Mill.) plants was examined over the first 48 h after inoculation of tomato roots with the antagonist. One of the most significant effects was the quick colonisation of cortical and vascular root areas by P. oligandrum (until 9 h post-inoculation); it was similar to invasions by the major pathogens of Pythium genus, and much faster than those by Pythium-minor pathogens. Despite the multiplication of hyphae in the root areas, fungal colonisation was associated with neither host wall disruption nor host cell alterations. The colonising hyphae looked healthy till the ninth hour after inoculation, then, they progressively became highly vacuolated. Cytological observations showed that, over the first 14 h of experiment, oomycete invasion was accompanied with rare host-induced defence reactions. Biochemical analysis evidenced an accumulation of phenolic compounds starting 3 h after inoculation. The 14th hour corresponded to the beginning of rishitin (phytoalexin) synthesis. Accumulation of biochemical host defence compounds was concomitant with early signs of hyphae alterations. During the next 34 h several host reactions were regularly amplified as evidenced by the plugging of invaded host cells with heterogeneous osmiophilic or high electron-dense (ED) materials. Fungal cell decay was accompanied with the formation of oogonia in the cortex, vascular parenchyma and xylem vessels. All these early events suggest a peculiar relationship established between P. oligandrum and the plant.

Cytochemical labeling for fungal and host components in plant tissues inoculated with fungal wilt pathogens.

Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.

Irregular growth forms and cell wall modifications, polygalacturonase detection, and endocell formation in Fusarium oxysporum f. sp. radicis-lycopersici infecting tomato plants, as studied ultrastructurally and cytochemically

Pathogen cells of Fusarium oxysporum f.sp. radicis-lycopersici infecting container-grown tomato plants were characterized ultrastructurally, using gold-complexed probes, chitinase and wheat germ agglutinin to localize chitin, and polyclonal antibodies to a polygalacturonase to localize this enzyme. It was isolated and purified from the pathogen growing in culture. Many fungal cells were of irregular forms (microhyphal, frondose) with modified, thin or imperceptible lucent wall layers, in which were often included components seemingly of host origin. Gold particles of the polygalacturonase probe were concentrated on portions of penetration hyphae and in areas of associated altered host wall. Fine filamentous-like structures, often linked to fungal cells, reached into extracellular matter and into host walls. Examination of 0.2–0.25 μm-thick sections at 120 kV, and tilted at various angles, indicated that fungal cells frequently had a pronounced wavy contour. Labelling of thin walls for chitin was mostly nil, particularly in contact with host walls, as of also thicker walls in similar situations, or it was then associated with the outside opaque layer. Cells of diverse dimensions with thin or thicker walls and with altered or normal content, contained endocells. Walls of the encodcells and of the enclosing cells often labelled differently for chitin with both probes. Endocells mostly did not originate from proliferation of a living into a dead cell but often ensuing as an apparent fragmentation of the cell content or following its retraction. The bearing of these observations on the host-pathogen relationship, particularly concerning the role of thin-walled hyphae and irregular forms, is discussed.

Morphological characterization of the mycorrhiza formed by Helianthemum almeriense Pau with Terfezia claveryi Chatin and Picoa lefebvrei (Pat.) Maire.

This work presents the first anatomical description of the mycorrhizal systems of Helianthemum almeriense, and of the structure and ultrastructure of the mycorrhizae formed by this plant species with the ascomycetes Terfezia claveryi and Picoa lefebvrei. Four different mycorrhizal systems are described, the club-shaped mycorrhiza being the most abundant. The type of mycorrhiza formed depended on the mycorrhiza culture conditions, but not on the fungal species. For both fungal species, H. almeriense formed an endomycorrhiza in natural field conditions, an ecto- and ectendomycorrhiza without a sheath in pot cultures, and an ectomycorrhiza with a characteristic sheath and Hartig net in in vitro cultures. This is the first report of a typical sheath in Helianthemum-desert truffle mycorrhizal associations. The results support the idea that culture conditions can induce changes in mycorrhiza morphology and that there is no clear barrier between the two main types of mycorrhiza organization in Helianthemum species. The ultrastructural study confirmed the regular presence of T. claveryi intracellular hyphae in direct contact with the host wall, a localization which seems to be a characteristic of the T. claveryi mycorrhiza organization. The P. lefebvrei mycorrhiza organization was characterized by intracellular hyphae with large amounts of electron-dense globules, probably with a lipidic content, and a warty ornamentation on the wall of the root external hyphae.

Dodder hyphae invade the host: a structural and immunocytochemical characterization.

Dodder (Cuscuta pentagona) hyphae are unique amongst the parasitic weeds for their ability to apparently grow through the walls of the host plant. Closer examination reveals, however, that the hyphae do not grow through the host but rather induce the host to form a new cell wall (or extend the existing wall) to coat the growing hypha. This chimeric wall composed of walls from two species is even traversed by plasmodesmata that connect the two cytoplasms. Compositionally, the chimeric wall is quite different from the walls of either the host or in other cells of the dodder plant, on the basis of immunocytochemical labeling. The most striking differences were in the pectins, with much stronger labeling present in the chimeric wall than in either the host or other dodder walls. Interestingly, labeling with monoclonal antibodies specific to arabinan side chains of rhamnogalacturonan I pectin fraction was highly enriched in the chimeric wall, but antibodies to galactan side chains revealed no labeling. Arabinogalactan protein antibodies labeled the plasma membrane and vesicles at the tips of the hyphae and the complementary host wall, although the JIM8-reactive epitope, associated with very lipophilic arabinogalactan proteins, was found only in dodder cells and not the host. Callose was found in the plasmodesmata and along the forming hyphal wall but was found at low levels in the host wall. The low level of host wall labeling with anticallose indicates that a typical woundlike response was not induced by the dodder. When dodder infects leaf lamina, which have more abundant intercellular spaces than petioles or shoots, the hyphae grew both intra- and extracellularly. In the latter condition, a host wall did not ensheath the parasite and there was clear degradation of the host middle lamellae by the growing hyphae, allowing the dodder to pass between cells. These data indicate that the chimeric walls formed from the growth of the host cell wall in concert with the developing hyphae are unique in composition and structure and represent an induction of a wall type in the host that is not noted in surrounding walls.

Antagonism of the Two-Needle Pine Stem Rust FungiCronartium flaccidumandPeridermium pinibyCladosporium tenuissimumIn Vitro and In Planta

ABSTRACT Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degrees C. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wall and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host cell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta-1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

Ultrastructural and cytochemical study of colonization of xylem vessel elements of susceptible and resistant Dianthus caryophyllus by Fusarium oxysporum f.sp. dianthi

The colonization processes of the xylem in the susceptible carnation cv. Early Sam and the resistant cv. Novada were studied ultrastructurally following inoculation with Fusarium oxysporum f.sp. dianthi. Samples from 1 to 3 cm above the incision were collected over 5 weeks and processed following conventional procedures as well as with probes for cellulose, N-acetyl-glucosamine, and pectin. The fungus grew profusely in the vessel lumina of the susceptible cultivar. Some of the colonized vessels were lined with coating material connected to the fungal cell wall and extending into the host cell wall through microfilamentous-like structures. Coatings did not label for pectin or cellulose. The pathogen crossed from one vessel element to another (and at times to parenchyma cells) usually directly through pit membranes; often the invading structures of the fungus appeared to be either only membrane-bound or formed solely of microfilamentous-like entities. The fungus subsequently invaded extensively, generally by means of microhyphae, the vessel intercalary walls from the pit membranes and vessel wall junctures. Microhyphae had thin or imperceptible walls and contained only some of the normal cytoplasmic components. Initially, the invading hyphae dislocated the host cell walls, apparently mechanically more than by lysis; however, more pronounced lysis occurred following general tissue invasion. Host parenchyma cells seemed relatively unaffected, even after the surrounding walls had undergone severe degradation. Colonization of resistant plants was restricted. Degradation of tissues did not occur and microhyphae were not observed. Inoculated vessel elements in the 'Novada' plants contained numerous fungal cells and little occluding material, whereas the surrounding vessels were almost completely occluded. The initially invaded xylem became tangentially compartmentalized by parenchyma cell wall thickenings and by hyperplastic parenchyma. Occasionally, hyperplastic tissues were slightly re-invaded, forming secondary invasion pockets. Vessel-occluding material varied in structure and opacity, not only from vessel to vessel but also within the same vessel, and contained microfilamentous-like structures and other types of fine fibrillar material. Some vessel elements in or near the secondary invasion pockets contained only the finer fibrils that reacted strongly with an antibody specific for pectin. Vessel elements rarely contained tyloses.Key words: cellulose, chitin, Dianthus caryophyllus, Fusarium wilt, gels and gums, host wall degradation, microhyphae, pectin, tyloses.

Immunogold localization of an extracellular β-1,3-glucanase of the ergot fungus Claviceps purpurea during infection of rye

Immunogold labelling and electron microscopy were used to investigate β-1,3-glucanase secretion by Claviceps purpurea during ergot disease of rye in situ. Molecular cytology allowed us to explore the hypothesis that this enzyme might degrade host phloem callose to maintain flow of assimilates for fungal nutrition and therefore β-1,3-glucanase could play a role in pathogenicity. An extracellular endo-β-1,3-glucanase was purified from axenic culture and an antibody was raised. Enzyme activity staining and immunoblotting showed that the antibody was monospecific for β-1,3-glucanase present in fungal protein populations. Mycelia printings of plate-grown cultures displayed spot-like and streaky immuno-signals suggesting a secretion of β-1,3-glucanase at hyphal tips and young hyphae. The enzyme was immuno gold localized predominantly in cell walls of mycelia from axenic culture. In Western blots of honeydew fractions, one β-1,3-glucanase was immunoreactive. In inoculated plants, immunogold labelling was found in all infection stages and limited to the host-pathogen interface. Gold labelling was detected over fungal protoplasts in vacuoles and in tubularvesicular complexes and multivesicular bodies, which fused with the fungal plasma membrane, indicating that they are part of the secretion pathway. The labelling of the fungal secretory organelles and lack of labelling in any host area apart from the interface verified the fungal origin of β-1,3-glucanase immuno-detected in infected ovaries. Antigenic sites were located in external, subcuticular, penetrating and intercellular hyphae, indicating that the enzyme was secreted throughout the colonization process in planta. Intense labelling regularly associated with fungal cell walls extended into adjacent host walls, indicating a migration of the fungal β-1,3-glucanase into the host apoplast. The gold labelling over host periplasmic spaces showed that the enzyme reached the deposition sites of callose, pointing to an enzymatic suppression of putative plant defense reactions. The host phloem was colonized inter- and intracellularly. Hyphae penetrated into the pectic middle lamella of sieve plates and intense immuno-labelling for β-1,3-glucanase in this area supports a phloem unblocking hypothesis.

Fine structure of the extracellular sheath and cell walls inOphiostoma novo-ulmigrowing on various substrates

The presence of microfilamentous-like structures of tubular appearance (MFS) in cell walls and extracellular sheath material (ES) in a number of isolates of Ophiostoma novo-ulmi Brasier grown on various substrates and following various treatments is reported. Standard fixation or high-pressure freezing methods were used, and cytochemical tests were carried out to detect fungal and host wall components and, in some cases, fungal DNA. In some cases, serial 0.2-μm-thick sections were examined at 120 kV and tilted to obtain stereoscopic images. Whether the fungal cell walls were thick and composed of an outer opaque and inner more electron-lucent layers, or thin and barely perceptible, MFS were observed to extend from the cell cytoplasm as parallel structures across the walls into the surrounding medium, including host cell components in infected elm tissues. MFS were associated (in samples from inoculated trees) with cleavage and desquamation of fungal walls. ES and MFS did not label for cellulose or chitin, but generally labelled slightly for β-(1-3)-glucan and mannose, and strongly for galactose. Only the lucent, inner fungal wall layer labelled for chitin and cellulose. DNA labelling was confined to nuclei and mitochondria in fungal cells from cultures on agar medium; in cells from cultures on millipore membranes, it was pronounced over imprecisely delimited cell regions. The possible ontogeny of MFS components and their importance are discussed. Key words: chitin, Dutch elm disease, fungal fimbriae, fungal walls, gold-complexed probes, microfilamentous structures (MFS).