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Microdochium majus and Microdochium nivale are two of fungal pathogens that cause Fusarium head blight (FHB) in wheat, and have also caused pink snow mold in eastern Hokkaido, Japan. With the aim of assessing levels of genetic variation and population structure, 172 isolates of these Microdochium species obtained from five populations of infected wheat seeds were first classified into each species using polymerase chain reaction (PCR) amplification with specific primers. In total 165 (95.9 % of all isolates) and seven isolates (six of Tokachi populations and one of Abashiri populations) were identified as M. majus and M. nivale, respectively, indicating that M. majus was predominant and the main causal pathogen of FHB in this area. Inter-simple sequence repeat (ISSR) analysis showed that the total genetic diversity was 0.023 when estimated by Nei's gene diversity index within the five populations dominated by M. majus. An AMOVA analysis also showed that 86.74 % of the total genetic variation was within populations and 13.26 % among populations. These results indicated that little genetic differentiation occurred among the five populations of M. majus. Based on the unweighted pair group method of cluster analysis using the ISSR data, all isolates were identified as one of eight haplotypes in M. majus or six haplotypes in M. nivale, allowing the construction of a dendrogram with two clades corresponding to each species. There was no correlation between the clustering of isolates and their geographic distribution on the tree. These findings show that migration is likely playing an important role in the population biology of M. majus, providing some support for the prediction of epidemics of fungicide resistant strains within populations of the FHB pathogen.

Creeping bentgrass (Agrostis stolonifera L.) is the most widely cultivated and high-value turfgrass species. Genetic linkage maps of creeping bentgrass were constructed for quantitative trait loci (QTL) analysis of gray snow mold (Typhula incarnata) resistance, recovery and leaf width. A segregating population of 188 pseudo-F2 progeny was developed by two-way pseudo-testcross mapping strategy. Amplified fragment length polymorphism, new developed Agrostis specific expressed sequence tag-single sequence repeat (SSR), random amplified polymorphic DNA and genomic SSR markers corresponding to DNA polymorphisms heterozygous in one parent and null in the other, were scored and placed on two separate genetic linkage maps, representing each parent. In the male parent map, 100 markers were mapped to 14 linkage groups covering a total length of 793 cM with an average interval of 8.2 cM. In the female parent map, 146 markers were clustered in another 14 linkage groups spanning 805 cM with an average distance of 5.9 cM between adjacent markers. We identified three putative QTL for leaf width and one QTL for snow mold disease resistance. The construction of a linkage map and QTL analysis are expected to facilitate the development of disease resistant creeping bentgrass cultivars by using molecular marker-assisted selection.

Numerous studies have demonstrated exceptionally high temperature sensitivity of the beneath-snow respiratory flux in cold-winter ecosystems. The most common, but still untested, explanation for this high sensitivity is a physical one based on the observation that water availability in soils increases exponentially as soils warm from −3 to 0°C. Here, we present evidence for a biological hypothesis to explain exponential kinetics and high Q 10 values as beneath-snow soils warm from −3 to 0°C during the early spring in a high-elevation subalpine forest. First, we show that some of the dominant organisms of the beneath-snow microbial community, “snow molds”, exhibit robust exponential growth at temperatures from −3 to −0.3°C. Second, Q 10 values based on growth rates across the temperature range of −2 to −0.3°C for these snow molds vary from 22 to 330. Third, we derive an analytical equation that combines the relative contributions of microbial growth and microbial metabolism to the temperature sensitivity of respiration. Finally, we use this equation to show that with only moderate snow mold growth (several generations), the combined sensitivities of growth and metabolism to small changes in beneath-snow soil temperature, create a double exponential in the Q 10 function that may explain the extremely high (~1 × 106) Q 10 values observed in past studies. Our biological explanation for high Q 10 levels is supported by several independent studies that have demonstrated build up of microbial biomass under the snow as temperatures warm from −2 to 0°C.

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.

Four hundred and eighty one accessions of species and subspecies of genera Triticum and Aegilops were evaluated for resistance to speckled snow mold caused by Typhula ishikariensis and for freezing tolerance. All Triticum and seven of Aegilops species were severely affected by snow mold. Among surviving Aegilops spp., only the Ae. cylindrica accessions exhibited resistance similar to that of the most resistant winter wheat cultivar, PI 173438. After repeated screening of accessions of Ae. cylindrica, 12 accessions were identified as having resistance similar to that of PI 173438; eleven of those were considered more freezing tolerant than PI 173438, but less than wheat cv. Valjevskaya, the freezing tolerant check. Accessions of Ae. cylindrica with snow mold resistance exceeding that of PI 173438, and with freezing tolerance, are currently being screened.

Pretreatment with sodium alginate (20 g/m2) prior to application of a biocontrol agent, Pseudomonas fluorescens strain A11RN, enhanced the control turfgrass snow mold caused by Typhula ishikariensis biotype B. Disease severities in two fields relative to untreated control plots were 53% and 61% in plots treated with both the strain and sodium alginate, which were significantly lower than 69% and 77% in plots treated only with the strain. The antagonist population remained above the threshold for effective control (ca. 1×107 cfu/cm3) at least for the first month under snow in pretreatment with sodium alginate. The population of the biocontrol agent after snowmelt on turfgrass was five times higher in sodium alginate-amended plots than in unamended plots. Sodium alginate alone did not reduce disease severity. Sodium alginate might have supported the survival of the applied strain.

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.

A water-insoluble, extracellular polysaccharide was isolated from the culture medium of the snow mold fungus, Microdochium nivale, that had been cultivated in potato/dextrose broth. The polysaccharide consisted of glucose only. Its Fourier transform infrared spectrum showed a beta configuration of the C1 position of glucose. Linkage analysis of the polysaccharide showed that it had a linear structure of β-(1→4)-linked glucose. The polysaccharide was therefore identified as cellulose. This is the first report of extracellular cellulose occurring in fungi.

An extracellular lipolytic enzyme active at low temperature was purified from the culture filtrate of the snow mold fungus, Typhula ishikariensis group III, strain 6-1-1. The molecular mass of enzyme was approximately 83 kDa (SDS-PAGE). The lipolytic enzyme was most active for p-nitrophenyl palmitate at 30 –C (pH 9.0). The lipolytic activity was 23.4% of the maximum at 4 –C, the temperature of culture. Thus, the isolated T. ishikariensis lipolytic enzyme is thought to represent a new member of a group of enzymes active at low temperature.

Isolates ofTyphula ishikariensis, a snow mold fungus, were collected from five localities in Norway. They were divided into three groups according to genetics, cultural morphology, etc. Group I grew normally at 10 °C. Its mating patterns with Japanese taxa were variable: compatible with both biotypes A and B; compatible with biotype A but incompatible with biotype B; and incompatible with both biotypes. Group I was prevalent in southern inland districts such as Buskerud, Oppland, and Hedmark. Group II had smaller sclerotia as compared to other groups, and its sclerotia were often covered with white mycelium in nature and in culture. Group II was compatible with biotype B only. Group III was characterized by irregular growth at 10 ° C and genetic incompatibility with biotypes A and B. Cultural morphology of group III resembled that of group I at 0 °C. Rind cell patterns of sclerotia did not separate these two groups or biotypes. Isolates of groups II and III were often obtained from coastal regions in Finmark. Mating reactions were variable: monokaryons were compatible with their respective dikaryons, and monokaryons of groups II and III occasionally mated with group I dikaryons. Dikaryons of groups II and III, however did dikaryotize monokaryons of other groups. Norwegian isolates ofT. ishikariensis were highly variable, and the orthodox nomenclature system seemed inapplicalbe at the infraspecific level.

A method was developed for assessing the resistance of wheat to speckled snow mold (Typhula ishikariensis) under controlled conditions in all steps. We modified the method of Nakajima and Abe and estimated the optimum conditions consisting of 2 weeks each for prehardening and hardening, 6 weeks for incubation at 8°C, and 2 weeks for regrowth in a greenhouse for screening resistance. The present method reduced the experimental period to 12 weeks and facilitated repeated experiments throughout the year.

The pink snow mold, Microdochium nivale (syn. Fusarium nivale) SUF 1377 strain produced an extracellular low temperature active lipase during growth at 4°C. The lipase had the highest activity at 20°C, and retained 19% of its maximum activity at 0°C.