This research is supported by  the International Program of the U.S. National Science Foundation (grant INT-9902175) and the Department of Science & Technology of the  Government of India  Research project  Investigators The Fernow Experimental Forest in West Virginia Pauri-Gahrwal area in Uttaranchal Narkanda and Dharamshala areas in Himachal Pradesh Fungi from Uttaranchal and Himachal Pradesh

Project Summary Comprehensive studies of the communities of ectomycorrhizal fungi associated with particular forest types are generally lacking.  Although geographically separated, the forests of eastern North America and northwestern India share a number of ecologically important ectomycorrhiza-forming tree genera in common.  Prominent examples include Quercus(oak), Pinus(pine), Picea(spruce), and Abies(fir).  The objectives of the proposed research project are (1) to compare the species composition, species diversity, taxonomic diversity, fruiting phenology, and sporocarp productivity of selected groups of ectomycorrhizal fungi (with particular emphasis being placed on members of the Amanitaceae, Boletaceae, Cantharellaceae, and Russulaceae) associated with upland coniferous and oak-dominated forest communities in northwestern India and the mid-Appalachians of the eastern United States and (2) to elucidate, using the morphological, anatomical, and other types of data available from direct comparisons of material collected from study sites in eastern North America and northwestern India, the biological and taxonomic relationships that exist between populations of fungi that appear to represent the same taxonomic entity (i.e., morphospecies) in these two different regions of the world.  The primary study sites to be used in the proposed research are the Narkanda and Dharamshala areas of Himachal Pradesh and the Garhwal area of Uttar Pradesh in northwestern India and the USDA Fernow Experimental Forest near Parsons in Tucker County, West Virginia, in the United States.     Introduction Objectives Preliminary Research Methods Significance of the Proposed Research

 

Introduction

          The dependence of certain forest trees on ectomycorrhizal fungi has long been known, but only recently has the biological nature of the fungus-tree association been examined in some detail.  On the basis of the studies that have been carried out, it would appear that ectomycorrhizal fungi have a limited capability to enzymatically degrade the complex carbohydrates of most organic detritus and, instead, rely on their hosts (i.e., forest trees) for their energy needs.  In return, they are able to take up such metabolites as phosphorus, nitrogen, sulfur, and zinc from thesoil and translocate these to their host.  In this way, they greatly extend the functional root system of the host (Allen 1991).  Most ectomycorrhizal fungi are basidiomycetes, with Amanita, Cortinarius, Lactarius, Russula,and Suillusamong the best known ectomycorrhizal genera (Hacskaylo 1972).  Ectomycorrhizal associations are widespread, particularly in temperate regions, and involve many of the ecologically important trees making up theforests characteristic of these regions.  Prominent examples include hemlock (Tsuga), spruce (Picea), pine (Pinus), fir (Abies), willow (Salix), oak (Quercus), birch (Betula), and beech (Fagus).
          Although geographically separated, the upland forests of eastern North America and northwestern India share a number of ecologically important ectomycorrhiza-forming tree genera in common.  In both regions, Piceaand Abiesare the usual dominants in forests found at higher elevations (generally >1200 m in the mid-Appalachians of eastern North America and >2500 m in northwestern India), whereas Pinusand especially Quercusare often major components of forests located at somewhat lower elevations (Kumar et al. 1990; Adams and Stephenson 1991; Stephenson and Adams 1991; Stephenson and Saxena 1994).  Relatively few studies have considered the ectomycorrhizal associations represented in these forests, and most such studies have been rather limited in scope.  In northwestern India, Bakshi (1974) reported some larger fungi forming mycorrhizal associations with forest trees, Sharma and Lakhanpal (1981) recorded 20 species in the Boletaceae forming mycorrhizal associations with Pinus wallichiana, Quercusspp., Rhododendronspp., and Betulaspp.  Saini and Atri (1984) listed 24 species of Russulaand 5 species of Lactariusassociated with deciduous, coniferous, and mixed forests.  Natarajan (1987) reported a number of mycorrhizal fungi from southern India associated with Pinus patulaand reported in vitro synthesis of mycorrhizae of this tree species with Amanita muscaria, Laccaria laccata, Suillus brevipes,and S. subluteus.  Lakhanpal et al. (1989) presented an account of the systematics and ectomycorrhizal relationships of some mushroom families in the N. W. Himalayas.  Sharma and Singh (1990) reported a number of ectomycorrhizal fungi associated with different forest trees of Himachal Pradesh.  In the single most detailed ecological study carried out to date, Kumar et al. (1990) provided data on the fruiting periods and ecological associations [i.e., with respect to certain species of trees and/or different forest types] of macrofungi belonging to five families (Agaricaceae, Amanitaceae, Hygrophoraceae, Pluteaceae, and Tricholomataceae); many of the fungi included in this study are species known to be ectomycorrhizal.  In the mid-Appalachians of eastern North America, Bills et al. (1986) compared species composition, richness, equitability, diversity, abundance, and fruiting phenology of ectomycorrhizal fungi in two forest types in the state of West Virginia.  Except for this one study, published information on the ectomycorrhizal fungi of the region tends to be limited to notes and comments contained in taxonomic treatments of particular taxa (e.g., Bills and Miller 1984).  This situation is certainly not unique, since comprehensive studies of the communities of ectomycorrhizal fungi associated with particular types of forest communities are generally lacking for most regions of the world.  In fact, virtually all such studies have been carried out in Europe (e.g., Lisiewska 1974; Petersen 1977; Arnolds 1981, 1982; DeDominicis and Barluzzi1983; Jansen 1984; Tyler 1985).  The lack of quantitative data on sporocarp production, species abundance, fruiting phenology, and community structure for ectomycorrhizal fungi limits our understanding of the role these organisms play in forest ecosystems.  Recently, a decrease in species diversity and the abundance of sporocarps has been reported for ectomycorrhizal fungi in some areas of continental Europe (Arnolds 1991).  It has been suggested that this "fungal decline" can be attributed to the indirect effects of air pollution, although it is possible that other factors may be involved.  Whether or not similar changes are now occurring or could occur at some point in the future in other regions of the world cannot be determined, simply because the prerequisite data do not yet exist.
          In India, research on fungal systematics has been slow to develop. For the most part, the species concepts used in Europe and North America have been applied to what appear to represent the same taxonomic entities (i.e., morphospecies) in India.  However, recent studies that have used data from sexual incompatibility tests and other experimental approaches to characterize more completely the systematics of particular taxa (e.g., Fries and Mueller 1984) suggest that species circumscriptions cannot be based solely on morphological criteria.  As such, it seems likely that more detailed comparative studies of the ectomycorrhizal fungi of the mid-Appalachians of eastern North America and northwestern India will yield a considerable body of new information on their taxonomic relationships.
 

Objectives

         In brief, the objectives of the proposed research project are (1) to compare the species composition, species diversity, taxonomic diversity, fruiting phenology, and sporocarp productivity of selected groups of ectomycorrhizal fungi (with particular emphasis being placed on members of the Amanitaceae, Boletaceae, Cantharellaceae, and Russulaceae) associated with upland coniferous and oak-dominated forest communities in northwestern India and the mid-Appalachians of the eastern United States and (2) to elucidate, using the morphological, anatomical, and other types of data available from direct comparisons of material collected from study sites in eastern North America and northwestern India, the biological and taxonomic relationships that exist between populations of fungi that appear to represent the same taxonomic entity (i.e., morphospecies) in these two different regions of the world.  A considerable body of data on phenology, productivity, and diversity of ectomycorrhizal fungi already exists for thestudy site eastern North America, and primary emphasis of the proposed project will be directed towards producing a similar body of data from northwestern India.  Comparative studies of material from the two regions will use both collections already available from eastern North America and additional material to be collected during the three years of the proposed project.

Preliminary Research

          The proposed research project is an outgrowth of studies already initiated in the mid-Appalachians of the eastern United States and northwestern India.  Stephenson (USA) has collected and studied the fungi of the primary study site (the Fernow Experimental Forest) in the eastern United States since 1989.  During a four-year period (1989-1993), collections were made at approximately two week intervals from a series of sixteen 100 m2 plots located at four different localities on the Fernow Experimental Forest (Iskra et al. 1995).  This research effort yielded more than 1000 collections of macrofungi along with a considerable body of data on the fruiting phenology, sporocarp productivity, and occurrence of ectomycorrhizal fungi at this study site.  In 1992, Bhatt (India) and Kumar (India) spent several months working with Stephenson in the United States, and one of the results of the research carried out during their visit was a preliminary checklist of the ectomycorrhizal fungi of the Fernow Experimental Forest.  This checklist then served as the basis for a more comprehensive publication on the mycoflora of this primary study site (Stephenson et al. 1994) and as the starting point for a detailed taxonomic treatment of members of the genus Amanitareported from or known to occur in the mid-Appalachians (Tulloss et al. 1995).  In addition, Stephenson has accumulated a considerable body of baseline data on soils and vegetation for both this study site and other potential study sites located at higher elevations in the same region (e.g. Stephenson and Adams 1986; Adams and Stephenson 1991).  Kumar and Bhatt have collected and studied the fungi of the study areas in northwestern India for much of the past decade.  A major portion of this effort (e.g., Bhatt 1986; Kumar 1987; Kumar et al. 1990; Bhatt et al., in press) has been directed toward the families of basidiomycetes (e.g., Amanitaceae, Boletaceae, Cantharellaceae, and Russulaceae) to be considered in the proposed project.
          The two primary study sites are considered especially appropriate for elucidating relationships between ectomycorrhizal fungi and their associated trees because species richness of all ectomycorrhiza-forming trees is relatively low while the level of dominance of a particular species is usually high.  For example, Piceaoften occurs in nearly pure communities in both regions, and the major species of Quercuspresent are usually the primary dominants in what are otherwise communities dominated by endomycorrhiza-forming taxa.
 

Methods

         Most previous studies of macrofungal community structure (e.g., Hering 1966, Bills et al. 1986, Hunt and Trappe 1987, Villeneuve et al. 1989) have used sporocarp density and/or biomass to quantify productivity; this same approach was used in the research already carried out on the Fernow Experimental Forest and also will be used in the proposed project.  In order to obtain quantitative data on sporocarp abundance and fungal community attributes, 3 to 5 permanent 100 m2 study plots will be established in each different forest type and/or at each different locality being studied in northwestern India.  This plot size was used in the research already carried out on the Fernow Experimental Forest (Iskra et al. 1995), and thus it would seem appropriate to continue using plots of the same size for the portion of the project to be carried out in India.  Stephenson has used both 1,000 m2 plots and 100 m2 plots in field studies of fungal biodiversity (e.g., Stephenson 1988, Iskara et al. 1995), and his own experience suggests that the use of a series of the smaller plots is more effective (i.e., fewer "missed" fruitings) and less time consuming (with respect to carrying out an intensive survey of a particular unit of forest) than the use of a single larger plot.  Study plots will be monitored at approximately two week intervals throughout the fruiting seasons (typically from mid-July until early October in eastern North America [Stephenson, unpublished data] and from late June until late October in northwestern India [Kumar et al. 1990]) during the three years of the proposed research.  All sporocarps present within each study plot will be recorded on each visit, and separate counts will be made for each different species.  After being tallied, all sporocarps will be removed from each plot so that they will not be counted again on a subsequent visit.  In addition, indiscriminate collecting (i.e., collections not made from study plots) will be carried out throughout each different forest type (1) to document more completely the assemblage of ectomycorrhizal fungi present and (2) to supplement the material collected for a particular species occurring in the plots with the additional material necessary for positive identification.  All field collections will be taken back to the laboratory, where full macroscopic descriptions of fresh material, proper labeling, and proper preserving of specimens for future study of microscopic anatomical features be carried out.  Whenever possible, the appearance of fresh material will be documented with color slides, since having this type of information is a definite aid to identification.  In addition, habitat/substrate/host associations will be noted and recorded for each collection.  For agarics, spore deposits will be obtained whenever possible; these will be used in determining spore color in mass, microscopic (as viewed under oil immersion) features and length-width measurements, and for SEM studies of spore ornamentation where appropriate.  A color guide (Kornerup and Wanscher 1961) will be used to record all combinations of sporocarp color in the fresh condition.  Sporocarps will be preserved by drying on shelves of a standard mushroom dryer (Smith and Smith 1973) by convective warm air drying.  Once fully labeled and dried, all material will be placed in small paper boxes or wax paper bags for storage in a standard herbarium cabinet. Later, detailed microscopic examination of preserved sporocarps will be carried out.  This portion of the proposed research will be carried out using the laboratory facilities at Fairmont State College in the United States and H. N. B. Garhwal University (Campus Pauri) and the Himachal Pradesh Agriculture University (HPAU) Oilseeds Research Station in India.  Two major reference collections of ectomycorrhizal fungi are housed at institutions located only about 4 1/2 hours driving distance from Fairmont State College.  The first of these is the National Fungus Collections (mentioned above) and the second is the herbarium of Virginia Polytechnic Institute and State University.  It is anticipated that the PIs from India would make extensive use of these collections when they visit the United States.  Because of the problems inherent in the identification of species in some taxonomically difficult groups (e.g., the genus Russula), a number of specimens will be passed along to other mycologists for help in determination.  Voucher specimens of all species encountered in the study will be deposited in the herbaria of Fairmont State College (FWVA), H. N. B. Garhwal University, or the Department of Plant Pathology, HPKV, Palampur.  In addition, duplicate material (and all specimens of any species new to science) will be sent to the National Fungus Collections (BPI) in Beltsville, Maryland.
          Ectomycorrhizal relationships among forest trees and their fungal associates have often been postulated from the basis of field observations (e.g., Malloch and Malloch 1981, Kumar et al. 1990).  Although a similar approach will be used in the proposed research, an effort also will be made to test potential hosts and their postulated fungal symbionts by means of in vitromycorrhizal synthesis.  The methods used are those described by Miller et al. (1986).  In order to evaluate host specificity in particular taxa of ectomycorrhizal fungi (Jacobson and Miller 1991), some of the synthesis experiments to be carried out will involve tree hosts from one region (e.g., India) and fungi from the other region (e.g., the United States).  Moreover, in an effort to evaluate the extent and nature of ectomycorrhizal associations for particular species of trees, root samples will be collected in the field, fixed in FAA, sectioned in the laboratory, and then characterized both macroscopically (i.e., noting such things as color, branching type, extent of extramatrical hyphaeand rhizomorphs) and microscopically (i.e., describing such features as mantle thickness, mantle appression, hartig net penetration, and characteristics of extramatrical hyphae).  This component of the proposed research project will be carried out in collaboration with Dr. Roland Treu.  The methods used will be those described by Treu et al. (1996).
          Whenever possible (many mycorrhizal fungi can not be cultured) an effort will be made to start tissue cultures from fresh material of species common to the two regions.  The cultures obtained will be compared for similarities in such characteristics as growth, color, texture, and relative amounts of aerial and submerged hyphae (Miller et al. 1983; Hutchison 1990, 1991; Hutchison and Summerbell 1990).  A number of techniques of modern molecular biology have been demonstrated to be a valuable tool in elucidating genetic variation and relatedness in many different types of organisms, including fungi (Micales et al. 1986).  For example, it has been shown that the DNA of eukaryotes contains base sequences which are useful in discriminating variations among closely related species and among different genetic strains within a species (Vilgalys and Hester 1990, Lee and Taylor 1992, Egger and Sigler 1993).  Polymerase chain reaction amplification, when coupled with restriction fragment length polymorphism analyses and direct sequencing of ribosomal RNA gene spacers, has been used with considerable success in a number of recent studies that have examined the genetic variation that exists for what appear to be populations of the same morphospecies on two different hosts or collected in two different regions of the world.  Some very limited preliminary research along these lines has already been carried out on material collected in the two primary study sites to be used in the proposed research.  However, the fact that the two PIs in India have no access to laboratory facilities for molecular studies precludes the inclusion of DNA analysis as a major component of the proposed research.  However, it is anticipated that some of the material collected in the project will be subjected to this type of analysis at some point in the future.
          It is anticipated that several thousand collections of fungi will be made during the three years of the proposed research.  The data represented by these collections and also those already available from studies already carried out in the Fernow Experimental Forest will be analyzed and the various community attributes (e.g., species composition, richness, diversity, equitability, and abundance) will be derived or calculated using the methods described by Stephenson (1988) and Stephenson et al. (1993).  In addition, the species lists compiled for the two regions will be compared (using taxonomic diversity and coefficient of community indices) with those available for other temperate regions of the world in order to assess patterns of distributions and biogeographical relationships (Redhead 1989) of the assemblages of fungi present in each instance.
 

Significance of the Proposed Research

          The data resulting from the proposed research project would have important implications for timber management, watershed protection, and reforestation.  Moreover, defining the ectomycorrhizal associates for forest trees in the two regions would contribute to our understanding of fungal biology, taxonomy, and evolution while at the same time yielding a body of data from which further studies could be launched.  In addition, the data obtained during the course of the proposed research will be used to assess biogeographical relationships and patterns of biodiversity (including the linkage that presumably exists between fungal diversity in a given community and the diversity of the community of plants with which these fungi are associated) for the groups of fungi being studied.  At present, very little is known about fungal biodiversity in either the eastern United States or northwestern India.  Finally, the proposed research would permit an already initiated cooperative research arrangement between American and Indian mycologists to expand so as to include a number of other mycologists in both countries.