Biotrophic Mycoparasite Research Articles

Mjuua agapanthi gen. et sp. nov., a biotrophic mycoparasite of Fusarium spp.

Fusarium agapanthi is newly reported from the centre of origin of Agapanthus in South Africa, where it is associated with dead flower stalks of Agapanthus praecox. Mjuua agapanthi, a rare hyphomycete with a morphology corresponding to asexual morphs of Pyxidiophora, was isolated as mycoparasitic on F. agapanthi, along with bacteria that co-occurred in synnematal heads of M. agapanthi. Germinating conidia of M. agapanthi were observed to parasitise germinating conidia of F. agapanthi. Although M. agapanthi could not be cultivated on its own, the association with Fusarium proved to not be restricted to F. agapanthi, as it could also be cultivated with other Fusarium spp. Mjuua agapanthi is a member of Pyxidiophorales, an order of obligate insect parasitic microfungi. The exact role of the bacteria in synnematal heads of M. agapanthi remains to be further elucidated, although one bacterium, Alsobacter metallidurans, appeared to cause lysis of the synnematal conidial cell walls. This discovery suggests that many unculturable obligate biotrophic microbes can probably be cultivated if co-cultivated with their respective hosts. Citation: Crous PW, Dijksterhuis J, Figge M, Sandoval-Denis M (2024). Mjuua agapanthi gen. et sp. nov., a biotrophic mycoparasite of Fusarium spp. Fungal Systematics and Evolution 13: 153-161. doi: 10.3114/fuse.2024.13.09.

Early transcriptomic response of the mycoparasite Sphaerodes mycoparasitica to the mycotoxigenic Fusarium graminearum 3-ADON, the cause of Fusarium head blight

Mycoparasites are an assemblage of biotrophic and necrotrophic fungi that occur on plant pathogenic fungal hosts. Biotrophic mycoparasites are often overlooked in transcriptomic-based biocontrol studies. Sphaerodes mycoparasitica (S.m.) is a specific biotrophic mycoparasite of plant pathogenic Fusarium graminearum (F.g.), a devastating Fusarium head blight (FHB) disease in small-grain cereals. To understand the biotrophic mycoparasitism comprehensively, we performed Illumina RNA-Seq transcriptomic study on the fungus–fungus interaction in vitro. The aim is to identify the transcript-level mechanism related to the biotrophic S.m. mycoparasitism, particularly its ability to effectively control the F.g. 3-ADON chemotype. A shift in the transcriptomic profile of the mycoparasite was triggered in response to its interaction with F.g. during recognition (1.5 days) and colonization (3.5 days) steps. RNA-Seq analysis revealed ~ 30% of annotated transcripts with "function unknown". Further, 14 differentially expressed genes functionally linked to the biotrophic mycoparasitism were validated by quantitative real-time PCR (qPCR). The gene expression patterns of the filamentous haemagglutinin/adhesin/attachment factor as well as cell wall-degrading glucanases and chitinases were upregulated by host interaction. Besides, mycoparasitism-associated antioxidant resistance genes encoding ATP-binding cassette (ABC) transporter(s) and glutathione synthetase(s) were upregulated. However, the thioredoxin reductase was downregulated which infers that this antioxidant gene can be used as a resistance marker to assess S.m. antifungal and antimycotoxigenic activities. The interactive transcriptome of S. mycoparasitica provides new insights into specific mycoparasitism and will contribute to future research in controlling FHB.Graphic

Adaptability of mitosporic stage in Sphaerodes mycoparasitica towards its mycoparasitic-polyphagous lifestyle

ABSTRACTSphaerodes mycoparasitica Vuj. is a Fusarium-specific mycoparasite. Some recent discoveries recognize its biotrophic polyphagous lifestyle as an interesting biocontrol property against a broad spectrum of mycotoxigenic Fusarium hosts. Secondary metabolites such as mycotoxins produced by Fusarium spp. may play an important role in the signaling process, allowing an early mycoparasite-host recognition. A multiple-paper-disc assay has been conducted to test S. mycoparasitica hyphal adaptability to filtrates of 12 Fusarium spp. This study shows that shifts of adapted and nonadapted hyphal migration towards different Fusarium-host filtrates may partly explain S. mycoparasitica polyphagous lifestyle, and its adaptability depending on host preference or compatibility. In terms of host compatibility, the current findings suggest that S. mycoparasitica tends to prefer native Fusarium hosts more related to its origin and propose that the mycoparasite could possess diphasic interactions such as biotrophic-attraction and antagonistic-inhibition relationships based on relative radial growth. This implies that the mycoparasite may use a group of mycotoxins produced by specific Fusarium spp. as an adaptive selective mechanism that facilitates a parasite-host recognition and further successful mycoparasitism. In particular, relative polarity or hydrophilicity/hydrophobicity of mycotoxins may be related to solubility and absorption properties in hyphae of the mycoparasite. Taken together, the studies of host compatibility and adaptability depending on host filtrates will aid in understanding complex mechanisms of S. mycoparasitica, as a promising model organism for a specific biotrophic mycoparasite to enhance and improve biocontrol efficacy against Fusaria.

QPCR quantification of Sphaerodes mycoparasitica biotrophic mycoparasite interaction with Fusarium graminearum: in vitro and in planta assays

Sphaerodes mycoparasitica, a biotrophic mycoparasite of Fusarium species, improved wheat seed germination and seedling growth in vitro compared to Trichoderma harzianum, a necrotrophic mycoparasite. However, under phytotron conditions, both S. mycoparasitica and T. harzianum had positive impact on wheat seedlings growth in the presence of F. graminearum. Once exposed to the mycoparasites, the DNA quantity of F. graminearum in wheat root decreased. Observed shifts in DNA quantity using qPCR, a set of newly designed Sphaerodes-specific SmyITS primers, as well as Trichoderma-TGP4 and Fusarium-Fg16 N primers, demonstrated the mycoparasite's biocontrol effectiveness in planta. In the presence of F. graminearum, the concentration of S. mycoparasitica DNA remained stable in the root, whereas the amount of T. harzianum DNA decreased. The toxicity assays indicated that S. mycoparasitica's mycelia withstand higher concentrations of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone mycotoxins than T. harzianum mycelia. This study compares the ability of two fungi to improve the wheat growth, decrease the root colonization of Fusarium, and withstand mycotoxins.

Sphaerodes quadrangularis biotrophic mycoparasitism on Fusarium avenaceum

Sphaerodes quadrangularis García, Stchigel & Guarro was shown to be a facultative contact biotrophic mycoparasite of Fusarium avenaceum (Fr.) Sacc. (teleomorph: Gibberella avenacea Cooke) that established an intimate relationship with its host by producing hook-shaped and clamp-like attachment structures that appeared to derive nutrients and other essential factors from living host cells. The contact cells of the mycoparasite surrounded and envelop host hyphae and, although they do not appear to penetrate the host, the parasitized hyphae were significantly smaller than unparasitized controls. Sphaerodes quadrangularis did not form specialized attachment structures when cultured together with strains representing F. oxysporum and F. graminearum, indicating some degree of host specificity in the parasite.

Biotrophic mycoparasitic interactions between Sphaerodes mycoparasitica and phytopathogenic Fusarium species

The objective of this study was to investigate the relationships between the biotrophic mycoparasite Sphaerodes mycoparasitica and pathogenic Fusarium strains. To study the interactions between S. mycoparasitica and four different phytopathogenic Fusarium strains, macroscopic observations were performed using dual-culture assays and microscopic examinations in combination with light and fluorescent microscopy. Both macroscopic and microscopic techniques were also vital in determining the host specificity of S. mycoparasitica with F. avenaceum, F. oxysporum, F. proliferatum, and F. sporotrichioides. Our results suggest that S. mycoparasitica established haustorial contact with F. avenaceum and F. oxysporum. Data obtained from the dual-culture assay and parasitism interactions revealed that this newly described contact biotrophic mycoparasitic fungus was capable of reducing F. avenaceum and F. oxysporum linear growth and size of hyphal cells through infection and penetration.

Alberta and Saskatchewan Joint Regional Meeting, 2008

Alberta and Saskatchewan Joint Regional Meeting, 2008

Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface

Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.

Immunocytochemical and cytochemical localization of chitinase and chitin in the infected hosts of a biotrophic mycoparasite, Piptocephalis virginiana

Localization of chitinase and chitin in the uninfected and infected mucoraceous hosts Choanephora cucurbitarum and Phascolomyces articulosus of the mycoparasite, Piptocephlis virginiana, was investigated immunocytochemically with an antibody raised against purified chitinase and cytochemically with wheat germ lectin for the detection of N-acetyl-D-glucosamine oligomers. In the uninfected susceptible host (C. cucurbitarum), chitinase was found in cytoplasmic vacuoles, whereas in the resistant host (P. articulosus) chitinase was observed in the cell wall, as well as in vacuoles. In both hosts, infection by the mycoparasite destroyed vacuolar integrity and the chitinase was dispersed into the cytoplasm. Only in the resistant host, was accumulation of chitinase observed at the penetration sites. N-acetyl-D-glucosamine oligomers were detected in the cell wall of both host fungi, as well as in the mycoparasite and its haustorium. In the infected resistant host, papillae and the extrahaustorial matrix also contained N-acetyl-D-glucosamine, indicating their cell wall-like composition. Differences in the spatial localization of chitinases and N-acetyl-D-glucosamine oligomers in the two fungal hosts are implicated in their compatible and incompatible interactions with the mycoparasite.

Immunocytochemical and Cytochemical Localization of Chitinase and Chitin in the Infected Hosts of a Biotrophic Mycoparasite, Piptocephalis virginiana

Immunocytochemical and Cytochemical Localization of Chitinase and Chitin in the Infected Hosts of a Biotrophic Mycoparasite, Piptocephalis virginiana

Olpitrichum tenellum as a biotrophic contact mycoparasite

Olpitrichum tenellum was shown to be a biotrophic mycoparasite, contacting but not penetrating its host hyphae by means of hook-shaped contact cells, which apparently absorb nutrients and growth factors from living host cells. The known hosts are Fusarium moniliforme, Alternaria alternata, and Cladosporium sp. The hyperparasite failed to grow in the absence of the host on glucose – yeast extract medium unless a water extract of the host was added. Keywords: Olpitrichum tenellum, mycoparasite, axenic culture, hyperparasite.

Biotrophic mycoparasitism byVerticillium biguttatum onRhizoctonia solani

Verticillium biguttatum cannot utilise cellulose or nitrate-nitrogen and it requires biotin for growth, yet it grew and sporulated abundantly onRhizoctonia solani on cellulose, obtaining at least organic carbon, nitrogen and biotin fromR. solani. Videomicroscopy of inter-hyphal interactions on films of water agar showed thatV. biguttatum behaved as a biotrophic mycoparasite. From germinating spores, it penetrated the hyphae ofR. solani and formed haustorium-like branches without killing the host cells, and the haustoria supported an external mycelial network of the mycoparasite. Later the mycoparasite sporulated, and the infected host cells died. On cellulosic substrataV. biguttatum did not reduce the growth ofR. solani, and often enhanced the rate of cellulose degradation. However,V. biguttatum drastically reduced the production of sclerotia byR. solani, often completely suppressing sclerotium production when the mycoparasite infected only a localized region of the host colony. This is ascribed to the creation of a nutrient sink by the parasite, consistent with biotrophy. On plates of cellulose agar the suppression of sclerotia was not confined to parasitized colonies but extended to adjacent colonies ofR. solani that had successfully anastomosed with the parasitized colony. There was no effect on adjacent vegetatively incompatible colonies, where attempted anastomoses caused cytoplasmic death. In comparable experiments the necrotrophic mycoparasiteGliocladium roseum had no long-distance effect on sclerotium production byR. solani.

Mechanisms of recognition in necrotrophic and biotrophic mycoparasites

Mechanisms of recognition in necrotrophic and biotrophic mycoparasites

Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, of the mycoparasite Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 agglutination units/mg) compared with that of the nonhost extract (21 agglutination units/mg). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the crude extract of the host revealed four bands, a, b, c, and d, with respective Mr of 117 000, 100 000, 85 000 and 64 000; these bands except for a faint band c, were absent from the nonhost surface. Deletion of proteins b or c from the crude protein extract of the host significantly reduced its agglutinating activity. Proteins b and c, purified by a series of procedures, were shown to be glycoproteins with glucose and N-acetylglucosamine as major saccharides. The agglutinating activity of a mixture of pure proteins b and c was over 500 times that of either glycoprotein alone, suggesting an involvement of both glycoproteins in the agglutination process. Further characterization showed that the two glycoproteins were heat-resistant with respect to their agglutinin function, which could be totally inhibited by three sugars: arabinose, glucose and N-acetyglucosamine. It is suggested that glycoproteins b and c are the two subunits of a carbohydrate-binding agglutinin present at the host cell surface and involved in agglutination and attachment of the mycoparasite germ tubes. Key words: agglutinin, attachment, cell surface, sugars, glycoproteins, mycoparasitism.

Infection of Pythium and Phytophthora species by Olpidiopsis gracilis (Oomycetes)

Zoospores from a culture of the biotrophic mycoparasite Olpidiopsis gracilis were inoculated on to vegetative hyphae of a range of Pythium and Phytophthora species. Twenty species of Pythium and two of Phytophthora were infected. The process of infection is described, and the optimum temperature for infection of Pythium sylvaticum was determined. Measurements of the expansion of infected portions of the hyphae of the Pythium host showed that despite the apparent hypertrophy, the rate of volume increase of the infected portions was not significantly different from that of uninfected hyphal apices.

Partial Purification of the Growth Factor Mycotrophein

Mycotrophein, a growth factor required by some biotrophic contact mycoparasites, was isolated from Arthrobotrys musiformis. Mycotrophein was partially purified using a combi? nation of silicic acid and high performance liquid chromatographies. The partially purified compound promoted axenic growth of Stephanoma phaeospora, Gonatobotrys simplex, Calcarisporium parasiticum and Gonatorrhodiella highlei, contact mycoparasites which require this factor for axenic growth. There was essentially no growth-promoting activity with any of the many authentic biochemicals tested. Electron impact mass spectral measurements on the purified mycotrophein indicated only the presence of a mixture of tetra- and pentaethyleneglycol mono-(nonylphenyl) ethers that could possibly act as carriers ofthe biologically active factor.

Host-Parasite Relations in a Mycoparasite. VIII. Age-Related Host Resistance inChoanephora Cucurbitarum

Isolated cell walls obtained from young (1 da) and mature (5 da) cultures of Choanephora cucurbitarum, susceptible and resistant, respectively, to the biotrophic mycoparasite, Piptocephalis virginiana, were analyzed by microchemical and electron microscopic techniques. Comparison of the chemical composition of both young and mature cell walls revealed no qualitative difference. However, as the cultures aged, an increase in chitin, protein, neutral sugars (especially glucose and mannose), and a decrease in chitosan content were noted. Glutaraldehyde-osmium-fixed thin sections and metal shadow replicas revealed that the cell walls of hyphae in young cultures had a single layer of randomly distributed microfibrils of chitin, whereas the hyphal walls in mature cultures possessed two layers, randomly- and parallel-oriented microfibrils. The presence of a secondary layer of cell wall in mature hyphae coincided with the ability of the host to form a papilla at the infection site when challenged by the parasite. The role of the host cell wall in age-related resistance to a mycoparasite is discussed.

Parasitism and Nutrition of the Contact Mycoparasite Acladium Tenellum

Acladium tenellum is a contact, biotrophic mycoparasite deficient for thiamine and biotin but not mycotrophein. Nine Deuteromycetes, of 31 fungi tested, were hosts for two isolates of A. tenellum. Hook-shaped contact cells that partially encircled but did not penetrate host hyphae were formed by the parasite on all hosts. Generally, parasitism had no visible effect on host hyphae or colony growth. The mycelium of Bispora sp., a host fungus, contained thiamine and biotin, suggesting growth of the parasite on certain fungi may depend on its ability to obtain these required nutrients from host mycelium. Carbon sources, glucose, mannose, and galactose supported optimal axenic growth of both A. tenellum isolates. Fructose and cellobiose, however, supported optimal growth of one isolate, but less than optimal of the second. Nitrogen sources, casein hydrolysate, alanine, asparagine, aspartic acid, glutamic acid, and glutamine supported optimal growth of both isolates.

Host–parasite relations in a mycoparasite. VI. Leakage of electrolytes, amino acids, and sugars after infection with Piptocephalis virginiana

The leakage of electrolytes, amino acids, and sugars from Choanephora cucurbitarum infected by a biotrophic haustorial mycoparasite, Piptocephalis virginiana was investigated and compared with published reports of leakage from plant hosts infected by biotrophic pathogens. During early stages of infection (24 h after inoculation), the mycoparasitic system showed a significant increase in the leakage of electrolytes, amino acids, and sugars contrary to plant host–pathogen systems which exhibit pronounced leakage during later stages of disease development. Among the 26 amino acids and amino compounds, aspartic acid, glutamic acid, and alanine exuded in higher amounts. No new species of amino acid or amino compound was detected in the leachate of the infected host. Sugar analysis of leachate showed the presence of eight different sugars among which glucose was found in significant amounts (50–53%). Comparison between leachate sugars and internal pool sugars of the infected and control hosts suggested that the leakage of sugars was selective. The nutritional implication of this preferential leakage is discussed.

Role of lipids in host-parasite relations of a mycoparasite

Analyses of total lipids extracted from the axenic culture of Piptocephalis virginiana , a biotrophic mycoparasite of mucoraccous fungi, revealed lack of (i) γ-linolenic acid, a polyunsaturated fatty acid characteristic of the order Mucorales and of (ii) diphosphatidylglycerol. Host infection resulted in an increase of total lipids, up to 25% or more, and this increase was mainly due to the polar lipid fraction since the neutral lipid fraction remained fairly constant in the infected and uninfected hosts. The percentage of γ-linolenic acid in phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol increased considerably in the infected host. Unequivocal evidence is presented to show that the degree of parasitism in P. virginiana is related directly to the level of γ-linolenic acid in the host. Neither the degree of unsaturation nor any other polyunsaturated fatty acid could replace γ-linolenic acid. The significance of the absence of γ-linolenic acid and diphosphatidylglycerol from the parasite's lipids is discussed.