Snow Mold Fungus Research Articles

Identification and characterization of extracellular GH3 β-glucosidase from the pink snow mold fungus, Microdochium nivale.

Glycoside hydrolase family 3 (GH3) β-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its β-glucosidase has not been identified. In this study, a GH3 β-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl β-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis=1.6m m), and d-glucose caused competitive inhibition (Ki=0.5m m). MnBG3A acted on β-glucobioses with β1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to β1-6 linkage. MnBG3A has similar features to those of β-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.

An Apoplastic Defensin of Wheat Elicits the Production of Extracellular Polysaccharides in Snow Mold.

TAD1 (Triticum aestivum defensin 1) is a plant defensin specifically induced by low temperature in winter wheat. In this study, we demonstrated that TAD1 accumulated in the apoplast during cold acclimation and displayed antifungal activity against the pink snow mold fungi Microdochium nivale. When M. nivale was treated with TAD1, Congo red-stainable extracellular polysaccharides (EPS) were produced. The EPS were degradable by cellulase treatment, suggesting the involvement of β-1,4 glucans. Interestingly, when the fungus was treated with FITC-labeled TAD1, fluorescent signals were observed within the EPS layer. Taken together, these results support the hypothesis that the EPS plays a role as a physical barrier against antimicrobial proteins secreted by plants. We anticipate that the findings from our study will have broad impact and will increase our understanding of plant–snow mold interactions under snow.

Characterization of microbial antifreeze protein with intermediate activity suggests that a bound-water network is essential for hyperactivity

Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein’s ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

The endophyte Allantophomopsis cytisporea is associated with snow blight on Calluna vulgaris in the Alps—An effect of climate change?

ABSTRACTShoots of Calluna vulgaris, Erica carnea, Juniperus communis subsp. nana, Picea abies, and Pinus mugo subsp. mugo covered with felty, melanized epiphytic mycelia typical for brown felt blight caused by Herpotrichia pinetorum were collected at several locations in the Swiss Alps. Most cultures prepared from the mycelia on J. communis subsp. nana and P. abies were H. pinetorum, whereas the majority of cultures from P. mugo subsp. mugo and C. vulgaris were identified by internal transcribed spacer ITS1-5.8S-ITS2 sequencing and morphology as Allantophomopsis cytisporea. The fungus tolerates low temperatures, has an optimum between 16°C and 24°C, and ceases to grow at 28°C. 35°C is lethal. A. cytisporea is known as the causal agent of cranberry black rot on Vaccinium macrocarpon but has never been described as a snow mold. A. cytisporea is an endophyte in C. vulgaris but seems able to kill leaflets and whole shoots during winter. The epiphytic mycelium can expand from C. vulgaris to neighboring shoots of P. mugo subsp. mugo and J. communis subsp. nana below the snow where it forms epiphytic mycelial mats reminiscent of H. pinetorum. H. pinetorum has a strong antibiotic effect against A. cytisporea at 4°C and 20°C, whereas A. cytisporea grows faster at these temperatures. The effects of climate change on the interaction between the two snow mold fungi and their consequences on regeneration of woody plants at timberline are discussed.

雪腐病菌における進化・適応(共生・寄生微生物の進化と環境適応,シンポジウム)

雪腐病菌における進化・適応(共生・寄生微生物の進化と環境適応,シンポジウム)

Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus.

Snow mold fungus, Typhula ishikariensis, secretes seven antifreeze protein isoforms (denoted TisAFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, TisAFP8, at 1.0 Å resolution is presented. TisAFP8 folds into a right-handed β-helix accompanied with a long α-helix insertion. TisAFP8 exhibited significantly high antifreeze activity that is comparable with other hyperactive AFPs, despite its close structural and sequence similarity with the moderately active isoform TisAFP6. A series of mutations introduced into the putative ice-binding sites (IBSs) in the β-sheet and adjacent loop region reduced antifreeze activity. A double-mutant A20T/A212S, which comprises a hydrophobic patch between the β-sheet and loop region, caused the greatest depression of antifreeze activity of 75%, when compared with that of the wild-type protein. This shows that the loop region is involved in ice binding and hydrophobic residues play crucial functional roles. Additionally, bound waters around the β-sheet and loop region IBSs were organized into an ice-like network and can be divided into two groups that appear to mediate separately TisAFP and ice. The docking model of TisAFP8 with the basal plane via its loop region IBS reveals a better shape complementarity than that of TisAFP6. In conclusion, we present new insights into the ice-binding mechanism of TisAFP8 by showing that a higher hydrophobicity and better shape complementarity of its IBSs, especially the loop region, may render TisAFP8 hyperactive to ice binding.

The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat

TAD1 (Triticum aestivum defensin 1) is induced during cold acclimation in winter wheat and encodes a plant defensin with antimicrobial activity. In this study, we demonstrated that recombinant TAD1 protein inhibits hyphal growth of the snow mold fungus, Typhula ishikariensis in vitro. Transgenic wheat plants overexpressing TAD1 were created and tested for resistance against T. ishikariensis. Leaf inoculation assays revealed that overexpression of TAD1 confers resistance against the snow mold. In addition, the TAD1—overexpressors showed resistance against Fusarium graminearum, which causes Fusarium head blight, a devastating disease in wheat and barley. These results indicate that TAD1 is a candidate gene to improve resistance against multiple fungal diseases in cereal crops.

Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense)

Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to −3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment

The snow mold fungus, Sclerotinia borealis, shows optimal growth at 4°C on potato dextrose agar (PDA) and can grow even at subzero temperature. Its mycelial growth was improved on frozen PDA at -1°C and on PDA containing potassium chloride (KCl) (water potential, -4.27 to -0.85 MPa) or D(-) sorbitol (-3.48 to -0.92 MPa). Its optimal growth temperature shifted from 4 to 10°C on PDA amended with KCl or sorbitol, indicating that inherent optimal growth occurs at high temperatures. These results suggest that S. borealis uses concentrated nutrients in the frozen environment and that such physiologic characteristics are critical for the fungus to prevail at subzero temperatures.

Sclerotia of Typhula ishikariensis biotype B (Typhulaceae) from archaeological sites (4000 to 400 BP) in Hokkaido, northern Japan

Despite their close association with human activities, plant pathogenic fungi have rarely been found in archaeological excavations. We report here that a fungus was closely associated with human activities even in prehistoric times. Sclerotium-like objects were found at historical sites (4000 to 400 BP) on the island of Hokkaido, northern Japan. They were spherical, 0.3-1.0 mm in diameter, and had a medulla and rind. Some had leaf fragments on the surface or a protuberance that resembled emerging sporocarp primordia. These traits indicated that they were sclerotia of the snow mold fungus, Typhula ishikariensis biotype B.

Mycelial growth of the snow mold fungus, <i>Sclerotinia borealis</i>, improved at low water potentials: an adaption to frozen environment

Mycelial growth of the snow mold fungus, <i>Sclerotinia borealis</i>, improved at low water potentials: an adaption to frozen environment

Aggressiveness of Three Snow Mold Fungi on Creeping Bentgrass Cultivars under Controlled Environment Conditions

Snow molds are the most important winter diseases of turfgrass in the United States and Canada. Eight isolates of three snow mold fungal species (three isolates of Typhula ishikariensis, three of T. incarnata, and two of Microdochium nivale) were collected from infected turfgrasses on golf courses. The isolates were evaluated for their relative aggressiveness on three cultivars (L-93, Penncross, and Providence) of creeping bentgrass (Agrostis palustris) under the same controlled conditions. Four plant ages (15, 19, 23 and 27 week-old plants from germination to inoculation) were evaluated for their susceptibility to the three pathogens and for the recovery of the plants. Regardless of age or cultivar of the host plant, M. nivale was found to be more aggressive and faster to infect and colonize than Typhula species. After three weeks recovery, M. nivale-inoculated plants showed higher disease severity than plants inoculated with the two Typhula species. Plants infected by Typhula species displayed no significant difference in disease severity. As creeping bentgrass plants get older, the severity of disease caused by three snow molds gradually decreases. This effect was observed in all cultivars tested, suggesting expression of age-related resistance as the bentgrass plants matured.

Crystal structure of an antifreeze protein from snow mold fungi

Crystal structure of an antifreeze protein from snow mold fungi

Snow mold fungus, Typhula ishikariensis group III, in Arctic Norway can grow at a sub-lethal temperature after freezing stress and during flooding

Snow mold fungus, Typhula ishikariensis group III, in Arctic Norway can grow at a sub-lethal temperature after freezing stress and during flooding Isolates of the snow mold fungus Typhula ishikariensis group III, which is predominant in Finnmark (northern Norway) and Svalbard, are more resistant to freezing stress than group I isolates from the southern part of Norway. Group III isolates showed irregular growth on potato dextrose agar (PDA) plates when subjected to heat stress at 10°C. However, group III isolates showed relatively good growth on PDA at 10°C after freezing treatment. The optimal temperatures for mycelial growth were 5°C on PDA and 10°C in potato dextrose broth (PDB), and group III isolates showed normal mycelial growth at 10°C in PDB. Mycelium of group III isolates cultivated in water poured into PDA plates, and normal hyphal extension was observed only in the liquid media. Hyphal growth became irregular when mycelia had extended above the surface of the liquid media. These results suggested that group III isolates can grow at a sub-lethal temperature after freezing stress and during flooding. Soil freezing and thawing occurs regularly in the Arctic, and physiological characteristics of group III isolates are well adapted to climatic conditions in the Arctic.

New findings of snow mold fungi from Greenland

Three species of snow mold fungi, Myriosclerotinia borealis (syn. Sclerotinia borealis), Typhula incarnata and T. ishikariensis (syn. T. borealis), were collected from Nuuk, Sisimiut, Ammassalik and Kulusuk in Greenland. Myriosclerotinia borealis and T. incarnata are new records from Greenland. Some isolates of T. ishikariensis were collected from Nuuk and Ammassalik and all isolates from Sisimiut showed irregular growth as hyphal extensions at 10°C in potato dextrose agar plates. These physiological characteristics of some isolates from Greenland are similar to those of psychrotrophisolates (group III strains) from Finnmark, northernmost Norway and Svalbard.

A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, Microdochium nivale

A novel cold-induced cystatin cDNA clone (TaMDC1) was isolated from cold acclimated winter wheat crown tissue by using a macroarray-based differential screening method. The deduced amino acid sequence consisted of a putative N-terminal secretory signal peptide of 37 amino acids and a mature protein (mTaMDC1) with a molecular mass of 23 kDa. The mTaMDC1 had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited partial similarity to the cystatin domain. The recombinant mTaMDC1 was purified from Escherichia coli and its cysteine proteinase inhibitory activity against papain was analyzed. The calculated Ki value of 5.8 x 10(-7) M is comparable to those reported for other phytocystatins. Northern and western blot analyses showed elevated expression of TaMDC1 mRNA and protein during cold acclimation of wheat. In addition to cold, accumulation of the TaMDC1 message was induced by other abiotic stresses including drought, salt and ABA treatment. Investigation of in vitro antifungal activity of mTaMDC1 showed strong inhibition on the mycelium growth of the snow mold fungus Microdochium nivale. Hyphae growth was totally inhibited in the presence of 50 mug/ml mTaMDC1 and morphological changes such as swelling, fragmentation and sporulation of the fungus were observed. The mechanisms of the in vitro antifungal effects and the possible involvement of TaMDC1 in cold induced snow mold resistance of winter wheat are discussed.

Genotypic Variation for Snow Mold Reaction among Creeping Bentgrass Clones

Creeping bentgrass (Agrostis stolonifera L.) is currently the most desirable grass for golf courses in temperate climates. In temperate climates, creeping bentgrass is highly susceptible to snow mold fungi which can cause significant injury and mortality. The objectives of this study were to survey a collection of creeping bentgrass clones for reaction to snow mold fungi (Typhula spp.), to identify clones with potential resistance to snow mold fungi, and to identify ecological factors related to necrotic reactions of creeping bentgrass clones to snow mold fungi. Three hundred sixty creeping bentgrass clones, selected from old golf courses in Wisconsin, were evaluated for necrosis reaction during incubation and recovery periods following inoculation with Typhula ishikariensis Imai or as noninoculated controls. Genotypic variation was observed for tolerance to snow mold and cold, dark (noninoculated) conditions but the two tolerances were uncorrelated with each other. Clones from fairways were more tolerant of snow mold, most likely due to long-term natural selection in the absence of fungicide applications. In a second experiment involving 72 selected clones, selection was successful in identifying divergent groups of clones, although differences between experiments indicated that future selection and breeding should make use of multiple inoculation runs. Resistance to snow mold in creeping bentgrass appears to be nonspecific with respect to race and species of snow mold isolates.

Antifreeze proteins from snow mold fungi

The psychrophilic fungi Coprinus psychromorbidus and Typhula ishikariensis produced unique antifreeze proteins (AFPs) in the extracellular space. Molecular masses of purified fungal AFPs of C. psychromorbidus and T. ishikariensis were approximately 22 and 23 kDa, respectively. Cloned genes of AFPs from T. ishikariensis do not have any similarity with known proteins. Purified fungal AFPs from cultural filtrate of T. ishikariensis and recombinant fungal AFP from methylotrophic yeast formed specific ice crystals resembling "Stone Age knives". These observations indicate that fungal AFPs do not form proper hexagonal ice crystals to inhibit their growth and that fungal AFPs can probably bind to surfaces of ice crystals in an irregular manner.Key words: antifreeze protein, snow mold fungi, Coprinus psychromorbidus, Typhula ishikariensis.

Distribution of Typhula ishikariensis Biotype A Isolates Belonging to a Predominant Mycelial Compatibility Group

The distribution pattern and frequency of isolates of a snow mold fungus, Typhula ishikariensis biotype A belonging to a predominant MCG (mycelial compatibility group, referred to as super MCG), were surveyed throughout its habitat from northern Honshu to eastern Hokkaido. About 38 and 14% of isolates examined belonged to super MCG in eastern and central Hokkaido, respectively ; however, super MCG was never found in southern Hokkaido or northern Honshu. These findings imply that T. ishikariensis biotype A consists of two populations in Japan, i.e., one that is distributed in Honshu and southern Hokkaido and lacks super MCG isolates, and the other that includes super MCG isolates and exists in central and eastern Hokkaido. The difference in distribution pattern of the two populations is discussed in terms of geological history during the Pleistocene (2 million to 10 thousand years ago). The tendency of global warming, which alleviates freezing damage, was considered to be responsible for the outbreak of this fungus in eastern Hokkaido.