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Speckled snow mold caused by Typhula ishikariensis is one of the most devastating diseases of winter wheat in Hokkaido, Japan and parts of the Pacific Northwest region of USA. Munstertaler is a winter wheat landrace from Switzerland that has very high resistance to snow mold and superior freezing tolerance. Quantitative trait loci (QTL) for resistance to speckled snow mold were identified in a doubled haploid population derived from a cross between Munstertaler and susceptible variety Ibis, both under field conditions and controlled environment tests. Composite interval mapping analysis revealed a major QTL on chromosome 5D from Munstertaler, and on chromosome 6B from Ibis. Flanking microsatellite marker cfd 29 for the QTL on chromosome 5D was about 5 cM distant from vernalization requirement gene Vrn-D1, suggesting that the QTL on chromosome 5D is located on a cold-stress-related gene cluster along with Vrn-D1 and freezing tolerance gene Fr-D1. The QTL on chromosome 6B from Ibis was located on the centromere region flanking QTn.mst-6B, which is reported to increase plant tiller number.

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.

In previous testing, we found that gray snow mold, caused by Typhula ishikariensis or T. incarnata, could be controlled by applying T. phacorrhiza onto turf grass prior to snowfall. To test for phytopathogenicity of this biocontrol agent, the five most disease-suppressive isolates were cultured on mixed grains and applied to 12 turfgrass cultivars and 2 winter wheat cultivars in December 1995 and 1996. After snow melt in April 1996 and 1997, significantly greater winter injury was visible on plots treated with the pathogens compared to T. phacorrhiza-treated plots or untreated plots. Except for one cultivar in 1996, there were no significant differences between T. phacorrhiza-treated plots and untreated plots. Pelletized alginate formulations of T. phacorrhiza containing kaolin clay with various nutritional amendments were tested for viability and efficacy. After 64 weeks of storage, viability remained >85% at -15°C and >70% at 4°C, but 2) was equivalent to using larger amounts of mixed grain inoculum (200 g/m2) or wheat bran inoculum (100 g/m2) in 2 years of field testing.

Speckled snow mold, caused by Typhula ishikariensis, is one of the most important Typhula snow molds in subarctic zones of the Northern Hemisphere. Nine isolates of three T. ishikariensis varieties (var. ishikariensis, var. canadensis, and var. idahoensis) isolated from infected turfgrasses on golf course fairways throughout Wisconsin were evaluated for their aggressiveness toward nine cultivars of three bentgrass species (three creeping, three colonial, and three velvet cultivars) under controlled environmental conditions. Speckled snow mold severity increased as inoculum concentration of T. ishikariensis was increased. In general, bentgrass susceptibility increased between 9 and 11 weeks after seeding but gradually decreased thereafter, suggesting expression of age-related resistance as plants matured. Significant differences in aggressiveness were detected within and among T. ishikariensis varieties. Significant interactions between T. ishikariensis varieties or isolates and bentgrass species were detected, but there was no interaction between pathogen isolates and bentgrass cultivars. Disease severity evaluations showed significant differences among bentgrass cultivars and species in their response to T. ishikariensis. Since bentgrass species exhibit differential responses to T. ishikariensis varieties, representative isolates of each variety should be employed for screening of bentgrass germplasm for resistance to speckled snow mold.

Nishio, Z., Iriki, N., Takata, K., Ito, M., Tabiki, T., and Murray, T. D. 2008. Influence of coldhardening and soil matric potential on resistance to speckled snow mold in wheat. Plant Dis. 92:1021-1025. The influence of soil matric potential, cold-hardening temperature, and duration on resistance to speckled snow mold caused by Typhula ishikariensis in wheat was investigated. Six winter wheat lines were subjected to cold-hardening temperatures of 2 or 4°C for 1, 2, 3, or 4 weeks with soil matric potential of –0.1 or –0.01 MPa. Plants were inoculated with T. ishikariensis after cold-hardening, incubated at 10°C for 25 days in the dark, and then evaluated for regrowth. Overall recovery from snow mold was least when plants were hardened at 2°C for 1 week at –0.01 MPa and greatest when hardened at 4°C for 4 weeks at –0.1 MPa. Survival of plants following snow mold was greater when plants were cold-hardened at 4 than at 2°C and at –0.1 than –0.01 MPa soil matric potential. The greatest difference in survival among lines and correlation with field observations occurred when plants were hardened at 4°C at –0.1 MPa matric potential for 3 weeks. Understanding the influence of temperature and soil matric potential during coldhardening on speckled snow mold resistance will be useful to breeding programs developing snow-mold-resistant cultivars under controlled environment conditions.

Typhula ishikariensis Imai is a causal agent of Typhula snow mold, one of the most important turfgrass diseases in northern regions of the United States. Within Wisconsin isolates, there are three district groups clustered with known isolates of T. ishikariensis var. ishikariensis, var. canadensis and var. idahoensis as identified by RAPD markers. To further investigate the genetic relationship among these groups (varieties), monokaryon-monokaryon and dikaryon-monokaryon mating experiments were conducted. Mating types from var. ishikariensis, var. canadensis and var. idahoensis isolates were paired in all possible combinations. Pairings between var. canadensis and var. idahoensis were highly compatible, while no compatibility was detected between var. ishikariensis and either var. canadensis or var. idahoensis. These results indicate that var. ishikariensis is genetically separated from var. canadensis and var. idahoensis, whereas var. canadensis and var. idahoensis appeared to be genetically related to each other as a taxonomic unit. In the genetic relationship with the known biological species, var. ishikariensis and var. canadensis were genetically related to biological species I and II, respectively. However, var. idahoensis was not compatible with any of the biological species, suggesting that the pathogen may be in the process of biological speciation from var. canadensis.

Organisms that live in cold climates have to confront a lot of obstacles due to ice crystal formation. As the temperature dips lower than sub-zero, ice crystals gradually develop inside the cell which leads to cell rupture. Antifreeze proteins (AFPs) are produced as a specialized adaptation by these psychrophiles that permit their survival in subzero environment. Antifreeze proteins produced by fungi are thought to bind onto ice crystals quickly using a regular array of well conserved residues/motifs and alter the structure of ice in or around the host thereby hindering growth and re-crystallization of ice. In this investigation, seven different antifreeze proteins (AFPs) of Typhula ishikariensis retrieved from Uniprot database were analyzed and characterized in silico using various computational tools. Their physico-chemical properties, hydropathicity and secondary structure have been identified. Phylogenetic analysis was also performed using MEGA4. The study might be an initiation in understanding the underlying structural and functional aspects of these proteins, their common characteristics and other features for academic and industrial purposes.

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.

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.

Antifreeze proteins are structurally diverse polypeptides that have thermal hysteresis activity and have been discovered in many cold-adapted organisms. Of these, fungal antifreeze protein has been purified and partially characterized only in a species of psychrophilic basidiomycete, Typhula ishikariensis. Here we report a new fungal antifreeze protein from another psychrophile, Antarctomyces psychrotrophicus. We examined its biochemical properties and thermal hysteresis activity, and compared them with those of the T. ishikariensis antifreeze protein. The antifreeze protein from A. psychrotrophicus was purified and identified as an extracellular protein of approximately 28 kDa, which halved in size following digestion with glycosidase. The A. psychrotrophicus antifreeze protein generated bipyramidal ice crystals and exhibited thermal hysteresis activity (for example thermal hysteresis = 0.42 °C for a 0.48 mm solution) similar to that of fish antifreeze proteins, while a unique rugged pattern was created on the facets of the ice bipyramid. The thermal hysteresis activity of the A. psychrotrophicus antifreeze protein was maximized under alkaline conditions, while that of the T. ishikariensis antifreeze protein was greatest under acidic conditions. The T. ishikariensis antifreeze protein exhibited a bursting ice growth normal to the c-axis of the ice crystal and high thermal hysteresis activity (approximately 2 °C), as in the case of insect hyperactive antifreeze proteins. From these results, we speculate that the A. psychrotrophicus antifreeze protein is very different from the T. ishikariensis antifreeze protein, and that these two psychrophiles have evolved from different genes.

Two Gram-variable, aerobic, motile, rod-shaped, endospore-forming bacterial strains, M4-2T and M4-1, were isolated from soil samples collected from Oblast Magadan, Russian Far East, as micro-organisms antagonistic to the psychrophilic phytopathogenic fungus Typhula ishikariensis. Strains M4-2T and M4-1 were identified as members of the genus Paenibacillus by phenotypic and phylogenetic analyses based on 16S rRNA gene sequences. The strains contained anteiso-C15:0 as the major fatty acid (63.0-64.7%) and MK-7 as the major isoprenoid quinone. The DNA G+C contents were 42.8 and 41.7 mol%, respectively. 16S rRNA gene sequence analysis showed that strains M4-2T and M4-1 exhibited high similarities with Paenibacillus macquariensis DSM 2T (99.5 and 99.7%, respectively) and Paenibacillus antarcticus LMG 22078T (99.4 and 99.5%, respectively). There were no clear differences in the phenotypic characteristics and chemotaxonomic and phylogenetic data between the novel isolates and P. macquariensis DSM 2T. DNA-DNA hybridization experiments between strain M4-2T and P. macquariensis DSM 2T and P. antarcticus LMG 22078T revealed reassociation values of 56 and 49%, respectively. Multilocus sequence analysis confirmed the differences between the new isolates and reference strains that were observed with the DNA-DNA hybridization studies. On the basis of the results described above, it is proposed that the isolates represent a novel subspecies of P. macquariensis, Paenibacillus macquariensis subsp. defensor subsp. nov. The type strain is M4-2T (=JCM 14954T=NCIMB 14397T).

Hoshino, T., Tronsmo, A.M. & Yumoto, I. 2008. Snow mold fungus, Typhula ishikariensis group III, in Arctic Norway can grow at a sub-lethal temperature after freezing stress and during flooding. – Sommerfeltia 31: 125-131. ISBN 82-7420-045-4. ISSN 0800-6865. 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.

Accumulation of soluble carbohydrates plays an important role in enhancement of resistance to freezing and snow mold of plants during cold acclimation. Nevertheless, few studies have examined whether changes in cell wall properties are involved in enhancement of resistance during cold acclimation. In this study, four winter wheat cultivars were sown in a field on six different dates during August-October, and their resistance to freezing and snow mold were compared in relation to soluble carbohydrate content and cell-wall mass in leaves. Resistance to freezing and snow mold was much higher in the plants sown on 23 September than in those sown on 9 September. The percentage of cell-wall mass in leaf to total dry mass (%CW) and water-soluble carbohydrate content also increased considerably during 9-23 September. Multiple regression analyses revealed that %CW contributed significantly to freezing resistance, whereas total water-soluble carbohydrate content contributed significantly to snow mold resistance. These results suggest that increased %CW enhances freezing resistance during cold acclimation.

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.

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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.

Frost tolerance was determined in Norwegian varieties of groups I and III of the psychrophihc fungus Typhula mhikanensis. When myceha of group I and III strains were rapidly frozen to -40℃ in a program freezer at a cooling rate of 20℃/h, the regrowth of group I strains was delayed at their optimal growth temperature (10℃), whereas the regrowth of group III strains was not affected at their optimal growth temperature (2℃). Furthermore, when group I and III strains were rapidly frozen and regrew at 0℃, the average temperature during snowfall in the northernmost area of Norway, group I strains, in contrast to group III strains, did not regrow at all. These results suggest that group III strains are more resistant than group I strains to rapid freezing Group III strains are well adapted to climatic conditions in northernmost Norway Freezing points of myceha of group III strains were determined by calonmetenc analysis Mycelia of group III strains froze at temperatures higher than -10℃ Therefore, the freezing resistance of group III strains might be based on extracellular ice formation. In the cytosol of group III, a 30 kDa protein that strongly immunoreacted with the anti-antifreeze protein (AFP) I antibody was found, and the 30 kDa protein level in group III strains was higher than that in group I strains. These results suggest that the difference in the distribution pattern of both groups can be ascribed, at least partially, to the difference in freezing resistance of myceha.

Growth of two groups of strains (group I and III) of the snow mould fungus, Typhula ishikariensis from northern Norway, were studied in agar and liquid culture. All strains could grow from—5 to 15 (group III) or 20°C (group I) and the optimum temperatures for growth were 4 or 10°C, respectively. The group III strains showed irregular growth as the hypal extensions were inhibited at 15°C. Different temperature two‐dimensional electrophoresis revealed that in the group III strains some intracellular proteins were modified at the highest temperature. One of the reasons that group III strains did not grow or showed irregular growth at 20 or 15°C, was probably due to protein aggregation or dissociation and thereby loss of vital properties of intracellular proteins.

Speckled snow mould (caused by Typhula ishikariensis) is a potential threat to winter wheat (Triticum aestivum L.) production in eastern Canada. Information on the inheritance of snow mould resistance is needed to develop an effective breeding strategy. In this study, the inheritance of resistance to the speckled snow mould was examined using crosses of a resistant genotype, PI 173438, and four susceptible cultivars, Lennox, Kitami-2, Norin-8 and Ena. The parental lines, and the F1 and F2 populations from each of the four crosses were grown in the field and in an unheated plastic greenhouse and scored for snow mould resistance. Biomass yield, survival, plant height and number of tillers/plant, all expressed as percentage of check plants, were used as indices of snow mould resistance. Generation means analysis, combining data from the field and greenhouse experiments, indicated that snow mould resistance was largely influenced by environmental conditions, and that additive genetic effects were more important than epistatic effects in controlling the expression of the disease. Dominance effects oriented towards susceptibility were detected in only one of the four crosses. Estimates of broad sense heritability in the four F2 populations ranged from 0.62 to 0.96 and from 0.34 to 0.79, respectively, when biomass yield and number of tillers/plant were used as indices of snow mould resistance. When plant height was used as an index of snow mould resistance, the heritability estimates ranged from 0.10 to 0.53. Key words: Wheat (winter), Triticum aestivum, speckled snow mould, Typhula ishikariensis, heritability

Isolates of Typhula ishikariensis, a snow mould fungus, from Norway were divided into three groups, which, although differing in culture morphology and in mating reactions with testers of T. ishikariensis biotypes A and B from Japan, were, at least partially, interfertile. Population structures of this fungus at 23 sites in meadows and pastures were then determined, based on vegetative compatability group. Except for two sites dominated by a single large vegetative compatibility group, populations were generally diverse, regardless of the cropping history of the site.

Improved winter hardiness is an important objective in Norwegian grass breeding. Selection for winter hardiness based on performance in a controlled environment is a means to obtain this goal faster than relying on field trials. Progress in selection will depend on the genetic variation and the heritability of the traits. This investigation shows that artificial screening for winter survival traits reveals significant genetic variation in Dactylis glomerata in all three characters tested: resistance to Fusarium nivale, resistance to Typhula ishikariensis and resistance to freezing. The broad sense heritability of resistance to snow mould fungi between 0.4 and 0.5, and for freezing resistance between 0.5 and 0.7, indicates good possibilities for improving the traits by selection for resistance. The genotypic correlation calculated from the analysis of covariance of two half-sib families was found to be approximately 1. Although associated with contradictory data, this finding supports the theory t...

Low-temperature fungi were collected from plants just after snowmelt, and their antagonistic activity against Typhula ishikariensis, a snow mold fungus, was determined using orchardgrass seedlings. Isolates from gramineous plant debris, considered to beT. phacorrhiza, suppressed the disease caused by T. ishikariensis biotype A or B. Antagonists differed in their effectiveness against these biotypes. Isolates antagonistic to biotype A, which is the principal snow mold of perennial ryegrass in northern Hokkaido, were localized in this district. Despite prolonged snow, susceptible, perennial ryegrass is successfully grown there. These findings suggest the natural occurrence of biological control of the disease in perennial ryegrass pastures in northern Hokkaido. Ground tissues of orchardgrass or alfalfa reduced activities of antagonists when mixed in the inoculum. Plant litter such as fallen maple leaves and rice straw favored antagonism. Application of the antagonists in a naturally infested field planted with perennial ryegrass resulted in an yield increase of 26.5% compared with the untreated control where fall cutting favored the occurrence of snow mold. Where plants were not cut in fall and snow mold damage was slight, yield increase was insignificant.

Resistance to the pathogens of snow mold, especially to Typhula ishikariensis S. Imai is one of the most important traits in wheat breeding for the central region of Hokkaido. T. ishikariensis always occurs in wheat nurseries when snow melts, at Hokkaido Natl.. Agric. Ext. Stn., where the snow mold resistance of winter wheat is being studied. Since T. ishikariensis consists of biotype A, B and C, it is considered that the resistance of winter wheat to each biotype should be determined. T. ishikariensis biotypes A, B and C which were cultured in a vermiculite-wheat bran medium were used as inoculum source. Five wheat varieties, PI 173438 (highly resistant), Horoshirikornugi (resistant), Takunekomugi (moderately resistant), Chihokukomugi (moderately susceptible) and lbis (susceptible) were treated with the inocula after the onset of a continuous snow cover in 1985 and 1986. In the plots inoculated with biotype A, PI 173438 always exhibited higher resistance to the fungi than the other varieties, but the damage of the wheat varieties inoculated with biotype B or C was so serious that the difference in resistance among the wheat varieties could not always be detected (Table 2, 3 and 4). Snow mold damage of wheat varieties inoculated with T.

Speckled snow mold, caused by the basidiomycete Typhula ishikariensis Imai, is one of the most prominent winter diseases on perennial grasses and cereal crops in the northern hemisphere. The first linkage map of T. ishikariensis was constructed using a population of 93 sibling monokaryons derived from a single dikaryotic hybrid isolate that was created by a hyphal fusion of two monokaryotic parental isolates. The parental isolates were produced from a pathogenic dikaryotic isolate collected from a golf course in Wisconsin. The two parents exhibit significant differences in the production of aerial mycelium and sclerotia, and in their aggressiveness on creeping bentgrass ( Agrostis stolonifera L.). A total of 251 loci were mapped, comprising 89 inter-simple sequence repeat (ISSR) and 160 random amplified polymorphic DNA (RAPD) markers along with 2 phenotype-based mating-type (MAT) loci. The MAT loci were mapped on linkage groups (LGs) 1 and 7. The markers were evenly distributed over 7 LGs, covering 436 cM with an average marker interval of 2.2 cM. Seven chromosomes were cytologically observed using germ tube bursting methods with acetocarmine staining. This reference linkage map of T. ishikariensis should provide a framework for the mapping of quantitatively controlled traits such as fungal growth, survival, and virulence/avirulence under low temperatures. The map should also be utilized for studying the genome organization of the cold-loving plant-pathogenic Typhula spp. and for comparative genome analysis among fungal taxa.

This study evaluates the effectiveness of four different rates of Wormcast (23 kg, 46 kg, 116 kg and 231 kg/100m2) in comparison to several control treatments in the control of snow mold disease Separate plots were inoculated with Typhula incarnata, Typhula ishikariensis, and Microdochium nivale snow molds. Treatment was applied in the fall before the first snowfall. Phytotoxicity and Biotic plus abiotic injury were measured on a weekly basis one the snow melted till greenup. The highest rates of phytotoxicity were seen in the Wormcast treatment. Plots inoculated with Microdochium nivale saw significant suppression of injury, although higher rates also saw higher rates of disease. Reworks

Typhula ishikariensis isolates were collected from various parts of Hokkaido. There were two genetically different groups which did not mate with each other; one was termed biotype A, and the other was further divided into two by cultural characteristics, i.e., biotypes B and C. Biotype A was identical to T. ishikariensis and T. ishikariensis var. ishikariensis. Biotype B was not identical to T. idahoensis or T. ishikariensis var. idahoensis. Biotype C was similar to T. ishikariensis var. canadensis. Biotype A tended to occur inland where deep snow cover lasts for a long time. Biotype B was obtained mainly from the coastal regions where snow cover is thin and short in time. The distribution of biotype C was irregular. Taxonomic significance of the genetics and allopatry in the T. ishikariensis complex is discussed.