Ultrastructure of Mycoparasitism of Trichoderma, Gliocladium and Laetisaria Species on Botryodiplodia theobromae

Mycoparasitic activities of various isolates of Trichoderma viride, T. harzianum, T. hamatum, T. longibrachiatum, T. koningii, T. pseudokoningii, Gliocladium virens and Laetisaria arvalis were studied against a serious plant pathogen, Botryodiplodia theobromae by scanning electron microscopy (SEM). Macroscopic observations of fungal growth in dual-cultures revealed that most of the isolates made hyphal contact with the pathogen within 2 days after inoculation, leading to the inhibition in pathogen growth. However, T. viride Tv-4, T. hamatum and T. pseudokoningii inhibited pathogen growth before hyphal contact and exhibited an inhibition zone between the colonies of both fungi. SEM investigations demonstrated that in case of hyphal interaction, the firm binding of antagonists (T. viride Tv-1 & Tv-3, T. harzianum Th-1 & Th-2, T. longibrachiatum and L. arvalis) to B. theobromae hyphae established either by coiling around its hyphae, or by penetrating its hyphal cells by forming hooks, haustoria and appressoria-like structures which invariably led to cell disruption. Although T. koningii and G. virens Gv-2 & Gv-3 did not interact physically by way of coiling and penetration, they produced wall lytic enzymes or antifungal substances after coming in contact with B. theobromae which caused wrinkling, bursting and collapsing of pathogen mycelium. It is, therefore, suggested that the outcome of the interaction of antagonist and pathogen was most likely determined by initial hyphal contact that triggered a series of events in pathogen degradation.     

Biological control of root rot-root knot disease complex of tomato

Efficacy of Pseudomonas aeruginosa alone or in combination with Paecilomyces lilacinus was evaluated in the control of root-knot nematode and root-infecting fungi under laboratory and field conditions. Ethyl acetate extract (1 mg/ml) of P. lilacinus and P. aeruginosa,respectively, caused 100 and 64% mortality of Meloidogyne javanica larvae after 24 h. Ethyl acetate fractions of biocontrol agents were more effective than hexane extracts in the suppression of M. javanica larvae, indicating that active nematicidal compounds are intermediary in polarity. In field experiments, biocontrol fungus and bacterium significantly suppressed soilborne root-infecting fungi including Macrophomina phaseolina, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani and Meloidogyne javanica, the root-knot nematode. P. lilacinus parasitized eggs and female of M. javanica and this parasitism was not significantly influenced in the presence of P. aeruginosa. P. aeruginosa was reisolated from the inner root tissues of tomato, whereas P. lilacinusdid not colonize tomato roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

A molecular survey of ectomycorrhizal hyphae in a California Quercus–Pinus woodland

Ectomycorrhizal (ECM) hyphal communities have not been well characterized. Furthermore, there have been few studies where the ECM hyphal community is compared to fungi detected as sporocarps or ECM-colonized root tips. We investigated fungi present as hyphae in a well-studied California Quercus–Pinus woodland. Hyphal species present were compared to those found as sporocarps and ECM root tips at the same site. Hyphae were extracted from root-restrictive nylon mesh in-growth bags buried in the soil near mature Quercus douglasii, Quercus wislizeni, and Pinus sabiniana. Taxa were identified using PCR, cloning, and DNA sequencing of internal transcribed spacer and 28s rDNA. Among the 33 species detected, rhizomorph-forming ECM fungi dominated the hyphal community, especially species of Thelephoraceae and Boletales. Most fungi in soils near Quercus spp. and P. sabiniana were ECM basidiomycetes, but we detected two ECM ascomycetes and three non-mycorrhizal fungi. Many ECM species present as hyphae were also previously detected at this site as sporocarps (18%) or on ECM root tips (58%). However, the hyphal community was mostly dominated by different taxa than either the sporocarp or ECM root communities.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

Experimental infection of white mouse with chrysosporium and paecilomyces

SPF white mice were inoculated intraperitoneally with 5 strains of saprophytic fungi of the mycelial genera Chrysosporium (C. keratinophilum, C. tropicutn) and Paecilomyces (P. lilacinus, P. marquandii, P. victoriae). The fungi caused granulomatous lesions in the peritoneal cavity and they were recultured (except P. lilacinus and P. marquandii) two months after inoculation. Spores, short hyphae and budding cells of all the fungi were observed in the granulomas stained by periodic-acid Schiff (PAS) and methenamine-silver nitrate (Grocott) techniques.     

Hyperparasitism of Streptomyces albus on a Destructive Mycoparasite Nectria inventa

Using scanning electron microscopy, Streptomyces albus was proved to be a hyperparasite of Nectria inventa, itself a well-known mycoparasite on many fungi. The hyperparasite had no apparent antagonistic activity against N. inventa, but did have intense tropic response toward it. Upon contact with the host, the hyperparasite grew along, and formed appressorium-like structures on the host hyphae. The parasitism that led to the eventual collapse of the host cells was not necessarily accompanied by actual hyphal penetration. The hyperparasite could, however, readily penetrate the host hyphae, and its hyphae were frequently found inside the host hyphae.     

Contact‐induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae

Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano‐fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour‐following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of ～ 8.7 μN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue.     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

Larvicidal activity of some fungal extracts on Aedes caspius and Culex pipiens (Diptera: Culicidae)

A preliminary study was conducted to evaluate the effects of nine methanol fungal extracts on fourth instar larvae of Aedes caspius and Culex pipiens (Diptera: Culicidae). None of the extracts tested showed larvicidal activity except for the Paecilomyces lilacinus extract, which showed 100 % mortality against the larvae of Ae caspius after 24 h. The LC₅₀ values of the P. lilacinus extract after 24 h were 190.66 μg/mL against Ae. caspius and 254.25 μg/mL against Cx. pipiens, respectively. After 48 h, meanwhile, the LC₅₀ values were 65.70 and 164.13 μg/mL, respectively. Histological analysis of the midgut of the treated larvae of Ae. caspius and Cx. pipiens revealed changes such as cell destruction, spacing between the cells and disruption of the microvilli, resulting in an appearance of vacuolization in the midgut. This article is the first report of the use of P. lilacinus extract for the control of Ae. caspius and Cx. pipiens larvae and the data obtained may help to provide a better understanding of the mode of action of P. lilacinus as an insecticide against mosquito larvae.     

Mycoparasitism of Endophytic Fungi Isolated From Reed on Soilborne Phytopathogenic Fungi and Production of Cell Wall-Degrading Enzymes In Vitro

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of β-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of β-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.     

Mycoparasitism of Pythium ultimum by Antagonistic Binucleate Rhizoctonia Isolates in Agar Media and on Capsicum Seeds

Hyphal interactions between two antagonistic binucleate Rhizoctonia isolates (BNR) and the seedling dampingoff pathogen, Pythium ultimum var. sporangiiferum, were observed by both light-and scanning electron microscopy (SEM), on agar media and on capsicum seeds in sterilized potting mix. Both BNR isolates displayed similar mycoparasitic behaviour towards P. u. sporangiiferum on agar media. This included parallel growth along the pathogen hyphae, formation of hook-shaped hyphal tips and coils on the surface of P. u. sporangiiferum hyphae and penetration and growth within pathogen structures. Disruption of cytoplasmic streaming and disorganisation of pathogen cytoplasm were also observed. SEM observations revealed alterations in P. u. sporangiiferum cell wall structure and the presence of penetration holes apparently due to digestion by the BNR. P. u. sporangiiferum was also parasitised by both BNR isolates on capsicum seed coats, with parallel growth, hook formation and coils commonly observed. The above observations indicated that mycoparasitism is a possible mode of action of BNR against P. u. sporangiiferum.     

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.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

Stage Specific Assessment of Candida albicans Phagocytosis by Macrophages Identifies Cell Wall Composition and Morphogenesis as Key Determinants

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

The different morphologies of yeast and filamentous fungi trigger distinct killing and feeding mechanisms in a fungivorous amoeba

Size and diverse morphologies pose a primary challenge for phagocytes such as innate immune cells and predatory amoebae when encountering fungal prey. Although filamentous fungi can escape phagocytic killing by pure physical constraints, unicellular spores and yeasts can mask molecular surface patterns or arrest phagocytic processing. Here, we show that the fungivorous amoeba Protostelium aurantium was able to adjust its killing and feeding mechanisms to these different cell shapes. Yeast-like fungi from the major fungal groups of basidiomycetes and ascomycetes were readily internalized by phagocytosis, except for the human pathogen Candida albicans whose mannoprotein coat was essential to escape recognition by the amoeba. Dormant spores of the filamentous fungus Aspergillus fumigatus also remained unrecognized, but swelling and the onset of germination induced internalization and intracellular killing by the amoeba. Mature hyphae of A. fumigatus were mostly attacked from the hyphal tip and killed by an actin-mediated invasion of fungal filaments. Our results demonstrate that predatory pressure imposed by amoebae in natural environments selects for distinct survival strategies in yeast and filamentous fungi but commonly targets the fungal cell wall as a crucial molecular pattern associated to prey and pathogens.