Tom Hsiang the Battle Under Snow Between Fungal Pathogens

Naoyuki Matsumoto · Tom Hsiang Snow Mold The Battle Under Snow Between Fungal Pathogens and Their Plant Hosts Snow Mold ThiS is a FM Blank Page Naoyuki Matsumoto • Tom Hsiang

Snow Mold The Battle Under Snow Between Fungal Pathogens and Their Plant Hosts Naoyuki Matsumoto Tom Hsiang Graduate School of Agriculture School of Environmental Sciences Hokkaido University University of Guelph Sapporo, Hokkaido Guelph, Ontario Japan Canada

ISBN 978-981-10-0757-6 ISBN 978-981-10-0758-3 (eBook) DOI 10.1007/978-981-10-0758-3

Library of Congress Control Number: 2016937674

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This Springer imprint is published by Springer Nature The registered company is Springer Science+Business Media Singapore Pte Ltd. Preface

Climate change does not occur uniformly across the globe; its effects are considered most severe at higher latitudes in the northern hemisphere, and agricultural production is most likely to be influenced there (Murray and Gaudet 2013). This concern motivated scientists to convene an interdisciplinary forum, Plant and Microbe Adaptations to Cold (PMAC), in 1997 in Sapporo, Japan. Fifteen years later, PMAC 2012 met again in Sapporo, and this conference focused on sustainable agricultural production under changing climatic conditions. Scientists, as well as farmers and policy makers, participated the conference to discuss issues of importance affected by climate change. Although concrete countermeasures were not presented, available scientific knowledge and established techniques can be adapted to minimize overwintering problems of crops. For example, extended snow cover for up to 163 days in certain areas of Hokkaido in northern Japan in the 2011/2012 winter (S. Inoue, personal communication) made farmers anxious; however, the survival of winter wheat was better than expected. Both biotic and abiotic factors that affect agricultural production respond to changes in winter climate. Abiotic factors mainly consist of freeze damage due to low temperatures and of ice encasement and damage that occur after repeated cycles of freezing and thawing of soil. Biotic factors are comprised mostly of snow molds incited by low-temperature fungi that prevail on plants under snow cover and represent the main theme of this book. Winter damage to agricultural production, unlike drought in summer, is not well documented. Gudleifsson (2013) presented an example in Iceland, where analyses of ice cores from glaciers indicated that the temperatures around the time of settlement from 900 to 1200 were fairly high and then fluctuated until 1900 with frequent winter damage to forage grass. Over one-third of the years were characterized as having mild or severe winter damage of pastures, resulting in reduced livestock production and subsequent famine. Correlations between temperature measurements and hay yield from 1900 to 2010 revealed that winter temperatures affected forage production more significantly (r=0.57***) than summer temperatures (r=0.28***). Recent global warming is generally considered to favor forage production.

v vi Preface Frost depth (cm)

1987 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 Year

Fig. 1 Change in annual soil frost in nine spots in Memuro, Hokkaido (Hirota et al. 2006)

Climate change also affects agriculture in Hokkaido, Japan, where the quantity and quality of summer crops have been improved due to extended growth periods and increased cropping acreage, as well as reduced cool weather damage. The trends are not so apparent in forage crops or winter wheat that overwinters under snow. However, especially in eastern Hokkaido where deep soil frost is characteristic of the winter climate, early onset of snow cover and deep snow cover has attenuated the depth and duration of soil frost (Fig. 1, Hirota et al. 2013). Conse- quently, changes in winter climate have led to changes in snow mold ﬂora, affecting breeding strategy. In this book, we described a common phenomenon occurring in snowy regions, namely, snow mold. Snow mold is important not only as one of agricultural issues in cold regions but interesting as a biological phenomenon. This book is an English version of the book Snow Mold written in Japanese by NM (Matsumoto 2013) and represents a synthesis of ideas of both authors of this version.

References

Gudleifsson BE (2013) Climatic and physiological background of ice encasement damage of herbage plants. In: Imai R, Yoshida M, Matsumoto N (eds) Plant and microbe adaptations to cold in a changing world. Springer, New York, pp 63–72 Hirota T, Iwata Y, Hayashi Y, Suzuki S, Hamasaki T, Sameshima R, Takayabu I (2006) Decreasing soil-frost depth and its relation to climate change in Tokachi, Hokkaido, Japan. J Meteorol Soc Jpn 84:821–833 Hirota T, Yazaki T, Usuki K, Hayashi M, Nemoto M, Iwata Y, Yanai Y, Inoue S, Suzuki T, Shirahata M, Kajiyama T, Araki K, Maezuka K (2013) Soil frost control: its applications to volunteer potato management in a cold region. In: Imai R, Yoshida M, Matsumoto N (eds) Plant and microbe adaptations to cold in a changing world. Springer, New York, pp 51–61 Preface vii

Matsumoto N (2013) Snow mold. Hokkaido University Press, Sapporo (in Japanese) Murray T, Gaudet D (2013) Global change in winter climate and agricultural sustainability. In: Imai R, Yoshida M, Matsumoto N (eds) Plant and microbe adaptations to cold in a changing world. Springer, New York, pp 1–15 ThiS is a FM Blank Page Contents

1 Introduction ...... 1 1.1 SnowMold...... 2 1.1.1 Damage ...... 4 1.1.2 Life History ...... 5 1.1.3 Environment Under Snow ...... 6 1.2 Subnivean Environment ...... 10 1.2.1 Litter Decomposition ...... 10 1.2.2 Organic Matter Recycling ...... 11 1.2.3 Diversity Creation ...... 11 1.3 Microorganisms in the Cryosphere ...... 12 1.3.1 Physiological Issues ...... 14 1.3.2 Ecological Issues ...... 16 References ...... 16 2 Ecology and Physiology ...... 23 2.1 Survival Strategy ...... 24 2.1.1 Individualism ...... 26 2.1.2 Population Structure ...... 29 2.2 Predictability of Snow Cover ...... 33 2.2.1 Sclerotial Germination ...... 34 2.2.2 Sclerotial Size ...... 38 2.3 Psychrophily and Freeze Tolerance ...... 43 2.3.1 Metabolism ...... 44 2.3.2 Freeze Tolerance ...... 45 2.3.3 Mechanism of Freeze Tolerance ...... 46 References ...... 49 3 Snow Mold Fungi ...... 55 3.1 Typhula spp...... 56 3.1.1 Mating Incompatibility ...... 56 3.1.2 Niche Separation ...... 59

ix x Contents

3.1.3 Typhula incarnata ...... 60 3.1.4 Typhula ishikariensis ...... 66 3.1.5 Typhula phacorrhiza ...... 75 3.2 Sclerotinia spp...... 76 3.2.1 Sclerotinia borealis ...... 76 3.2.2 Sclerotinia nivalis ...... 79 3.2.3 Sclerotinia trifoliorum ...... 80 3.3 Microdochium nivale ...... 81 3.3.1 Taxonomic Issues ...... 81 3.3.2 Ecological Features ...... 82 3.4 Pythium spp...... 84 3.5 Other Basidiomycetous Snow Mold Pathogens ...... 85 3.5.1 Supponuke fungus (Athelia sp.) ...... 85 3.5.2 Low-Temperature Basidiomycete (LTB, Coprinus psychromorbidus)...... 87 References ...... 88 4 Resistance ...... 95 4.1 Field Observations ...... 96 4.2 Laboratory Trials ...... 99 4.3 Resistance Mechanism ...... 100 4.3.1 Hardening ...... 100 4.3.2 Reserve Materials ...... 101 4.3.3 PR Proteins and Other Substances ...... 102 4.4 Conclusions ...... 104 References ...... 104 5 Control ...... 109 5.1 Chemical Control ...... 112 5.1.1 Bordeaux Mixture ...... 113 5.1.2 Organic Mercury ...... 113 5.1.3 PCP, PCNB, and Thiophanate-Methyl ...... 114 5.1.4 New Fungicides and Mixture of Chemicals ...... 114 5.1.5 Resistance Activators ...... 115 5.2 Biological Control ...... 115 5.3 Cultural Control ...... 118 5.3.1 Cultural Practices ...... 118 5.3.2 Cultivar ...... 119 5.4 Conclusions ...... 124 References ...... 124 6 Concluding Remarks ...... 129 References ...... 131

Index ...... 133 Chapter 1 Introduction

Abstract In cold-temperate region, plants are more frequently damaged by biotic factors, primarily snow mold, than by abiotic factors such as freezing. Snow mold is used here as a generic name for plant diseases that occur under snow cover. Many different fungi may be involved, and each snow mold fungus has its own ecological and physiological features. They normally infect and can prevail on plants under snow, but generally are dormant during other seasons. Their habitat under snow is characterized by constant low temperature, darkness, and high moisture. Snow mold fungi are, in general, opportunistic pathogens, which, in the absence of antagonists, attack plants depleted of reserve material. Such organisms which not only tolerate cold temperatures but thrive under such conditions are called “psychrophiles.” In the ﬁnal section of this chapter, ambiguities in the use of the common term “psychrophile” are illustrated, and these are ascribed to the complex life cycles of different fungi. Another term, “cryophile,” may be more appropriate, to denote fungi, including snow mold fungi that prevail in the cryosphere.

Dr. Takao Araki who showed NM the signiﬁcance of ﬁeld observations and led him to a whole new world under snow; photograph taken circa 1978.

In cold-temperate region, agricultural ﬁelds are often covered with snow for more than 4 months, and plant tops look dormant even before snow cover and are yellowed after snow cover. From the point of view of the plant, half of the year is winter. However, despite harsh winter climates in such regions, plant growth is promoted due to longer day lengths in summer. Survival through winter months is the greatest challenge for such organisms. Wild plants have consequently developed diverse winter survival strategies (Sakai 2003). Overwintering domesticated plants such as winter cereals and forage crop have been selected and protected by humans, resulting in yield increases. Today, we are facing another difﬁculty, namely, global warming.

1.1 Snow Mold

In the temperate region, plants are more likely to be killed by snow mold (a biotic factor) than freezing or ice encasement (abiotic factors). We are generally unable to observe the process of pathogenesis under snow, but can see the consequences after snowmelt. Snow mold damage becomes evident by a delay in plant growth or by outright plant death. Frequently, disease surveys have been made in northern hemisphere countries on overwintering crops, e.g., cereals and forage crops in Idaho, USA, by Remsberg and Hugerford (1933); forage crops in Alaska, USA, by Lebeau and Logsdon (1958); winter wheat (Triticum aestivum) in Ontario, Canada, by Schneider and Seaman (1987); turfgrass in Alberta, Canada, by Vaartnou and Elliott (1969); winter cereals in Alberta, Canada, by Gaudet and Bhalla (1988); forage crops and winter cereals in Saskatchewan, Canada, by Smith (1975); winter cereals in Saskatchewan, Canada, by Gossen and Reiter (1989); forage crops in northern Finland by Ma¨kela¨ (1981); forage crops in Norway by Årsvoll (1973); forage crops in northern Norway by Andersen (1992); and forage crops in Iceland by Kristinsson and Gudleifsson (1976). These surveys documented the fungi Sclerotina borealis, Typhula ishikariensis, T. incarnata, and Microdochium nivale as important snow mold pathogens. Smith (1975) emphasized the need for more detailed studies on these pathogens in terms of environmental factors and disease incidence. Plants are seldom killed during overwintering with a few exceptions (Hakamata et al. 1978, Fig. 1.1). In regions of long severe winters, plants have been naturally or artiﬁcially selected for winter hardiness and survival at the expense of productivity. Plants often look dead just after snowmelt, but soon resume growth because the crowns have remained alive (Fig. 1.2). Phenomena referred to as “winter kill” or “winter damage” may have biotic or abiotic sources, but sometimes the causal agent remains unidentiﬁed or may be obscure. “Winter kill” and “winter damage” in certain regions are mostly caused by snow mold. Using 12 orchardgrass cultivars, Abe and Matsumoto (1981) analyzed factors involved in their overwintering in 1.1 Snow Mold 3

Fig. 1.1 Freezing damage on perennial ryegrass planted in a square in Løken, Norway. Timothy surrounding the perennial ryegrass plot survived and remained green. The ﬁeld was located on a slope where ice encasement was unlikely to occur

Fig. 1.2 Turfgrass damage in a golf course nursery induced by Typhula ishikariensis. Fungicide applications prior to snow cover protected plants from damage, and the plants greened up quickly after snow melt (left-side plot). Leaves were killed, but later green shoots appear indicating that crowns and roots were not also entirely killed (right-side plot) 4 1 Introduction

Table 1.1 Major snow molds on agricultural cropsa Disease Taxonomic position Scientiﬁc name Chromalveolata Oomycota Snow rot Pythium iwayamai P. okanoganense P. paddicum Fungi Ascomycota Pink snow mold Microdochium nivale Sclerotinia snow mold Sclerotinia borealis Snow mold S. nivalis Clover rot S. trifoliorum Supponuke disease Basidiomycota Athelia spb Cottony snow mold Coprinus psychromorbidus Gray snow mold Typhula incarnata Speckled snow mold T. ishikariensis aSnow molds on forest trees are excluded bInferred from rDNA ITS sequence (A. Kawakami, personal communication)

Sapporo, Hokkaido, Japan, to reveal that the resistance to Typhula spp. was the most important survival factor. Snow mold is a generic term referring to diseases that are incited by fungal pathogens prevailing under snow, and it does not indicate a speciﬁc taxonomic group of fungi (Table 1.1). Many ascomycetes and basidiomycetes are known as snow mold pathogens. Oomycetes, which are not fungi but belong to Chromalveolata, can also cause snow mold. Zygomycetes or Chytridiomycetes have not been identiﬁed as snow mold pathogens so far but need critical examination.

1.1.1 Damage

Several fungal species are known to attack plants under snow. They are divided into two types: ones occurring regularly year after year and the others irregularly (Nissinen 1996). The occurrence of the latter type is unpredictable, and speciﬁc winter climate conditions favor their outbreak. Sclerotinia borealis caused serious damage to orchardgrass in 1975 in eastern Hokkaido, and farmers resorted to importing feedstuff from abroad (Araki 1975). Of 15,000 ha of grasslands in this area, 62 % required reseeding, 28 % needed renewal, and 10 % were planted with other crops. The outbreak of Sclerotinia snow mold is ascribed to the two climatic factors in the winter of 1974/ 1975: (i) delayed onset of snow cover predisposing orchardgrass to disease with low-temperature stress and (ii) deep snow cover in late March prolonging thaw and 1.1 Snow Mold 5 hence providing the pathogen with an extended active phase. As a consequence, timothy (Phleum pratense) with a higher level of resistance to Sclerotinia borealis (freezing) replaced orchardgrass. Speckled snow mold caused by Typhula ishikariensis is at the other end of the spectrum and shows a consistent occurrence in many regions. In Japan, fungicides are sprayed on winter wheat prior to snow cover as a reliable control measure. However, persistent snow cover in eastern Hokkaido occurred 1 month earlier than normal in 1998, and farmers did not have the opportunity to spray fungicides. T. ishikariensis badly damaged winter wheat, and a survey made the following spring in the Abashiri district indicated that winter wheat crop had to be abandoned in 30 % of the ﬁelds (Matsumoto et al. 2000). Crop rotation and the use of resistant cultivars, along with chemical control, have successfully suppressed the damage ever since. In North America, fungicides are rarely used for controlling snow mold on winter wheat, but they are often used to control snow mold on golf courses, especially putting green areas (Hsiang et al. 1999; Gossen et al. 2001).

1.1.2 Life History

Typical life cycle of Typhula snow mold fungi is illustrated in Fig. 1.3. A more detailed life cycle can be found in Hsiang et al. (1999), and one for Microdochium nivale can be found in Tronsmo et al. (2001). 1. Spring to autumn The pathogens pass the dormant phase from spring to autumn in the form of resting structures such as sclerotia, while plants are growing. Sclerotia are attacked by various mycoparasites during dormancy (Harder and Troll 1973; Matsumoto and Tajimi 1985). The difference in survival pattern of sclerotia reﬂects survival strategy of each pathogen (Matsumoto and Tajimi 1988). Woodlice were found to feed on Typhula sclerotia (Hoshino 2003). 2. Late autumn Sclerotia germinate to develop sporocarps after dormancy, and triggers include temperatures falling to freezing levels with abundant moisture (Yang et al. 2006). Ascocarps produced by S. borealis discharge ascospores to infect plants. Basidiospores produced by T. incarnata are infective, but not those of T. ishikariensis (Cunfer and Bruehl 1973). 3. Early winter Snow mold pathogens usually start to infect plants under snow cover (Ozaki 1979; Oshiman 1999), but T. incarnata can infect plants before snow cover (Jackson and Fenstermacher 1969; Matsumoto and Araki 1982), and M. nivale can cause abundant disease without snow cover (Tronsmo et al. 2001). Mycelia developed from weathered sporocarps are the major inoculum especially in T. ishikariensis (Cunfer and Bruehl 1973). 4. Winter 6 1 Introduction

spores sclerotia (a)

(b)

sporocarps

Fig. 1.3 Life cycle of sclerotial snow mold fungi. The solid line separates dormant phase (right) from active phase (left). Life stage above the dotted line represents that under snow cover, and the stage without snow is shown below the dotted line. a (Typhula) and b (Sclerotinia)

The pathogens attack plants under snow cover, particularly as the plants become weaker at the end of a longer winter, and ultimately produce resting structures to pass dormancy.

1.1.3 Environment Under Snow

Three critical factors for snow mold development, namely, low temperatures, darkness, and high moisture, are constantly maintained under snow. Snow mold pathogens with low-temperature tolerance cause opportunistic infections under such circumstances. In plant ecology, Grime (1977) distinguished three strategies based on the permutations of high and low stress that limits biomass and with high and low disturbance that destroys biomass (Table 1.2). The subnivean habitat of snow mold is considered to be an extreme stress environment and typically only for stress-tolerant organisms. Since snow molds are low-temperature tolerant, the key issue is not low-temperature stress but the predictability of persistent snow cover; in other words, the snow cover duration from the start of snow cover until it disappears has the largest effect on snow mold development. This issue will be discussed later in more detail (see Chap. 2). 1.1 Snow Mold 7

Table 1.2 Three primary strategies in plants Stressa Low High Disturbanceb Low Competitive strategy Stress-tolerant strategy High Ruderal strategy No viable strategy Grime (1977) aStress consists of conditions that restrict production, e.g., shortage of light, water or mineral nutrients, and suboptimal temperatures bDisturbance is associated with the partial or total destruction of the plant biomass and arises from the activities of herbivores, pathogens, and man and from phenomena such as wind damage, frosts, desiccation, soil erosion, and ﬁre

Fig. 1.4 Winter damage of conifers in a nursery. Lower parts of plants were covered with snow and escaped from the damage; however, plant tops (arrow) were exposed to freezing temperatures and winter desiccation

Snow cover, when deep enough, insulates the ground from the open air temperature (Fig. 1.4) and keeps the soil surface at approximately 0 C. Although low temperature under snow inactivates most microorganisms, snow mold fungi can prevail at subzero temperatures. Also, their optimal growth temperature is generally low, ranging between 5 and 20 C (see Table 2.1). However, when the ambient temperature is around 10 C, snow mold fungi seldom attack plants even though the plants resume growth after dormancy. Exceptions are pink snow mold caused by Microdochium nivale (Asuyama 1940) and rot of clover (Trifolium repens) caused by Sclerotinia trifoliorum (Scott 1984). These pathogens have higher optimal growth temperatures (see Table 2.1) and can also attack growing plants. Organic soils with high microbial activity suppress the mycelial growth of Typhula spp. more strongly than those with low microbial activity (Jacobs and 8 1 Introduction

Fig. 1.5 Agar culture experiments to demonstrate the nature of snow mold fungi, escaping from antagonism in subnivean environments. Mycelial growth of Typhula ishikariensis at 0 C(upper right) is half that at its optimal growth temperature of 10 C(upper left); however when cultures were covered with unsterile soil, mycelial growth is greatly reduced at 10 C(lower left) but not at 0 C(lower right) because of the activity of antagonists that can grow at 10 C (Matsumoto 2005)

Bruehl 1986). Matsumoto and Tajimi (1988) ascribed the reduced activity of Typhula snow mold at optimal growth temperature to antagonism from other microorganisms: Typhula ishikariensis grew optimally at 10 C and to about half optimal growth rate at 0 C in culture, but when culture plates were covered with unsterile soil, mycelial growth was almost completely inhibited at 10 C, but at 0 C was as vigorous as that in axenic culture (Fig. 1.5). Snow mold fungi are regarded as fugitive microorganisms, escaping from antagonism to the subnivean habitat. Low temperatures under snow limit the activity of organisms, and only low-temperature-adapted organisms can flourish. Consequently, microflora under snow is simple; Bruehl et al. (1966) isolated 89 fungi, including 34 unidentified fungi, from winter wheat in winter and early spring, and Årsvoll (1975) described 33 fungi from grasses just after snowmelt. These fungi are, however, mostly mesophiles and unlikely to be actively growing under snow. Forty fungal species are known to cause diseases on wheat seedling, but only five of them attack overwintering plants under snow (Wiese 1977). Of 29 fungal pathogens of orchardgrass illustrated in the disease inventory of Japan, only four kinds of snow molds were incited by nine fungal species (Anonymous 2000). The severe conditions under snow thus restrict the diversity of fungal pathogens. Snow cover occurs for more than 140 days per year in some areas, and only a few fungal pathogens exclusively exploit plant resources. Recent advances in molecular techniques have brought about new findings about microbial activity beneath snow cover. Conventional methods to isolate fungi from plants under snow or just after thawing revealed many mesophiles, leading to the 1.1 Snow Mold 9 assumption that there was no marked difference in fungal flora between summer and winter. Lipson et al. (2002) collected soil DNA from grassland in North America to find that winter soil microflora greatly differed from those in summer and that fungi increased more in winter than did bacteria. Unlike forest soils in which basidiomycetes dominated, the fungal flora from grasslands was diverse and included various, unknown ascomycetes (Schadt et al. 2003). The role of these ascomycetes is yet to be elucidated. Plant photosynthesis is impaired under snow, and plants become depleted of reserve materials through respiration, predisposing them to attack. Nakajima and Abe (1994), using three winter wheat cultivars, demonstrated that decline in resistance to pink snow mold corresponded to the depletion of reserve materials. These cultivars similarly exhausted reserve materials at 15 C in the darkness (Fig. 1.6a), and the resistance likewise declined (Fig. 1.6b). Varietal differences in disease resistance correlated with food reserve depletion rates. In conclusion, snow mold fungi monopolize plants deteriorating under snow, exploiting low-temperature environments when antagonists are functionally absent. These features characterize snow mold fungi as opportunistic parasites. ab

(mg) growth

LI50 (days) Etiolated

Duration of snow cover (days)

Fig. 1.6 Exhaustion of reserve material under snow as determined by etiolated growth in three winter wheat cultivars (a) and their decline in resistance to pink snow mold caused by Microdochium nivale as determined the number of days required to kill 50 % of plants (b) (T Nakajima and J Abe (1994) ©Canadian Science Publishing) 10 1 Introduction

1.2 Subnivean Environment

Sufficient snow cover keeps the soil surface at a constant low temperature, insu- lating against fluctuating air temperatures. For example, in southern Finland, when ambient temperature was below À30 C, soil surface temperature was maintained at À2 C or above with snow cover deeper than 25 cm (Ylima¨ki 1962). In a grassland at high elevation in Central Utah, USA, soil surface temperatures ranged from À2.2 to 1.1 C with snow depth greater than 60 cm (Bleak 1970). Oke (1978) stated that 10 cm of fresh snow in southern Ontario, Canada, was sufficient to insulate soil surface temperatures from air temperatures. Sharratt et al. (1992) in a more extensive study involving 16 years of data in Minnesota, USA, found that snow cover of over 40 cm was required to uncouple air temperature from soil surface temperature which stayed near 0 C. Under snow cover, snow mold fungi play a primary role as pathogens to attack and decompose live organic matter (plant tissues). Other saprophytic microorganisms are more important in decomposition of dead organic materials and nutrient cycling during the growing season.

1.2.1 Litter Decomposition

In cold-temperate regions, the production of plant litter such as dead leaves and twigs culminates in the autumn, and litter may become substantially decomposed under snow (Bleak 1970; Moore 1983). Plant debris is broken apart by invertebrates (Aitchison 1979) and decomposed by microorganisms (Bleak 1970) in subnivean environments as is the case with other ecosystems. This process represents the first step of organic matter recycling, and many studies indicate significant litter decomposition under snow. Fifty-five percent of decomposition of oak litter occurred under snow in Min- nesota, USA (MacBrayer and Cormack 1980). Pine needle litter lost 9 % of its total weight during the first year, and 80 % of the first year’s weight loss took place under snow in an alpine forest, Nevada, USA (Stark 1972). Forty to 60 % of balsam fir litter became decomposed under snow in Quebec, Canada (Taylor and Jones 1990). In a subarctic woodland, where the ground was covered with snow from late October or early November to late May, 60–90 % of first-year litter became decomposed in winter (Moore 1984). The amount of litter broken down under snow in abandoned grasslands in central Finland was equivalent to one-third of annual foliage production (Torma€ ¨la¨ and Eloranta 1982). In the Rocky Mountains in Utah, coarse grasses under snow cover showed 30–39 % weight loss from fine mesh bags, while broadleaf forbs showed around 50 % weight loss (Bleak 1970). The rate of CO2 evolution from first-year litter was 6.6 times greater under snow than that prior to snowfall in a Minnesota forest (MacBrayer and Cromack 1980). The magnitude of winter respiratory CO2 evolution was equivalent to 60 % of annual biomass input into aspen woodland litter in southwestern Alberta, Canada 1.2 Subnivean Environment 11

(Coxson and Parkinson 1987). The aspen forest floor experienced 60 freeze–thaw cycles between December and March, but microbial activity remained high deep in the forest floor where the freeze–thaw cycle did not influence the activity. Sapro- phytic fungi were found to propagate rapidly at subzero temperatures (Schmidt et al. 2013). They are considered to play a major role in organic matter recycling under snow.

1.2.2 Organic Matter Recycling

Microbial biomass culminated by winter in an alpine ecosystem in Colorado, USA, with its accumulated nitrogen (Lipson et al. 1999). Observations made on CO2 evolution as an indicator of microbial activity under snowpack revealed that CO2 evolution was ﬁrst detected in early March, continued to increase until thawing in mid-May, and then declined when thaw water saturated the snowpack (Brooks et al. 1996). While soil surface temperature under snow was À14 C in January and 0 C on May 4th, CO2 dynamics corresponded to the change in inorganic nitrogen in soil. Dead leaves colonized by basidiomycetous mycelia under snow contained ﬁve times greater nitrogen than uncolonized leaves (Hintikka 1964). Low-temperature zygomycetes grew rapidly, using nutrients exuded in the soil– snow interface in late winter, and they left substantial mycelia on the surface just after snowmelt (Schmidt et al. 2008). Nitrogen was then released from the mycelial debris in spring. Allelochemicals present in plant tissues were also decomposed by microorganisms until plants resumed growth the following spring (Schmidt and Lipson 2004). Psychrophilic decay fungi removed most of the cell contents of pine needles and mineral elements such as Ca, Mg, etc. accumulated in their hyphae (Stark 1972). The hyphae disappeared within 2 or 3 days after snowmelt, due presumably to the decomposition by bacteria, and the elements were leached into the surface layer, adhering to organic colloids or possibly taken up by microorganisms or by plant roots (Stark 1972).

1.2.3 Diversity Creation

While saprophytic fungi play an important role in recycling under snow in the ecosystem of cold-temperate regions, pathogenic fungi attacking seeds and seedlings under snow are considered to contribute to the maintenance of diversity in the forest. Trees produce a large number of seeds to extend their habitat, but not all the seeds germinate or grow into seedlings or trees. Natural forest regeneration may usually be attained within the gap where large trees have fallen, providing a space for the next generation with abundant sunlight. Fungal pathogens present in the litter layer cause pre- and postemergence seedling damping-off, and the rate of damping-off increases with decreasing distance of seeds from mother tree. 12 1 Introduction

Consequently, seedlings of different species are more likely to establish. This phenomenon is considered as one of the mechanisms to maintain diversity in tropical and temperate forests (Packer and Clay 2000). Many fungi are known to attack seeds and seedlings to maintain the diversity of forest in snowy regions, too. Nuts of beech (Fagus crenata) are rotted by Rhizoc- tonia solani and Cylindrocarpon magnusianum under snow (Ichihashi et al. 2005). Even if they successfully germinate, most seedlings are killed by Colletotrichum dematium (Sahashi et al. 1995). Ciboria batshiana causes nut rot of Quercus mongolica var. crispula and Q. serrata under snow (Ichihashi et al. 2008). Racodium therryanum present in the forest ﬂoor attacks seeds of conifers, Picea jezoensis (Cheng and Igarashi 1987) and Larix kaempferi (Igarashi and Cheng 1988). In Korea, Abies koreana is similarly attacked by the fungus, and the damage becomes serious with increasing duration of snow cover (Cho et al. 2007). Further studies are necessary to reveal the mechanism of succession and diversiﬁcation by elucidating the effect of snow cover and by investigating host–parasite interactions at different growth stages of host trees. Similar phenomena are found in polar regions; mosses are important as primary colonizers in these regions, and Pythium spp. can damage them resulting in patches (see Fig. 3.20). These fungi seldom cause fatal damage but seem to promote regeneration by degrading moribund parts of the moss colonies (M. Tojo, personal communication). Since grasses and other mosses are often found in the centers of disease patches, Pythium spp. are able to promote plant succession in polar regions (Hoshino et al. 2001).

1.3 Microorganisms in the Cryosphere

Mycology, which was earlier placed in botany because of perceived relationships between fungi and plants, occupies a significant position in microbiology along with bacteriology, and these two disciplines are often regarded as similar because of the use of similar laboratory methods for their isolation and cultivation. However, bacteria with their simple prokaryotic life cycle propagating through binary cell division are different from fungi with their more complex methods of eukaryotic propagation and life cycles. Fungi have evolved different growth–temperature relations at different stages of growth; e.g., in Sclerotinia borealis, while its optimal mycelial growth (asexual stage) occurs at 4–10 C, fructification (sexual stage) is best at temperatures fluctuating between 5 and 25 C (see below). Hoshino and Matsumoto (2012) illustrated discrepancies in the use of the term “psychrophile” when it was used for fungi with complex life cycles and proposed a new term “cryophilic fungi” for those that spend a certain life stage or whole life cycle (sexual and/or asexual reproductive stages) in the cryosphere. The term cryosphere collectively describes the portions of the Earth’s surface where water exists as the frozen state such as snow cover, glaciers, ice sheets and shelves, freshwater ice, sea ice, iceberg, permafrost, and ground ice. Many kinds of 1.3 Microorganisms in the Cryosphere 13

Table 1.3 Low-temperature microorganisms Name Microorganism Growth temperature relations Authors Psychrophile Bacteria, Yeasts Grows at 0 C Schmidt-Nielsen (1902) Low Wood-decaying Optimum at 20–24 C Humphrey and temperature fungi Siggers (1933) Intermediate Optimum at 24–32 C High Optimum higher than 32 C temperature Phychrotoleranta Microfungib Grows at subzero temperatures Panassenko (1944, Mesotolerant Grows at 5–30 C 1967) Thermotolerant Able to grow at 40–50 C Psychropile Yeasts Optimum at 20 C or below Baxter and Gibbons (1962) Obligate Yeasts Unable to grow at 20–25 C Stokes (1963) psychropile Psychrophilea Fungi Optimum at 10 C or below Deverall (1968) Psychrophile Bacteria Optimum at 15 C or below maxi- Morita (1975) mum at 20 C aAlso referred to as cold-adapted (see the text) bFungi other than those producing large fruit bodies (mushrooms) fungi have been reported from the cryosphere other than snow mold fungi, e.g., Robinson (2001), Ludley and Robinson (2008), Hoshino et al. (2009), and Tojo and Newsham (2012). The presence of cryophilic fungus was first recorded in 1788 on snow scald of winter cereals in Toyama, Japan (Hori 1934). Various terms have been used for low-temperature microorganisms (Table 1.3). Morita (1975) compared previous reports to conclude that psychrophiles were defined as bacteria having an optimal temperature for growth at 15 C or lower and a maximum at about 20 C. He mentioned in his review, “yeasts that meet the above definition are known to exist but they will not be discussed.” Morita’s definition of psychrophile is widely recognized in modern microbiology, and the same concept was adopted for fungi by many scientists. However, Vidal-Leiria et al. (1979) pointed out that the maximal growth temperature of the psychrophilic yeast group was below 24 C. The presence of many exceptions was generally recognized. Fungi growing at subzero temperatures are also referred to as cold-adapted fungi or cryophiles (cold, freezing-loving organisms). Cold-adapted fungi by the definition include two distinct groups of organisms, i.e., psychrotolerants and psychrophiles: the former is distributed in habitats with temperature fluctuations and grow over a wide temperature range with an optimal at higher than 20 C, and the latter persists in permanently cold habitats such as polar regions and year-round snow-covered mountain tops (Margesin et al. 2007). The word cryophile is ambig- uous in modern mycology, on the one hand, often equated with psychrophile (e.g., 14 1 Introduction

Jennings and Lysek 1999) but, on the other hand, indicating fungi that grow under snow (Eckblad 1978). These terms thus far mentioned have exceptions. The primary cause of the confusion is likely because the words were deﬁned based on growth–temperature relations of fungi compared with those of bacteria and yeasts, but irrespective of complex life stages of fungi which have diverse types of morphogenesis or reproduce sexually and asexually to adapt different environmental conditions. Among cryophilic fungi, snow mold fungi are best studied due to their economic importance and interesting physiology and ecology. These issues will be discussed using the phenology of snow mold fungi (Fig. 1.3) and the deﬁnitions of the terms (Table 1.3).

1.3.1 Physiological Issues

Sclerotinia borealis grows optimally in a temperature range between 4 and 10 C, and its maximum growth temperature is at 20 C (Smith 1987; Hsiang et al. 1999; Hoshino et al. 2009, 2010), although the fungal growth is strongly limited at the upper temperature. Doubling ingredients of growth medium (PDA) promoted mycelial growth of S. borealis (Tomiyama 1955). The same results were obtained by adding sucrose or KCl (Bruehl and Cunfer 1971) or D-mannitol (Namikawa et al. 2004). In addition, Hoshino et al. (2010) showed that the optimal growth temperature on PDA shifted from 4 to 10 C by adding KCl or sorbitol, and after the addition, the fungus grew well even at 20 C. These results indicate that mycelial growth–temperature relations are changeable by regulating osmotic pressure of media. Optimal temperature for fructification is higher than that for mycelial growth in S. borealis, and fluctuation in temperature plays a key role in fructification (Saito 2001). According to Itsuki (1984), sclerotia failed to germinate to develop ascocarps at a constant temperature of 9 C, and 76.7 % of sclerotia germinated at the conditions simulating autumn climate (25 C light/15 C darkness for 28 days and subsequently 20 C light/5 C darkness for 38 days). Germination rates for sclerotia after incubation for 66 days at 25 C light/15 C darkness and at 20 C light/5 C darkness were 53.3 % and 16.7 %, respectively. Thus, S. borealis should not be regarded as a psychrophile in terms of sclerotial germination. The life cycle of the Typhula ishikariensis complex with a high degree of intraspecific diversity is similar to that of S. borealis. Group III, one of the taxa in the complex, exists in coastal regions in Norway (Matsumoto et al. 1996), Svalbard (Hoshino et al. 2003), and Greenland (Hoshino et al. 2006). The fungus shows irregular mycelial growth on PDA at 10 C with gnarled hyphae (Fig. 1.7, Matsumoto et al. 1996), and sclerotia are large with exuded droplets and are produced in aggregates. Mycelial growth is regular at 0 C, but shows abnormalities at 10 C. The anomalous growth at 10 C is alleviated when cultures are covered with water (Hoshino et al. 2008). The fungus also grows well on PDA fortified with 1.3 Microorganisms in the Cryosphere 15

Fig. 1.7 Cultural morphology of Typhula ishikariensis group III isolates grown on PDA at 10 C for 2 weeks (left two rows) and at 0 C for a month (right two rows). The position of isolates correspond in the left and right sides

Fig. 1.8 Cultural morphology of a Typhula ishikariensis group III isolate grown at 10 C. Left, PDA, right, PDA + carotene (Courtesy, T. Hoshino) carotene or ascorbic acid or on cornmeal agar (Fig. 1.8, T. Hoshino, unpublished data). T. ishikariensis group III is unable to exhibit its full growth potential at 10 C on PDA due to oxidation (T. Hoshino, personal communication). 16 1 Introduction

Snow rot fungi (Pythium iwayamai, P. okanoganese) which attack grasses grow best at 20 C on cornmeal agar (Lipps and Bruehl 1978; Lipps 1980). Zoospores are released at 1–15 C for P. iwayamai and at 1–10 C for P. okanoganese but not at all above 20 Cor15C, respectively (Lipps 1980). Oospores of P. iwayamai germinate at temperatures lower than 15 C but never at temperatures higher than 20 C (Takamatsu 1989). Mycelial growth of P. iwayamai and P. okanoganese at different temperatures place them as psychrotolerant, but they are regarded as psychrophiles on the basis of optimal temperatures for zoospore release and oospore germination.

1.3.2 Ecological Issues

Growth–temperature relations are mostly studied under axenic conditions to reveal physiological potentials of microorganisms, including snow mold fungi. These experiments often ignore the fact that, in nature, microorganisms have close interactions with other microorganisms and plant host tissues to complete their life cycle. The signiﬁcance of microbial interactions is clearly illustrated by the experiments with Typhula ishikariensis (Fig. 1.5); the fungus failed to grow at 10 C on PDA when cultures were covered with unsterile soil but were not affected by microbial activity in the soil at 0 C. Optimum mycelial growth occurs at 20 CinSclerotinia nivalis, categorizing the fungus as psychrotolerant. Saito (2001) inoculated the mycelia of the fungus onto carrot and Artemisia cina and incubated the plants at 0, 3, and 15 C. Plants were infected by S. nivalis at 0 and 3 C but not at 15 C. The sclerotia, when placed on unsterile soil at 0 C, developed mycelia (myceliogenic germination) to infect carrot roots; however, sclerotia neither germinated nor infected at 20 C. At 20 C, microﬂora on and around sclerotia is thought to suppress sclerotial germination. Such features of S. nivalis indicate that the fungus is a psychrophile.

References

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Abstract The subnivean environment represents a habitat of snow mold fungi where their resources, i.e., living plant tissues, are limited and do not increase. The fungi have adopted two major contrasting life history strategies in terms of resource

Buller (1931)wasimpressedwiththewayinwhichmanycoloniesofCoprinus sterquilinus coexisted within a horse dung ball, helping each other to develop a single fruit body. The phenomenon was later expounded by Todd and Rayner (1980) based on their idea of fungal individualism. These ideas lead us to further explore the ecology of snow mold fungi. Figure reproduced from Buller (1931).

© Springer Science+Business Media Singapore 2016 23 N. Matsumoto, T. Hsiang, Snow Mold, DOI 10.1007/978-981-10-0758-3_2 24 2 Ecology and Physiology utilization: facultative snow mold fungi can become abundant even without snow cover, and obligate snow mold fungi require snow cover for activity. Some obligate snow mold fungi particularly ascomycetes share plant tissues with other genotypes of the same species and coexist, while basidiomycetous obligate snow mold fungi often exclude even other genotypes of the same species and monopolize resources by individualism. The duration of snow cover is, of course, critical to snow mold fungi for survival and propagation, but the beginning of persistent snow cover and its end are other important issues. Snow mold fungi developed diverse life history strategies to adapt to the predictability of snow cover, which is typically represented by the germination rate and the size of sclerotia. Snow mold fungi also exhibit diverse metabolic reactions to tolerate freezing stress by producing antifreeze proteins, exhibiting osmophily, and altering fatty acid composition. Some snow mold fungi protect themselves from freezing by colonizing deeper into plant tissues.

The cryosphere where snow mold fungi exist differs from the habitat of the great majority of organisms because the survival of snow mold fungi depends on the physical condition of snow cover and because plant tissues are usually dormant under snow, with no new growth. Each snow mold fungus has developed its own unique survival strategy. The cryosphere habitat occurs regularly in some areas, while it is unpredictable in other areas; snow mold fungi cope with the predictability of snow cover using different strategies. The cryosphere is an environment at zero to subzero temperatures, and the fungi must tolerate low temperatures, using freeze tolerance mechanisms.

2.1 Survival Strategy

Niche separation facilitated the coexistence of different snow mold fungi. The phenomenon is realized as a difference in distribution patterns on a macroscale (Tomiyama 1955; Matsumoto and Sato 1983; Takamatsu 1989b) and also on the microscale, as a difference in tissues colonized within a plant (Takenaka and Arai 1993). Here, we discuss intraspecies interactions within a species that occur on the same resources in the same habitats. When typical aboveground plant pathogens ﬁnish exploitation of plant tops during the growing season, they may migrate through airborne spores, causing secondary infection. This type of epidemiology does not apply to snow mold fungi; they must reach plant resources through airborne propagules prior to snowfall and/or infect adjacent plants under snow through mycelia emerging from soilborne propagules, such as sclerotia. An exception is the facultative snow mold fungus, Microdochium nivale, which may remain associated throughout the entire growth stages of winter cereals and grasses (for details, see Chap. 3). Secondary infection is unlikely to occur in the cryosphere because of snow cover. Also, plant biomass does 2.1 Survival Strategy 25 not increase since photosynthesis is impaired under snow and most plants are dormant. Thus, the subnivean environment where snow mold fungi prevail represents a habitat where resources are limited and constantly diminish. Each fungus has developed a unique strategy under such circumstances. Limited resources may be utilized by two contrasting methods, i.e., collectivism and individualism. Facultative snow mold fungi (Matsumoto 1994) partake of the same plant tissues as typical plant pathogens. Since strains differing in genetic background coexist on single resources, different strains are often isolated from the same lesions. On the contrary, resources are allocated to each strain of individualistic fungi, and each strain exclusively occupies its own territory (Fig. 2.1). Although plant biomass does not increase under snow, it is replenished in the summer. Strains of individualistic snow mold fungi compete and antagonize each other under snow, but cease hostilities in summer. Cultural practices such as tillage and ridging promote inter-strain interactions even during the truce in summer; territories become intermingled partially or entirely. Snow mold fungi that do not necessarily depend on the cryosphere can prevail on growing plants and are referred to as facultative snow mold fungi. Their growth temperatures are higher, and its distribution range is wider in time and space (Matsumoto 1994, Table 2.1). Microdochium nivale, a representative of facultative snow mold fungus, is associated with every growth stage of winter cereals as a plant pathogen (Cook 1981). M. nivale from ears of wheat showing head blight can cause pre- and post-emergence damping-off, attacks lower sheaths, and incites pink snow mold under snow (Nakajima and Nemoto 1987). The fungus produces lesions on leaves in summer and ultimately incites head blight. Pathogens of barley such as

Fig. 2.1 Disease patches on turfgrass caused by Typhula ishikariensis biotype B. There were 72 patches in a 2 Â 2-m plot, consisting of 31 individuals (assessed by the MCG testing), and the most predominant one was found in nine patches 26 2 Ecology and Physiology

Table 2.1 Obligate and facultative snow mold fungi and their growth-temperature relations Growth temperature( ̊C)b Fungusa Minimum Optimum Maximum Obligate snow mold Pythium iwayamai <0 18–22 25–30 Racodium therryanum <À5 15–20 nd Sclerotinia borealis <À7 10–15 <20 Sclerotinia navalis <020nd Typhula incarnata <À7 10–15 <20 Typhula ishikariensis <À7 5–10 <20 Typhula phacorrhizac <0 10–15 <25 Supponuke fungusd nd 10 <25 Facultative snow mold Pythium paddicum <02230 Microdochium nivale >À5 10–20 30 Sclerotinia trifoliorum <0 15–19 nd aObligate or facultative tropisms modiﬁed from Matsumoto (1994) bMostly from Hoshino et al. (2009), nd not determined. cWu and Hsiang (1999) dGrowth temperatures from Simizu and Miyajima (1990). Inferred to be Athelia sp. by A. Kawakami (personal communication)

Ceratobasidium gramineum, causing foot rot (Takamatsu 1990), and Rhynchosporium secalis, causing scald (Suzuki and Arai 1990), are known to incite snow mold under a covering of damp snow. They also may be regarded as facultative snow mold pathogens. Obligate snow mold fungi grow at a lower temperature range (Table 2.1) and, as a rule, prevail exclusively under snow.

2.1.1 Individualism

Todd and Rayner (1980) presented the idea of “fungal individualism” based on the mechanism of self–nonself recognition in fungi. Typical examples may be found in stumps colonized by wood-decaying basidiomycetes. These develop many fruit bodies on colonized stumps, and each fruit body originates from the territory of an individual strain, known as a genet. Colonized stumps are divided into two parts, i.e., rotted wood (decay column) and black, undecayed interaction zones surrounding decay columns, leading to a marble-like pattern on the cut surface. Each decay column is colonized by an individual of a wood-decaying basidiomycete. Hyphae of different genets may anastomose, but since most are vegetatively incompatible, this results in death of fused cells. 2.1 Survival Strategy 27

Cytoplasmic incompatibility is the basis of self–nonself recognition, which is homologous to organ transplant rejection in humans. For basidiomycetous fungi, when different individuals are confronted on agar plates, dark demarcation lines may occur between the colonies (Fig. 2.2). This method is useful to determine vegetative cytoplasmic compatibility. Isolates compatible with each other do not produce demarcation line and are regarded as belonging to the same mycelial compatibility group (MCG). Individuals belonging to the same MCGs do not always share the entire same genetic background (i.e., may not necessarily be identical genetically) and hence may be distinguishable by DNA sequence or marker methods (Fig. 2.3, Matsumoto et al. 1996b). Lebeau (1975) was the ﬁrst to report antagonism between individuals of snow molds, and when different strains of low-temperature basidiomycete (LTB) were mixed and inoculated onto alfalfa, disease severity was reduced. Also, Årsvoll (1976) found the same phenomenon for basidiomycetous snow molds but not for ascomycetous snow molds (Table 2.2). Plant damage was much less on plants inoculated with a mixture of four strains of T. ishikariensis as compared to plants inoculated with single strains, and disease was reduced to half in the case of T. incarnata (Årsvoll 1976). Inoculum mixtures of neither Microdochium nivale nor Sclerotinia borealis showed disease reduction, indicating that individuals of the ascomycetous snow molds did show this antagonism. The idea of fungal individualism (Todd and Rayner 1980) led Matsumoto and Tajimi (1983) to conﬁrm that intraspecies antagonism in Typhula incarnata and T. ishikariensis was the outcome of individualism. Overall antagonism between strains is considered to have occurred within thorough mixtures of inoculum, resulting in decreased inoculum potential; inoculation with a mixture of different

Fig. 2.2 Distinction of individuals (MCG) by pairing. Dark demarcation lines (arrow) occur between individuals differing in cytoplasmic compatibility but not between identical individuals (asterisked) 28 2 Ecology and Physiology

Fig. 2.3 RAPD patterns of Typhula ishikariensis biotype A isolates belonging to the same (lanes 1–10) and different individuals based on mycelial compatibility. Isolates 1–10 belong to the same MCG (mycelial compatibility group), and the rest are other MCGs. Isolate 10 was distinguished from isolates 1–9 by DNA tests (Matsumoto et al. 1996b © Canadian Science Publishing)

Table 2.2 Disease severity on plants inoculated with different snow molds singly or in mixture of strainsa Inoculated with a single strain mixture Snow mold Strain A Strain B Strain C Strain D AþBþCþD Typhula ishikariensis 96.1 99 98.7 93.7 <5.0 Typhula incarnata 96.2 70.6 94.9 88.3 46 Microdochium nivale 75.9 82.9 96.5 86.8 87.8 Sclerotinia borealis 89.6 81.9 78.4 89.3 87.1 Årsvoll (1976) aFigures indicate disease severity of timothy, and strains A–D were designated arbitraly

strains escalates antagonism between individuals. Individualism may be found in disease patches of turfgrass caused by T. ishikariensis biotype B (Fig. 2.1, Matsumoto and Tajimi 1993b). Each individual genotype of the pathogen monop- olized its own territory (patch), excluding others; predominant individuals were found repeatedly in many patches, while a large number of individuals occupied single patches. Plants survived in a line between patches occupied by different individuals, which corresponded to dark demarcation line in paired culture. Mas- sive hyphal cell death should have occurred along the interface between territories. 2.1 Survival Strategy 29

2.1.2 Population Structure

As illustrated in the previous section, the populations of T. ishikariensis on turfgrasses consist of dominant individuals found repeatedly from many patches and others occurring in a few or single patches. A series of inoculation experiments were subsequently conducted to elucidate the bases of superiority or inferiority of individuals. Orchardgrass was inoculated with mixtures of two strains, and resultant sclerotia were collected from diseased plants to identify their origin based on MCG. Stains of T. ishikariensis biotype A were often indistinguishable based on pairing experiments; they belonged to the same MCGs. Molecular techniques often failed to separate these strains (Fig. 2.3), and they were regarded as belonging to the same genet.

Typhula ishikariensis Biotype A

The occurrence of a limited number of alleles for mating incompatibility leads to the prediction of low genetic diversity in T. ishikariensis biotype A (Matsumoto and Tajimi 1993a; for details, see Chap. 3). This also implied the presence of common MCGs among the fields, and subsequent field surveys showed that there were at least six major common MCGs of this fungus in the experimental grass fields at the Hokkaido National Agricultural Research Center, Sapporo (Fig. 2.4), over an area of ca. 5 ha. Common MCGs were further found throughout Hokkaido, excluding the southwestern part. MCGs S, 1, 2, 3, and 5 were ubiquitous, and super MCG (MCG S) was the most prevalent. Ten isolates randomly chosen from super MSG were separated into five sub-MCGs by the random amplified polymorphic DNA (RAPD) analysis. These sub-MCGs shared all the common mating incompatibility alleles and were presumed to represent genets originating from sibling crosses. The low genetic diversity in T. ishikariensis biotype A was in contrast to biotype B and T. incarnata (Matsumoto and Tajimi 1989). Extensive surveys revealed that MCG S was found to be prevalent in eastern Hokkaido (Matsumoto et al. 2000); of 697 biotype A isolates collected from eastern Hokkaido, 264 (37.9 %) belonged to MCG S, while 14.4 % (67 out of 466 isolates) were identified as MCG S in northern and central Hokkaido. MCG S was not found in southern Hokkaido or Tohoku when 97 isolates were examined from these localities. Change in winter climate in eastern Hokkaido, where winter climate has changed most drastically (Hirota et al. 2013), promoted biotype A over biotype B, and this has had an impact on agricultural production (Matsumoto and Hoshino 2013). MCG S is considered to play a major role among the biotype A populations in eastern Hokkaido. The distribution pattern of MCG S implies the route of entry into Hokkaido. Geological evidence indicates that Hokkaido was connected to Sakhalin, Russia, three times in the past and only once it was connected southerly with Honshu (Fig. 2.5, Ono 1990). Biotype A is considered to have first reached Hokkaido from 30 2 Ecology and Physiology

Field 23a (meadow fescue) S, 4 200m Field 23b (alfalfa) S, 1, 5

1500m

Field 9 (timothy) S, 2, 3, 4, 5

Fig. 2.4 Distribution of MCGs of Typhula ishikariensis biotype A in grass ﬁelds of the Hokkaido National Agricultural Research Center, Sapporo. S and 1–5 represent MCGs

Sakhalin

Sohya Straigt

Hokkaido

Sappor Tsugaru Straight o

Honshu

1.2 million 160,000 120,000 12,000䡚7,000 years ago

Fig. 2.5 Landbridges (S1, S2, S3, and T) during the Pleistocene. Hokkaido was connected to other islands three times and dimidiated by coastline transgression 120,000 years ago (Modiﬁed From Matsumoto et al. 2000)

Sakhalin when the So¯ya Strait ceased to exist by a landbridge (possibly S1, Fig. 2.5), and it moved down further to Honshu through landbridge T. MCG S isolates were found also in Sakhalin, and sub-MCG groupings were in common between Sakhalin and Hokkaido populations (Hoshino et al. 2004c). The absence of MCG S from Honshu and southern Hokkaido suggests its immigration to Hokkaido through landbridge S3 (Fig. 2.5). 2.1 Survival Strategy 31

Typhula ishikariensis Biotype B

On the managed turfgrass swards where the plants form a uniform horizontal surface, snow molds are seen growing radially from the primary inoculum and manifest their activity as homogenous circular disease patches after thawing (Fig. 2.1). Often only the foliage and stems of turfgrass plants in disease patches are killed, and growth can resume from the crown and roots for full recovery by the summer. In the case of T. ishikariensis biotype B, sclerotia produced on underground plant parts the previous year play a major role as the primary inoculum to extend patches the following year. Since each MCG occupies a single patch, the extension of patches is regarded as the growth of individuals. We found extraordinarily large patches in an orchardgrass (Dactylis glomerata) grassland in Iwate, northern Honshu; one exceeded 60 m in diameter, and several were 20 m in diameter (Fig. 2.6). Orchardgrass plants, presumed to have invaded after disease occurrence or escaped infection, were growing in the center of the largest, torus-shaped patch. Diseased plants had T. ishikariensis biotype B sclerotia. We named this particular patch “Godzilla patch” after the special effect ﬁlm revived in Hollywood. An estimate was made to infer the age of Godzilla patch, assuming that patches extended 0.125 m/year:

60 m Ä 2 Ä 0:125 m ¼ 240 years

The estimate seemed dubious since the grassland was developed rather recently, and this motivated us to determine the population structure of the patch, where we uncovered many different MCGs in single patches. Godzilla patch was not

Fig. 2.6 Godzilla patch (arrow) and other patches (asterisk) in an orchardgrass grassland, Iwate, northern Honshu 32 2 Ecology and Physiology produced by the growth of a single individual of T. ishikariensis biotype B. Instead, a fairy ring fungus was considered to have caused the primary infection, debilitating plants and predisposing them to infection by biotype B. Fairy rings are caused by basidiomycetous fungi and known to both kill plants and stimulate their growth through their organic matter degrading activities, and some will produce mushrooms on their periphery. Large fairy rings can measure several hundred meters across.

Effect of Agricultural Practices on Population Structure

The population structure of Typhula spp. varies according to their life history strategies and is also affected by environmental conditions. Matsumoto and Tajimi (1993b) assessed the population structure of T. incarnata and T. ishikariensis biotypes A and B in diverse habitats (Table 2.3). Uncultivated lands such as roadsides and preservation fields for plant and cultivar collections represent stable habitats with little or no disturbance (Table 2.3 habitats G, I, J, and H). Fields of winter wheat (Triticum aestivum) and first-year breeding fields of forage crops are highly disturbed environments for Typhula species (Table 2.3 habitats A, B, and C). Disturbance is intermediate in agricultural fields 2–3 years after preparation (Table 2.3 habitats D, E, and F). The population structure of T. incarnata was always diverse irrespective of the extent of habitat disturbance, reflecting its survival strategy of high reproduction and high mortality rates. While its basidiospores are effective as propagules (Hindorf 1980; Matsumoto and Araki 1982) to produce new genets every winter, many genets perish since most sclerotia are attacked and killed by mycoparasites during dormancy in summer (Matsumoto and Tajimi 1985). Among the Typhula ishikariensis complex with the strategy of low reproduction and low mortality rates, the effects of disturbance were most obvious in biotype B. In an experimental field where winter wheat had been continuously grown for more than 30 years, the biotype B population was simple, consisting of four MCGs (Table 2.3 habitat C), half of the isolates tested belonged to the most prevalent MCG (Fig. 2.7). The harshest environment in Sendai, northern Honshu (habitat B), strictly limited population diversity; only the small sclerotial form of biotype B survived (see Chap. 3). Half of the populations from uncultivated lands representing the most undisturbed habitats belonged to unique MCGs (Table 2.3 habitats I and J). Since Typhula ishikariensis biotype A exists exclusively in snowy areas (Matsumoto et al. 1982) and is unable to survive in less snowy areas, it has no clear relationship between population diversity and habitat disturbance. However, populations from habitat G with low disturbance had a few unique MCGs as compared with those from habitats A and D with high and moderate disturbance, respectively (Table 2.4). Populations from habitat G tended, as a whole, to be more virulent and to grow more slowly. Since individual plants were grown separately with a distance of 50–60 cm in forage grass breeding fields, keen competition on separate grass plants is considered to select for virulent individuals. 2.2 Predictability of Snow Cover 33

Table 2.3 Population strucure of Typhula incarnata (TN) and T. ishikariensis (TS) biotypes A and B in habitats with different levels of disturbance No. % Disturbance isolates unique Diversity Taxon Habitat Plant levela examined MCGsb indexc TN A Meadow fescue High 24 91.7 22.65 G Timothy Low 15 100.0 15.00 TS A Meadow fescue High 36 25.0 9.59 biotype A D Perennial ryegrass Moderate 50 30.0 12.42 E Alfalfa (grown in Moderate 60 33.3 14.53 rows) F Orchardgrass Moderate 17 23.5 7.64 G Timothy Low 86 4.9 8.51 I Uncultivated land Low 11 72.7 8.15 TS C Winter wheat (con- High 30 3.3 2.88 biotype B tinuous monoculture) F Orchardgrass Moderate 76 22.4 11.24 I Uncultivated land Low 20 50.0 10.90 J Uncultivated land Low 13 61.5 7.00 Small B Winter wheat High 19 5.3 1.23 sclerotium (converted from B paddy) Bþsmall H Golf course turfgrass Low 72 26.4 20.25 sclerotium B Revised from Matsumoto and Tajimi (1993b) ©Canadian Science Publishing aHigh:tilled the previous year; moderate:tilled 2–3 years ago; and low: tilled 5–8 years ago or uncultivated bMCGs consisting of single isolates cHill’s diversity index

2 2 2 2 1 1 2 2 3 3

2 2 2 1 1 2 1 1 1 4

1 1 2 1 1 1 1 1 1 3

Fig. 2.7 Population structure of Typhula ishikariensis biotype B in a ﬁeld where winter wheat had been monocultured for more than 30 years in Kunneppu, Hokkaido. Figures in rectangles indicate MCGs. There were four MCGs, and a half of the population belonged to MCG 1

2.2 Predictability of Snow Cover

Since snow cover is prerequisite for the survival of snow molds, the longer the duration of snow cover, the better the habitat for the pathogens. Another issue that concerns snow cover is predictability; when snow cover begins and ends is as 34 2 Ecology and Physiology

Table 2.4 Virulence and mycelial growth rate of Typhula ishikariensis biotype A populations from habitats differing in the level of disturbancea Habitat A (1)b Habitat D (2)b Habitat G (8)b Growth Growth Growth Virulencec rated Virulence rate Virulence rate Mean 2.88 1.14 2.39 1.23 3.41 1.02 Least significant dif- 0.77 0.08 0.74 0.08 0.63 0.16 ference (5%) Coefficient of variation 31.0 17.6 29.6 6.1 19.8 30.5 (%) Matsumoto and Tajimi (1993b) ©Canadian Science Publishing aEighteen isolates were randomly selected for comparison bA: planted with meadow fescue; D: perennial ryegrass; and G: timothy. Each grass plant was cultivated individually. Figures in parentheses indicate the number of years after disturbance cBased on the damage of inoculated orchardgrass (0, healthy; – 6, killed) dMycelial growth rate (mm/day) on PDA incubated for 21 days at 0 C important as its duration. Most snow molds diapause during summer in the form of sclerotia. At temperatures near freezing (Yang et al. 2006), sclerotia of Typhula phacorrhiza and presumably other Typhula spp. start to recover from dormancy before the onset of snowfall, and the time to resume growth is vital to snow molds irrespective of species (Matsumoto 2005). Even the generalist, Typhula incarnata , exhibits specific adaptations since different populations of T. incarnata react differently to snow cover predictability (Matsumoto et al. 1995). T. ishikariensis biotypes A and B cope with the predictability of snow cover by infraspecific differentiation of specialists (Matsumoto and Tajimi 1990). The differences in the mode of adaptation to the habitat predictability are reflected in the sclerotial size of isolates from different populations (Fig. 2.8). Sclerotinia borealis is also known to show environmental adaptation; in inland Alaska, USA, where weather changes drastically, the fungus does not produce ascospores to infect plants, but mycelia germinate directly from sclerotia infect plants so that the high inoculum potential may secure infection and subsequent pathogenesis (McBeath 1988).

2.2.1 Sclerotial Germination

Snow mold may cause severe damage to plants when 60–70 Typhula sclerotia/kg soil are found in the ﬁeld from the previous season (Jacobs and Bruehl 1986). The sclerotia, however, fail to infect plants if snow cover does not occur very soon after germination, and then the germinated sclerotia subsequently die. T. ishikariensis is known to produce secondary sclerotia (Christen 1979), which is regarded as an adaptation for germinated sclerotia to diapause under inappropriate conditions (Fig. 2.9). The fungus attacking underground parts of tulip in Russia also produces 2.2 Predictability of Snow Cover 35

Fig. 2.8 Variation in Typhula ishikariensis sclerotial size in Typhula ishikariensis biotypes A and biotype A B and in T. incarnata. Each crescent indicates individual isolates. Thickness of individual crescent indicates frequency distribution in sclerotial size. Sclerotia of T. ishikariensis biotype A (top) are uniform, while those of biotype B (middle 5) are as variable as those of biotype B T. incarnata (bottom)asa taxon, but variability within each isolate is small. T. incarnata sclerotia are variable within single isolates and correspond to that of T. ishikariensis biotype B as a taxon (Modiﬁed from Matsumoto 2005)

Typhula incarnata

small large Size one to seven secondary sclerotia (Tkachenko 1995) to cope with environmental change (Tkachenko 2013). Although information on sclerotial germination of snow mold fungi is limited (Saito 2001), low temperature and high humidity in the autumn promote germination (Detiffe and Maraite 1985). Sclerotinia borealis sclerotia, for example, start to germinate from September to October in nature, and its ascospore discharge culminates from early to mid-November. Details have been illustrated on the environmental conditions by Itsuki (1984); thermocycling was essential to obtain maximal germination of sclerotia, and constant temperature at its optimal mycelium growth temperature of 9 C failed to induce apothecial formation. Sclerotia of T. ishikariensis started to germinate 2 weeks after incubation under the experimental regime of 8 h/day at 10 C and 16 h/night at 5 C and mostly germinated 4 weeks after (Kawakami et al. 2004). Sclerotia of Typhula phacorrhiza begin to germinate in the fall as temperatures approach 0 C (Yang et al. 2006). 36 2 Ecology and Physiology

Fig. 2.9 Production of a secondary sclerotium (left, arrow)byTyphula ishikariensis biotype A. Normally, stipes ﬁrst emerge from its sclerotia, and then fertile heads develop on their tip (left)

Maraite et al. (1981) referred to isolate variability in sclerotial germination in T. incarnata . Matsumoto et al. (1995) further investigated the variability to correlate the environmental threshold for germination and fungal population in terms of habitat difference, i.e., predictability of snow cover. Sclerotia were collected from six isolates, each representing six populations differing in the predictability of snow cover and subjected to the germination regime of 10 h/day at 8 C and 14 h/night at 6 C. Sclerotia were passed through a 2-mm mesh sieve, and only those remaining on the 1-mm mesh sieve were used to minimize the effect of size. Emergence of sporocarp stipes was the criterion for sclerotial germination, which usually ﬁrst occurred 2 weeks after incubation in each population, and half of the sclerotia germinated 23 days after (Fig. 2.10a). There was no difference in the rate of sclerotial germination among the populations under the controlled conditions. However, population differences were evident when sclerotia were left outdoors in the shade where ambient temperature ranged between À4 and 14 C (Fig. 2.10b). Sclerotia of populations from Nayoro (150 days with snow cover p.a.) and Sapporo (120 days) in snowy, predictable habitats more readily germinated than those from Toyama (60–90 days) and Yamaguchi (10–20 days); rate of sclerotial germination after the experiment was more than 50 % for the former populations and was 40 % and 20 % for populations from Toyama and Yamaguchi, respectively. These results indicate that optimal condition for sclerotial germination is the same among T. incarnata populations, but threshold differs among populations. In other words, a faint environmental signal may trigger sclerotial germination for populations from snowy, predictable habitats since snow cover occurs with high certainty after such a 2.2 Predictability of Snow Cover 37

b % germination %

Incubation in days

Fig. 2.10 Sclerotial germination by Typhula incarnata under controlled conditions (a) or left outdoors in late autumn. (b) Sclerotia passed through a 2-mm mesh and remained on a 1-mm mesh were used. Sclerotia with stipe primordia were regarded as germinated. Na Nayoro, Sa Sapporo, To Toyama, and Ya Yamaguchi (Matsumoto et al. 1995) signal. However, populations from less snowy, unpredictable habitats have a higher threshold since germination in response to a faint signal may lead to the death of sclerotia before snow cover allows the activity and subsequent survival and propagation of the fungus. 38 2 Ecology and Physiology

2.2.2 Sclerotial Size

Leguminous plants produce hard seeds to cope with the change in soil water level. Hard seeds take time to germinate since the testa absorbs water slowly. Sclerotinia borealis sclerotia smaller than 1.5 mm across are hard to germinate (Ma¨kela¨ 1981). Similarly, sclerotia of the small sclerotium form of Typhula ishikariensis biotype B seldom germinate (Matsumoto and Tajimi 1990). Fastidious germination of these sclerotia is not ascribed to their rind, which is equivalent to the testa of plant seeds, but size plays a key role. The sclerotium represents both sexual and asexual propagules in many fungi, capable of producing sexual spores or vegetative hyphae depending on the circumstance. The size of sclerotia reﬂects the life history strategies of T. ishikariensis biotype B and T. incarnata (Fig. 2.8). Inter-isolate variation is great in T. ishikariensis biotype B to adapt to diverse habitats which differ in the predictability of snow cover. T. incarnata, on the contrary, is a versatile fungus and shows much less inter-isolate variation for sclerotial size, but much more intra-isolate variation than does T. ishikariensis.Evena single isolate of T. incarnata may have highly variable sclerotial sizes covering more than a whole range of Typhula ishikariensis biotype B isolates. According to Imai (1937), T. incarnata sclerotia range between 0.5 and 4.5 mm in diameter.

Typhula ishikariensis Biotype B

Matsumoto and Tajimi (1990) compared nine isolates, each from seven localities differing in snow cover conditions, for sclerotial size, virulence, and sclerotial germination (Table 2.5). Sclerotia from snowy habitats tended to be large with an exception of Oomagari. The Oomagari population had smaller sclerotia than those of populations from Yakumo, Abashiri, and Sapporo despite that all four localities had 120 days with snow cover p.a. The high coefficient of variation of Oomagari implied that snow cover was more unpredictable than other habitats, and this was demonstrated by the snow cover index, calculated as mean number of days with snow cover p.a./coefficient of variation, which was 6.4 for Oomagari, while it was 17.1, 13.0, and 12.0 in Yakumo, Abashiri, and Sapporo, respectively. These figures indicate that, in Oomagari, snow cover lasts long in some years, whereas it is not very persistent in other years, but averages 120 days with higher variation. In Oomagari, it is hard to predict the beginning and end of the permanent snow cover period. It is intuitive that, within species, sclerotia may be larger in snowy localities since snow mold fungi grow well under snow cover (Fig. 2.11), and with a longer duration of vegetative growth under snow, the fungus can store larger amounts of reserve materials in sclerotia. Then, how do populations with small sclerotia survive in less snowy, unpredictable habitats? A clue was obtained from an outbreak of the small sclerotium form of biotype B on winter wheat in Sendai (Honkura et al. 1986; Honkura 1991). Snow cover normally thaws in a week in Sendai but lasted for about 60 days in the 1983–1984 winter to reveal the presence of the fungus. In normal winters, winter wheat plants killed by the small sclerotium . rdcaiiyo nwCvr39 Cover Snow of Predictability 2.2

Table 2.5 Features of Typhula ishikariensis biotype B populations differing in in snow cover conditionsa Parameter Hamatonbetsu Yakumo Abashiri Sapporo Oomagari Morioka Sendai No. days with snow coverb 146.1 (8.5) 117.8 (6.9) 120.5 (9.3) 122.2 (10.2) 119.6(18.7) 95.7 (20.6) 41.5 (34.0) Snow cover indexc 17.2 17.1 13.0 12.0 6.4 4.7 1.2 Sclerotial sized 1.09 (23.0) 0.79 (19.4) 0.87 (15.8) 0.80 (10.5) 0.62 (18.5) 0.70 (12.8) 0.42 (13.6) Virulencee 4.25 (10.5) 4.99 (10.2) 4.60 (14.8) 4.21 (13.5) 4.56 (13.3) 4.56 (12.2) 5.53 (6.5) Sclerotial germinationf 1.18 1.24 0.39 0.65 0.62 0.48 0.05 Modified from Matsumoto and Tajimi (1990) ©Canadian Science Publishing aNine isolates were examined for each population bNo. of days with snow cover p.a. (coefficient of variation) cNo. of days with snow cover p.a./coefficient of variation, indicating the predictability of snow cover. dMean diameter mm (coefficient of variation) eVirulence level: 0, ¼ no damage; 6, ¼ plants killed f0 ¼ no sclerotial germination, 3 basidiocarps developed. Incubated for 35 days under the regime of 8 C, 10 h day/6 C, 14 h night 40 2 Ecology and Physiology

Fig. 2.11 Sporophore development in Typhula ishikariensis biotype B. The longer the duration of snow cover, the larger the sclerotium and sporophore. Large sclerotia of an isolate from snowy Hamatonbetsu (bottom). Isolates from unpredictable, least snowy Sendai are specialized as the small sclerotial form and practically asexual (top). Its small sclerotia seldom produce sporophores, and its basidiospores are, even if produced, mostly inviable. Sclerotia and sporocarps of isolate from moderately snowy Sapporo (middle) (Matsumoto and Tajimi 1991)

form are damaged exclusively in underground parts, showing damping-off symptoms with sclerotia only on underground parts (Fig. 2.12). More surprisingly, the fungus produces sclerotia inside lower sheaths of rice plants (Fig. 2.13). The fungus had originally been referred to as biotype C (Matsumoto et al. 1982), but was later considered as an ecotype of biotype B adapted to the unpredictable habitat (Matsumoto and Tajimi 1991). Biotypes B and C were interfertile, producing fertile progenies with robust virulence. The above features of the small sclerotium form of Typhula ishikariensis biotype B implied that the fungus was soilborne. The subterranean environment is a habitat with constant temperature and humidity and without sunlight. Also, microbial activity is lowered in winter. The small sclerotium form was selected in this subterranean habitat in Sendai as a substitute to the subnivean habitat, attacking underground plant parts with strong virulence. Its sclerotia are small and rarely develop basidiocarps (Fig. 2.11, Table 2.5). In such environments, the number of mating compatibility alleles was decreased compared to other habitats (Table 2.6). These isolates are thought to have originated from Cyperaceae growing on ill-drained lands, where paddy ﬁelds were later developed. Rice was another host since its lower stems remain viable for some time after harvest. The fungus had not been recognized in Sendai until the spring of 1984 when winter wheat showed damping-off symptoms with small sclerotia on damaged leaves and sheaths after a 2.2 Predictability of Snow Cover 41

Fig. 2.12 Underground parts of winter wheat plants were damaged by the small sclerotium form of Typhula ishikariensis biotype B, showing damping-off symptoms. No signs were evident on plant tops after normal winters

Fig. 2.13 Sclerotia of the small sclerotium form of Typhula ishikariensis biotype B produced on rice stubble (arrows). Sclerotia are produced exclusively on underground plant parts. Rice plants do not die after harvest and develop sheaths in autumn which are predisposed to the fungal infection due to decreasing temperature 42 2 Ecology and Physiology

Table 2.6 Size variation of sclerotia and mating compatibility alleles in Typhula ishikariensis biotype B populations Mating compatibility allelesb Isolate Sclerotial sizea Locality AB 11 Large Hamatonbetsu 1 212 61 Normal Yakumo 3 4 34 64 Normal Yakymo 5 6 3 5 48 Normal Abashiri 7 8 6 7 35 Intermediate Sapporo 9 10 8 9 55 Intermediate Oomagari 10 11 10 11 58 Intermediate Oomagari 1 4 9 11 3 Intermediate Morioka 12 13 512 8 Intermediate Morioka 5 11 513 21 Small Sendai 5nd10 nd 23 Small Sendai 6nd 3 10 25 Small Sendai 2nd10 nd Matsumoto and Tajimi (1991) aHighly predictable habitat such as Hamatonbetsu often produce isolates with sclerotia more than 2 mm in diameter, whereas Sendai, the most unpredictable habitat, exclusively has an ecotype with small sclerotia bAlleles A and B were designated arbitrarily. Those found repeatedly were underlined. nd: not determined due to anomalous results, indicating that the Sendai popolation is practically asexual and propagating clonally severe winter. Putting greens on golf courses represent another harsh habitat for snow mold. Greens are mowed a low height which reduces the amount of organic matter in the thatch and belowground plant biomass for the fungus to survive on. Green may also receive repeated fungicide applications during the growing season to possibly reduce sclerotial survival, as well as heavy fungicide applications before snowfall to control snow mold. Spray application of fungicides was commonly used at rates of up to 1 L/m2, which is equivalent to only 1 mm precipitation, and this would fail to penetrate deeply into the soil unless there was heavy irrigation or heavy rainfall right afterward. Subterranean inocula of T. ishikariensis biotype B would then remain untouched by fungicide sprays, and this has led to the selection of the small sclerotium form on golf courses.

Typhula incarnata

Typhula incarnata is found in warm regions in the western parts of Japan such as Tokushima (Tasugi 1936) and Yamaguchi (Matsumoto et al. 1995), and its ubiquity may be inferred from the variability of sclerotial size, covering the intrataxon variability of T. ishikariensis biotype B. Reserve materials such as carbohydrates and proteins stored in sclerotia (Newsted and Huner 1988; Willetts et al. 1990) support the development of basidiocarps which produce infectious basidiospores (Matsumoto and Araki 1982). The larger the sclerotium, the more abundant the 2.3 Psychrophily and Freeze Tolerance 43 reserve materials for fructiﬁcation. Basidiocarp production is effective for the long- distance dispersal of T. incarnata through airborne propagules although inoculum potential of basidiospores is limited. Airborne infection is successful only when snow cover subsequently occurs after spore dispersal. Small sclerotia, on the contrary, do not produce basidiocarps (Tra¨nkner and Hindorf 1982) due presumably to the shortage of reserve materials, but they develop mycelia to infect plants in the vicinity. Mycelia have higher inoculum potential than basidiospores to cause infection. Thus, T. incarnata has a hedging strategy in terms of sclerotia: large sclerotia for long-distance dispersal through basidiospores and small sclerotia producing mycelia to secure persistence in the vicinity. Hoshino et al. (2004a) found T. incarnata isolates that had small sclerotia in Radzikow, Poland, with irregular snow cover, e.g., snow cover was normally less than 10 cm and lasted for a total of 20–90 days. Their mycelia grew optimally at the same temperature as others, i.e., 10 C, but faster. These isolates produced sclerotia of normal size at 10 C and became indistinguishable from other isolates. They were considered to be adapted to the unpredictable habitat in Poland, but much remains to be investigated.

2.3 Psychrophily and Freeze Tolerance

Crops may not be cultivated everywhere in the cryosphere, but snow mold fungi are even distributed in very frigid areas such as polar regions (Fig. 2.14). They have been reported from coastal regions of the North Atlantic islands including Iceland

Fig. 2.14 Snow rot of moss caused by Pythium polare in Svalbard, Norway (Courtesy, M. Tojo) 44 2 Ecology and Physiology

(Hoshino et al. 2004b), Greenland (Hoshino et al. 2006a, 2006b), northern Scandi- navia (Matsumoto et al. 1996a), and Svalbard (Hoshino et al. 2003a) and also in Antarctica (Bridge et al. 2008; Tojo et al. 2012). These regions have maritime climates, and winter temperatures occasionally rise followed by refreezing of thaw water (Årsvoll 1973; Coulson et al. 1995; Gudleifsson 2013). Snow mold fungi can survive also in areas with severe soil frost in Alaska, inland Canada (Lebeau and Logsdon 1958; Smith 1987), Moscow, and Siberia (Hoshino et al. 2001). Although snow mold fungi prevail where ambient temperatures are usually insulated by snow cover, they can persist under freezing conditions. They tolerate freezing temperatures, using different mechanisms. Hodkinson and Wookey (1999) illustrated ecophysiological features of winter-hardy organisms in tundra, e.g., capacity to dehydrate, production of antifreeze proteins, supercooling, chill tolerance, freeze tolerance, selection of microhabitat, subnivean activity, and tolerance to anoxia. In fungi, trehalose, polyol, unsaturated lipids in membrane, antifreeze protein, and low-temperature enzymes are involved (Robinson 2001). Snow mold fungi also have various freezing tolerance strategies to survive in the cryosphere (Hoshino et al. 2009, 2013).

2.3.1 Metabolism

Characteristics of snow mold fungi that allow them to grow at low temperatures have led to investigation of low-temperature enzymes (Hoshino 1997), but their optimum temperatures mostly do not differ from enzymes of mesophiles. For example, lipase from Microdochium nivale (Hoshino et al. 1996), cellulase and xylanase from Coprinus psychromorbidus (Inglis et al. 2000), and polygalac- turonase from Sclerotinia borealis (Takeuchi et al. 2002) retain their activity at low temperatures, but their optima do not differ from those of mesophiles. Although a prerequisite for active growth of snow mold fungi at low temperatures is that every biochemical and physiological step must function at these temperature range (Cairns et al. 1995), their metabolism is usually enhanced at high temperatures. Experimental evidence revealed that, at higher temperatures, some steps are speciﬁcally inhibited in metabolic pathways of snow mold fungi and, consequently, they prevail only at low temperatures. Typhula idahoensis (¼ T. ishikariensis) with an optimum growth temperature at 5 C consumes oxygen maximally at 20 C (Dejardin and Ward 1971). Although protein synthesis by Microdochium nivale occurs up to 25 C, certain physiological abnormalities in an asporogenous strain occurred at temperatures higher than 12 C, which shifted its optimal growth to between 9 and 12 C (Cairns et al. 1995) from the regular 14 to 22 C (Snider et al. 2000). A heat shock protein was produced in this isolate at 25–35 C (Cairns et al. 1995), which was 10 C lower than in mesophilic fungi. Oxidation incites irregular growth of T. ishikariensis group III at 10 C, but growth is normal when oxidation is alleviated by antioxidants (Fig. 1.9). 2.3 Psychrophily and Freeze Tolerance 45

2.3.2 Freeze Tolerance

Hoshino et al. (2009) compared mycelial growth of various snow mold fungi at À1 C after their growing mycelia had been frozen at À20 C for 24 h (Table 2.7). Facultative snow molds such as Microdochium nivale and Sclerotinia trifoliorum and some obligate snow molds such as Pythium iwayamai, Sclerotinia nivalis, and Athelia sp. failed to survive freezing at À20 C with little or no subsequent growth at À1 C. Only obligate snow molds such as Racodium therryanum, S. borealis, Typhula incarnata , and T. ishikariensis survived the treatment. These results clearly demonstrated that snow mold fungi differed in the level of freezing tolerance; each fungus has its own survival strategy with a unique mechanism, reﬂecting habitat differences. Typhula ishikariensis group III (Matsumoto et al. 1996a) is prevalent in coastal regions of northern Norway where plants are often killed by freezing (Årsvoll 1973). Freezing tolerance was compared between groups I and III of T. ishikariensis (Hoshino et al. 1998). Actively growing mycelia were subjected to a freezing regime: temperature was decreased at a rate of 20 C per hour to À40 C and gradually risen to 2 C in 16 h. They were then incubated at the optimum growth temperatures of 10 C and 2 C for groups I and III, respectively. Mycelial growth of group I isolates was impaired; however, group III isolates grew normally after the treatment. Group III mycelia froze at À9.8 to À6.1 C and had antifreeze protein more abundantly than group I. Isolates of T. ishikariensis group III were found also in Novosibirsk, Russia, where soil froze to 3 m deep, and they

Table 2.7 Freezing tolerance of snow mold fungi Mycelial growth (mm/month) Snow mold fungi Frozena Untreated controlb Oomycetes Pythium iwayamai 0.0 15.2 Ascomycetes Microdochium nivale 0.0 7.2 Racodium therryanum 17.5 14.5 Sclerotinia borealis 13.2 20.4 Sclerotinia nivalis 0.2 8.2 Sclerotinia trifoliorum 0.1 12.8 Basidiomycetes Typhula incarnata 7.5 28.3 Typhula ishikariensis biotype A 18.0 36.9 Typhula ishikariensis biotype B 11.9 15.5 Athelia sp. 0.0 36.9 Hoshino et al. (2009) aPregrown on PDA at 10 C, subjected to freezing at À20 C for 24 h, and then returned to À1 C to observe regrowth of mycelia bPDA was not frozen at À1 C. 46 2 Ecology and Physiology showed the same level of freezing tolerance (Hoshino et al. 2001). These ﬁndings indicate that the extreme freezing tolerance of T. ishikariensis group III facilitates its survival in coastal regions of northern Norway where freezing and thawing occur repeatedly to reduce winter survival of forage grasses and in inland Siberia where soil frost restricts the activity of most organisms (Hoshino et al. 2008).

2.3.3 Mechanism of Freeze Tolerance

Antifreeze Protein

Antifreeze protein production is the most common feature of freeze-tolerant organisms (Snider et al. 2000); unregulated growth of ice crystals within cells is fatal, destroying the cell. Some fungi are known to produce antifreeze proteins (Duman and Olsen 1993; Snider et al. 2000; Hoshino et al. 2003c) to moderate ice crystal formation and growth. Water freezes and thaws at 0 C, and antifreeze proteins can decrease freezing temperature through hysteresis. Antifreeze proteins are produced in response to decreasing incubation temperature (Newsted et al. 1994). In snow mold fungi, basidiomycetous Coprinus psychromorbidus (LTB) and Typhula spp. produce antifreeze protein but not ascomycetous or oomycetous snow molds (Snider et al. 2000; Hoshino et al. 2003c). Antifreeze proteins produced inside and outside the cell protect it from freezing, supporting subnivean activity, but are not considered directly involved in pathogenesis (Snider et al. 2000). Since fungi secrete extracellular enzymes to absorb nutrients, this process is inhibited by freezing. Antifreeze proteins may play an important role in nutrient intake in snow mold fungi (Snider et al. 2000). Extracellular antifreeze proteins alone may not be enough for snow mold fungi to intake nutrients. Possibly, Typhula spp. utilize antifreeze proteins when trapped in extracellular polysaccharides (Hoshino et al. 2009). Many organisms other than fungi have antifreeze protein (Duman et al. 1993). In the presence of fungal antifreeze proteins, ice crystals grow into “Stone Age arrowhead” (Hoshino et al. 2003b) or maple leaf shapes (Snider et al. 2000). Gene sequence analysis indicated that an antifreeze protein of T. ishikariensis was a novel one (Hoshino et al. 2003c).

Osmophily

An ascomycetous snow mold, Sclerotinia borealis, tolerates freezing using a mechanism different from that of basidiomycetous snow mold fungi. S. borealis survives freezing through osmophily (Tomiyama 1955) where mycelial growth was greater on frozen media than on unfrozen ones at the same temperature. Hoshino et al. (2010), under more strict experimental conditions, reproduced the ﬁnding and concluded that improved mycelial growth on frozen media was ascribed to enhanced osmotic pressure (Table 2.8). Mycelial growth of S. borealis at À1 C 2.3 Psychrophily and Freeze Tolerance 47

Table 2.8 Mycelial growth on frozen PDAa PDA frozen Unfrozen control Oomycetes Pythium iwayamai 0.0b 15.2b Ascomycetes Microdochium nivale var. nivale 0.0 9.9 Racodium therryanum 14.5 17.5 Sclerotinia borealis 30.0 17.3 Sclerotinia nivalis 0.2 9.2 Sclerotinia trifoliorum 0.1 12.8 Basidiomycetes Typhula incarnata 8.0 26.9 Typhula ishikariensis biotype A 18.0 36.9 Typhula ishikariensis biotype B 11.9 15.5 Typhula ishikariensis group III 19.0 32.7 Athelia sp. 0.0 36.9 Modified from Hoshino et al. (2010) aPDA cultures were frozen at À20 C prior to incubation at À1 C bMycelial growth, mm/month on frozen PDA was twice as that on unfrozen PDA; however, Microdochium nivale and Pythium iwayamai failed to grow on frozen PDA. Freeze-tolerant T. ishikariensis as well as T. incarnata had reduced mycelial growth on frozen PDA. Mycelial growth of S. borealis was similarly enhanced on PDA amended with sorbitol or KCl. Growth–temperature relations were modified in this fungus, too; optimal mycelial growth temperature on PDA was 5 C, but shifted to 10 C when the medium was fortified with 0.2–0.3 M sorbitol. These results show that the optimal mycelial growth of organisms such as S. borealis may remain hidden if tested under conventional growth conditions. Each snow mold fungus grows to survive in its own habitat interacting with both biotic and abiotic factors. The mechanism of S. borealis to survive freezing thus differs from that of Typhula spp.; S. borealis avoids freezing of cytoplasm by enhancing osmotic potential in the cell. The osmophily of this fungus enables it to exploit winter- hardened plant tissues which resist invasion by T. ishikariensis and Microdochium nivale. The hardening process of accumulation of reserve materials and consequent enhanced water potential (Tronsmo 1986) provides even better conditions for exploitation by S. borealis. This is analogous to the case with osmophilic bacteria in permafrost which reproduce even at À20 C, utilizing dense solutes in unfrozen water in permafrost (Rivkina et al. 2000). 48 2 Ecology and Physiology

Fatty Acids

Microdochium nivale remains active during cooler, wetter summer weather and lacks in speciﬁc mechanisms for freezing resistance, but the fungus can adapt to low temperatures by changing fatty acid composition both in quantity and quality (Istokovics et al. 1998). When incubation temperature was reduced from 25 to 10 C, the content of unsaturated fatty acids, linolenic acid (18:3), increased and linoleic acid (18:2) and oleic acid (18:1) decreased. This change is effective to maintain membrane ﬂuidity. When incubation temperatures were further lowered to 4 C, total biomass production diminished, but there was no change in fatty acid composition, and there was an increase in triacylglycerol, a major component of neutral lipids. Triacylglycerol is inferred to be used as a reserve material. In conclusion, M. nivale is considered to shift its metabolism to accumulate lipids rather than to increase unsaturated fatty acids in response to low temperature. This explains why the fungus is unable to better adapt to freezing conditions. Furthermore, Microdochium nivale extracellularly secretes cellulose (Schweiger-Hufnagel et al. 2000) and fructans (Cairns et al. 1995) instead of antifreeze proteins. These compounds may bind antifungal peptides from plants to nullify their effects (R. Imai, personal communication).

Survival in Living Plant Tissues

Freeze tolerance has little been studied in oomycetes including Pythium spp. which can cause snow molds. Mycelia of P. iwayamai do not survive freezing at À20 C (Table 2.7, Hoshino et al. 2009). Pythium iwayamai and P. paddicum cause snow mold of winter cereals in converted paddy fields in warm areas of Japan (Takamatsu 1989a), and P. polare (Tojo et al. 2012) occurs on moss in the Arctic regions such as Greenland (Hoshino et al. 2006b) and Svalbard (Hoshino et al. 1999) and in Antarctica (Bridge et al. 2008). Freezing tolerance is critical to survive especially in polar regions. Plant substrates in permafrost colonized by pathogenic fungi provide them with a habitat for survival as natural cryoprotectants (Ozerskaya et al. 2009). Ancient seeds of higher plants constitute a specific habitat for microorganisms in frozen soil, favoring their preservation for millennia (Stakhov et al. 2008). Hoshino et al. (2013) were the first to demonstrate the phenomenon by experiments with P. iwayamai and P. polare. Although hyphae of these fungi failed to survive after repeated freeze–thaw (À20/2 C) cycles, they survived the treatment when incubated within the leaves of infected Kentucky bluegrass (Poa pratensis) (Murakami 2012). While freezing is detrimental to survival structures such as oospores and hyphal swellings, remaining propagules are activated and ready to germinate (M. Tojo, personal communication). Free mycelia of Pythium spp. cannot tolerate freezing, but zoospores are abundantly released from surviving propagules when plants are covered with thaw water and can rapidly grow on plants. Thus, Pythium spp. are considered to recognize freezing temperature as a signal to resume growth under snow. References 49

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Newsted W, Polvi S, Papish B, Kendall E, Saleem M, Koch M, Hussain A, Culter AJ, Georges F (1994) A low molecular weight peptide from snow mold with epitopic homology to the winter flounder antifreeze protein. Can J Bot 72:152–156 Ono Y (1990) The northern landbridge of Japan. Quat Res 29:183–192 (in Japanese) Ozerskaya S, Kochkina G, Ivanushkina N, Gilichinsky DA (2009) Fungi in permafrost. In: Margesin R (ed) Permafrost soils, Soil biology 16. Springer, Berlin, pp 85–95 Rivkina EM, Friedmann EI, McKay CP, Gilichinsky DA (2000) Metabolic activity of permafrost bacteria below the freezing point. Appl Environ Microbiol 66:3230–3233 Robinson CH (2001) Cold adaptation in Arctic and Antarctic fungi. New Phytol 151:341–353 Saito I (2001) Snow mold fungi in the Sclerotiniaceae. In: Iriki N, Gaudet DA, Tronsmo AM, Matsumoto N, Yoshida M, Nishimune A (eds) Low temperature plant microbe interactions under snow. Hokkaido Nat Agric Exp Stn, Sapporo, pp 37–48 Schweiger-Hufnagel U, Ono T, Izumi K, Hufnagel P, Morita N, Kaga H, Morita M, Hoshino T, Yumoto I, Matsumoto N, Yoshida M, Sawada MT, Okuyama H (2000) Identification of the extracellular polysaccharide produced by snow mold fungus Microdochium nivale. Biotechnol Lett 22:183–187 Simizu M, Miyajima K (1990) Supponuke snow mold on winter wheat. Ann Phytopath Soc Jpn 56:141–142 (in Japanese) Smith JD (1987) Winter-hardiness and overwintering diseases of amenity turfgrasses with special reference to the Canadian Prairies. Agriculture Canada Tech Bull 1987-12E, Saskatoon Snider CS, Hsiang T, Zhao G, Griffith M (2000) Role of ice nucleation and antifreeze activities in pathogenesis and growth of snow molds. Phytopathology 90:354–361 Stakhov VL, Gubin SV, Maksimovich SV, Rebrikov DV, Savilova AM, Kochkina GA, Ozerskaya SM, Ivanushkina NE, Vorobyva EA (2008) Microbial communities of ancient seeds derived from permanently frozen Pleistocene deposits. Mikrobiologia 77:348–355 Suzuki H, Arai M (1990) Occurrence and control of barley scald. Shokubutsu Boueki 44:214–219 (in Japanese) Takamatsu S (1989a) Ecological study of Pythium snow rot of wheat and barley. Spec Bull Fukui Agric Exp Stn 9:1–135 (in Japanese) Takamatsu S (1989b) Distribution of Pythium snow rot fungi in paddy fields and upland fields. JARQ 23:100–108 Takamatsu S (1990) A new snow mold of wheat and barley caused by foot rot fungus, Ceratobasidium gramineum. Ann Phytopath Soc Jpn 55:233–237 Takenaka S, Arai M (1993) Dynamics of three snow mold pathogens Pythium paddicum, Pythium iwayamai, and Typhula incarnata in barley plant tissues. Can J Bot 71:757–763 Takeuchi K, Ikuma T, Sagisaka K, Saito I, Takasawa T (2002) Cold adaptation of polygalac- turonase activity from the culture of the psychrophilic snow mold Sclerotinia borealis. Res Bull Obihiro Univ 24:7–13 Tasugi H (1936) Snow mold of winter wheat. Ann Phytopath Soc Jpn 6:155–156 (in Japanese) Tkachenko OB (1995) Adaptation of the fungus Typhula ishikariensis Imai to the soil inhabitance. Mycol Phytopathol 29:14–19 (in Russian) Tkachenko OB (2013) Snow mold fungi in Russia. In: Imai R, Yoshida M, Matsumoto N (eds) Plant and microbe adaptations to cold in a changing world: proceedings of the plant and microbe adaptation to cold conference 2012. Springer, New York, pp 293–303 Todd NK, Rayner ADM (1980) Fungal individualism. Sci Prog Oxf 66:331–354 Tojo M, Van West P, Hoshino T, Kida K, Fujii H, Hakoda H, Kawaguchi Y, Mu¨hlhause HA, Van den Berg AH, Ku¨pper FC, Herrero ML, Kemsdal SS, Tronsmo AM, Kanda H (2012) Pythium polare, a new heterothallic Oomycete causing brown discoloration of Sanionia uncinata in the Arctic and Antarctic. Fungal Biol 116:756–768 Tomiyama K (1955) Studies on the snow blight disease of winter wheat. Hokkaido Natl Agric Exp Bull 47:1–234 (in Japanese) Tra¨nkner A, Hindorf H (1982) Beitrage zur morphologie der sporophoren von Typhula incarnata Lasch ex. Fr. Med Fac Landbouww Rijksuniv Gent 47:847–853 References 53

Tronsmo AM (1986) Host water potential may restrict development of snow mould fungi in low temperature-hardened grasses. Physiol Plant 68:175–179 Willetts HJ, Bullock S, Begg E, Matsumoto N (1990) The structure and histochemistry of sclerotia of Typhula incarnata. Can J Bot 68:2083–2091 Wu C, Hsiang T (1999) Mycelial growth, sclerotial production and carbon utilization of three Typhula species. Can J Bot 77:312–317 Yang Y, Chen F, Hsiang T (2006) Fertile sporophore production of Typhula phacorrhiza in the ﬁeld is related to temperatures near freezing. Can J Microbiol 52:9–15 Chapter 3 Snow Mold Fungi

Abstract Snow mold fungi include a diverse group of fungi and are not conﬁned to a speciﬁc taxon, since they can be found in the Basidiomycota, Ascomycota, and

Sclerotia of Typhula ishikariensis (top) and Sclerotinia borealis (bottom, courtesy, T. Hoshino) produced on diseased plants. Fungal cultures are easily established in snow mold fungi; sclerotial samples are persistent and represent the best source for fungal isolation, and mycelia readily develop from sclerotia when incubated at right temperatures.

© Springer Science+Business Media Singapore 2016 55 N. Matsumoto, T. Hsiang, Snow Mold, DOI 10.1007/978-981-10-0758-3_3 56 3 Snow Mold Fungi even the non-fungal Oomycota. We cannot directly observe the process of pathogenesis under snow cover, but do see the consequences of infection after snowmelt to gain inferences about the infection process. Such difﬁculties of direct observation seem to hinder their ecological characterization. However, investigations on snow cover conditions facilitate ecological characterization since snow mold fungi depend almost exclusively on snow cover for survival. Each pathogen may spe- cialize locally both ecologically and pathologically, giving rise to taxonomic confusion because of the scarcity of comparative studies. The confusion has been addressed through mating studies and the development of molecular analysis techniques. In this chapter, we emphasized more on what snow mold fungi do (ecology) than what they are (taxonomy).

Snow cover, a requisite for snow mold growth and development, is a barrier to direct observations of pathogenesis by snow mold fungi, and this hindered progress in understanding the ecology of such organisms. Also, some of the pathogens stay dormant after snowmelt in the form of sclerotia, providing limited features for taxonomic resolution. However, since snow cover is essential for snow molds to establish, survive, and propagate, snow cover conditions in specific habitats can be used to characterize features of the pathogens. High levels of genetic variation have led to taxonomic confusion (Remsberg 1940b). McDonald (1961), upon request from the Associate Committee on Plant Diseases in Canada, made a literature survey on low-temperature grass pathogens, especially in the genera of Typhula and Sclerotinia. Consequently, T. incarnata and S. borealis were recognized as legitimate, but he concluded that T. ishikariensis required international comparisons although T. ishikariensis had priority. In this chapter, we minimized the listing of taxonomic features of the pathogens, with greater emphasis on their ecological aspects. How they respond to the ongoing and future climate change is, we believe, more significant in terms of agricultural production as well as scientific interest.

3.1 Typhula spp.

3.1.1 Mating Incompatibility

Basidiomycetes, including species of Typhula, are typically outbreeders. They try to extend their genetic variability through sexual reproduction and, on the other hand, exclude other strains that differ genetically through vegetative incompatibility (Todd and Rayner 1980). The genus Typhula is no exception. Since the ecology of each species is largely reﬂected in its reproduction strategy, the mating incompatibility system is ﬁrst explained here. Typhula spp. are usually dikaryotic, containing two haploid nuclei in a cell. The two nuclei are different in mating incompatibility alleles. They fuse in the basidium 3.1 Typhula spp. 57

A1B1 A1B2 A2B2 A2B1

Fig. 3.1 Process of sexual reproduction in basidiomycetes, showing recombination of the mating alleles and nuclear conditions. 1 Two haploid nuclei with mating alleles (A1B1 or A2B2) present in a basidium; 2 nuclei fuse and turn into diploid (A1A2B1B2); 3 mitosis occurs to produce two diploid nuclei; 4 four haploid nuclei (A1B1, A1B2, A2B1, and A2B2) are produced through meiosis; and 5 each haploid nucleus migrates into each basidiospore (Modiﬁed from Buller 1931) to develop diploid state, followed by meiosis. Four haploid basidiospores are then produced in most species (Fig. 3.1). T. ishikariensis basidiospores are mostly monokaryotic, and mitosis occurs before spore germination (Cunfer 1974). Single basidiospores usually develop into monokaryotic mycelia. Matings between monokaryons (mon–mon matings) are useful to identify mating incompatibility alleles (Figs. 3.2 and 3.3). In mon–mon matings, dikaryons are produced only when all the mating incompatibility alleles are different, and pairing is unsuccessful even when both monokaryons just have single alleles in common. In Typhula spp., mating incompatibility alleles may be found as follows: when single basidiospore monokaryons derived from a single basidiocarp are paired in all possible combinations, they are divided into four groups on the basis of mating pattern (Fig. 3.3). For example, monokaryons 1 and 2 with mating allele A1B1 can mate with monokaryons 5 and 9, having A2B2 but not with others since other monokaryons share either the A or B allele or both. Common alleles were found to present in isolates of T. ishikariensis from Japan, Finland, and the USA (Bruehl and Machetmes 1979). Basidiomycetes have a unique mating system, i.e., di–mon mating, where monokaryons can receive a compatible nucleus from dikaryons to become dikaryons (Buller 1931, Fig. 3.2). Di–mon matings are effective to determine genetic relationships, using tester monokaryons (Tkachenko et al. 1997). When testers are dikaryotized, dikaryons may be regarded as having the same genetic background. Røed (1969) may have been the earliest to use the mon–mon mating technique to reveal the conspeciﬁcity of Typhula graminum and T. itoana to T. incarnata. Bruehl et al. (1975) demonstrated the genetic separation between T. ishikariensis and T. idahoensis by di–mon matings and regarded them as separate species. 58 3 Snow Mold Fungi

mon-mon mating

di-mon mating

Fig. 3.2 Two modes of mating in basidiomycetes. As a result from mon–mon mating, two nuclei differing in mating alleles coexist in a cell to form a dikaryon. In di–mon mating, a monokaryon is dikaryotized, receiving an unlike nucleus from a dikaryon. Dikaryons have clamp connections (arrow)

A1B1 A2B2 A1B2 A2B1

1 2 5 9 4 7 8 10 3 6 A1B1 1 䠉䠉䠇䠇䠉䠉䠉䠉䠉䠉 2 䠉䠉䠇䠇䠉䠉䠉䠉䠉䠉 5 䠇䠇䠉䠉䠉䠉䠉䠉䠉䠉 A2B2 9 䠇䠇䠉䠉䠉䠉䠉䠉䠉䠉 4 䠉䠉䠉䠉䠉䠉䠉䠉䠇䠇 7 䠉䠉䠉䠉䠉䠉䠉䠉䠇䠇 A1B2 8 䠉䠉䠉䠉䠉䠉䠉䠉䠇䠇 10 䠉䠉䠉䠉䠉䠉䠉䠉䠇䠇 3 䠉䠉䠉䠉䠇䠇䠇䠇䠉䠉 A2B1 6 䠉䠉䠉䠉䠇䠇䠇䠇䠉䠉

Fig. 3.3 Bifactorial mating pattern of mon–mon matings. Ten monokaryons selected arbitrarily from a single basidiocarp are paired in all possible combinations. Four different mating patterns are obtained. þ (dikaryotized) (clamp connections present) and À (not dikaryotized). The mating patterns are clariﬁed on assumption that paired monokaryons are compatible only when they do not have any alleles in common 3.1 Typhula spp. 59

3.1.2 Niche Separation

Three fungi, i.e., T. incarnata and T. ishikariensis biotypes A and B, are known to cause snow mold in Japan, and they are not interfertile. Also, they do not overlap in niche, considering their distribution pattern and plant resources they utilize. Matsumoto and Sato (1983) revealed the ecological features to separate their niches (Fig. 3.4). T. incarnata or T. ishikariensis biotype B was inoculated onto winter wheat plants with different levels of winter hardiness that were either unhardened in the shade or hardened by supplemental lighting. While unhardened plants were more severely injured by both pathogens, hardened plants were not damaged by T. incarnata but substantially diseased by biotype B (Table 3.1). These results indicate that unhardened plants are more predisposed to the attack of the pathogens and represent the resources easy to exploit. Typhula ishikariensis biotype A was also included in inoculation experiments with hardened orchardgrass to investigate intertaxon interactions of the three pathogens (Table 3.2). Biotype B was most virulent, followed by biotype A. T. incarnata was least virulent. Two pathogens were then inoculated in pairs, and plant damage was compared with results from plants inoculated singly.

A Mode of life

in Saprophytic Pathogenic

Dead Susceptible Resistant

Cline of resources (physiolosical condition of host plant)

Fig. 3.4 Niche separation in the pathogenic species of Typhula (Modiﬁed from Matsumoto and Sato 1983). In: T. incarnata.A:T. ishikariensis biotype A. B: T. ishikariensis biotype B. : physiological potential; : ideal resources; and : realized niche 60 3 Snow Mold Fungi

Table 3.1 Damage of winter wheat plants differing in the level of winter hardiness inoculated with Typhula incarnata or T. ishiakriensis biotype B Growth conditions in autumna Lightenedb Shadedb Regrowth of plants after inoculation with Typhula incarnata 84.1 16.3 T. ishikariensis biotype B 22.7 7.9 Uninoculated control 74.2 61.5 LSD(1%) 20.8 29.1 Winter hardiness level as revealed by % dry matter of leaf sheaths 27.7 20.0 % TNC in lower leaf sheathsc 27.1 10.5 Matsumoto and Sato (1983) aSown in early September. Half of the plants received supplemental lightening from early November. The other half was placed in the shade in early November bInoculated in mid-December and incubated for 70 days under snow. Figures indicate plant regrowth cTNC, total non-structural carbohydrates

Morphological features of sclerotia on diseased leaves were used as the criterion to determine which fungus was the principal cause of damage in mixed inoculation. Biotype A was not affected by co-inoculants in terms of plant damage and sclerotial formation, and T. incarnata was not affected by biotype B. Biotype B was affected both by biotype A and T. incarnata. In conclusion, T. incarnata was the least virulent but can survive as a saprophyte (Matsumoto and Sato 1982). Its versatility helps the fungus exploit both living and dead substrates and survive in wide-ranging conditions in the cryosphere. Biotype A is restricted to very snowy regions. Although the fungus is moderately virulent, strong competitive capacity enables it to monopolize the best resources that are readily available to any snow mold pathogens. Biotype B can survive in less snowy regions. Its strong virulence facilitates the exploitation of resistant plants. Chang et al. (2006) also demonstrated niche separation in T. incarnata, T. ishikariensis, and T. phacorrhiza in Wisconsin golf courses, USA, based on statistical analyses of the distribution patterns of the fungi and environmental factors such as annual number of days with snow cover and mean air temperature.

3.1.3 Typhula incarnata

Taxonomic Issues

Typhula incarnata had been regarded as synonymous with T. graminum until Røed (1969) revealed their genetic identity by mating experiments. Berthier (1976), however, distinguished both species by sclerotial characteristics. Conidiogenesis on the sclerotium was never observed in T. incarnata but evident in T. graminum. 3.1 Typhula spp. 61

Table 3.2 Pathogenic interactions of Typhula incarnata and T. ishikariensis biotypes A and B Inoculated with Disease severitya Percent colonizationb Biotype B 5.25 Biotype A 3.45 Biotypes A + B 3.43 A 97.5/B 12.5 Biotype A + in 3.33 A 100/in 5.0 T. incarnatain +biotype B 1.6 B 80.0/in 36.5 T. incarnata 1.18 Uninoculated control 0.00 Matsumoto and Sato (1983) aInoculated onto orchardgrass: 0 ¼ no damage, 6 ¼ plants killed bFigures indicate frequencies of fungal colonization on leaf blades whose sheaths were also dead. Fungal identiﬁcation was based on the features of sclerotia. A: biotype A, B: Biotype B, and in : T. incarnata

These two species may also be distinguished by the color of basidiocarps. Subse- quently, Olariaga et al. (2008) coined a new name, T. berthieri, for T. graminum which is characterized by white and smaller basidiocarps and conidial production on the sclerotium. However, the status of these putative species should be con- ﬁrmed by mating experiments and molecular analysis.

Ecological Features

Typhula incarnata is distributed in snowy and also less snowy areas throughout the world, e.g., warm southwestern parts of Japan such as Yamaguchi (Matsumoto et al. 1995) and Tokushima (Tasugi 1936) and low-altitude parts of Europe such as Belgium (Cavelier and Maroquin 1978), Germany (Hindorf 1980a), and the UK (Woodbridge and Coley-Smith 1991). The fungus is also known to occur in the northwestern, mountainous parts of Iran (Hoshino et al. 2007). It is also found in parts of southern Ontario (Smith 1992) which often experience less than 6 weeks of snow cover. T. incarnata has been observed to attack plant tissues in winter in the absence of snow cover (Bruehl and Cunfer 1971; Detiffe et al. 1981). The ubiquity of T. incarnata is ascribed to its ecophysiological versatility. The fungus survives not only as a parasite but as a saprophyte (Matsumoto and Sato 1982; Jacobs and Bruehl 1986), colonizing plant debris such as dead leaves (Fig. 3.5a) and even cattle dung. Basidiospores are dispersed in late autumn and can infect plants from a distance (Hindorf 1980b; Matsumoto and Araki 1982). Sclerotia are produced on underground plant parts deep enough to prevent basidiocarp production (Fig. 3.5b). Sixty-four percent of sclerotia buried 19 cm deep in the soil germinated to develop mycelial strands (Detiffe et al. 1985). In areas of little to no snow cover, winter wheat seedlings were attacked only in the roots and lower sheaths to exhibit damping-off symptoms, as also found with the small sclerotium form of T. ishikariensis biotype B (see Fig. 2.12). Luxuriant foliage of Italian ryegrass (Lolium multiﬂorum) maintained humidity, resulting in 62 3 Snow Mold Fungi

Fig. 3.5 Production of sclerotia (arrows) as a result of saprophytic colonization on a dead leaf of Betula platyphylla (a) and soilborne pathogenesis on winter wheat roots (b, courtesy S. Ikeda) foliage blight by T. incarnata in winter even where there was no snow cover (Fig. 3.6) (Matsumoto, unpublished). Sexual reproduction is effective in T. incarnata. The fungus develops relatively large basidiocarps (Fig. 3.7), and basidiospores are used to extend genetic variability (Bruehl and Machtmes 1978). Matsumoto and Tajim (1989) analyzed mating incompatibility alleles from basidiospores of 23 basidiocarps collected from grassweeds along a 50-m-long path: there were 32 A and 18 B alleles (Table 3.3). Four A alleles were common in three basidiocarps and another four A alleles in two basidiocarps. B alleles were more commonly shared. These results indicate that genetic exchange is common among individuals of a T. incarnata population, with related individuals in close proximity. Basidiospores are infective in T. incarnata, and the resulting monokaryons are dikaryotized to produce new genets. Cavelier (1986) conﬁrmed the dikaryotization of monokaryons inoculated onto plants by airborne basidiospores. On the other hand, lower survival rate of this fungus was inferred by ﬁeld observations on sclerotial survival in summer (Fig. 3.8, Matsumoto and Tajimi 1985). Percent viability of sclerotia was about 90 % in early May and reduced to 24.2 % and 5.0 % in late July for those placed on the ground surface and those buried 1 cm deep in the soil, respectively. Sclerotial viability was not observed to change even until the end of experiments in late November. Diverse mycoparasites were obtained from the sclerotia (Fig. 3.9), and their inoculation onto T. incarnata 3.1 Typhula spp. 63

Fig. 3.6 Foliage blight of Italian ryegrass by T. incarnata. Dense growth of pant tops keeps moisture high enough to allow the fungus to colonize without snow cover

Fig. 3.7 Basidiocarps of Typhula incarnata. Large, pink basidiocarps develop readily from sclerotia of the fungus sclerotia confirmed that the sclerotia lost viability through mycoparasitism in the field. Typhula incarnata is r-selected as compared to T. ishikariensis and characterized by high birth and high death rates which has selective advantage in ecological vacuums such as agricultural fields with frequent tillage facilitating recolonization each year (Pianka 1970). The ecophysiological versatility of the fungus allows it to establish in new habitats. Its high birth and high mortality strategy shortens 43So odFungi Mold Snow 3 64

Table 3.3 Diversity in mating incompatibility alleles in a Typhula incarnata population from non-agricultural landa Basidiocarp designation 0 3 4 5 6 8 11 13 15 16 18 19 29 34 38 39 40 41 43 44 45 46 48 A allele 1 3 5 795513151017191491712251992830nd12 2 4 6 8 10 11 12 14 16 17 18 20 21 22 23 24 26 24 27 29 31 nd 32 B allele 1 1 1 53192213543156148373nd3 2 3 4 6 7 8 10 11 12 14 15 6 9 16 13 11 17 18 8 9 9 nd 18 Matsumoto and Tajim (1989) aBasidiocarps were collected at 90 cm intervals along a 50 m-long path where orchardgrass predominated. Four monokaryons from each basidiocarp differing in alleles were paired in all possible combination to identify mating incompatibility alleles. A and B alleles were arbitrarily determined. nd not determined 3.1 Typhula spp. 65 viability (%)

May 6 24 Jun 23 25 Aug 24 Sept27 Oct 5 Nov 25 Sampling date

Fig. 3.8 Change in the viability of sclerotia of Typhula incarnata (open circles) and T. ishikariensis biotype A (closed circles) placed on the soil surface (broken lines) and 1 cm deep in the soil (solid lines) Matsumoto and Tajimi (1985) ©Canadian Science Publishing

Fig. 3.9 Survival of sclerotia of T. incarnata (left) and T. ishikariensis biotype A (right). Only one sclerotium of T. incarnata survived to extend mycelium (arrow, high mortality), whereas most sclerotia remained viable in T. ishikariensis biotype A (low mortality) generation intervals to allow rapid development of genotypes adapted to novel environments. Vergara et al. (2004) determined genetic relationships of T. incarnata isolates collected form 40 localities in northern USA, and three local populations were found to have developed recently from a common ancestor. Isolates of T. incarnata from the world have mating incompatibility alleles in common, and no population has so far been found as genetically separate (Røed 1969; Bruehl and Machtmes 1978). Different capacities are used in different habitats, and possibly the high versatility prevents the development of subspeciﬁc differentiation. This is in contrast to T. ishikariensis which has differentiated into 66 3 Snow Mold Fungi infraspeciﬁc taxa. As mentioned in the previous chapter, T. incarnata populations seem only to differ in the germination rate of sclerotia so that each population may adapt to the different levels of snow cover predictability (Matsumoto et al. 1995).

3.1.4 Typhula ishikariensis

Taxonomic Issues

Isolates of the Typhula ishikariensis complex are highly variable and have been studied in isolation by individual scientists without exchange of materials for comparison, which resulted in coining unique epithets. In describing T. idahoensis, Remsberg (1940a) overlooked the paper of Imai (1930) who described a new species, T. ishikariensis, leading to taxonomic confusion in the T. ishikariensis complex. American scientists were the first to try to settle the problem. Bruehl and Cunfer (1975) separated T. idahoensis from T. ishikariensis based on morphology, host range, and distribution. They further revealed the genetic isolation of the two species, although T. ishikariensis and T. idahoensis mated rarely (Bruehl et al. 1975). Of 992 interspecies combinations, 19 % produced culturable offspring (Christen and Bruehl 1979). These offspring included hybrids that were virulent and considered capable of survival in nature. Extensive surveys made subsequently in nature indicated the presence of such hybrids that could mate with both T. ishikariensis and T. idahoensis, and this led Bruehl and Machtmes (1980)to conclude that these two species had intermediates, as well as being indistinguishable based on culture morphology alone. Årsvoll and Smith (1978), studying genetic compatibility, regarded T. ishikariensis, T. idahoensis, and a new taxon, Typhula FW, as a single species and established three varieties. Varieties ishikariensis and idahoensis were distinguishable by sclerotial rind cell patterns. Matsumoto et al. (1996a) noted the high variability in rind cell patterns of isolates from Japan (Fig. 3.10) and Norway and suspected the significance of rind cells as a taxonomic criterion. Var. canadensis (Typhula FW) could be separated from the two former varieties by its smaller sclerotia. A similar fungus with small sclerotia was also present in Japan, which was formerly referred to as T. ishikariensis biotype C but later as the small sclerotium form of biotype B (Matsumoto and Tajimi 1991). Random amplified polymorphic DNA analyses using 11 isolates of the three varieties from North America indicated that var. idahoensis was genetically distant from the other two but that these varieties were not different enough to be regarded as separate species when ITS sequences were taken into account (Hsiang and Wu 2000). Two taxa of T. ishikariensis were described in Japan with taxonomically illegit- imate terms, i.e., biotypes A and B (Matsumoto et al. 1982). The term biotype is often used for insect pests and denotes a group of individuals having the same genotype. Biotype has often been used when details are not fully known (Claridge and Den Hollander 1983). Biotypes A and B may be regarded as separate species in 3.1 Typhula spp. 67

Fig. 3.10 Sclerotial rind cells of Typhula ishikariensis biotype A

terms of genetics, morphology, and ecology, when foreign taxa were not involved in the comparison. However, both biotypes can exchange genetic material through the media of North American taxa (Matsumoto et al. 1983) as well as Norwegian taxa (Matsumoto et al. 1996). It is obvious that T. ishikariensis is made up of several taxa that differ genetically and morphologically. Monokaryons of biotypes A and B, which are absolutely incompatible with each other (Matsumoto et al. 1983), represent good testers to reveal the genetic background of all the taxa of the T. ishikariensis complex of the world. North American taxa were divided into two groups: var. ishikariensis that was compatible with biotype A and var. idahoensis and var. canadensis, both compatible with biotype B (Matsumoto et al. 1983). Norwegian isolates were separated into three groups: groups I and II were compatible with biotypes A and B, respectively, and group III was not compatible with either biotype (Matsumoto et al. 1996). Isolates from European Russia dikaryotized monokaryons of biotype A and group I (Tkachenko et al. 1997). Consequently, Matsumoto (1997) proposed two tentative biological species based mainly on mating reactions of monokaryons of biotypes A and B (Matsumoto 1997). Biological species are groups of interbreeding natural populations that are reproductively isolated from other groups (Mayr 1963) and recognized in other basidiomycetous sensu lato species complex such as Heterobasidion annosum (Korhonen 1978) and Armillaria mellea (Anderson and Ullrich 1979). Millett (1999) supported the idea of biological species when analyzing ITS sequences of T. ishikariensis isolates from Wisconsin golf courses, in the USA. These two biological species were found also in Iceland (Hoshino et al. 2004a) and in Russia (Tkechenko 2013). It is logical to distinguish three biological species in the T. ishikariensis complex based strictly on mating compatibility with biotypes A and B (Table 3.4) although molecular phylogeny based on ITS regions indicates the afﬁnity of var. idahoensis to biological species I (S. Millett and T. Hoshino, personal communications). 83So odFungi Mold Snow 3 68

Table 3.4 Biological species belonging to the Typhula ishikariensis complex and their ecological featuresa Biological species I Biological species III (AþBÀ) Biological species (AÀBþ) (AÀBÀ) Distribution Taxa Habitat Taxa Habitat Ecotype Taxa Habitat Japan Biotype A Snowy Biotype B Less snowy regions Small sclerotium form (highly regions unpredictable) Norway Group I Snowy Group II Sloping lands Sclerotia with fluffy surface Group IIIc (low Severe regions mycelia (windborne?) temperature) soil frost Russia Biological Often in Biological species Coastal region in the Group IIIc (low Severe species I soil II North Pacific temperature) soil frost North var. Former for- var. idahoensisb Former grasslands America ishikariensis est lands var. canadensis Severe soil freezing Modified from Matsumoto (1997) aDistinction of biological species is based on mating compatibility with biotypes A and B. Mating reactions of monokaryon testes of biotypes A and B are indicated in parentheses after each biological species bIndistinguishable in culture morphology from biological species I. Molecular phylogeny of the ITS regions also indicates affinity to biological species I (T. Hoshino, personal communications). However, on the basis of mating compatibility, var. idahoensis should be included in biological species II cMolecular phylogeny also suggests that group III should be treated as a separate entity. Also present in Greenland (T. Hoshino, personal communications) 3.1 Typhula spp. 69

Ecological Features

Sexual reproduction is apparently much less effective in T. ishikariensis as compared to T. incarnata, and outbreeding is practically impaired in this species, promoting clonal development to adapt to local environments. Table 3.4 shows comparisons between ecological features of the taxa belonging to the T. ishikariensis complex. Biological species I is widespread in snowy regions and exploits diverse host plants. Biotype A attacks both monocots and dicots, and sclerotia are produced abundantly on dead plants (Fig. 3.11). Its distribution is restricted to very snowy areas in Japan (Matsumoto et al. 1982). Analysis of mating incompatibility alleles of biotype A isolates from both monocots and dicots revealed its low genetic diversity (Table 3.5, Matsumoto and Tajimi 1993) and that single genotypes often dominate populations (see Chap. 2). But the wider host range of biotype A to utilize both dicots and monocots compensates for its limited distribution range. The diversity of biological species I is evident when isolates from all over Russia are examined; the fungus attacks pine (Pinus sylvestris) in Yekaterinburg, Ural (Hoshino et al. 2004b), extending its host range to gymnosperms. In Moscow, the fungus causes damage to tulip (Sinadoskii and Tkachenko 1981), which was never observed in Japan (Matsumoto, unpublished). In North America, var. ishikariensis is found in regions of previous forest cover, and it is also pathogenic to dicots (Bruehl and Cunfer 1975). Biological species II seems to be specialized to exploit monocots. The host range of biotype B is restricted to monocots, although the fungus attacks Stellaria spp. (chickweed) whose tops remain green but are moribund in late autumn (Matsumoto 1989). The fungus also kills alfalfa (Medicago sativa) and canola (Brassica napus) when inoculated experimentally, but sclerotia are seldom produced (Matsumoto 1989). In the less rainy regions of North America where gramineous plants dominate, var. idahoensis is widely distributed and attacks monocots (Bruehl and Cunfer 1975). Var. canadensis is a major turfgrass pathogen in western Canada and the most cold tolerant (Årsvoll and Smith 1978). The fungus produces small

Fig. 3.11 Sclerotia of Typhula ishikariensis biotype A produced on canola. Sclerotia are easily detached from diseased tissues 70 3 Snow Mold Fungi

Table 3.5 Mating Perennial ryegrass isolatesa Alfalfa isolatesb incompatibility alleles of Allelesc Allelesc Typhula ishikariensis biotype A isolates from perennial Isolate ABIsolate AB ryegrass and alfalfa in PR 4 2, 3 1, 3 AL 1 1, 2 1, 3 Hokkaido PR 6-7 1, 2 1, 2 AL 2 1, 2 1, 2 PR 7-6 1, 2 1, 2 7AL 1 1, 2 1, 2 PR 9-4 2, 6 2, 4 23AL 1, 5 1, 3 PR 10-8 1, 2 1, 2 23AL 1 2, 4 1, 3 HP 1, 2 1, 2 23AL 2R 1, 2 1, 2 TP 14 1, 2 1, 2 23AL 3 2, 4 1, 3 7PR 1, 2 1, 2 23AL 4 2, 4 1, 3 Matsumoto and Tajimi (1993) aIsolates collected from Sapporo except PR4 and 7PR from Hamatonbetsu. Sapporo and Hamatonbetsu are 250 km distant bAll from Sapporo cA and B alleles were arbitrarily determined

Fig. 3.12 A certain strain of Typhula ishikariensis group II from Norway with “tennis ball” sclerotia covered with thick mycelia (left) and knobby, heavily ramiﬁed hyphae (center) and normal hyphae of biotype A (right) sclerotia suspended in aerial mycelia as is the case with the small sclerotium form of biotype B. Some isolates of group II develop knobby, highly ramiﬁed hyphae and have sclerotia covered with thick mycelia that resemble tennis balls (Fig. 3.12, Matsumoto et al. 1996). The fungus also produces mycelia abundantly on diseased tissues. Since such a genotype occurs in sloping lands, the wind may carry sclerotia along with mycelia when plant debris dries off in spring. Biological species III consists exclusively of low-temperature-tolerant group III (Matsumoto et al. 1996) and has not been reported from North America or Japan. 3.1 Typhula spp. 71

The fungus is distributed along the coastal regions in Norway where plants are often killed by low temperature (Årsvoll 1973) and inland Russia (Hoshino et al. 2001). The fungus has so far been collected from monocots in a restricted area of the world, and its precise host range and other ecological features are yet to be elucidated. Basidiospores are regularly produced in T. ishikariensis as in T. incarnata with an exception of the small sclerotium form of biotype B (see Chap. 2) but do not function as infection source in any fungi of the complex. Cunfer and Bruehl (1973) inoculated basidiospores of T. idahoensis onto winter wheat (50 pots) and barley (32 pots), whose leaves had been wounded with scissors; consequently, sclerotia were found in 7 pots for each crop, and plants were healthy in 19 and 12 potted plants, respectively, for winter wheat and barley. Monokaryons of T. idahoensis were observed to be nonpathogenic (Kiyomoto and Bruehl 1976). Matsumoto (1989) found that virulence was low in monokaryons, but enhanced after dikaryotization in biotypes A and B. Monokaryons may not survive long in nature unless they are dikaryotized. Thus, T. ishikariensis basidiospores are less likely to establish alone. Mycelia developed from basidiocarps and those directly from sclerotia play a major role in epidemiology. Although basidiospores are mostly wasted, the fungus utilizes energy stored in sclerotia effectively by developing mycelia from weathered basidiocarps and directly from sclerotia to infect plants. These features indicate that T. ishikariensis is not an airborne pathogen. The nature of T. ishikariensis as a soilborne pathogen varies globally among taxa belonging to this species. Sclerotia of T. idahoensis do not function as inoculum when buried in soil 2 cm below the surface or deeper (Davidson and Bruehl 1972). T. ishikariensis biotype A was not found on underground plant parts in Japan; however, in Moscow, roots of tulip (Sinadoskii and Tkachenko 1981) and hop (Hoshino et al. 2004b) were badly damaged by a fungus resembling biotype A. The sclerotia of the Russian fungus mostly lost viability when buried in soil for 13 months, but survivors produced secondary sclerotia (Tkachenko 1984). Biolog- ical species I was commonly present (10 isolates out of 46) in Wisconsin golf courses (Millett 1999). On the other hand, only one isolate of biotype A has so far been discovered from golf courses in Japan (Matsumoto 1989). Although intensive fungicide applications in golf courses favor biological species II that can survive on plant roots, biological species I may be able to utilize underground parts in Wisconsin. Foliar application of fungicides sometimes fails to control biotype B since its sclerotia deep in the soil are not exposed directly to fungicides (Saito 1988). Populations of T. ishikariensis reflect habitat dynamics such as changes in geology and climate. T. idahoensis in Washington, USA, may be regarded as an outbreeder with diverse mating incompatibility alleles; however, populations in Idaho and Utah were not sexual and strongly virulent (Bruehl et al. 1978). Washington populations exist in relatively new habitats where the ground had been covered with glacier until 15,000–12,000 years ago, and Idaho and Utah 72 3 Snow Mold Fungi represent older habitats of the pathogen. Geological differences in both habitats might have allowed evolution of populations that differ pathogenically. Climatic change in eastern Hokkaido created a new habitat available for biotype A and led to a founder effect of a predominant clone (super MCG, see Chap. 2, Matsumoto and Hoshino 2013). Most biotype A sclerotia are not colonized by other fungi and successfully pass the dormancy until autumn (Matsumoto and Tajimi 1985, Fig. 3.8), but how sclerotia are affected by other fungi after dormancy has little been studied. By the end of November which was 6.5 months after the start of the experiment, no biotype A sclerotia had germinated. However, sampling of sclerotia at the end of October revealed that sclerotia buried in the soil were found colonized more frequently by other fungi and viability was reduced to 70 % (Fig. 3.8). These findings suggest that sclerotia are more vulnerable to the attack of mycoparasites just before germination. Sclerotia are hollow during dormancy, and medullas contain tightly interwo- ven hyphae; however, germinating sclerotia are filled with hyphae which are presumed to have grown from the medulla (Matsumoto et al. 2010, cf. next section). Microbial activity may be enhanced by the exudates from such sclerotia. Out of 645 biotype A sclerotia collected from an alfalfa field in late autumn, 75 % were found to be germinated (Fig. 3.13a, Matsumoto 2008). Basidiocarps were detached from half of these germinated sclerotia, leaving rind shells (Fig. 3.13b arrow). Ungerminated sclerotia were divided into two types. Type one sclerotia were covered with white mycelia but remained firm and apparently intact (Fig. 3.13b). Such sclerotia were not viable, and an unidentified fungus with white mycelium and no clamp connections always developed from them (Fig. 3.13c). Type two sclerotia were tough and wrinkled, resembling raisins (Fig. 3.13d). These sclerotia always produced mycelia of other mostly Dematiaceae fungi differing from well-known mycoparasites found during dormancy in summer. Possibly, germinating sclerotia are more vulnerable to the attack of other fungi that cause opportunistic infection. These findings indicate that, in Japan where it is humid in late autumn, biotype A sclerotia are less likely to survive for years. Most sclerotia successfully survive the dormancy in summer and start to germinate in late autumn during which sclerotial survival is affected by the attack of weak mycoparasites. These mycoparasites have not been studied and should be compared with the well-known mycoparasites in summer. Thus, T. ishikariensis biotype A sclerotia are annual and unlikely to remain dormant, surviving for years. The fungus may be controlled in the upland cropping system through crop rotation of winter wheat with summer crops. Residues of these crops and weeds such as Poa annua and Stellaria spp. that survive winter should be removed so that the inoculum potential of the fungus may be lowered for the subsequent winter. 3.1 Typhula spp. 73

Fig. 3.13 Diverse Typhula ishikariensis biotype A sclerotia collected from an alfalfa ﬁeld in late autumn. (a) Viable sclerotia with elongated stipes. Mycelia developing on the tip; (b) sclerotia covered with ﬂuffy mycelia of other fungus. Sclerotial rind (arrow) is detached from the basidiocarp. (c) White mycelia develop always form such sclerotia; (d) shrunk sclerotia that resemble raisins and are not viable

Sclerotia Recovered from Archaeological Sites

Although problems arising from plant pathogenic fungi are closely related to human activities, the record of their occurrence from archaeological sites is extremely limited. During excavations of historical sites estimated at 4000 years before present in Hokkaido, Japan, sclerotium-like objects (SLOs) were abundant from ﬁreplace vestiges along with plant remains such as cereals, beans, and walnuts. SLOs were also recovered from inside and outside houses of the Ainu which were buried by volcanic eruption on September 27, 1667. The SLOs were spherical, ranging 0.3–1.0 mm in diameter, and totally carbonized. Their shape and size lead Matsumoto et al. (2010) to assume that they were sclerotia of Typhula ishikariensis. Scanning electron microscopic observations 74 3 Snow Mold Fungi

Fig. 3.14 Sclerotia of Typhula ishikariensis biotype B recovered from historical sites. Carbonized sclerotia with (a) or without rind (b), germinating sclerotium (c), and sclerotium with a leaf fragment (d). (a, b) From a fireplace vestige at 4000 years before present; (c, d) from outside houses buried in volcanic ash at 400 years before present (Matsumoto et al. 2010) revealed a pattern on the surface of an SLO from a fireplace vestige that was mostly covered with ash (Fig. 3.14a, circled). The pattern resembled sclerotial rind cells of T. ishikariensis. After a slight sonication treatment to remove ash, the rind was peeled, and the medulla was exposed (Fig. 3.14b). The SLOs were hollow inside, and the medulla did not fill the entire objects. In contrast, samples buried in volcanic ash were all filled with medulla, although they also lacked distinct rinds or rind patterns. Some of them had protuberances that resembled emerging sporocarp primordia (Fig. 3.14c) or had leaf fragments on the surface (Fig. 3.14d). Sclerotia of T. ishikariensis, especially large ones, are generally hollow in spring and filled with growing medulla before germination in autumn. The volcanic eruption occurred in late September, which coincided with the time when Typhula sclerotia begin to germinate. Leaf fragments attached to SLOs provided a further clue to distinguish biotypes. T. ishikariensis biotype B sclerotia are firmly attached to plant tissues, and detached sclerotia often have plant fragments on the surface. 3.1 Typhula spp. 75

Consequently, SLOs were identified as sclerotia of T. ishikariensis biotype B. They were found prevalent in other historical sites in Hokkaido and also recovered from historical sites of the Primorye region, east Russia, that date back to 2500 before present. Archaeological excavations in these sites all revealed the presence of cultivated plants as well as the Caryophyllaceae weed Stellaria sp. (Yamada 1993). T. ishikariensis biotype B occurs almost exclusively on monocots, and among dicots, Stellaria sp. is the sole known host of the fungus. The Ainu cultivated various crops, and Setaria italica and Echinochloa sp. were most important in early modern times (Yamada 1998). It is not clear how the snow mold fungus could have grown on them. Gramineous summer crops are unlikely to be attacked by the fungus; however, if the grain readily falls on the ground when mature in late summer or autumn, and start to germinate, their seedlings may be attacked by snow molds. Snow molds are seldom obvious unless plants are cultivated as is the case with other plant diseases. The abundance of sclerotia of the snow mold fungus T. ishikariensis biotype B provides further circumstantial evidence that agriculture was practiced even in prehistoric days in northern Japan. The Ainu may have disposed of plant materials in fireplaces along with freshly gathered sclerotia in spring on plant debris used to start fires.

3.1.5 Typhula phacorrhiza

Typhula phacorrhiza may easily be identified solely by the features of sclerotia (Schneider and Seaman 1986; Hsiang et al. 1999). The sclerotia often have stalks by which they are attached to plant tissues. The stalk is inconspicuous in large sclerotia. Digitate, interlocking rind cells are characteristic and facilitate identification. Although humidity and low temperatures near freezing are essential for fructification (Yang et al. 2006), some isolates seldom produce basidiocarps. Typhula phacorrhiza is a potential antagonist to snow molds (see Chap. 5), but is pathogenic to winter wheat under snow in Canada (Schneider and Seaman 1986); however, isolates from corn debris screened in Canada as biocontrol agents were not found to attack creeping bentgrass (Burpee et al. 1987; Wu and Hsiang 1998), although they did differ in their ability to antagonize snow mold (Wu et al. 1998). Japanese isolates did not cause damage to perennial ryegrass in the field (Matsumoto and Tajimi 1992). Inoculation experiments with orchardgrass seedling revealed isolate variability in antagonism against T. ishikariensis biotypes (Matsumoto and Tajimi 1992): isolates collected from the biotype A area in Hokkaido were antagonistic to biotypes A and B of T. ishikariensis, while those from other areas where biotype B was present or where T. ishikariensis was absent suppressed biotype B, only. Wu et al. (1998) found that an isolate of T. phacorrhiza collected from corn stubble was able to antagonize T. ishikariensis and T. incarnata to reduce gray snow mold disease on turfgrass while not causing disease on the plant. Wu and Hsiang (1999) examined specialization of these three Typhula species and found that the 76 3 Snow Mold Fungi radial growth of T. phacorrhiza was significantly greater than T. incarnata or T. ishikariensis on a variety of defined carbon sources and speculated that this greater ability to utilize these structural and storage carbohydrates, combined with higher mycelial growth and sclerotial production over a wider range of temperatures, may help explain how some isolates of T. phacorrhiza are able to outcompete gray snow mold in field tests. Hsiang and Cook (2001) later found that this isolate is also able to suppress the development of pink snow mold caused by M. nivale in field tests across Canada. At low temperatures, T. phacorrhiza was able to outgrow M. nivale and grew better than other Typhula species tested at higher temperatures (Snider et al. 2000).

3.2 Sclerotinia spp.

Most species of the genus Sclerotinia are mesophiles as typiﬁed by S. sclerotiorum, but S. borealis, S. nivalis, and S. trifoliorum can cause damage to plants under snow cover (Saito and Tkachenko 2003), attacking gramineous plants, dicots, and forage legumes, respectively. Saito (2001) delimited the species concept of S. borealis and S. nivalis and investigated their host range to clear the ambiguity and confusion over both fungi.

3.2.1 Sclerotinia borealis

Taxonomic Issues

The variability of Sclerotinia borealis was poorly studied until critical examination by Saito et al. (2011). Isolates of S. borealis from nonagricultural lands in various parts of Eurasia differed from those from agricultural crops in cultural morphology, mycelial growth rate, sclerotial size, and ascocarp size. Consequently, the fungus was placed in three groups (Saito 2006), and molecular analyses supported their differences (I. Saito, personal communication). S. borealis as a species is characterized by psychrophily and zerophily and shares common morphological features such as the lack of intracellular space in the sclerotial medulla (Fig. 3.15b), eight nuclei in the ascospore (Fig. 3.15c), and the textura globulosa constituting ascocarp ectal excipulum (Fig. 3.15a), Saito et al. 2011). S. borealis was once designated as Myriosclerotinia borealis (Kohn 1979), but the fungus needs further taxonomic examination and is treated here as S. borealis. Infraspecies variability should be elucidated after molecular comparisons. The complete genome sequence of S. borealis indicated the similarity with S. sclerotiorum and also revealed genome regions and genes speciﬁc to S. borealis presumed responsible for adaptation to cold (Mardanov et al. 2014). 3.2 Sclerotinia spp. 77

Fig. 3.15 Three features to characterize Sclerotinia borealis. Ascocarp with ectal excipulum (ee) consisting of spherical cells (a), sclerotial medulla lacking in intercellular space (b), and hyaline ascospores each having eight nuclei (c) (Courtesy, I. Saito)

Ecological Features

Sclerotinia borealis occurs in areas with severe soil freezing (Tomiyama 1955; Nissinen 1996). However, soil freezing does not determine its distribution; resistance to the fungus depends largely on the freezing resistance of plants, and consequently, the pathogenesis of S. borealis is affected by plant freezing damage. Ozaki (1979) revealed the signiﬁcance of plant freezing resistance by sequentially exposing forage grasses to low temperatures to incite the fungal damage (Fig. 3.16). The disease was most severe on perennial ryegrass (Lolium perenne), followed by meadow fescue (Festuca pratensis), orchardgrass (Dactylis glomerata), and Ken- tucky bluegrass (Poa pratensis). Timothy (Phleum pratense) was the most resistant. S. borealis does occur even on perennial ryegrass in mid- and northern Hokkaido where early and deep snow cover protects plants from freezing (Ozaki 1979). Matsumoto (unpublished) observed severe damage of perennial ryegrass by S. borealis in spots where snow was blown by the wind in a mountainous region in Iwate, Honshu. Resistance of winter wheat varieties to the disease was correlated to 78 3 Snow Mold Fungi

100 PR

OG 50

KB Ti

0 0 C) Diseased plants (%) ̊ mean -10

minimum -20 Temperature (

Nov/7 14 21 28 Dec/5 12 19 control Date of treatment (left outdoors)

Fig. 3.16 Effect of plant predisposition to cold on the incidence of Sclerotinia borealis. Potted grasses grown outdoors were taken in an incubator controlled at 4 C at week interval from November 7 to keep them from freezing. They were then buried under snow on January 20. Control plants were kept outdoors. The later plants were taken in, the more severely plant damage increased. Plants had received enough airborne inoculum before treatment. PR perennial ryegrass, MF meadow fescue, OG orchardgrass, KB Kentucky bluegrass, and Ti timothy (Drawn from the data of Ozaki 1979) the level of freezing tolerance (Amano and Ozeki 1981). Resistant winter wheat varieties may be screened by “high ridge culture” (Yamana 2012), where soil surface temperatures drop to about À3 C under snow and remain around À1 C for a long period (Fig. 3.17). Tomiyama (1955) revealed that S. borealis mainly occurred in eastern Hok- kaido, but the significance of the disease has been overlooked. According to Nissinen (1996), its damage greatly fluctuates from year to year and is serious in years with deep soil freezing with delayed occurrence of persistent snow cover. The severe outbreak of S. borealis on orchardgrass in eastern Hokkaido in the spring of 1975 was ascribed to delayed snow cover and to deep snowfall in March, protracting the activity of the pathogen (Araki 1975). Orchardgrass was replaced with more freeze-tolerant timothy since then. Since young plants of timothy are vulnerable to attack by S. borealis, they are required to grow large enough before snowfall to overwinter in first-year grasslands. In 2008, the fungus occurred with severity in 1281 ha of grasslands newly sown with timothy. Field surveys by Sato et al. (2009) indicated that the later the sowing date, the more severe damage to timothy stands (Fig. 3.18). Seeding was delayed due to the dry summer and to farmers postponing seeding of timothy to keep corn in the field longer to improve the quality of silage. 3.2 Sclerotinia spp. 79

Fig. 3.17 Decrease in soil surface temperature in high ridge culture in Kunneppu, Hokkaido. Top: 2010 and bottom: 2011 (Yamana 2012) high ridge C)

̊ level row

days after snow cover

Soil surface temperature ( high ridge level row

Days after snow cover

Although S. borealis mainly attacks gramineous plants (Groves and Bowerman 1955;Roed€ 1960), its host range needs further investigations (Saito 2001). Infected gramineous plants exhibit severe symptoms since the fungus grows into crowns, resulting in poor regrowth the following spring or even the death of whole plants. These features leave an impression that S. borealis is highly virulent; however, its pathogenesis depends on the predisposition of hosts with low temperatures, i.e., the fungus can prevail only on moribund tissues at low temperatures. S. borealis is a facultative parasite and at best a weak pathogen (Årsvoll 1976) or should even be considered a saprophyte. Saito (1998) found its sclerotia on senescent leaves of non-gramineous plants such as species of Iris, Brassica, Allium, and Campanula, which were considered to result from saprophytic growth. The fungus occurs also on Juncaceae and Cyperaceae in Northern Europe and coastal regions of the North Paciﬁc (Kohn 1979). By the start of winter, these plants seldom have green tissues, but rather moribund or dead tissues by the time of snowfall.

3.2.2 Sclerotinia nivalis

Snow mold of overwintering dicots was reported many years ago (Tochinai and Sugimoto 1958), and the pathogen was identiﬁed as Sclerotinia intermedia which had previously been poorly described (Sugimoto et al. 1959). Saito (1997) renamed the pathogen S. nivalis. The fungus can attack a wide range of overwintering plants such 80 3 Snow Mold Fungi

Fig. 3.18 Outbreak of Sclerotinia borealis on ﬁrst-year grassland of timothy in Tokachi, Hok- kaido, in 2008. Timothy plants barely survived damage (a, arrow) but replaced by weeds such as Stellaria sp. and Capsella bursa-pastoris (b) as in the Asteraceae, Brassicaceae, Lamiaceae, and Polygonaceae. Its optimal temperature for mycelial growth is around 20 C and was pathogenic to Chrysanthemum coronarium and other summer crops at this temperature, but was less virulent than the mesophile, S. sclerotiorum (Tochinai and Sugimoto 1958). Although S. nivalis also has been reported from China, Korea, and Russia (Tkachenko et al. 2003), its signiﬁcance in agricultural production is not understood, and more study is needed.

3.2.3 Sclerotinia trifoliorum

Sclerotinia trifoliorum is closely related to S. sclerotiorum and S. minor. It was thought that S. trifoliorum causes damage only to forage legumes (Purdy 1979). Scott (1981) conﬁrmed species separation of these fungi based on differences in esterase isozyme patterns. The fungus causes signiﬁcant damage to alfalfa (Medicago sativa) and clover (Trifolium spp.) in snowy regions, reducing their persistence, especially of clover. A practical control measure is the use of resistant cultivars (Scott 1984), and resistance to S. trifoliorum is the top breeding objective for red clover (T. pratense) in Hokkaido. 3.3 Microdochium nivale 81

3.3 Microdochium nivale

3.3.1 Taxonomic Issues

The causal pathogen of pink snow mold has been known under several scientific names and with a seemingly variable biology. The fungus used to be treated as Fusarium nivale (teleomorph, Calonectria nivalis), but was designated as Gerlachia nivalis due to the mode of conidiogenesis and further divided into two varieties based on spore size (Gams and Mu¨ller 1980). Until recently, these varieties were referred to as Microdochium nivale var. nivale and var. majus (teleomorph, Monographella nivalis; Samuels and Hallett 1983). Gams and Mu¨ller (1980) did not recognize clear differences between the varieties on winter wheat in terms of ecology and growth–temperature relations. Smith (1983), however, reported significant differences between isolates from winter cereals and grasses in Saskatchewan, mid-western Canada; isolates from winter cereals produced large conidia and developed a teleomorph, while isolates from grasses had small conidia with one or no septum and failed to produce a teleomorph. None of the isolates from grasses in Norway produced perithecia (Smith 1983). Litschko and Burpee (1987) collected isolates from Ontario, eastern Canada, and found that both varieties were present in turfgrass and winter wheat isolates. Their isolates were mostly var. nivalis. Four winter wheat isolates (two varieties each) were homothallic and did not require opposite strains for mating, but turfgrass isolates were heterothallic. In a separate study in the same general region, Mahuku et al. (1998) identified all their turfgrass isolates as var. nivale and, using molecular techniques such as RAPD and IGS-RFLP, found that populations showed detect- able host specialization on different turfgrasses but with a general level of genetic diversity that implied recombination. RAPD analysis of British isolates indicated that winter wheat isolates consisted of the two varieties which agreed prior reports on the difference in conidial size and the presence or absence of teleomorph (Lees et al. 1995). PCR using specific primers indicated that isolates of var. nivale constituted a single group despite the fungus containing both homo- and heterothallic isolates (Nicolson et al. 1996). ITS-RFLP analysis of 70 isolates from Hokkaido, Japan (Terami and Kawakami 2006), revealed only four isolates from winter wheat identified as var. majus, and all others were var. nivale (16 isolates from winter wheat and 50 isolates from forage grasses). Similarly, Hokkaido isolates collected from a total of 45 fields (mostly planted with winter wheat and one with Italian ryegrass) had small conidia and were identified as var. nivale, but were found to be homothallic. Thus, M. nivale is obviously diverse in terms of conidial size and teleomorph production, as well as host preference, i.e., annual or perennial. Glynn et al. (2005) proposed recognition of the two varieties as separate species, M. nivale and M. majus, based on the comparison of the elongation factor 1-alpha gene involved in protein synthesis. Jewell and Hsiang (2013) used four genomic regions (RNA polymerase II, beta-tubulin, elongation factor 1-alpha, and the 82 3 Snow Mold Fungi internal transcribed spacer region of ribosomal DNA) on a variety of isolates from North America and Europe and from wheat and turfgrass hosts and supported the elevation of the varieties to species level. Whole-genome sequencing (Hsiang, unpublished) revealed that the two taxa have greater variation than found intraspe- cifically. Because most research on these Microdochium species, causing pink snow mold on grasses and cereals, was done before they were recognized as distinct species, they then collectively continue to be referred to in this review as M. nivale (sensu lato), and when the variety is specified, it is referred to as M. nivale (sensu stricto) or M. majus.

3.3.2 Ecological Features

Plant damage caused by M. nivale under snow is referred to as pink snow mold. Pink snow mold occurs widely throughout the world. Plant damage is often light with lower, senescent leaves damaged, but the disease is found with greater severity in neutral volcanic ash soil (Nakajima and Naito 1995) or on intensively managed turfgrasses (Smiley et al. 2005; Tronsmo et al. 2001). The causal fungus is associated with the hosts at every growth stage (Cook 1981), and this is one of the reasons why it can be so severe. Seed-borne infestations represent the primary inoculum for winter wheat seed (Hewett 1983). Seedlings are more prone to attack at low temperatures (Millar and Colhoun 1969). Since the fungus can grow mycelia through unsterile soils at temperatures 10 C or below (Booth and Taylor 1976b), mycelia in plant debris such as straw can be more signiﬁcant as primary inoculum than infested seeds (Booth and Taylor 1976a). M. nivale can remain latent endophytically in barley seedlings (Perry 1986). Conidia densely produced on diseased leaves under snow turn infected plants pink, inciting secondary infection. Cool and damp weather from spring to summer promotes disease on turfgrasses (Smith 1987; Tani and Beard 1997) and cereals (Asuyama 1940). In eastern Washington, USA, ascocarps are seldom produced because the air is generally dry during snow thaw and the soil is dry during summer (Cook and Bruehl 1968). Such ascospores can infect plants to cause pink snow mold but these infections are not important as secondary inoculum in this region (Cook and Bruehl 1968). However, in Europe, ascospores are discharged onto the inﬂorescense of oats and cause head blight to produce infested seeds (Noble and Montgomerie 1956). Since M. nivale is associated with all the growth stages of host plants, then interruption of the life cycle of the pathogen may be an effective control measure. For example, until the 1950s in Japan, wheat seedlings that suffered from soil heaving were covered with soil to minimize ineffective tillers, and consequently, this cultural practice promoted decomposition of diseased leaves. Using this method in a 4-year trial, Nakajima (2007) almost completely suppressed ascocarp production and reduced the incidence of wheat head blight by 76 %. Microdochium nivale sensu stricto and M. majus can attack both annual and perennial plants, and both species are present on both types of plants. However, 3.3 Microdochium nivale 83

Fig. 3.19 Pink snow mold occurred exclusively on a breeding line of meadow fescue. Cottony mycelia are abundantly produced on diseased plants which make them look like shaved ice there seems to be an association between the heterothallic form on perennial plants and the homothallic form on annual plants. Homothallism is speculatively a more effective strategy on annual hosts since the fungus has less chance to encounter opposite strain as compared to perennial hosts which persist for years long enough to wait for the arrival of opposite strain. Specialized genotypes are more likely to occur in perennial hosts as reported by Mahuku et al. (1998) on creeping bentgrass. Since host–parasite interactions last for years in perennial hosts, M. nivale is always subjected to selection pressure from the host. An example is illustrated by the emergence of a “ﬂuffy” genotype on a certain breeding line of meadow fescue in the late 1980s at the Hokkaido National Agricultural Experiment Station. The fungus produced “cottony” mycelia on diseased plants (Fig. 3.19), but disappeared during the process in which the meadow fescue line was excluded from the breeding program (Matsumoto unpublished data). Microdochium nivale is a facultative snow mold pathogen (Matsumoto 1994) and capable of attacking plants during their growing season and of saprophytic growth throughout the year. Its versatility is ascribed to its ecophysiological capabilities, extending its distribution in time and space. These features, along with its ease of handling, characterize the fungus as an appropriate model organism to study evolution and host–parasite interactions (Ergon et al. 1998; Ergon and Tronsmo 2006). 84 3 Snow Mold Fungi

3.4 Pythium spp.

Snow mold caused by Pythium spp. (often referred to as snow rot) was first found in Toyama, Japan. Winter cereals have been grown as rotation crops in paddy fields throughout Japan, and where snow cover remains for months, damage by Pythium spp. has been confused with moisture damage. Disease damage and the significance of these pathogens have not been well elucidated. Although Pythium spp. are commonly present in fields with no cropping history of barley or wheat as well as in forests (Takamatsu and Ichitani 1987b), Pythium spp. as snow molds are not well studied. Some limited findings on Pythium snow mold are also available from the USA (Lipps and Bruehl 1978). Pythium spp. can infect plants through zoospores released in water. Several species in this genus are known to cause disease under snow (Hirane 1960; Lipps 1980; Ichitani et al. 1986), and P. iwayamai is the most important. Different species are present in soils with different levels of drainage. Three species were detected in 122 winter cereal fields converted from rice paddy: P. paddicum was most frequently recovered from ill-drained fields, and P. iwayamai was as prevalent as P. paddicum in well-drained fields (Table 3.6, Takamatsu and Ichitani 1987a; Taka- matsu 1990). P. okanoganese was recovered exclusively from well-drained fields, and its potential for damage in the upland cropping system is yet to be determined, but it has also been found in forest soils (Takamatsu and Ichitani 1987b). P. paddicum can tolerate the low-oxygen high-carbon dioxide conditions of paddy fields and is recovered most frequently in September (Takamatsu 1993). These features characterize P. paddicum as a facultative snow mold. P. vanterpoolii and P. volutum are weakly pathogenic to barley under snow (Ichitani et al. 1986). Pythium spp. often coexist with Typhula incarnata on diseased plants. Using ELISA, Takenaka and Arai (1993) studied dynamics of P. iwayamai and T. incarnata on barley. When both pathogens were found together in the field, P. iwayamai was the first to infect plants, and then T. incarnata replaced P. iwayamai. The succession of both pathogens is typical of primary and cellulose-decomposing fungi in saprophytic succession (Garret 1970): the former rapidly colonizes fresh substrates to utilize sugars and readily accessible carbohydrates, and the latter can degrade cellulose. While P. iwayamai was frequently recovered from leaf sheaths and leaf blades, T. incarnata was detected from whole plant, including roots (Takenaka and Arai 1993). In polar regions, Pythium spp. attack indigenous mosses, and the damage occurs in patches (Fig. 3.20). P. ultimum var. ultimum (Hoshino et al. 1999), Pythium HS

Table 3.6 Pythium species Number of ﬁelds Species presenta detected from winter cereal Drainage Investigated Diseased Pp Pi Po ﬁelds converted from paddy the previous year Poor 69 37 36 2 0 Good 53 27 19 15 8 Takamatsu (1990) aPp Pytium paddicum,PiP. iwayamai,PoP. okanoganese 3.5 Other Basidiomycetous Snow Mold Pathogens 85

Fig. 3.20 Snow rot of moss (Sanionia sp.) caused by Pythium polare in the polar regions (Courtesy, M. Tojo) groups (Hoshino et al. 2000), and P. polare (Tojo et al. 2012) were recovered from diseased tissues. These pathogens are considered involved in plant succession since other plants are often established in the disease patch (see Chap. 1). P. ultimum var. ultimum is a ubiquitous fungus, but polar isolates are different from those from temperate isolates such as those found on the main island of Japan in growth– temperature relations (Hoshino et al. 1999). Isolates of P. polare are distributed at both poles and show mating compatibility (Tojo et al. 2012). Their principal moss host, Sanionia, is also present at both poles. The most remarkable feature of Pythium spp. causing snow rot is that they are phylogenetically close (Le´vesque and De Cock 2004; Tojo et al. 2012): they mostly constitute a single clade despite the occurrence of Pythium spp. practically in all habitats including the cryosphere and their diversity, composed of both parasites and saprophytes. Their evolutionary pathways may have many critical steps that differed from other snow mold pathogens and from other Pythium spp.

3.5 Other Basidiomycetous Snow Mold Pathogens

3.5.1 Supponuke fungus (Athelia sp.)

An undescribed snow mold has been known from the late 1970s on winter wheat in eastern Hokkaido. "3.5 Other Basidiomycetous Snow Mold 86 3 Snow Mold Fungi

Pathogens",6,1Diseased plants exhibited decay in lower leaf sheaths, resulting in damping-off. The name “supponuke” comes from the diagnostic feature of diseased plants: plant tops were easily pulled up. The causal fungus was infrequently isolated from diseased tissues, and mycelia with clamp connections developed from thin scab-like sclerotia. The isolates were pathogenic to winter wheat but left unidentified (Simizu and Miyajima 1990). Subsequent surveys by Simizu and Miyajima (1992a) revealed that the distribution overlapped with that of Sclerotinia borealis in eastern Hokkaido (Tomiyama 1955) and that its damage tended to be severe in winters when S. borealis damage was commonly observed. The pathogen differed from Typhula spp. in cultural morphology and did not mate successfully with them in pairing tests. The fungus was subsequently subjected to mating experiments with LTB (low-temperature basidiomycetes) from Canada, but pairing was also unsuccessful (Simizu and Miyajima 1992b). DNA sequencing and phylogenetic analyses indicated that the fungus was a species of Athelia (A. Kawakami, personal communication). Athelia spp. are common in forest litter, and Matsumoto (unpublished) obtained a circumstantial evidence to confirm its presence on a first- year golf green planted with creeping bentgrass. Forest soil had been brought in to construct the green the previous summer, and disease patches occurred the following spring (Fig. 3.21). The trend of supponuke incidence clearly coincides with the change in winter wheat cultivars planted in eastern Hokkaido. Until the 1980s, the disease was commonly found on wheat cultivar “Horoshiri-komugi” but almost disappeared with increasing acreage of more freeze-tolerant cultivar “Chihoku-komugi” (Simizu 1993, 2014). These observations indicate the significance of breeding in controlling the snow mold. The cultivar Chihoku-komugi, which was relatively

Fig. 3.21 Supponuke disease on creeping bentgrass in a lawn constructed with forest soil the previous year 3.5 Other Basidiomycetous Snow Mold Pathogens 87 resistant to Sclerotinia borealis but susceptible to other snow molds, was found to be resistant also to supponuke, and Horoshiri-komugi was more susceptible. Another factor was the change in winter climate there, since the early onset of persistent snow cover was considered to have suppressed disease incidence.

3.5.2 Low-Temperature Basidiomycete (LTB, Coprinus psychromorbidus)

LTB is important in western Canada and infects plants by hyphae (Cormack 1948). LTB infects turfgrass, forage crops, and winter cereals as well as apples in storage and can grow saprophytically on dead plant tissues (Gaudet 2001). Such versatility may be ascribed to our poor knowledge on LTB which is likely a species mixture consisting of diverse groups. Spores are not known in LTB, and the presence of clamp connections indicates that the fungus is basidiomycetous (Broadfoot and Cormack 1941). LTB was separated into three groups based on culture morphology (Ward et al. 1961). Basidiocarps developed on diseased alfalfa indicated that LTB belonged to the genus Coprinus (Traquair 1980). The fungus was later identified as C. psychromorbidus (Readhead and Traquir 1981). Mating compatibility and DNA analyses showed that C. psychromorbidus was divided into at least four groups (Laroche et al. 1995). Taxonomic distinction and ecophysiologic characterization of each group should provide us with more precise information. There are recent attempts to sequence the genome of this fungus, but assembly results, as is the case with many multinucleate basidiomycetes, have given many short sequences (Hsiang, unpublished). LTB can persist in fields where winter wheat has not been planted for two seasons (Piening et al. 1990). Optimal temperature for pathogenesis on wheat, alfalfa, and forage grasses is 2 C (Cormack and Lebeau 1959), but some strains cause most severe damage at À7toÀ3 C (Gaudet 1986). LTB grows best at 10–15 C (Smith 1987). Winter wheat is exposed to low temperatures of À10 to À5 C in less snowy years in the Canadian Prairies, which is not fatal to plants, but the damage due to LTB culminates under such conditions (Gaudet and Chen 1988). Although the exposure to À7.5 C for 1–5 weeks was not lethal, the viability and regrowth of first-year alfalfa were badly reduced by LTB under such conditions (Hwang and Gaudet 1998). The evolution of cyanide was shown to be the critical factor in alfalfa–LTB interactions: cyanide accumulated under the snow in the crowns of infected alfalfa, leading to plant death (Lebeau and Dickson 1955). 88 3 Snow Mold Fungi

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Abstract Plants generally have active countermeasures against invasion by pathogens, and the physiological condition of host plants is critical to fungal infection. On the other hand, overwintering plants have long been thought to passively endure snow mold attack, storing reserve materials for dormancy under snow to allocate for spring ﬂush. Recent studies, however, revealed that overwintering plants can actively respond to snow mold attack. The unique feature of overwintering plants is that they utilize reserve materials as the energy source for active resistance. Since snow cover inhibits plant photosynthesis, overwintering plants need to store sufﬁ- cient reserve materials prior to the occurrence of persistent snow cover. Hardening is the process of plant acclimation to survive winter, and freeze tolerance and cold adaptations are increased during the process of hardening. Some of these

The most reliable evaluation method of snow mold resistance is to observe and compare winter survival of plants in naturally infested ﬁelds. Many alfalfa cultivars resistant to Typhula ishikariensis biotype A were developed from ﬁeld research at Hokkaido National Agricultural Research Center.

© Springer Science+Business Media Singapore 2016 95 N. Matsumoto, T. Hsiang, Snow Mold, DOI 10.1007/978-981-10-0758-3_4 96 4 Resistance mechanisms have been elucidated to the molecular level, and such information is essential to resistance breeding.

Snow mold resistance directly contributes to yield increases of winter cereals and forage crops and to extending the persistence of pastures. Even where fungicides can be applied, the cultivation of resistant crops and cultivars contributes to environmental conservation by reducing the amount of fungicide needed. Also, cultural practices, such as fertilizer application, seeding date, and the timing of final cutting of forage crops, all can affect resistance level. Sugar content and percent dry matter in the stems of winter wheat plants were found to be associated with resistance to Typhula incarnata, and sugars became depleted with time, predisposing plants to fungal attack (Kakizaki 1936; Hirai et al. 1952). Tomiyama (1955) showed the difference in resistance among leaves of individual winter wheat plants: the fungus prevailed more rapidly on lower leaves than upper leaves. Varietal differences were evident in lesion size (Tomiyama 1955) and in the rate of mycelial spread on wheat leaf blades (Takenaka and Yoshino 1987). More recent studies revealed active resistance mechanisms of plants to snow mold as is the case with other diseases (Ergon et al. 1998; Kawakami 2004; Gaudet et al. 2003b). Physiological conditions of plants influence host–parasite interactions and are extremely critical in snow mold pathogenesis. Decreasing ambient temperatures trigger plant hardening (Paulsen 1968). Freezing tolerance and snow endurance were improved during hardening, as well as snow mold resistance (Ergon et al. 1998; Gaudet et al. 1999). When hardened, timothy seedlings were exposed to 12–18 C for 2 weeks (dehardened); freezing tolerance was much reduced, but resistance to T. ishikariensis remained unchanged, implying that plant hardening and disease resistance involved two separate mechanisms (Fig. 4.1, Tronsmo 1985). Reserve materials are consumed in overwintering plants to cope with snow mold attack under snow where plants are unable to obtain energy through photosynthesis to resist. Plants must store enough reserve material before winter and conserve throughout the winter to have sufficient wherewithal for spring growth.

4.1 Field Observations

Seeding at the right time is an effective cultural practice for snow mold control (Bruehl et al. 1975; Kunii 1980; Takamatsu et al. 1985), although resistance to snow mold varies among many crops and cultivars (e.g., Vargas et al. 1972; Smith 1975; Adachi et al. 1976; Kunii 1978; Litschko et al. 1988; Nakayama and Abe 1996; Wang et al. 2005; Bertrand et al. 2009). The outbreak of Sclerotinia borealis in ﬁrst-year timothy grasslands in 2007 in Tokachi, Hokkaido, illustrates the signiﬁcance of seeding date (Sato et al. 2009): the cultural practice established for timothy could not be carried out as recommended due to circumstances where the farmers postponed seeding timothy to improve the maturity of corn crop, which 4.1 Field Observations 97 Freeze tolerance

T. ishikariensis Resistance to Resistance

control H2 H2 + DH1 H2 + DH2 Treatments

Fig. 4.1 The effects of dehardening of timothy on freeze resistance and resistance to Typhula ishikariensis. H2, hardened for 2 weeks; H2 þ DH1, hardened for 2 weeks and then dehardened for a week; H2þDH2, hardened for 2 weeks and then dehardened for 2 weeks (Drawn from the data of Tronsmo 1985) was grown as a rotation crop prior to timothy. This is one of the examples in which an established cultural practice becomes unavailable as an option because of changing climatic circumstances. During the course of investigations to determine the right time for seeding in Washington, USA, Bruehl (1967c) found that winter 98 4 Resistance

Fig. 4.2 Influence of 100% seeding date on the survival of winter wheat in Washington, USA, when attacked by severe snow ab mold. Plants sown in August successfully survived, but mostly killed when sown in September Survival (curve A). Small plants sown in late October also showed good survival (curve B) (Modified from 0 Bruehl et al. 1975) August September October Seeding date wheat seedlings survived better when sown earlier in August. Although small plants sown late in the autumn escaped from snow mold damage (Fig. 4.2), they concluded that late sowing diminished the advantage of winter crops, specifically a longer growing season, resulting in yield loss. The idea of disease escape was exploited for spring wheat by Sasaki et al. (1991) and Sato and Sawaguchi (1998) to develop a method to seed spring wheat just before snow cover. Seeds of spring wheat did not germinate or barely geminated under snow, escaping from snow mold damage. Among different snow mold fungi, cultivars or lines of winter wheat responded similarly to Microdochium nivale, Typhula incarnata, and T. ishikariensis (Bruehl 1967a). These findings suggest that breeding and selection for resistance to one disease also include resistance to others. Since physiological specialization was not evident in these fungi (Bruehl 1967b), race differentiation is unlikely to occur. However, in the case of M. nivale, a fungal strain occurred specifically on a certain line of meadow fescue (see Chap. 3). Amano and Ozeki (1981) found that some lines of winter wheat resistant to Typhula spp. were heavily attacked by S. borealis, and Amano (1987) was able to divide winter wheat into four groups based on the results from field trials: A, non-winter hardy; B, freeze tolerant; C, intermediate; and D, snow tolerant (Typhula resistant). Despite the general association of resistance to S. borealis with freeze tolerance, group B included winter wheat that was vulnerable to S. borealis and also winter wheat that was resistant to S. borealis but not freeze tolerant. Resistance to winter Pythium spp. does not involve all the same mechanisms as resistance to other snow mold fungi. Lipps and Bruehl (1980) reported that plants resistant to Typhula spp. with abundant reserve materials were susceptible to Pythium spp. and that small plants were more resistant. The same results were obtained by Takamatsu et al. (1985). 4.2 Laboratory Trials 99

4.2 Laboratory Trials

Low temperatures are essential for snow mold fungi to infect and to prevail on plants. Wernham and Chilton (1943) examined responses of grasses to Typhula spp. under controlled temperatures at 2–8 C. Such laboratory trials were effective to screen for resistance, since results from the laboratory inoculation tests agreed with those from ﬁeld observations (Cormack and Lebeau 1959). They were successful in inoculation with Typhula spp. and LTB but not S. borealis, although there are reports of successful inoculation with S. borealis using ascospores (Årsvoll 1977). Using the snow mold chamber method, Bruehl et al. (1966) distinguished resistant winter wheat cultivars from susceptible ones. Since then, many crops have been screened in laboratory trials because the experiments can be made throughout the year (e.g., Blomqvist and Jamalainen 1968; Gaudet and Kozub 1991; Tronsomo 1993; Miedaner et al. 1993; Chang et al. 2006; Bertrand et al. 2009; Bertrand et al. 2011). These experiments required plant hardening and low temperatures and, consequently, still took months to obtain results. Nakajima and Abe (1990) reduced the duration of experiments by using a quicker pathogenicity testing method. Winter wheat seedlings grown in a commercial potting medium were hardened outdoors, inoculated with M. nivale or T. incarnata, and incubated at 5–18 C. Varietal differences were detected 7–10 days after inoculation at 18 C (Fig. 4.3). Inoculation with M. nivale at 15 C showed differences between seven cultivars. If plants were grown exclusively indoors in controlled chambers to minimize contamination, this method was applicable to the more psychrophilic T. ishikariensis (Kawakami and Abe 2003). Tiller survival (%)

Incubation period (days)

Fig. 4.3 Percent survival of tillers of winter wheat cultivars following inoculation with Microdochium nivale and Typhula incarnata at 18 C. ○, PI173438 (resistant); ~, Nanbukomugi (intermediate); ●, Fukuhokomugi (susceptible) (Nakajima and Abe 1990) © Canadian Science Publishing 100 4 Resistance

4.3 Resistance Mechanism

4.3.1 Hardening

Abe and Yoshida (1997) evaluated the level of hardiness by freeze tolerance, using ﬁeld-grown winter wheat to reveal that hardening was promoted with decreasing minimal daily temperature. Plant hardening is induced and develops with decreasing ambient temperature, and many physiological changes occur in the plant (Paulsen 1968). Hardening is not only affected by soil fertility (Freyman and Kaldy 1979) but by the timing of harvest in forage crops. The term “critical period” is commonly recognized among grassland farmers and scientists: if a perennial grass were harvested during the critical period, grass winter survival could be greatly impaired, and snow mold disease could be extensive (Sakamoto and Okumura 1973; Andersen 1992). If plants were harvested before they completed the hardening process, they could continue to allocate reserve materials for regrowth depending on growth conditions and consequently become vulnerable to snow mold because accumulated resistance would decline with regrowth. In short, agricultural practices critical to winter survival depend how much reserve material plants can store during the process of hardening (Abe 1995b). Temperate grasses (Moriyama et al. 1995) and winter wheat (Yoshida et al. 1997) were hardened at temperatures below 10 C, with accompanying water loss which plateaued in early November (Fig. 4.4a). Freeze tolerance reached a peak in midwinter (Fig. 4.4b). The process of hardening may be divided into two stages in terms of water content and freeze tolerance of plants (Fig. 4.5, Abe 1995a). Grass plants ﬁrst lose water to a level of 3 g water/g plant dry matter (stage 1), and, subsequently, only bound water is retained with increased freeze tolerance (stage

ab O/g dry tissue) C) 2 ̊ ( 50 LT Water content (g H Water content Month Month

Fig. 4.4 Seasonal changes in crown water content (a) and in freeze tolerance (b) in orchardgrass (○●), timothy (△), and perennial ryegrass (□) (Moriyama et al. 1995) 4.3 Resistance Mechanism 101

Free water

Bound water Stage 1 C) ̊ ( 50 Hardening LT Stage 2

Water content (g H2O/g dry tissue)

Fig. 4.5 Two stages in the process of hardening in orchardgrass (○●), timothy (△), and perennial ryegrass (△) (Moriyama et al. 1995). Physical states of water and two stages in hardening process were designated following Abe (1995b)

2). Stage 1 terminated when ambient temperatures fell to the freezing level, during which plants continued photosynthesis and dehydration with loss of free water. They continued to accumulate reserve materials and assumed a more rosette appearance with leaves appressed to the ground. Water content remained constant in plants during stage 2, and the sugar component varied among plant species and was a distinguishing characteristic for freeze-tolerant vs. snow-tolerant winter wheat cultivars. Snow-tolerant cultivars continued to accumulate fructans, which were degraded into mono- and oligosaccharides to enhance osmosis in freeze- tolerant cultivars (Yoshida et al. 1998a, b). These two tolerance types differed in sugar metabolism in stage 2 (Yoshida et al. 1998a, b).

4.3.2 Reserve Materials

Total nonstructural carbohydrates (TNC) are stored in plants before overwintering (Smith 1968; Tamura et al. 1985; Yoshida et al. 1985). Resistance to Typhula ishikariensis was not correlated with the amount of TNC in winter wheat but to its consumption rate (Kiyomoro and Bruehl 1977). TNC is mostly fructans in winter cereals and grasses. Yukawa and Watanabe (1991), using sib-related winter wheat 102 4 Resistance cultivars and lines, found a positive relationship between fructan content and winter hardiness. Fructan dynamics as a mechanism of snow mold resistance in winter wheat has been elucidated more recently by Kawakami (2004), Kawakami and Yoshida (2012), and Gaudet and Laroche (2013). In the ﬁrst two studies, cultivars used were PI173438, resistant to Typhula ishikariensis, and Valuevskaya that was freeze tolerant but not resistant to the fungus. The reduction rate of fructan content 50 days after incubation under snow was 35 % in PI173438, while it was 74 % in Valuevskaya. The level of fructose, which had been degraded from fructan, was found to remain constant, implying that plants degraded fructan to produce fructose and maintained it at a constant level to consume as an energy source. Fructan content in T. ishikariensis-inoculated crowns reduced to 25 % in PI173438 and 50 % in Valuevskaya as compared to the uninoculated control at 20 days after inoculation. The fungus started to infect 10 days after that and developed extensive mycelia on leaves 20 days after that. Fructan was rapidly decomposed in leaves, and fructose content was also reduced. The reduction of fructose in leaves was thought to stem from enhanced metabolism to resist the fungus, actual consumption by the fungus, or leakage from dead cells due to infection. Since the fungus did not infect crowns until 20 days after inoculation, fructose reduction in crowns was not ascribed to fungal consumption, but speculatively that wheat plants actively degraded fructan in crowns to use fructose as an energy source to resist fungal attack. Fructan metabolism should further be studied to reveal molecular bases of hardening and snow mold resistance. To date, characterization has only been made on genes involved in the synthesis and degradation of fructan (Kawakami and Yoshida 2002, 2005; Kawakami et al. 2005), and more recently, Kawakami and Yoshida (2012) obtained further evidence: transcripts of a fructan exohydrolase gene (wfhsm3) increased in wheat leaves infected with T. ishikariensis. Chilling tolerance of rice was enhanced when fructan synthesis genes from winter wheat were introduced to rice (Kawakami et al. 2008).

4.3.3 PR Proteins and Other Substances

Energy generated through active degradation of fructan by plants is used to resist snow mold possibly by producing pathogenesis-related (PR) proteins (Van Loon 1985) and other compounds, and some PR proteins have both antibiotic and antifreeze activities (Kuwabara and Imai 2009). According to Ergon et al. (1998), various PR proteins were induced after infection by Microdochium nivale, which was faster and more intense in hardened wheat plants. A comparison of cold- induced and M. nivale-induced proteins in winter rye showed that both groups of PR proteins were electrophoretically identical and contained glucanase-like, chitinase-like, and thaumatin-like proteins with similar levels of activities for the former two and that only the PR proteins induced by cold exhibited antifreeze activity (Hiilovaara-Teij et al. 1999). PR proteins and transcripts of phenylalanine 4.3 Resistance Mechanism 103 ammonia lyase (PAL) were detected from winter wheat during the processes of hardening and dehardening, and their changes in quantity corresponded with snow mold infection in late winter (Gaudet et al. 2000). Also, PAL activity was enhanced in hardened barley leaves in response to Pythium paddicum infection (Watanabe et al. 2008). Plants produce various compounds in addition to PR proteins to respond to snow mold infection. Cold shock proteins are known to occur in plants in response to low temperature. Karlson et al. (2002) demonstrated that a cold-regulated nucleic acid- binding protein of winter wheat was similar to the major cold shock protein of Escherichia coli in structure and expression. Kim et al. (2009) revealed that a gene, CSP3 of Arabidopsis thaliana, was activated at 4 C and it enhanced freeze tolerance. A defensin-like gene was recovered in winter wheat specifically during hardening, and the protein product inhibited bacterial growth, but lacked antifreeze activity (Koike et al. 2002). Five cultivars of winter wheat differing in snow mold resistance and freeze tolerance showed differences in the pattern and level of expression of defensin and lipid transfer protein (LTP, Gaudet et al. 2003a). Since LTP expression was much higher in snow mold-resistant PI181268 than moderate Norstar, LTP was regarded as most directly involved in snow mold resistance (Gaudet et al. 2003b). Winter wheat produced cystatin during hardening that specifically inhibited cystein proteinase and mycelia growth of Microdochium nivale (Christova et al. 2006), but its mechanism remains unknown (R. Imai, personal communication). Wheat plants exposed to low temperatures produced a low molecular weight antibiotic, feruloylagmatine (Jin and Yoshida 2000). This antibiotic was not detected from wheat plants under snow, but two kinds of hydroxycinnamic acid amides were recovered (Jin et al. 2003). These two compounds were found to inhibit M. nivale, and their concentrations increased under snow cover (Jin et al. 2003). Resistance to snow mold has been found to involve specific mechanisms, but the overall picture showing how each mechanism interacts in plants is yet to be elucidated. Gaudet et al. (2003b) speculated that the consecutive events in plant disease resistance occur as follows: (1) plants respond to a mild abiotic stress of decreasing temperature in autumn to activate chitinase and β-1–3 glucanase genes, and these enzymes are involved in both freeze tolerance and disease resistance; (2) infection by snow mold fungi further induces and enhances gene transcription for defense in plants; and (3) mechanisms induced by both biotic and abiotic factors interact each other to support winter survival of plants in response to the environmental change under snow. 104 4 Resistance

4.4 Conclusions

Physiological conditions of plants, i.e., winter hardiness, indirectly regulate resistance against snow mold. Since disease is incited by opportunistic infections of often moribund tissues, direct genetic interactions are not likely to be involved between hosts and pathogens. The key issue of snow mold resistance is to enhance the hardiness level of plants. Molecular studies on the development of hardiness and overwintering mechanisms should elucidate the genetic background of snow mold resistance to identify target genes for further study (e.g., Gaudet and Laroche 2013; Kang and Park 2013; Mazzucotelli et al. 2013; Yoshida and Kawakami 2013). These genes would certainly be useful as molecular markers in disease resistance breeding (Bertrand and Castonguay 2013). However, ﬁeld observations on snow mold resistance are essential, especially under the ongoing changes and shifts in environmental conditions.

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Abstract Microorganisms play the major role in the decomposition of plant debris. Plant pathogens are among them, and some can exploit tissues which are still living, causing economic damage to crops. Consequently, humans have developed many countermeasures to control plant diseases. Fungicide application has been the most practical and easiest method, even against snow mold. Biological control using low-temperature antagonists can be effective, although reduction of costs related to production and efﬁcacious timing of application requires more work. Results from research on cultural practices have contributed to improved productivity. Risks from changing climate, however, may pose a threat because cultural practices and cultivars for each region were selected before the most recent climate changes. The intrinsic capacity of plants to resist or tolerate snow mold and other stresses and perhaps greater phenotypic plasticity in responses should further be selected and improved to cope with changing environments for future sustainable production.

Control of gray snow mold by Typhula phacorrhiza applied at a rate of 100 g/m2 the previous autumn before snowfall (October 1999), and observed after snowmelt (May 2000) in the Rocky Mountains, British Columbia, Canada. The background shows fungicide trials.

The life cycle of most snow mold fungi is divided into two phases, i.e., active phase under snow and dormant phase without snow during the growing season of plants (Hsiang et al. 1999b; Matsumoto 1985, 1988, 2009). Different control measures are available against snow mold fungi, according to the phases of the life cycle (Table 5.1). Chemical control such as fungicide sprays is most effective when it is made just before snow cover. Cultural practices for forage crops to improve winter hardiness include fertilizer application in late autumn and the proper timing of the ﬁnal cut. The choice of resistant cultivars is, of course, essential. Seeding date affects winter survival. Some biological control agents have shown success because they were chosen for their antagonism against snow mold and also their low-temperature tolerance (Matsumoto 1998). Some of these agents have been formulated commercially; for example, the fungus Typhula phacorrhiza was registered against turfgrass snow molds in 2011 in both the USA (nepis.epa.gov) and Canada (pr-rp.hc-sc.gc.ca). Snow mold diseases of economic concern occur mainly on forage crops, winter cereals, and turfgrasses. These plants differ in the particular tissues used by humans, economic value per area, and the extent of disturbance due to cultural practices such as tillage. These factors are keys to select control measures (Table 5.2). Since a single disease patch after snowmelt usually exceeds the aesthetic tolerance for putting green turf on golf courses, near absolute disease control is required, and furthermore the putting greens are given limited time for recovery in the spring. In forage crops, snow mold damage may be ameliorated by fertilizer application until the ﬁrst cutting. Winter cereals are cultivated for grains and are given the longest period of time for recovery, which is until autumn, and surviving plants may compensate by increasing biomass regrowth to make up potential yield losses.

Table 5.1 Control measures against snow mold based on its ecological features and growth stages of plants Spring/Summer Autumn Winter Growth Dormant in the Resuming growth after Invading plants stage of forms of sclerotia, dormancy pathogens etc. Plant Flush Vegetative Hardening in process Dormancy growth growth stage Control Cultural practises Cultural practises (fertilizer Cultural practises measure (seeding at the right application at the right time and (snowmelt accelerated time) ﬁnal cutting at the right time) by black carbon deposition) Biological control Chemical control (suppression of Biological control (reducing dormant infection and mycelial growth) (microbial antagonism) propagules) Effective control measures in italic 5 Control 111

Table 5.2 Control measures based on the length of time for recovery and the intensity of cultural practice, and the persistence of antagonists Control measures Forage crops Winter cereals Turfgrass Cultural practice þþ þ À Chemical control Àþ þþ Biological control þÆ þþ Time for recovery þþþÆ Intensity of cultural practice Æþ þþ Persistence of antagonists (stability of the habitat) þþ Æ þ Modiﬁed from Matsumoto (1998)

Turfgrass putting greens are the most intensively cultivated, whereas forage crops are given the least intense cultivation, and winter cereals are intermediate. The extent of cultural practices is well correlated with economic importance per unit area. It pays to invest extensively on snow mold control in turfgrasses since the aesthetics of the plants is of much higher value than the seed or forage yield of other overwintering plants. As for biological control, the golf course habitat is not tilled annually, and thatch with relatively undisturbed, dying, and dead plant tissues is available for antagonists although practices such as de-thatching and aeration will tend to reduce the levels of thatch. In comparison, fields planted with winter cereals are usually tilled each year and rotated with nonhost, summer crops, and hence the habitat is highly disturbed, and antagonists face greater barriers against their persistence. Control measures against the same pathogens vary, according to the intensity of cultural practices or economic significance for each crop (Table 5.2). Fungicides are seldom applied for forage crops, whereas cultural practices and breeding show sufficient efficacy and return on investment. In winter cereals, crop rotation should be practiced along with the use of resistant cultivars. Chemical control is essential for high-intensity turfgrass culture, especially on golf course putting greens (Hsiang et al. 1999a). Fungicide applications can effectively reduce the incidence of Typhula ishikariensis biotype B by suppressing the development of disease patches normally observed just after snowmelt, but fungicides are unable to control fungal growth on underground plant parts even with higher rates. Chemical control may not always be essential in winter wheat, but the failure to use fungicides when necessary could result in severe damage (Fig. 5.1, Matsumoto et al. 2000). Biolog- ical control is effective for forage crops and turfgrasses whose damage is estimated directly by the survival and regrowth of plant tops. Biological control requires substantial inoculum of antagonists and is not economically justified in the case of most forage crops. 112 5 Control

Fig. 5.1 Winter wheat ﬁelds suffered from serious damage from Typhula ishikariensis biotype A. Snow cover occurred much earlier than usual, and farmers were unable to spray fungicides the previous year

5.1 Chemical Control

Turfgrass and winter cereals often receive fungicides to protect them from the attack by snow mold fungi such as Sclerotinia borealis, Typhula ishikariensis, T. incarnata, and Microdochium nivale. In general, opportunistic parasites may be suppressed by changing the environment, but this does not always apply to snow mold. Removal of snow, although not practical, removes the requisite environment for severe snow mold development, but allows for another problem, namely, freeze damage to the host plants. Fungicide sprays can protect plant tops from snow mold, but do nothing against abiotic damage. Microdochium nivale is a seed-borne pathogen (Hewett 1983) and may be controlled by seed treatment with fungicides or other disinfestation agents. Control of S. borealis requires annual fungicide applications since airborne propagules reach plants from outside the ﬁeld. Soilborne T. ishikariensis also requires annual treatment due to the presence of sclerotia in the soil, even in spots where fungicides were applied the previous year. Turfgrasses receive heavy fungicide treatments, which are applied diluted in water at typical rates of 100 ml/m2 in Canada and allowed at up to 1 L/m2 in Japan. The latter application rate appears very high, but is only equivalent to 1-mm precipitation. Such a limited amount of precipitation is unlikely to reach past the foliage and to the soil. Thus, chemical control is just to protect aboveground parts since belowground parts are not exposed to fungicides, and the common fungicides used do not have the ability to systemically travel downward in plants. This is why T. ishikariensis biotype B predominates in the turf 5.1 Chemical Control 113

(Matsumoto and Tajimi 1993). Chemical control of this fungus often fails in winter wheat fields due to its soilborne nature to cause damping-off (Saito 1982, 1988). Sclerotia of T. incarnata are also often observed on roots. However, recent trials revealed that tebuconazole and fluazinam were found to suppress soilborne infection even though they were sprayed onto plant tops (Yamana and Jinno 2014). Chemical control of snow mold fungi is made even more difficult with a prolonged interval between fungicide spray and the onset of snow cover, where the residual efficacy of the fungicides decreases with time. The amount of precipitation before snow cover was critical in reducing residual efficacy and differed among fungicides. It is, for example, recommended that fluazinam should be sprayed again after 150 mm of precipitation, and 85 mm for tebuconazole to control T. incarnata on winter wheat (Souma and Nagahama 2016). Cook and Hsiang (2003) found that granular PCNB applied 3 weeks before snowfall could still provide efficacy against Fusarium patch and snow molds on turfgrasses, whereas an application 6 weeks before snowfall (along with simulated rain applied as irrigation) showed reduced efficacy. Saito (2006) reviewed the history of chemical control of winter wheat snow mold in Japan and distinguished four stages in the history. Many chemicals have been used to diminish the damage so far, and some of them are no longer available due to environmental pollution, persistence in the environment, and the emergence of resistant fungal strains.

5.1.1 Bordeaux Mixture

Bordeaux mixture is a combination of a mixture of copper sulfate and lime used as a fungicide. Its effectiveness against snow mold was ﬁrst recognized in 1928 in Japan and has been used even after organic mercury was banned. It was reportedly effective especially against Pythium spp. and is still registered and considered a low-risk fungicide in many jurisdictions worldwide.

5.1.2 Organic Mercury

Phenylmercuric acetate was recommended for field application in late autumn and seed disinfestation of Microdochium nivale. Several organic mercurial salts were developed and became essential for sustainable production of wheat and prevention of snow mold in turf. However, the residue in rice grain attracted public concern and motivated the authority to ban the use of organic mercury for field application in 1970 and for seed disinfestation in 1973 in Japan. In North America, mercurial fungicides lost registration in the USA in 1987 and in 2000 in Canada. Organic mercury had a wide spectrum, with efficacy against many different snow mold 114 5 Control pathogens, and mercurial residues were still present in soils many years after usage was discontinued (Matthews et al. 1995).

5.1.3 PCP, PCNB, and Thiophanate-Methyl

The herbicide pentachlorophenol (PCP) was known to have efﬁcacy against Sclerotinia borealis snow mold since 1962, and the fungicide pentachloroni- trobenzene (PCNB) since 1964. These chemicals were used as alternatives to organic mercury after its removal, but were not as effective against M. nivale. Field experiments in 1970 and 1971 in Japan found the fungicide thiophanate- methyl to be more effective than PCNB against the Ascomycetes, S. borealis and M. nivale, and thiophanate-methyl was registered and combined with PCP as a major formulation for the control of snow mold in Hokkaido in 1973. PCP was, however, not effective against T. incarnata and often caused damage to wheat plants. PCP was also regarded as a water pollutant. Some strains of Microdochium were found to be resistant to thiophanate-methyl in 1981 (Tanaka et al. 1983) and against iprodione in 1982 in the USA (Chastagner and Vassey 1982) and in 1990 in New Zealand (Pennucci et al. 1990). PCNB lost its registration for use against turfgrass diseases in Canada in 2010 because of concerns with environmental safety, particularly the possibility of contamination by hexachlorobenzene, a suspected human carcinogen, and a precursor to PCNB.

5.1.4 New Fungicides and Mixture of Chemicals

In Japan, triadimefon, trichlophosmethy, and organic copper were found effective for control of especially basidiomycetous snow mold fungi to replace PCP by 1985, in addition to mepronil and flutolanil. Propiconazole was subsequently registered, and guazatine replaced thiophanate-methyl. Since multiple fungal species might occur simultaneously to cause snow mold, single applications of specific fungicides often did not work, where mixtures of multiple fungicides had greater efficacy. Fluazinam with a wide spectrum was then used, after that. Consequently, many types of fungicides have been used singly or in combination for foliar application. Guazatine for seed disinfestation of Microdochium nivale is also available in combination with organic copper in Japan. In North America, for turfgrass snow mold, the trend has been toward mixtures of chemicals such as Instrata (containing chlorothalonil, propiconazole, and fludioxonil) and Trilogy (containing iprodione, tryfloxystrobin, and triticonazole). In many snowy regions, both pink snow mold and gray snow mold can occur together, or it is not known which one will be dominant, so a mixture allows for activity against both types of diseases. 5.2 Biological Control 115

5.1.5 Resistance Activators

Plants utilize reserve materials to actively resist snow mold under snow (Kawakami 2004), but over the course of winter, the wherewithal to resist decreases. Plants possess many resistance mechanisms against diseases that are triggered following pathogen attack. There are also compounds with little direct antifungal activity that can pre-trigger and increase resistance against pathogens of plants. During activated resistance, a stimulus is recognized by the plant, and the signaling pathways eventually promote the expression of defense genes that may lead to the production of antimicrobial proteins (Edreva 2004). The enhanced resistance is expressed locally at the site of infection and, in some cases, systemically throughout the plant (Me´traux et al. 2002). Hsiang et al. (2013) summarized some research on activating disease resistance in turfgrasses against fungal pathogens, but found that although these resistance activators could signiﬁcantly reduce damage observed at snowmelt, they did not show as much efﬁcacy against winter diseases such as snow molds compared to summer diseases and speculated that the dormant or moribund tissues before or under snow cover did not respond strongly to resistance activation compared to actively growing tissues in summer.

5.2 Biological Control

Biological control of plant diseases was extensively studied from the mid-1970s, especially on soilborne diseases. Soilborne diseases were generally regarded as difﬁcult to control with fungicides except for the use of fumigation. Biological control using antagonists seemed promising as an alternative control measure. Antagonists were expected to establish in the soil and/or rhizosphere and to control diseases for a long time; however, most trials were unsuccessful due mainly to the failure in the establishment of antagonists, which had been effective in laboratory experiments. Most biocontrol agents failed to establish possibly because the soil habitat was colonized by diverse microorganisms and no niche was available for them. Most studies did not fully take into consideration of the soil habitat ecology with diverse and complex microﬂora as well as the possible ecological relationships of the indigenous and introduced microorganisms. Matsumoto (1985, 1988, 1993, 1998) discussed the ecological features of pathogen–antagonist interactions for snow mold. He distinguished the dormant and active phases of the pathogens and how they interacted with antagonists and their effects on them (Table 5.3). Antagonists parasitizing dormant structures such as sclerotia face little active opposition from the parasitized host in summer (Matsumoto and Tajimi 1985). In the active phase of pathogenic growth and attack under snow, interactions are more complex. Many antagonists compete with the pathogens for plant tissues, resulting in disease reduction. This interaction 116 5 Control

Table 5.3 Relationships between snow mold pathogens and antagonists Effect of pathogens on antagonistsa) Phase of pathogens Àþ Dormant NA Parasitism (mycoparasite) Active Competition (saprophyte, parasite) Parasitism (nematode) Modified from Matsumoto (1985) aAgents are shown in parentheses mechanism was exploited for biological control of snow molds but only partly elucidated for T. phacorrhiza (Wu and Hsiang 1999). The environment under snow plays a key role in the success of field experiments: microflora under snow is of relatively low abundance with only psychrophilic microorganisms showing activity. The habitat under snow is sparsely colonized, and many antagonists can establish if they are low-temperature tolerant. Low-temperature fungi such as T. phacorrhiza (Burpee et al. 1987; Matsumoto and Tajimi 1992; Wu et al. 1998), Acremonium boreale (Smith and Davidson 1979), and Humicola grisea (Sato et al. 1999) were reported as antagonists of snow mold fungi. No mycoparasites have so far been reported for snow mold fungi, but several nematodes are known to parasitize the hyphae of Microdochium nivale and Typhula ishikariensis and were regarded as promising agents (Shchukovskaya et al. 2012). A bacterium, Pseudomonas fluorescens from the hyphae and sclerotia of Typhula ishikariensis and T. incarnata, is also known to inhibit their mycelial growth (Matsumoto and Tajimi 1987). Biological control usually works only where disease occurrence is mild and is generally more expensive than comparable chemical control measures. Snow mold occurs on various crops with different economic significance. Of these, turfgrass is a strong candidate for biological control because of its high economic significance (Matsumoto 1998, Table 5.2). Although the level of biocontrol must be very high with no disease patches visible just after snowmelt and with little time available for recovery, intensive management on golf courses allows for expensive forms of disease control. Typhula phacorrhiza has been the most intensively studied as a biocontrol agent of snow mold. Typhula phacorrhiza is congeneric with T. ishikariensis and T. incarnata. Biocontrol with T. phacorrhiza has extensively been studied in Canada (Burpee et al. 1987; Lawton and Burpee 1990; Wu et al. 1998; Hsiang and Cook 2001). Although the mechanism of antagonism was not fully elucidated (Burpee 1994), the antagonism by T. phacorrhiza is ascribed to its ability to grow over a wide range of temperature and to utilize diverse carbohydrates (Wu and Hsiang 1999). Wu et al. (1998) collected several hundred isolates from across southern Ontario and tested them for ability to grow and produce sclerotia. Thirty of these strains were chosen for local field testing starting in 1994, and this testing was extended to sites across Canada. Effective control was obtained when the agent was formulated and applied at a rate of 20 g/m2 on a putting green of creeping bentgrass (Wu et al. 1998). Typhula phacorrhiza successfully established, exhibited residual control over the 5.2 Biological Control 117 following 2 years (Hsiang et al. 1999a), and was also effective against Microdochium nivale (Hsiang and Cook 2001). The biocontrol agent became registered with the EPA in the USA and the PMRA in Canada in 2011 by Agrium, although the company has not further developed the product commercially, citing high production costs. Independent of the work in Canada, Matsumoto and Tajimi (1992) confirmed the effectiveness of T. phacorrhiza on forage grasses and revealed variability in antagonism among 36 isolates collected throughout northern Japan. These isolates differed in the extent and range of antagonism against T. ishikariensis biotypes A and B on orchardgrass. While the damage by biotype B on orchardgrass plants was greatly alleviated by most T. phacorrhiza isolates regardless of their locality, those from the snowy areas where biotype A prevailed were strongly antagonistic to both biotypes. An effective isolate could increase herbage productivity of the first cut by 26.5 % in a perennial ryegrass field where biotype A was prevalent and produced many sclerotia on plant debris. The antagonist tested originated from the Tenpoku district, northern Hokkaido, where snow cover lasted for more than 140 days p.a. This implied that biological control occurred spontaneously there. Despite heavy snow cover, perennial ryegrass is grown regularly in Tenpoku (Tezuka and Komeichi 1980). Humicola grisea is effective as a snow mold antagonist and also shows residual effect (Y. Sudate, personal communication) similar to T. phacorrhiza. The fungus is thought to be present within plant tissues as an endophyte, but little is known about its survival. H. grisea is known to produce a heat-tolerant substance to destroy the cell wall of Microdochium nivale (Sato et al. 1999). Acremonium boreale was observed to be antagonistic to snow mold fungi when paired in culture (Smith and Davidson 1979), but failed to suppress the disease by T. ishikariensis biotype B on orchardgrass (Matsumoto and Tajimi 1992). A Neotyphodium endophyte that was known to enhance tolerance to drought and diseases predisposed infected plants to attack by T. ishikariensis (Wa¨li et al. 2006). A bacterium, Pseudomonas fluorescens, associated with the hyphae and sclerotia of Typhula spp. and Sclerotinia borealis, suppressed mycelial growth in culture (Fig. 5.2, Matsumoto and Tajimi 1987) and was found to effectively control T. ishikariensis biotype B on golf course turfgrass (Oshiman et al. 1998). The effect was ascribed to an antibiotic produced by the bacterium (Oshiman et al. 1996). In eastern Canada, sclerotia of T. ishikariensis collected just after thawing were often contaminated with bacteria and failed to survive the dormancy in summer (E. Schneider, personal communication). In a sclerotial survival test, buried sclerotia of T. ishikariensis and T. incarnata were found to have low survival rates in summer (Hsiang, unpublished). Mycoparasites can penetrate sclerotial rinds; however, bacteria are not likely to do so in summer and may normally be excluded from sclerotia when they are produced under snow. This phenomenon of sclerotial colonization before summer maturity and dormancy should be studied in greater detail to reveal the dynamics of field sclerotial survival and possibly to uncover a clue to natural biological control. 118 5 Control

Fig. 5.2 Antagonistic bacterium associated with the sclerotia of Typhula incarnata. Upon isolation of snow mold fungi, contaminant bacteria often inhibit mycelial growth on isolation media (arrow)

5.3 Cultural Control

5.3.1 Cultural Practices

Crops grown in cold-temperate regions all require a certain level of snow mold resistance (e.g., Jamalainen 1974; Tezuka and Komeichi 1980; Kunii 1987; Litschoko et al. 1988). Soil fertility and availability of nutrients are also critical factors affecting winter survival (Årsvoll 1977; Freymand and Kaldy 1979). In forage crops, many studies in Hokkaido, Japan, concentrated on the interactions between cultural practices in autumn such as fertilizer application and the ﬁnal date of mowing and agronomical traits such as winter hardiness to improve the yield of ﬁrst cutting (e.g., Kondo 1973; Sakamoto and Okumura 1973; Sakamoto and Okumura 1974; Noshiro and Hirashima 1974, and Yamagami and Okumura 1976). These studies revealed that winter hardiness was most greatly reduced when grasses were harvested in early October, exhausting much reserve materials for subsequent regrowth, and that autumn fertilization enhanced winter hardiness and contributed yield increase the following spring. However, in winter wheat, autumn fertilizer application resulted in decreased winter hardiness (e.g., Kunii 1980b). What caused the contradiction? It seems that forage crops require more nutrition to attain the appropriate level and that winter wheat had more than necessary (Fig. 5.3). In turfgrasses, applications of fertilizer with soluble nitrogen are known to delay dormancy, and enhanced susceptibility to snow molds has been observed (Smiley et al. 2005). Recommendations are made for control of gray and pink snow molds 5.3 Cultural Control 119

forage crop winter wheat winter hardiness winter low low high

deficient appropriate excessive Nutritional level

Fig. 5.3 Effect of fertilizer application in autumn on the winter hardiness level of forage crops and winter wheat. Forage crops are grown at low fertilizer levels, and their winter hardiness is improved by fertilizer application. However, in winter wheat, fertilizer levels and fertility are high, and excessive application reduces winter hardiness on turfgrasses including balanced soil fertility, avoiding heavy fall nitrogen fertilization, prevention of snow cover into the growing season, and promoting drainage and drying at snow mold melt (Smiley et al. 2005). Cultural techniques may reduce the severity of snow mold diseases, but cannot prevent them entirely (Hsiang et al. 1999a). Snow mold resistance is also affected by seeding date (Bruehl 1967; Bruehl et al. 1975; Kunii 1980a). In other words, when to seed is crucial to snow mold control. While late sown, small plants of winter cereals are more resistant to Pythium spp. (Takamatsu et al. 1985), early sown, large plants are generally resistant to snow mold (Bruehl and Cunfer 1971; Kunii 1980a). During the experiments to determine the proper seeding dates in Washington, USA, Bruehl et al. (1975) found that very small wheat plants which had been sown late in mid-October were free from the attack of Microdochium nivale and Typhula spp. (Fig. 4.2).

5.3.2 Cultivar

Alfalfa in Japan

Alfalfa cultivation started in the 1950s in Hokkaido. Cultivated areas steadily increased up to more than 10,000 ha in 1986 and then plateaued; however, suitable alfalfa cultivars were not available in eastern Hokkaido, the largest dairy farming area. The following describes how agricultural scientists made efforts to introduce alfalfa there. Eastern Hokkaido is characterized by severe soil freezing in winter and by cool, damp summer, hindering the introduction of alfalfa. Takeda et al. (1990) ﬁrst tested freeze tolerance, using 60 cultivars from North America and Europe. The cultivars were grown in the ﬁeld to compare winter survival of 120 5 Control

first-year seedlings and other agronomic traits. Canadian cultivars with high freeze tolerance and strong autumn dormancy suffered from soil heaving more seriously than moderately tolerant cultivars (Takeda and Nakajima 1991a). There was a negative correlation between freeze tolerance and soil heaving. They concluded that vigorous tap root growth was essential for first-year seedlings to establish in eastern Hokkaido. There was, however, a problem that hampered the growth of alfalfa seedlings. The summer climate in eastern Hokkaido predisposed alfalfa to Lepto leaf spot caused by Leptosphaerulina briosiana, reducing winter hardiness. Cultivars screened were all from the USA and western Canada with dry summers, and these were unable to resist the disease (Takeda and Nakajima 1991c). The vigorous growth of 1-year-old seedlings in cool and damp summers was identified as the most critical feature in eastern Hokkaido for winter survival. The significance of Lepto leaf spot was subsequently revealed after fungicide testing. Young seedlings of three cultivars ranging in non-treated winter survival rates were sprayed with thiophanate-methyl to control Lepto leaf spot and compared in terms of percent winter survival (Fig. 5.4, Takeda and Nakajima 1991c). The percentage of plants killed for cultivars rated as low survival was 45 % without chemical control, and this was decreased to 25 % with fungicide application. About 1 % of plants failed to survive in the cultivars rated as winter hardy. Freezing resistance was more essential for established plants after 2 years, and the resistance to Lepto leaf spot was also essential (Takeda and Nakajima 1991b). Typhula ishikariensis biotype A was seldom found during their field experiments from 1988 to 1992.

fungicide sprayed

no spray percent dead plants percent

low high winter hardiness

Fig. 5.4 Improvement of winter survival in ﬁrst-year seedlings of alfalfa cultivars with low winter hardiness by fungicide application to control Lepto leaf spot in eastern Hokkaido (Takeda and Nakajima 1991c) 5.3 Cultural Control 121

In 1993, a total of 3,150 seedlings were transplanted in the ﬁeld to reveal that the resistance to Lepto leaf spot was highly heritable (Takeda et al. 1998). The selected plants were naturally infected by T. ishikariensis biotype A in the 1995–1996 winter and again more seriously damaged by the fungus in the spring of 1999. The fungus was known to reduce the productivity and persistence of alfalfa in snowy regions (Komatsu 1983), but alfalfa was free from the attack in eastern Hokkaido. T. ishikariensis biotype B attacking only monocots used to prevail in eastern Hokkaido (Matsumoto et al. 1982), and the recent climate changes have favored the occurrence of biotype A. The effect of the fungus was evident when the yield of Kitamidori alfalfa was compared at two experimental sites. Whereas the 1993 crop was as high as that in 1982 in Sapporo with consistently much snow cover, the yield reduction was 25 % in 1993 in Kunneppu where snow cover increased during the period (Fig. 5.5). Resistance to leaf diseases, including Lepto leaf spot, was one of the breeding objectives in Hokkaido National Agricultural Experiment Station (currently Hok- kaido National Agricultural Research Center), Sapporo, with sunny, dry summers and where T. ishikariensis biotype A predominated due to consistent deep snow. Collaboration with Konsen Hokkaido Prefectural Agricultural Experiment Station, using selected strains, produced Lepto leaf spot-resistant Hisawakaba, which was registered as a recommend cultivar in 1994 (Yamaguchi et al. 1995). The development of Hisawakaba made alfalfa cultivation possible in eastern Hokkaido. Haruwakaba, improved in productivity, persistence, and winter hardiness, was subsequently released (Hiroi et al. 2005). Ceres registered in 2005 is characterized by the vigorous growth of lateral roots to avoid soil heaving as well as Lepto leaf spot resistance (Takayama 2005; Yatsu 2009). yield (dry (dry matter kg/a) yield

Kun-neppu Sapporo

Fig. 5.5 Change in yield of Kitamidori alfalfa between 1982 and 1993 in Kunneppu and Sapporo, Hokkaido, Japan. Typhula ishikariensis biotype A has recently prevailed in Kunneppu due to climate change, while the fungus has consistently been present in Sapporo (Modiﬁed from data of Ueda et al. 1985; Yamaguchi et al. 1995) 122 5 Control

Winter Wheat in Japan

Extensive cultivation trials were first made in 1954, using Hokuei winter wheat which was first recognized as excellent in Hokkaido (Hasebe et al. 1970). The scheme was designed based on the finding of Tomiyama (1955) that Sclerotinia borealis occurred in eastern Hokkaido and Typhula spp. in western Hokkaido to accumulate the data on fertilization level for each area (Fig. 5.6). Consequently, winter wheat rapidly increased in acreage after 1975: Hokuei was followed by Horoshiri-komugi, which had a measurable level of snow mold resistance but was not suitable for noodle flour. A breeding project was then initiated to improve quality, and Chihoku-komugi was released in 1981 (Ozeki et al. 1987). Chihoku- komugi was inferior in resistance to Typhula spp. and Microdochium nivale. However, Chihoku-komugi was relatively freeze tolerant, and supponuke snow mold caused by Athelia sp., which had been prevalent in the Sclerotinia area in eastern Hokkaido, practically disappeared with the introduction of Chihoku- komugi (Simizu 2014). Then, snow mold resistant Hokushin (Yanagisawa et al. 2000) replaced Chihoku-komugi, and Kitahonami with the same snow mold resistance level was registered in 2006 after improvement in the resistance to rust and preharvest sprouting (Yanagisawa et al. 2007). Despite only having moderate resistance to snow mold, Yumechikara with resistance to wheat yellow mosaic virus was registered for very high-protein flour in 2008 (Tabiki et al. 2011). Optimal infection by the virus occurs at 10–15 Cin autumn (Ohto 2005), reducing winter hardiness of overwintering seedlings. In an experimental field naturally infested with wheat yellow mosaic virus, susceptible cultivars were all badly damaged by Typhula incarnata, and Yumechikara winter

Fig. 5.6 Damage of winter wheat caused by Sclerotinia borealis may be minimized by seeding at the right time and use of appropriate fertilizer levels 5.3 Cultural Control 123

Fig. 5.7 Spring ﬂush of Yumechikara winter wheat (lower right plot) in a ﬁeld infested with wheat yellow mosaic virus. Typhula incarnata proliferated on susceptible cultivars wheat was free from snow mold as well as viral damage (Fig. 5.7). Similar interactions were reported in Belgium in barley between T. incarnata and barley yellow dwarf virus (Cavelier and Maroquin 1978).

Winter Wheat in North America

According to Murray et al. (1999), snow mold (mostly incited by Typhula spp. and Microdochium nivale) occurred with greater severity on winter wheat in Washington State between the 1940s and 1970s and gradually declined. However, snow mold still caused substantial damage in some places in 1995 and 1996 and remains as important where snow cover lasts for more than 150 days. Snow mold control research was conducted mainly using cultural practices such as seeding date, crop rotation, fertilizer application, snowmelt acceleration with carbon black, and crop residue management. Resistance breeding was then recognized as the main research objective. Various fungicides were examined from 1944 to the late 1950s. Only organic mercury remained as promising, but chemical control was not accepted because of the cost and unpredictable onset of snow cover. No fungicide is registered against snow mold on wheat in the USA (Murray et al. 1999) or in Canada (Gossen et al. 2001). Resistance breeding extensively started from 1960, and Sprague winter wheat was bred in 1972, followed by Edwin and Bruehl in 1998 and 1999, 124 5 Control respectively. The wheat cultivar Bruehl is not as resistant as Sprague but more productive. Resistance breeding and cultural practices are essential for snow mold control also in the states of Alberta, Manitoba, and Saskatchewan, western Canada (Fowler 2002). Fungicides are not available for winter wheat to control snow mold, and snow cover is, on the contrary, considered as useful since it protects seedlings from freeze damage.

5.4 Conclusions

Snow mold no longer seems to be a serious issue for agricultural crops under normal environmental conditions, since cultural practices have been developed against snow mold, i.e., selection of appropriate cultivars (Tezuka and Komeichi 1980; Kunii 1987) and seeding at the right time (Kunii 1980a and Miyamoto et al. 1989), as well the use of fungicide-treated seed (Murray et al. 1999). And for the most part, cultural and fungicidal practices allow for adequate control of snow molds on turfgrasses. These techniques obviously alleviate the damage of snow mold, but may not be as protective under sudden environmental changes. In 1998, for example, snow cover occurred much earlier, and farmers could not spray fungicides for winter wheat in Abashiri, eastern Hokkaido. A subsequent outbreak of Typhula ishikariensis biotype A led farmers to abandon 30 % of the ﬁelds (Fig. 5.1, Matsumoto et al. 2000). Fungicides are used on winter wheat every year in Japan preventively. The amount of fungicides sprayed per ha is 1,340 ml which costs 6,582 yen (ca. 55 US dollars, according to Hokkaido Department of Agriculture, Anonymous 2005). Since winter wheat is grown over 100,000 ha, fungicide use is extensive. Perhaps Hokkaido is the only place in the world where fungicides are foliar applied to control snow mold of winter wheat. It is about time to reconsider such practices.

References

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Abstract Climate change should facilitate agricultural production further north in northern temperate regions, where temperatures and growing season duration were previously limiting. Cultivars with higher productivity and quality will likely be introduced in these areas. However, with warmer temperatures, the period available for hardening off may be shortened, and furthermore, plants may not have been selected for the combinations of warmer temperature and shorter day length for the winter hardening process. Also, the introduction of new crops especially in high latitudes may not be obtained without the improvement in photoperiod response. Snow cover lasting for months may partly be replaced by a long spell of wet and cool conditions. Facultative snow mold fungi such as Microdochium nivale and Pythium spp. may become even more important since they can prevail under conditions with greater magnitude of climatic variations and less seasonal predictability. Biodiversity plays a key role in natural ecosystems to cope with such phenomena, but modern agriculture has focused on homogeneity for efﬁciency. It is necessary to reconsider the role and use of heterogeneity and diversity into management of snow molds. Climate change poses a great challenge for farmers and scientists to adapt in an area of less predictability.

Diseases result from the interactions of three factors, i.e., pathogen, host, and environment. Climate change is predicted to reduce the incidence of snow mold (Boland et al. 2004). In fact, snow mold has disappeared in some areas of Washington, USA (Murray et al. 1999) and southern Norway (K. Årsvoll, personal communication) and is increasingly less predictable in southern Ontario in the last 20 years necessitating the use of tarps or green covers to simulate snow cover for snow mold fungicide tests (Hsiang unpublished). Since the development of snow mold relies on snow cover, the duration of cover is the key factor affecting severity. Its effect became manifest by the outbreak of Sclerotinia borealis in eastern Hokkaido, Japan, in the winter of 1974–1975: late onset of persistent snow cover predisposed plants to the attack and prolonged thawing extended fungal activity. We have not suffered such a disaster ever since. This is largely due to recent climate change, as well as to the development of countermeasures. How snow mold scientists have responded to the change was addressed in Chap. 5 and Matsumoto and Hoshino (2013).

Climate change should facilitate agricultural production further north in northern temperate regions, where temperatures and growing season duration were previously limiting. Cultivars with higher productivity and quality will likely be introduced in these areas, but they are often also less winter hardy. Tronsmo (2013) warned that in high latitudes like Norway, the introduction of new crops may not be obtained without the improvement in photoperiod response. With decreasing significance of S. borealis, less freeze tolerant but more productive perennial ryegrass (Lolium perenne) is being cultivated in pastures of eastern Hokkaido. Recent mild winters with deep snow cover there promoted trials to cultivate overwintering carrots, resulting in a new disease caused by Typhula variabilis and T. japonica (Ikeda et al. 2015). Facultative snow mold fungi such as Microdochium nivale and Pythium spp. may become even more important as snow cover is diminishing, since long duration snow cover may be replaced by long duration wet and cool conditions or more numerous alternations between snow cover and snow melt which is favorable for these pathogens. Also, the occurrence and duration of snow cover will be more unpredictable. In the winter of 2015–2016, Pythium spp., including P. iwayamai, caused substantial damage to barley in Fukui, central Japan, facing to the Sea of Japan, where they have so far had mild winters for decades but had 101 days with snow cover that winter (Yamaguchi et al. 2015). This outbreak illustrates the risk of facultative snow mold fungi which can persist as other agents than pathogens in mild winters. We can attempt to adjust to regular, predictable climate changes to minimize the reduction in productivity by cultural practices and the intrinsic capacity of crops. However, the rate of climate change may be increasing with greater magnitude of variations and with less seasonal predictability. Biodiversity plays a key role in natural ecosystems (Kreyling 2013), but modern agriculture, on the contrary, has focused on homogeneity for efficiency, and it is necessary to reconsider the role and use of heterogeneity and diversity into management of snow molds. Forage crops are cross-pollinated to develop cultivars, and consequently each cultivar consists of individuals differing in snow mold resistance. Cultivars are evaluated in terms of yield, heading date, etc. The range of variability, i.e., resilience to environmental fluctuation, is an important trait to cope with changing environment and requires further research and selection. It should also be interesting to investigate how mixtures of grasses and legumes or even cultivar mixtures react to environmental change to provide resilience. Such a scheme should work in forage crops since the plants may adjust and compensate for snow mold damage to obtain sustainable herbage production. Another aspect to consider is that with warmer temperatures, the period available for hardening off may be shortened, and furthermore, plants may not have been selected for the combinations of warmer temperature and shorter day length for the winter hardening process. Cultivar selection processes may need to be adjusted to account for potential future climate changes. For snow mold disease control in turfgrasses, the increasing societal resistance against fungicide use will necessitate the development of more resistant cultivars or improved cultural techniques to decrease the effects of snow mold damage. There is References 131 also extensive new research into the use of agents which have little direct effect against pathogens, but are able to cause the host plants to increase their inherent resistance against pathogen attack. Climate change poses a great challenge for turf managers and turf scientists to adapt in an area of less predictability. Winter wheat is an inbreeding crop, and snow mold resistance is maintained through breeding. Crop rotation should also be practiced. Resistance breeding (e.g., Bertrand et al. 2009; Bertrand and Castonguay 2013; Rognli 2013), combined with the elucidation of resistance mechanisms (e.g., Gaudet and Laroche 2013; Yoshida and Kawakami 2013), still remains important to adapt to the changing environment and to minimize the damage from snow mold using environmentally sustainable practices.

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A Carbohydrate, 42, 60, 76, 84, 101, 116 Abiotic factor, 1, 2, 47, 103 Carbon dioxide (CO2), 11, 84 stress, 103 Carotene, 15 Antagonist, 8, 9, 75, 109, 111, 115, 118 Cellulose, 48, 84 Antibiotic, 102, 103, 117 Ceratobasidium gramineum,26 Antifreeze, 24, 44–46, 48, 102, 103 China, 80 protein, 44–46, 48 Chitinase, 102, 103 Archaeological site, 73–75 Chytridiomycetes, 4 Ascomycete, 4, 9, 24, 45, 47, 114 Climate change, 56, 121, 129–131 Ascorbic acid, 15 Clone, 72 Ascospore, 5, 34, 35, 76, 77, 82, 99 Cold Athelia sp., 4, 26, 45, 47, 85–87, 122 acclimation, 95 shock protein, 103 B temperate region, 2, 10, 11, 118 Collectivism, 25 Bacteria, 9, 11–14, 47, 103, 117, 118 Compatibility, 27, 28, 40, 42, 56, 66–68, Barley, 25, 71, 82, 84, 103, 123, 130 85, 87 yellow dwarf virus, 123 Conidia, 81, 82 Basidiocarp, 39, 40, 42, 43, 57, 58, 61–64, Coprinus psychromorbidus, 4, 44, 46, 87 71–73, 75, 87 Coprinus sterquilinus,23 Basidiomycete, 4, 9, 26, 27, 45, 47, 56, 58, 86, 87 Cornmeal agar, 15, 16 Basidiospore, 5, 32, 40, 42, 43, 57, 61, 62, 71 Critical period, 100 Bentgrass, 75, 83, 86, 116 Crop rotation, 5, 72, 111, 123, 131 Β-1-3 glucanase, 103 Crown, 2, 3, 31, 87, 100 Biological control, 109–111, 115–118 Cryophile, 13 Biological species, 67–71 Cryosphere, 12–16, 24, 25, 43, 44, Biomass, 6, 7, 10, 11, 24, 42, 48, 110 60, 85 Biotic factor, 3, 47, 103 Cultural practice, 25, 32–33, 82, 96, 97, Breeding, 32, 80, 83, 86, 96, 98, 104, 111, 109–111, 118, 123, 124, 130 121–124, 131 Cutting, 96, 110, 118 ﬁnal, 96, 110 C Cyanide, 87 Canada, 2, 10, 44, 56, 69, 75, 76, 81, 86, 87, 110, Cystatin, 103 112–114, 116, 117, 120, 123, 124 Cytoplasmic (in)compatibility, 27

D Germany, 61 Damping-off, 11, 25, 40, 41, 61, 86, 113 Global warming, 2 Decay column, 26 Glucanase, 102, 103 Defensin, 103 Godzilla patch, 31 Dehardening, 97, 103 Golf course, 3, 5, 33, 42, 60, 67, 71, 110, Dematiaceae, 72 111, 116, 117 Dicot, 69, 75, 76, 79 Grass, 2, 4, 8, 29–32, 34, 56, 59, 75, 77, 78, Dikaryon, 57, 58 83, 86, 100, 101, 116, 117 Di-mon mating, 57, 58 Gymnosperm, 69 Disturbance, 6, 7, 32–34, 110 Diversity, 8, 11–12, 14, 29, 32, 33, 64, 69, 71, 81, 85, 129, 130 H D-mannitol, 14 Habitat, 6, 8, 11, 13, 24, 25, 32–34, 36–38, Dormancy (dormant), 5–7, 24, 25, 32, 34, 40, 42, 43, 45, 47, 48, 56, 63, 65, 56, 72, 95, 110, 115–118, 120 68, 71, 72, 85, 111, 115, 116 Hardening, 47, 96, 97, 99–103, 110, 130 Hard seed, 38 E Head blight, 25, 82 Ecotype, 40, 42, 68 Heat shock protein, 44 Endophyte, 117 Hedging, 43 Enzyme, 44, 46, 103 Heterothallism, 83 High birth and high mortality, 63 High ridge culture, 78, 79 F Hokkaido, 4, 5, 29, 30, 33, 70, 72, 73, 75, Facultative snow mold, 24–26, 45, 83, 129, 130 77–81, 83, 85, 86, 96, 114, Fairy ring, 32 117–122, 124, 129, 130 Fatty acid, 24, 48 eastern, 4, 5, 29, 72, 78, 85, 86, 119–122, Fertilizer, 96, 110, 118, 119, 122, 123 124, 129, 130 Feruloylagmatine, 103 Homothallism, 83 Finland, 2, 10, 57 Host-parasite interaction, 12, 83, 96 Foot rot, 26 Hydroxycinnamic acid amide, 103 Forage crop, 2, 32, 87, 96, 100, 110, 111, Hyphal fusion, 28, 48 118, 119, 130 Freeze tolerance (tolerant), 24, 43–48, 98, 100, 103, 119, 120 I Freezing, 2, 3, 5, 7, 13, 24, 34, 44–48, 68, Iceland, 2, 43, 67 75, 77, 78, 96, 101, 119, 120 Incompatibility, 27, 29, 56, 58, 62, 64, 65, Fructan, 48, 101, 102 69–71 exohydrolase, 102 Individualism, 23–28 Fructiﬁcation, 12, 14, 43, 75 Inoculum potential, 27, 34, 43, 72 Fructose, 102 Iran, 61 Fruit body, 26 Italian ryegrass, 61, 63, 81 Fungicide, 5, 42, 71, 96, 109–115, 120, 123, 124, 129, 130 Fungus, 12–14, 16, 24–26, 29, 32, 34, 37, 38, K 40, 45, 47, 48, 60–63, 66, 69, 71–73, KCl, 14, 47 75–83, 85–87, 96, 102, 110, 113, Konsen area, 121 117, 121 Korea, 12, 80

G L Generalist, 34 Landbridge, 30 Genet, 26, 29 Lepto leaf spot, 120, 121 Index 135

Leptosphaerulina briosiana, 120 P Life history, 5–6, 24, 32, 38 PAL. See Phenylalanine ammonia lyase (PAL) Linoleic acid, 48 Patch, 12, 25, 28, 29, 31, 85, 86, 110, 113, Linolenic acid, 48 116 Lipid transfer protein (LTP), 103 PDA, 14–16, 34, 45, 47 Litter, 10–11, 86 Permafrost, 12, 47, 48 Low temperature basidiomycete (LTB), 27, 46, Persistent snow cover, 5, 6, 24, 78, 87, 129 86, 87, 99 Phenylalanine ammonia lyase (PAL), 103 Pink snow mold, 4, 7, 9, 25, 76, 81–83, 114, 118 M Poland, 43 Mating incompatibility factor, 29, 56–58, 62, Polar region, 12, 13, 43, 48, 84, 85 64, 65, 70, 71 Population structure, 29–33 Meadow fescue, 33, 34, 77, 78, 83, 98 Pathogenesis-related (PR) protein, 102 Medula, 72, 74, 76, 77 Predictability, 6, 24, 33, 43, 66, 130, 131 Mesophile, 8, 44, 76, 80 Primary colonizer, 12 Microdochium nivale, 2, 4, 5, 7, 9, 24–28, 44, 45, Propagule, 24, 32, 38, 43, 48, 110, 112 47, 48, 81–83, 98, 99, 102, 112–114, Protein, 24, 44–46, 48, 81, 102, 103, 115, 122 116, 117, 119, 122, 123, 130 Pseudomonas ﬂuorescens, 116, 117 Minimal temperature, 100 Psychrophile, 12–14, 16 Mon-mon mating, 57, 58 Pythium Monocot, 69, 75, 121 P. iwayamai, 4, 16, 26, 45, 47, 48, 84, 130 Monokaryon, 57, 58, 62, 64, 67, 68, 71 P. okanoganese, 4, 16, 84 Moribund tissue, 79, 104, 115 P. paddicum, 4, 26, 48, 84 Mortality, 32, 63, 65 P. polare, 43, 48, 85 Moss, 12, 43, 48, 85 P. ultimum, 84, 85 Mycelial compatibility group (MCG), 25, 27–33, 72 R Mycelial growth minimal, 100 Racodium therryanum, 12, 26, 45, 47 optimal, 13, 47 Red colver, 80 Mycoparasite, 5, 32, 62, 72, 116, 117 Reserve material, 9, 38, 42, 43, 47, 48, 96, 98, 100–102, 115, 118 Resilience, 130 N Resistance, 4, 5, 9, 48, 77, 80, 95–104, 115, 118–124, 130, 131 Niche, 24, 59, 60, 115 Resource, 8, 24, 25, 59, 60 Nitrogen, 11, 118, 119, 124 Rhynchosporium secalis,26 Norway, 2, 3, 14, 43, 45, 46, 66, 68, 70, 71, Rind, 38, 66, 67, 72–75, 117 81, 129, 130 Russia, 29, 34, 45, 67–69, 71, 75, 80

O S Obligate snow mold, 24, 26, 45 Sackhlin, 29, 30 Oleic acid, 48 Scald, 13, 26 Oomycete, 4, 45, 47 Scandinavia, 44 Oospore, 16, 48 Sclerotial Opportunistic infection, 6, 72, 104 germination, 5, 14, 16, 34–37, 39 Optimal temperature, 7, 13, 14, 16, 80, 87 size, 34, 38, 42, 76 Orchardgrass, 33, 34, 61, 64 Sclerotinia Osmophily, 24, 46, 47 S. borealis, 4, 5, 14, 26–28, 34, 35, 38, Osmotic pressure, 14, 46 44–47, 56, 76–79, 86, 98, 99, 112, Outbreeder, 56, 71 114, 117, 122, 129, 130 Outbreeing, 56 S. intermedia,79 Oxidation, 15, 44 S. nivalis, 4, 16, 45, 47, 76, 79–81 136 Index

Sclerotium(a), 35, 38–42, 60, 61, 65, 66, 68, 70, T. incarnata, 2, 4, 5, 26, 27, 29, 32, 34, 71, 73, 74 36, 38, 42, 43, 45, 47, 56, 57, 59–63, Secondary sclerotia, 34, 35, 71 65, 69, 71, 75, 76, 84, 96, 98, 99, Seed, 82, 98, 111–114, 119, 124 112–114, 116, 117, 122, 123 Seeding date, 96, 98, 110, 119, 123 T. ishikariensis, 15, 26 Sendai, 32, 38–40, 42 biotype A, 29, 32, 33, 56, 60, 67, 69, 71, Snow cover 72, 120, 121, 124 duration, 33, 40, 129, 130 biotype B, 28, 29, 31, 32, 38, 40, 42, index, 38, 39 59–61, 75, 111, 112, 117, 121 Snow mold biotype B small sclerotium form, 38, 40, facultative, 25, 26, 45, 79, 83, 84, 130 41, 61, 66, 71 grey, 45 group I, 45 obligate, 24, 26, 45 group II, 70 pink, 4, 7, 9, 25, 76, 81, 82, 114, 118 group III, 15, 45, 47, 67, 70 sclerotinia, 4, 6 T. phacorrhiza, 26, 34, 35, 60, 75, 76, 110, supponuke, 122 116, 117 Snow tolerance (tolerant), 6, 46, 47, 69, 70, 88, 96, 98, 112, 116, 120, 122, 130 Soil U borne, 24, 40, 112, 113, 115 U.K, 61 fertility, 100, 118, 119 U.S.A., 2, 10, 11, 57, 60, 65, 67, 71, 82, 84, 97, freezing, 77, 78, 119 98, 110, 113, 114, 117, 119, 120, frost, 44, 46 123, 129 heaving, 82, 120, 121 Sorbitol, 14, 47 Specialist, 34 V Speckled snow mold, 4, 5 Varietal difference, 9, 96, 99 Sporocarp, 5, 6, 36, 74 Versatility, 60, 61, 63, 65, 83, 87 Spring wheat, 98 Strategy, 5, 7, 24, 33, 43, 45, 56, 63, 83 Stress, 4, 6, 7, 24, 103 W Succession, 12, 84, 85 Water Super mycelial compatibility group (MCG), bind, 48, 103 25, 27, 33, 72 content, 100, 101 free, 101 Wheat T spring, 98 Teleomorph, 81 winter, 2, 5, 8, 9, 32, 38, 40, 59, 61, 71, 72, Temperate region, 2, 118, 130 75, 78, 81, 82, 85–87, 96, 98–103, Temperature, 6, 8, 9, 11–14, 16, 26, 27, 35, 113, 118, 122–124, 131 36, 40, 41, 43–46, 48, 56, 60, 68, 70, yellow mosaic virus, 122, 123 79–81, 85–87, 100, 101, 103, 110, Winter 116, 130 cereal, 2, 13, 24, 25, 48, 81, 84, 87, 96, 101, Tester, 57 110–112, 119 Timothy, 5, 28, 34, 77, 78, 80, 96, 97, climate, 2, 4, 29, 87 100, 101 damage, 2, 7 Total nonstructural carbohydrate (TNC), 60, 101 hardiness, 2, 59, 102, 104, 110, 118, Tundra, 44 120–122 Turfgrass, 2, 3, 25, 28, 29, 31, 33, 69, 75, 81, kill, 2 82, 87, 110–119, 124 survival, 2, 46, 100, 103, 110, 118–120 Typhula FW, 66 T. berthieri,61 Z T. graminum, 57, 60, 61 Zoospore, 16, 48, 84 T. idahoensis, 44, 57, 66, 67, 69, 71 Zygomycete, 4, 11