Comparative Physiology and Behaviour of the Mycoparasites Pythium acanthophoron, P. oligandrum and P. mycoparasiticum

Pythium acanthophoron (two isolates) was as aggressive as P. oligandrum in hyphal interactions with Fusarium oxysporum on water agar films. It caused rapid post-contact lysis or cytoplasmic coagulation of the host, and often branched and penetrated the host at contact points. P. acanthophoron grew across a range of fungi on pre-colonized agar plates; the range was narrower than for P. oligandrum but broader than for P. mycoparasiticum. In chemically defined liquid media, P. acanthophoron was unique among the six known mycoparasitic Pythium spp. in requiring organic nitrogen but not thiamine. It also grew on mannitol as the sole carbon source in the presence of calcium or sterols or both. However, individual isolates of the three mycoparasitic species showed different responses to calcium and the sterols ergosterol, cholesterol and beta-sitosterol when grown on mannitol. All three mycoparasites required components of molasses, carrot extract or sunflower seed extract to produce oogonia in culture; they formed few oogonia on potato extract, with or without dextrose, even when supplied with sterols. The three mycoparasites were less tolerant of high NaCl levels in culture than were three phytopathogens (P. aphanidermatum, P. ultimum, P. graminicola), in contrast to a previous report for P. oligandrum and P. ultimum. These in vitro studies suggest that P. acanthophoron has potential for use as a biocontrol agent instead of, or in addition to, P. oligandrum.     

A new species of Leptobacillim,L. symbioticum,isolated from mites and sori of soybean rust

A verticillium-like fungus forming a whitish colony and mainly solitary phialides that produced fusiform to cylindrical conidia in chains was isolated from uredinia of Phakopsora pachyrhizi, a causal agent of soybean rust. In addition, two similar looking isolates were obtained from Prostigmata mites. Our taxonomic study based on morphology and phylogenetic analysis using ITS rDNA and LSU rDNA D1/D2 region sequences revealed that the three isolates were the same species and assigned to the genus Leptobacillium. These isolates represent a new species, morphologically and phylogenetically distinguished from the type species, L. leptobactrum, and we propose L. symbioticum sp. nov. In addition, Simplicillium chinense and S. coffeanum are assigned to the genus Leptobacillium.     

Strangler unmasked: Parasitism of Cystoderma amianthinum by Squamanita paradoxa and S. pearsonii

The mushroom-forming genus Squamanita comprises 10 described species, all parasitic on basidiomes of other members of the order Agaricales, including members of the genera Cystoderma, Galerina, Inocybe and Hebeloma. Here we report an anatomical investigation of the stipitate “mycocecidium’ (=fungus gall) formed on the basidiome of Cystoderma amianthinum (“powdercap”) by S. paradoxa (“powdercap strangler”), alongside the development of taxon-specific-PCR primer to localise the presence of S. paradoxa/C. amianthinum mycelia within mycocecidia, in associated plant tissues and apparently healthy host basidiomes. Dissection of fungarium samples also confirmed these findings, whilst ITS barcode sequencing of all available samples held at the RBG Kew and Edinburgh fungaria did not reveal any variation in ITS sequences within UK populations of S. paradoxa or the closely related S. pearsonii. The absence of any ¹³C or ¹⁵N isotopic differences between C. amianthinum and S. paradoxa suggests that S. paradoxa is nutritionally dependent on its host. The status of C. amianthinum as host of S. pearsonii is also confirmed.     

Pythium periplocum, an aggressive mycoparasite of Botrytis cinerea causing the gray mould disease of grape-vine

Pythium periplocum Dreschler has been found to be an aggressive mycoparasite of Botrytis cinerea, the causal agent of the gray mould disease of the grape-vine. When grown together, the former enters the latter's mycelium, branches freely within, coagulates its cytoplasm and finally tears its hyphae apart, bringing about widespread destruction of the grape-vine pathogen. Extensive coiling around the host, as reported in the case of other mycoparasites belonging to the genus Pythium, has not been observed here. The infected mycelium of B. cinerea fails to infect the grape-vine and does not induce the characteristic gray mould symptoms. Since P. periplocum is not a grape-vine parasite, it could be useful for the biological control of B. cinerea. A brief account of this mycoparasitism is discussed in this article.     

Potentially pathogenic and biocontrol Ascomycota associated with green wall structures of basket willow (<Emphasis Type="Italic">Salix viminalis</Emphasis> L.) revealed by phenotypic characters and ITS phylogeny

Ascomycota are among the fungi that cause serious willow diseases in all natural habitats worldwide. This study was conducted to determine if basket willow used in green wall structures (GWS) built of willow stems were infected by potentially important fungal diseases or their antagonists in urban areas of eastern Canada. In total, 13 different phenotypic genera belonging to eight families of ascomycetous fungi were isolated and identified according to their sexual and/or asexual forms. Venturia pathogenic species complex were represented by three different anamorphs: Fusicladium, Fusicladium-Cladosporium, and Pollaccia as anamorph. They were responsible for the highest incidence value on leaves (IF > 15%). Cryptodiaporthe, Drepanopeziza, and Glomerella dominated on bark (IF > 5%). A significantly higher incidence value of fungal communities was found on first year than on second year GWS. The correspondence analysis using χ² distance showed that communities of potentially pathogenic species are closely related to diseased plants, while healthy plants often contain biocontrol species such as Cladobotryum mycoparasite on healthy bark and Alternaria sp. antagonist on healthy leaves. The phylogenetic positions of the different fungal taxa and their relationship have been revealed by use of PCR amplified internal transcriber spacer (ITS) region of rDNA.     

Proteomic response of Trichoderma aggressivum f. europaeum to Agaricus bisporus tissue and mushroom compost

A cellular proteomic analysis was performed on Trichoderma aggressivum f. europaeum. Thirty-four individual protein spots were excised from 2-D electropherograms and analysed by ESI-Trap Liquid Chromatography Mass Spectrometry (LC/MS). Searches of the NCBInr and SwissProt protein databases identified functions for 31 of these proteins based on sequence homology. A differential expression study was performed on the intracellular fraction of T. aggressivum f. europaeum grown in media containing Agaricus bisporus tissue and Phase 3 mushroom compost compared to a control medium. Differential expression was observed for seven proteins, three of which were upregulated in both treatments, two were down regulated in both treatments and two showed qualitatively different regulation under the two treatments. No proteins directly relating to fungal cell wall degradation or other mycoparasitic activity were observed. Functions of differentially produced intracellular proteins included oxidative stress tolerance, cytoskeletal structure, and cell longevity. Differential production of these proteins may contribute to the growth of T. aggressivum in mushroom compost and its virulence toward A. bisporus.     

Screening Trichoderma species for biological control activity against Phytophthora ramorum in soil

Despite efforts of eradication and sanitation, Phytophthora ramorum persists in the United States and abroad. Fungicides have limited effectiveness, but there are concerns that they may only inhibit pathogen growth and hasten resistance development after repeated fungicide applications. Biological control is an active control measure that can work continuously as long as the agent is alive and active. The goal of this study was to examine whether Trichoderma spp. have the potential as a biological control agent against P. ramorum. Sixteen Trichoderma spp. isolates were screened for mycoparasitism of P. ramorum in a dual culture assay. The different Trichoderma spp. isolates demonstrated variable mycoparasitic activities with some isolates showing no activity while others completely eliminated the pathogen after 4 weeks. Seven isolates of T. asperellum were consistent among replicated trials in eliminating recovery of P. ramorum from the exposed agar plugs and preventing leaf disk necrosis. Further testing of six T. asperellum isolates against two different P. ramorum isolates (A1 and A2 mating types) resulted in the same high level of mycoparasitic activity. Soil assays involving P. ramorum-infested potting mix and selected Trichoderma spp. isolates demonstrated that two isolates (04-22 and 02-64) were consistent among the repetitions to eliminate P. ramorum propagules to non-detectable levels. Based on these results, specific T. asperellum isolates have the potential to remediate P. ramorum-infested soil and have the potential to be developed into a commercially-viable product.     

Peptaibol production by sepedonium strains parasitizing boletales

Fungi of the genus Sepedonium (anamorphic ascomycetes) are known to infect fruiting bodies of Basidiomycetes of the order Boletales. We have characterized twelve Sepedonium isolates by intact-cell mass spectrometry (IC-MS) with the help of respective biomarkers and their metabolite spectra focusing on peptaibol production. A strain of mycoparasitic S. chalcipori was grown in solid-state fermentation, and tylopeptin production was found, suggesting an ascomycete origin of these peptaibols, which were first described in the basidiomycete Tylopilus neofelleus. In addition, the structures of two new peptaibols, chalciporin A (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Ser-Leu-Ala-Leu-Aib-Gln-Leuol) and chalciporin B (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Gln-Aib-Ala-Leu-Aib-Gln-Leuol) are presented. The IC-MS technique was applied for in situ peptaibol analysis of Sepedonium strains growing on Boletales, in particular S. chrysospermum infecting Xerocomus cf. badius. We found chrysospermins at the surface and within basidiomycete tissue, as well as in the cultivated parasite.     

A molecular approach to explore the extent of the threatened fungus Hypocreopsis rhododendri within wood

Hypocreopsis rhododendri is a rare fungus that grows on woody stems in hyperoceanic climax scrub on the west coasts of Britain, Ireland, and France. Knowledge of the distribution and abundance of the fungus is based entirely on sporocarp records; it does not account for any occurrence as vegetative mycelia. To address this issue, a H. rhododendri-specific polymerase chain reaction (PCR) was developed and used to assay Corylus avellana (hazel) stems for the presence of H. rhododendri mycelia. The primers ITSHrF and ITSHrR were designed within the internal transcribed spacer 2 region, and their specificity to H. rhododendri was established by their failure to amplify DNA extracted from 14 other Hypocreaceae species. The sensitivity of the assay was demonstrated by amplifying DNA extracted from 4 mg C. avellana wood spiked with 0.0013 % H. rhododendri mycelium. Samples of wood and bark were then taken from around and directly underneath 11 H. rhododendri sporocarps and assayed for the presence of H. rhododendri. PCR products were obtained from a third of the surface bark samples, but only one faint product was obtained from 70 samples taken from beneath the outer bark. The results support the view that H. rhododendri does not form mycelia within stems. We suggest that H. rhododendri is not a saprotrophic fungus, but instead appears to be a parasitic on the wood decay fungus Hymenochaete corrugata, with which it always occurs. Evidence that tissue of H. corrugata is present within the sporocarps of H. rhododendri is discussed.