BOARD OF GOVERNORS, AMERICAN ACADEMY OF MICROBIOLOGY

R. John Collier, Ph.D. (Chair) Harvard University Medical School

Kenneth I. Berns, M.D., Ph.D. University of Florida Genetics Institute

E. Peter Greenberg, Ph.D. University of Washington

Carol A. Gross, Ph.D. University of California, San Francisco

Lonnie O. Ingram, Ph.D. University of Florida

J. Michael Miller, Ph.D. Centers for Disease Control and Prevention

Stephen A. Morse, Ph.D. Centers for Disease Control and Prevention

Edward G. Ruby, Ph.D. University of Wisconsin-Madison

Patricia Spear, Ph.D. Northwestern University

George F. Sprague, Jr., Ph.D. University of Oregon

Judy A. Wall, Ph.D. University of Missouri-Columbia

COLLOQUIUM STEERING COMMITTEE

Arturo Casadevall (Co-Chair) Albert Einstein College of Medicine

Joseph Heitman (Co-Chair) Duke University Medical Center

Scott Baker Pacific Northwest National Laboratory

Gerald Fink Whitehead Institute for Biomedical Research, MIT

Christina Hull University of Wisconsin-Madison Medical School

Carol Colgan, Director American Academy of Microbiology

Merry Buckley, Science Writer Ithaca, New York

COLLOQUIUM PARTICIPANTS

Karen Bartlett University of British Columbia, Canada

Joan Bennett Rutgers University

Richard Bennett Brown University

Teun Boekhout CBS-KNAW, The Netherlands

Dee Carter University of Sydney, Australia

Leah Cowen University of Toronto, Canada

Melanie Cushion University of Cincinnati College of Medicine

Fred Dietrich Duke University Medical Center

Françoise Dromer Institut Pasteur, France

John Edwards Harbor-UCLA Medical Center

Marta Feldmesser Albert Einstein College of Medicine

Bettina Fries Albert Einstein College of Medicine

James Galagan Broad Institute, MIT

Barbara Howlett University of Melbourne, Australia

Alexander Johnson University of California, San Francisco

Nancy Keller University of Wisconsin-Madison

Bruce Klein University of Wisconsin-Madison

James Kronstad University of British Columbia, Canada

Jennifer Lodge St. Louis University School of Medicine

Joyce Longcore University of Maine

Aaron Mitchell Columbia University

Nicholas Money Miami University, Ohio

Eleftherios Mylonakis Massachusetts General Hospital

Joshua Nosanchuk Albert Einstein College of Medicine

John Perfect Duke University Medical Center

Nuri Rodriguez-del Valle University of Puerto Rico

David Soll University of Iowa

Gillian Turgeon Cornell University

Theodore White Seattle Biomedical Research Institute

Floyd Wormley University of Texas at San Antionio

SPONSOR REPRESENTATIVES

Victoria McGovern Burroughs Wellcome Fund

Charles Saunders Procter & Gamble

Paul Steed Serenex

NON-PATHOGENIC ACTIVITIES

Fungi are often overlooked in ecology, but their influence in the environment and their roles in biology are undeniable. Ecosystems rely on fungi to degrade organic matter and mineralize the products, making essential elements available for uptake by plants and bacteria. In broader terms, these fungal degradation activities have a profound influence on the carbon cycle, particularly with respect to degradation of lignin, cellulose, and other plant materials and the subsequent release of carbon dioxide into the atmosphere. In addition, many crops, forests, and other ecosystems rely on interactions with fungi to provide nitrogen and other nutrients. Roughly 60% of plants interact with fungi in ways that benefit the plant. Fungi are also consumed by many organisms, including animals, insects, worms, bacteria, and other fungi.

Fungi maintain a myriad of different symbiotic relationships with animals, plants, insects, amoeba, bacteria, and other fungi. The human gut, which is teeming with microorganisms, is one fungal habitat that is of particular interest to humans, but even here we know relatively little about the interactions between fungi and the host and between fungi and bacteria. Other key interactions include mycorrhizae, symbiotic associations in which fungi colonize the roots of plants and provide a linkage between plant and soil, an arrangement that often grants a plant a competitive advantage over plants that lack mycorrhizal fungi and grants the fungus much-needed carbon. Plants would not have been able to colonize land without mycorrhizal fungi, and most species of plants today continue to engage in these associations to extract nutrients from their environments. Bacteria, too, maintain symbioses with fungi. Certain fungal plant pathogens, for example, play host to endosymbiotic bacteria that regulate or enable the production of virulence factors and permit the fungus to infect key crop plants like rice or allow the fungus to produce spores during its lifecycle for dissemination and survival.

THE IMPACT OF ECOSYSTEM CHANGE ON FUNGI

Fungi as integral parts of diverse ecosystems are subject to the same kinds of stresses other populations experience during ecosystem change. Events and processes such as invasions, migration, climate change, outbreaks, or land use change (such as forest-to-agriculture transitions) can put ecosystems under stress and induce fungal imbalances as measured by disease prevalence, sampling, or even direct observation.

Example situations in which fungal populations have been manifestly impacted by changes in an ecosystem include:

• ▪ Black mold in the ecosystems around Chernobyl, Ukraine. In the wake of the 1986 disaster at the Chernobyl Nuclear Power Plant, areas surrounding the former power plant were contaminated by significant levels of radioactivity. These areas have seen increases in the incidence of a form of black mold that is destroying plant life and eroding environmental quality. Scientists hypothesize that melanin protects the mold from the radioactivity, but it may also serve as an energy-harvesting apparatus.
• ▪ Coral bleaching. Bleaching of corals due to ocean water temperature elevations promotes infection by Aspergillus species, resulting in coral death.
• ▪ A rise in Cryptococcus gattii infections. It has been suggested that C. gattii takes advantage of some genetic changes and possibly alterations in its ecosystem, and that recent dry conditions during the summers on Vancouver Island have encouraged an outbreak of C. gattii infections in the area. Prior to the outbreak of cryptococcal disease in British Columbia, Canada, the environmental niche of Cryptococcus gattii was thought to be restricted to tropical or semi-tropical climates. C. gattii as the causative agents of an emerging infectious disease is an example of the role of micro-environmental changes, including climate change, that will increase the potential for exposure to pathogenic fungi to humans, domestic and wildlife. How the organism came to Vancouver Island and is now spreading beyond to the British Columbia mainland and into the United States in Washington and Oregon cannot be known for certain, but likely has involved human activities.
• ▪ Mold in homes. Wet indoor environments encourage the overgrowth of Stachybotrys species and other molds, which has been associated with sick building syndrome. One prominent example of mold overgrowth can be found in homes and businesses flooded during hurricane Katrina in August 2005. Very little is known about the impacts of indoor mold growth on human health, but it is possible the effects of mold exposure are profound and lasting. The health impacts of mold contamination in flooded portions of New Orleans and surrounding areas cannot be predicted. Should affected houses be destroyed or rebuilt? Is any exposure to indoor mold unacceptable? The answers to these questions are not known. Natural disasters like hurricane Katrina provide natural laboratories for understanding the effects of perturbation on fungi and the subsequent impacts of fungi on human health. More effort should be devoted to testing and long-term monitoring of mold contamination and human health in Katrina-affected areas.
• ▪ Mold in the space station. Air in space vehicles is often contaminated with fungal spores. Mold has become a major problem for the Russian space station Mir, where it has been found growing in sensitive electronic equipment.
• ▪ Fungal outbreaks following earthquakes. Outbreaks of Coccidioides infections have occurred in California following earthquakes in the 1990s that disturbed soil leading to airborne spores and infections in humans.

It is common knowledge that global climate changes are constantly occurring and are probably more pronounced in our current era. These changes will impact the ecology of fungi and their relationship to the environment before we have been able to identify them and assess their role in current ecosystems. Global climate change will result in profound ecological alterations, and its impacts on fungi will inevitably affect humans as well. One example of these impacts that has already been witnessed is the emergence of fungal resistance in certain plant cultivars that were once sensitive. Also, in some areas of the world, fungal plant pathogens are spreading into new territories, and monoculture approaches to agriculture may prove to be particularly susceptible to outbreaks. If scientists can learn how ecosystem changes predispose an environment to fungal threats, they can prepare proactively for these threats in the future, rather than react to crises as they arise.

As illustrated above, environmental changes can and do bring about changes in fungal activities, but it is also possible these changes could be so profound as to threaten the existence of a fungal species. However, there is little information on the robustness of fungal populations in the environment, so the possibility of extinction is difficult to gauge. In Europe, some mushrooms are considered endangered, and in Eastern Europe acid rain has caused declines in mycorrhizae, but mushrooms and trees may be impacted by any number of stresses, and cause and effect are difficult to tease apart in these situations. Is a decline in mycorrhizae causing a decline in forests or vice versa? This has yet to be established. It may be possible to use fungi as biomarkers for environmental stress or as indicators of ecosystem health, but currently the links between various species of fungi and ecosystem health are vague.

FUNGAL DISPERSAL AND INVASIONS

The introduction of fungal species into the environment is often traced through the spread of fungal disease. Fungal spores can travel widely by air currents, and transcontinental spread of spores is known to occur. Although fungi can disseminate by means of wind, insects, and seeds, in recent history human activities have been primarily responsible for introducing a number of fungal diseases into new geographical regions, and these introductions have sometimes caused serious problems. Examples of human-mediated introductions include:

• ▪ The “chytrid invasion.” Humans have transported the African clawed frog, Xenopus laevis, all over the world for use in pregnancy tests and research. This frog species carries with it a species of fungus, Batrachochytrium dendrobatidis (Bd), a chytridiomycete, to which X. laevis is resistant but many other amphibians are not. The fungus is responsible for severe declines in the populations of amphibian species around the globe. Studies of population genetics of the pathogen, so far, do not confirm (or refute) the spread of chytridiomycosis by X. laevis. The American bullfrog (Lithobates catesbeiana) is another Bd-resistant candidate for spreading this disease. Although it has been shown that X. laevis carries the disease now, it is not known if it was the first to do so.
• ▪ Crop disease. All, or almost all, crop diseases are transported throughout the globe by human activity.
• ▪ Tree diseases. Dutch elm disease and chestnut blight were both spread by human activities from areas where trees had evolved some level of resistance to areas where most trees were sensitive to the respective fungal pathogens. In recent times, pitch canker, which is destroying the native stands of Monterey pines, and sudden oak death, which is decimating coastal oaks on the Pacific coast and caused by the pseudofungus (Straminopile) Phytophthora ramorum and spread by nursery trade, are among many invasive fungal species transported by human activity.
• ▪ Cryptococcus gattii and Eucalyptus trees. C. gattii, which causes cryptococcosis in humans, is primarily associated with Australian Eucalyptus trees and has likely been spread along with these trees or materials derived from them when used in plantation and reforestation programs in Africa, Southeast Asia, South and North America, India and parts of Europe. As one example, in the 1800s thousands of eucalypts were imported from Australia, where C. gattii is endemic, and planted to stabilize the soil in parks and land throughout California, and in some cases the organism has been recovered from trees in these new locales.
• ▪ Rose handler's disease. Sporothrix schenckii, which causes sporotrichosis (also called rose handler's disease), was widely dispersed when infected plant seedlings and their sphagnum moss packing were shipped from one area to another.
• ▪ Human travel and fungal diseases. Human movement can carry human fungal diseases from areas where a disease is endemic to new territories. C. gattii, for example, has spread from Vancouver Island, where an outbreak began in the late 1990s, to Washington State, thanks in part to the movement of vehicles and travelers.
• ▪ Molecular analysis suggests that humans may have been inadvertently responsible for the introduction of C. immitis into the South American continent as they migrated into that region in Paleolithic times. Furthermore, coccidioides species may infect travelers who visit endemic areas even for short periods of time.
• ▪ Nosocomial spread of Candida species on the hands or nails of health care workers to patients in intensive care units.
• ▪ Transmission of endemic mycoses with organ transplantation, leading to cases of infection in patients with no travel history to endemic areas.

Scientists hypothesize that melanin protects the mold from the radioactivity, but it may also serve as an energy-harvesting apparatus.

In cases in which pathogen dispersal is aided by human travel, interventions could conceivably be designed to contain the spread, although this would require increased knowledge of ecology and pathogenesis or avoidance of aerosols of dust in southwestern U.S. deserts. Another example is Synchytrium endobioticum, a chytrid disease called black wart of potato, which is on the U.S. bioterrorism list. Strict controls are in place to keep this pathogen from spreading out of Newfoundland. When this fungus has escaped, it caused the U.S.-Canada border to be temporarily closed to potatoes. http://maine.gov/agriculture/pi/whatsnew/potatowart_CA.htm

For effective management of fungal invasions, there needs to be a system for data collection, determination of the scope of the outbreak, and implementation of policy.

Historically, fungal diseases have not been reported or tracked. This has made it difficult to control the spread of fungal pathogens, because good epidemiological data on the scope of infection are not consistently available. Fungal infections are reportable in France since 2002; the French National Reference Center for mycoses and antifungals is charged with surveillance. Other public health agencies should implement formal programs to report cases, track disease progress, and design interventions in outbreaks of fungal disease. For effective management of fungal invasions, there needs to be a system for data collection, determination of the scope of the outbreak, and implementation of policy. In cases in which pathogen dispersal is aided by human travel, interventions could be arranged around highways and logging routes to try to contain the spread.

A standard protocol in the advent of a specific outbreak of a fungal disease of humans, animals or plants should be in place that will summon the available experts on the subject and make their expertise available to government agencies. In some instances, the necessary information and expertise are available, but for multiple reasons these resources are not utilized for shaping public health policy. A standard procedure should be developed to address outbreaks; interdisciplinary groups of public health officials, zoologists, and microbiologists must address this need.

USING FUNGI AND FUNGAL PRODUCTS IN INDUSTRY

The use of fungi in industry has two aspects to it. The first concerns the application of the enzymatic capacity of fungi to mediate industrial processes. Some of the processes fungi carry out in the environment have been appropriated by industry, and there are many more opportunities for exploiting other fungal capabilities that remain to be explored. Fungi decompose biomass, and they are particularly effective at breaking down celluloses and five-carbon sugars. Industry can capitalize on these metabolic capabilities by using fungi to produce fuel (including ethanol) from plant materials. In a recent development in this arena, the Biogreen compact in Europe discovered a zygomycete capable of producing ethanol more efficiently than Saccharomyces, and waste products from the process can be made into diapers and other products. These fungal degradative capabilities can also be used in bioremediation, the treatment of chemical contamination using microorganisms or microbial enzymes.

The other use of fungi in industry is related to the field of biotechnology. Biotechnology has emerged hand-in-hand with the availability of Saccharomyces as a “small factory” for the production of innumerable products that range from hormones to proteins for therapeutic uses. Fungal systems have an advantage over bacterial systems in that they can produce certain products with the correct post-translational modifications needed for activity.

RESEARCH NEEDS

Environmental sampling from a variety of niches (insects, animals, water, air, soil, and compost piles, to name a few) has revealed a remarkable diversity of fungal species and countless fungi that haven't been named yet. The current level of scientific knowledge about fungi is inadequate for managing exotic and introduced fungal diseases in the environment, for preventing the possible endangerment or extinction of species of fungi, or for coping with fungal contamination in homes and businesses. This shortfall in knowledge extends to even the most fundamental issue: we do not know where the known species of fungi live or how many are present in those niches, so it's difficult to appreciate how fungi interact with and influence their environments. With respect to environmental fungi, a global census of the various locations and species of fungi that exist in nature is a pressing research priority. A census of this kind is critical to our ability to establish the role of these fungi in normal and perturbed ecosystem function.

Global Fungal Census

A global fungal census should entail extensive sampling of the environment, followed by PCR amplification using specific primers for fungi; the ITS region of the rDNA genes should then be sequenced. ITS sequences will provide a kind of molecular bar code for identifying fungi within the environmental samples and their relationships to known species.

Diverse ecosystems across the planet should be sampled for this census, including both terrestrial and aquatic habitats. Scientists have recently discovered many novel and diverse species of fungi living in the oceans, for example, proving these habitats and the deep ocean, in particular, are ripe for further study. Fungi associated with insects are also extremely diverse and merit further research. Nearly every insect species examined to date is host to its own unique and diverse microbial community, and these communities are likely to include many new fungi. The fungi associated with mites could point the way to new biocontrol measures for mite pests, and beetles are likely to be a significant source of diverse yeasts, to name a couple of the potential avenues to explore. Tritrophic interactions of insects, fungi, and plants could be a good place to seek novel fungal antimicrobials. Animal gastrointestinal tracts contain numerous species of fungi, but these niches have been largely unexplored. Extreme environments, including hot springs, thermal vents in the oceans, and arctic ice, are also a mystery with respect to their fungal inhabitants. All of these habitats should be surveyed more thoroughly. Research should also explore possible changes in the distribution of fungal populations in response to various perturbations.

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FUNGI AND INDOOR AIR QUALITY: WHAT'S THE EVIDENCE?

FUNGI AND HUMAN HEALTH

The human “microbiome” includes all the microorganisms present on or in the human body; fungi are a minority constituent of this microbial community, numerically speaking. It's difficult to say how many fungi are associated with the human microbiome, but the number and species of fungi on and in a given human are likely to change with climate, diet, gender, age, immune status, prescription medications, and other factors. Recent surveys of bacteria associated with the human body show that these populations shift over time and are highly variable from person to person and, therefore, support this hypothesis.

Of the roughly 1.5 million fungal species estimated to be alive today, only some 200 have been associated with the human body so far, either as pathogens or as commensals, and of these, a dozen or so represent the most common fungal pathogens. This includes the Candida species C. albicans, C. tropicalis, C. parapsilosis, C. metapsilosis, C. orthopsilosis, C. krusei, C. guilliermondii, C. lusitaniae, C. keyfr, C. dubliniensis, and C. glabrata, which are components of the normal microbiota and can also cause superficial and systemic infections; molds (such as Aspergillus fumigatus and zygomycetes) that infect the lung; the basidiomycete fungi Cryptococcus neoformans and C. gattii, which infect the lung and central nervous system; and the dimorphic human fungal pathogens, of which there are several species (Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, Coccidioides posadasii, Penicillium marneffei, and Sporothrix schenckii). Other fungi commonly associated with human disease include the Malassezia species, common commensals that have evolved to specifically interact with human skin and secretions. In turn, these fungi secrete antigenic proteins that may play a direct causative role in skin disorders. Another successful group of human fungal pathogens are the dermatophytes, which cause athlete's foot and other common skin, nail, and hair infections. Finally, there are the black yeasts, Mucorales, Fusarium, Scedosporium and the black molds.

A great deal about human fungal pathogens remains unknown. Researchers do not know, for instance, how many fungal clones (genotypes/isolates) are present in an infected patient, and many of the details about the interactions between fungi and their human host, whether during commensal growth or infection, are not known. Much of the information about pathogens comes from experiments that tested cultures of fungi during logarithmic phase, a growth habit that is poorly reflective of growth in a host. Certain secondary metabolites, for example, function during infection, but secondary metabolites are generally not produced during log phase growth, so studies that use fast-growing cultures will often miss the effects of secondary metabolites.

Fungi do not restrict themselves to causing diseases in immune-compromised or otherwise debilitated hosts, although this is a common misconception. There are many known fungal infections that strike apparently healthy people, including fungal sinusitis and vaginitis, and there may be connections between fungi and such common conditions as asthma, allergies, and sick building syndrome. Even disseminated cryptococcosis can occur in hosts without an underlying disease or disorder. It is also important to note that infecting a human is not necessarily an evolutionary dead end for a fungus. Anthropophilic dermatophytes, for example, are transmitted from human to human, and as such have evolved to cause chronic diseases with little host immune response that are difficult to treat and completely resolve. Commensal fungi like Candida and Pneumocystis spp. are also passed from person to person, and they have co-evolved with humans and do little damage in their commensal roles. One factor involved in fungal interactions with humans is temperature—commensals must evolve to be able to thrive and grow at 37°C. There are probably other selective pressures, such as access to nutrients. Malassezia sp. are associated with human skin as commensals and play a role in common skin disorders, such as dandruff and eczema. Recent genomic studies reveal they lack the enzyme fatty acid synthase, explaining their requirement for lipids during laboratory culture and their known predilection to survive on human skin by scavenging lipids from sebaceous secretions.

It is important to note that, unlike viruses and some bacteria, person-to-person transmission of invasive fungal disease is rare. Since fungi are a minority of the microbes in the human body, it likely that their most important interactions are with their bacterial competitors. Examples of these exchanges are emerging from studies of the interplay between fungi and pathogenic bacteria (Pseudomonas species), probiotic bacteria (lactobacilli), and other cohabitants (streptococci).

TRANSKINGDOM PATHOGENIC FUNGI

Certain pathogenic fungi infect members of both the plant and animal kingdoms, overcoming significant differences between these types of hosts.

Fungi, as stated previously, are among the most important degraders of organic materials in the environment, yet as such they sometimes do not have a specific preference for the source of organic matter. Certain pathogenic fungi infect members of both the plant and animal kingdoms, overcoming significant differences between these types of hosts. Fungi that do not require a living host are more likely to pose a threat across kingdom lines, since their requirements for growth are not as specific as those of a fungus that needs the support of living host cells. Many fungi will consume resources regardless of whether the source is alive or dead; they are not selective. Factors that may determine the possibility of fungi to constitute a transkingdom threat include their ability to grow at higher body temperature and the ability to breach surface barriers on insects and plants, and sometimes knocking out a single fungal gene can turn a harmless endophyte into a pathogen.

Fungi that can infect hosts from different kingdoms may develop the ability to infect a given host by training and developing traits using another, related host. Amoeba, for example, are a kind of “boot camp” for Cryptococcus species, since amoeba internalize Cryptococcus in much the same way human macrophages (attack cells of the innate immune system) do.

High exposure to certain fungi may contribute to their ability to cross kingdom boundaries. There have been reports of farm workers becoming infected with the plant pathogen Fusarium after repeated exposure to that fungus.

Almost all fungi, including those that pose transkingdom threats, live in the environment at some point during their life cycle, and their time in this ecological niche could teach researchers about a fungus' vulnerabilities. Known transkingdom pathogens include Aspergillus flavus, which infects and damages oilseed crops, insects and humans; Fusarium species, which infect cereal crops and immune-compromised humans (and pose a particular public health threat due to their resistance to many anti-fungals); Sporothrix species, which infect both plants and animals; black fungi, which can infect cacti and humans; and Microsporidia, Zygomycetes, Alternaria species, and Cladosporium species. Cryptococcus species may be able to cross kingdom boundaries, but it is difficult for them to infect plants. Other transkingdom fungal pathogens may be waiting to be discovered. Fungi probably have many interactions with different kingdoms of organisms, including fish, frogs, insects, amoeba, mammals, and plants.

MODEL HOSTS

It is important to focus studies on current, well established heterologous host model systems, but it may also prove useful to develop new model systems for studying fungal pathogenesis. Researchers have used models such as Caenorhabditis elegans (a nematode), fruit flies, Arabidopsis species (plants), frogs, toads, and others, but selecting the right model system is important, and more model systems should be developed. An ideal model is manipulable, tractable, available, reproducible, and inexpensive to purchase and study. Many model hosts have innate immunity, but fewer hosts, including zebrafish and mammals, have adaptive immunity.

VACCINES AND THERAPEUTICS FOR FUNGAL INFECTIONS

Developing new vaccines and therapies for fungal diseases is a neglected area of research. Given the increasing incidence of fungal infections, it is important to develop new vaccines and new types of treatments. The antifungal therapies available today should also be improved.

In some parts of the world, endemic mycoses remain a problem for otherwise healthy, immune competent hosts. South American blastomycosis (caused by Paracoccidioides brasiliensis), is a target ripe for vaccine development. There have been significant strides in understanding immunology for other endemic fungal infections, including histoplasmosis and blastomycosis; this progress offers lessons for moving forward with vaccines in the future.

Some fungal diseases are better suited than others to control with vaccines, like coccidioidomycosis, which is limited to distinct geographical regions (the desert southwest of the United States and portions of South America) and poses a particular threat to known subpopulations (including the elderly, new residents, and prison inmates) and would be relatively easy to implement in the community. Likewise, implementing vaccination programs for fungal diseases that strike immune-compromised patients (including Candida, Aspergillus, and Cryptococcus neoformans infections) would also be straightforward, since immune-compromised individuals are usually under the care of a physician and, as such, are easily identified as targets for a vaccine prior to beginning an immunosuppressed state of treatment.

In other cases, it may be difficult to identify members of the target population for a vaccine, making it harder to implement a vaccination program. The effectiveness of adjuvants in vaccines for fungal diseases requires a great deal of study. Also, clinicians need more information about the appropriate length of time to treat fungal infections with antifungal drugs and the better use of fungal biomarkers in management strategies.

Researchers must explore new methods for addressing fungal infections, including new antifungal drugs, improved diagnostics, and alternative therapeutic methods like immunotherapy and antibody therapy for delivering radiation to the target pathogen. It may also be possible to develop vaccines that deliver cytokine therapy, as well as an immunogen. The success rate in managing invasive fungal infections remains mediocre and does not approach success rates with bacterial infections.

RESEARCH NEEDS

There are many unmet needs in terms of understanding the biology of pathogenic fungi. Filling these gaps in our understanding will have important practical implications for agriculture and medicine, as well as implications for studying the evolution of life. In addition, learning more about fungi and about ways to control and harness them will prove valuable for improving industrial applications like mining fungal genomes and metabolomes for useful chemicals and engineering fungi to produce materials (e.g. vaccines, therapeutic proteins).

Mycology needs access to fast-response funding mechanisms to head off outbreaks. The current outbreak of Cryptococcus gattii on Vancouver Island and Puget Sound, for example, demonstrates the need to respond quickly to an outbreak. This required a multi-disciplinary research team to put together the information coming independently from veterinarians, cetacean pathologists, respiratory physicians, epidemiologists, and environmental hygienists to identify an emerging infectious disease and initiate appropriate therapy in order to save lives. Many fungal diseases can muster only small clusters of experts, so assembling a task force to cope with an outbreak of corn blight, for example, would be problematic.

CENSUS OF THE FUNGAL PORTION OF THE HUMAN MICROBIOME

Millions upon millions of microorganisms, including many microscopic fungi, are found on and in the human body. However, the role the microbiome plays in human health is poorly understood, and the function of the fungal component is even more elusive. We recommend conducting a census of the fungi associated with the human body under a variety of conditions. A fungal census of the human microbiome has the potential to considerably advance our understanding of how fungi impact human health and disease.

To date, there has been no systematic effort to enumerate and describe the fungi of the human body. There is a clear need for the scientific community to pursue this research. The National Institutes of Health has undertaken a project to study the human microbiome, but these studies are heavily skewed in favor of bacteria, essentially disregarding the fungal component of this important ecosystem.

Before beginning a fungal census, it may be advisable to capture a broad overview of the scope of the matter by analyzing the diversity of the fungal ribosomal RNA genes in the human microbiome to provide a quick view of how many different species of fungi to expect. It is quite possible that many of the fungi associated with the human body will resist cultivation. Once an estimation of the fungal rRNA diversity is available, researchers can begin to design culture conditions to characterize the community over time, space, health, and disease. If two species of fungi are always found together, that would suggest experiments to define the interplay between them.

Sampling sites for the census should include the gastrointestinal tract, the mouth, the skin (including the feet, toes, and scalp), hair, nails, lungs, vagina, and the nasopharynx. The fungal census should include samples from normal, healthy individuals of both sexes from a range of different ages (babies, teens, adults, and elderly individuals), and possibly from individuals belonging to different ethnic groups.

Census studies should also be extended to individuals with diseases, including:

• ▪ Patients treated with high doses of antibiotics that are known to perturb the distribution of fungi in patients (these results could shed light on the longstanding hypothesis that antibiotic treatment kills off bacteria and enables fungi to flourish),
• ▪ Chemotherapy patients,
• ▪ Individuals with skin disorders, including atopic dermatitis, that are associated with different commensal fungi,
• ▪ Individuals with depressed immune systems, including AIDS patients and transplant recipients,
• ▪ Individuals with metabolic disorder/obesity (an increasingly predominant group in the population), and
• ▪ Individuals with exposure to aquatic environments, including sewage, which may increase exposure to chytridiomycetes and other fungi and pathogens.

It may emerge from this census that certain species of fungi are associated with a panoply of diverse, poorly understood diseases, possibly including obesity, autism, alcoholism, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, malabsorptive syndromes, and colon cancer.

A census of the fungi in the human microbiome would inevitably shed light on the interplay between fungi and bacteria in the gut—interactions that are thought to shape the persistence of bacteria in this environment.

Disease may be more complex than previously thought. Illness may be the product of an imbalanced microbial community. In this scenario, a single pathogen almost never acts alone. Instead, combinations of organisms contribute to illness, and the response of the host bears greatly on the outcome.

A fungal census like the one described here could reveal a great deal about the concept of pathogenicity as it applies to fungi. Fungi can be seen as opportunists waiting for hosts that have the right combination of susceptibilities and ready resources for a fungus to grow and multiply, and some fungi are more aggressive in their pursuit of these host resources than others. A census could enable scientists to name and describe new species of fungi and place them in their appropriate spots on the continuum between “pathogen” and “harmless commensal.”

Bacteria are a huge, potentially overwhelming, component of the human micro-biota, and they may make it more difficult for researchers to assess the fungal components and contributions in this environment. On the other hand, it is possible that the fungal metabolome will be easier and clearer to discern, despite the minority status of fungi.

Information about the fungal component of the microbiome can be integrated into the emerging systems biology field. Metabolic reconstruction will shed light on which microbes fungi interact with and how.

FUNGI AND THE SPECIES CONCEPT

As with any other category of organisms, it is important to establish a meaningful and consistent set of criteria for distinguishing one species of fungus from another. Prior to the advent of molecular biology, certain fungi were assigned two names: one for the sexual stage (teleomorph) and another for an asexual reproductive stage (anamorph). This convention is an anachronism that dates from a period in microbiology when, in the absence of molecular evidence, fungi were classified solely on the basis of their morphological form. Dual nomenclature confuses fungal systematics to the detriment of scientific discovery and, most importantly, impairs communication among scientists, hindering the development of the field and the implementation of efficient medical treatment. The difficulties of this system are felt most acutely in education and training and in attracting new talent to the field.

Efforts to assign a single name to each species are now underway, and we strongly encourage further progress. Defining fungal species allows scientists to ascertain fungal mating cycles, breed fungi effectively for desired traits, and ensure accurate representation when sequencing fungal genes. More broadly, a widely accepted definition of fungal species allows scientists to communicate their findings to other researchers and to the medical and agricultural communities.

A species designation should accurately convey the essential characteristics that identify an organism as a member of a particular species. These become particularly important in the case of the clinical environment where the identification of a fungal species is needed for determination of the correct therapeutic approach in sometimes life threatening conditions. With respect to a fungal isolate, clinicians are concerned with the potential properties of the fungus and its drug resistance. Farmers and plant breeders have similar concerns with respect to fungal pathogens in agriculture, but they also need to know about an isolate's dispersal mechanisms and the possibility of host resistance. A detailed analysis beyond a simple species identification is necessary to reveal some of these traits, which can vary between isolates of the same species.

In traditional terms, a species is described as a set of individuals that inter-breed, but this is a problematic definition in the case of fungi since sexual cycles are difficult to recreate under controlled conditions necessary to verify species boundaries and the sexual cycles for many fungi have yet to be discovered. As more and more fungi are discovered, it will become increasingly difficult (if not impossible) to determine the ability of these organisms to undergo sexual reproduction in or out of the laboratory setting.

Fungal species, like those of any eukaryote, may be recognized by phenotype (morphological species recognition), genetic isolation (phylogenetic species recognition) or reproductive isolation (biological species recognition. The default method is morphological species recognition, but these taxa typically contain more than one species when phylogenetic or biological methods are applied. Where mating tests have been possible, good agreement has been found between phylogenetic and biological species recognition. This finding is important because very few fungi can be mated to establish biological species, whereas phylogenetic species can be recognized for any fungus for which DNA can be obtained. However, phylogenetic species recognition is laborious because fungal individuals must be thoroughly sampled throughout their range. As a result, phylogenetic species recognition has been applied only to a relatively few, socially important species—that is, human, animal and plant pathogens, or model organisms. For the vast number of other fungi, we have a morphological species concept and, at best, sequence from the ribosomal DNA region.

The most variable of these rDNA regions, the ITS, has been chosen as the fungal “Barcoding” molecule and is useful for assessing fungal identity where well-recognized species identification is not essential (in ecological studies, for example). Following the lead of bacteriologists, mycologists will be able to apply a Bayesian phylogenetic method to add environmental sequences to databases with known sequences and assign the unknown sequences to species with statistical probabilities. Here, ecological studies can be broadened to include socially-important aspects such as quarantine or epidemiology of plant and animal pathogens. Essential to this approach will be a central, curated database of sequence from laboratory and environmental studies. These data will facilitate any method of rapid identification, from the current microarray technology to computational methods that will be applied to high throughput sequencing.

Historically, fungi have been identified by observation of macroscopic and microscopic morphology, relying on the production of typical colonial morphology and sporulation patterns.

When an isolate's detailed genetic sequence data is not available, it can be difficult (if not impossible) to know whether the fungus is a representative of a known species or something new altogether. Historically, fungi have been identified by observation of macroscopic and microscopic morphology, relying on the production of typical colonial morphology and sporulation patterns. Most of fungal identification in the clinical environment still depends on this approach that requires a specially-trained personnel because not all isolates present the typical structures that enable reliable identification. Nevertheless, DNA-based identification of fungi with the advent of molecular biology techniques presents itself as a more reliable, fast and accurate method for fungal identification. In order to achieve this goal, a fungal typing chip that allows rapid identification would be very useful for researchers, clinicians, and agricultural interests. Ideally, such a chip would allow users to identify a fungal isolate and place the isolate in the context of a detailed fungal database that provides information about the relevant characteristics of each organism. A detailed database would also aid in the classification of nonculturable fungi uncovered by surveys of fungal diversity. To assemble such a chip and an accompanying database, it is necessary to identify key fungal sequence signatures that could be used for isolate identification.

POPULATION GENETICS/GENOMICS

The study of fungal population genetics, which explores the diversity and history of alleles (genes or genetic loci) in a species, has proven valuable in a number of ways. With respect to pathogenesis, for example, population genetics can reveal where an organism arose, how it evolved during periods of high and low disease rates, and the history of the organism and its transmission and distribution habits.

In fungi, as in all other classes of organisms, evolution works at the population level, and natural variations within populations play a major role in the evolution of new traits that can pose a threat or a benefit for humans. For therapeutic purposes, researchers must recognize variation in fungal populations in order to understand the pressures that drive the evolution of new species and traits.

Genetic variation within fungal populations is essential to their evolution, and knowledge of it is equally essential to understanding to how to control fungi that cause disease in animal or plants. In microbes, population genetic studies address population structure in species, modes of reproduction, gene flow among populations, migration to new geographic regions, population bottlenecks and expansions, and the forces of drift and selection in promoting adaptation. Until now, all population genetic studies have relied upon, at best, sequence data for a handful of genes. Now, with inexpensive methods of resequencing genomes, population genomics is upon us. Human population genomics is paying huge benefits in understanding human susceptibility to disease and is just beginning to touch fungal biology. Populations of genomes from yeast are now published and, for a few pathogenic fungi, small populations of genomes are now available (five for Cryptococcus, 15 for Coccidioides, including at least two for each of five populations). The tremendous amount of sequence data that population genomics will provide must also be curated centrally in fungal database to be of broad and general utility.

GENOMICS: PAST DISCOVERIES AND FUTURE POTENTIAL

Many aspects of mycology, in particular, have been unlocked by sequencing the genomes of 100–150 fungal representatives of the estimated 1.5 million fungal species that exist.

Genomics—the study of all or part of an organism's genetic information—has had a profound impact on biology as a whole, introducing a new era of information-driven science. Many aspects of mycology, in particular, have been unlocked by sequencing the genomes of 100–150 fungal representatives of the estimated 1.5 million fungal species that exist. Although the sequenced representatives are heavily skewed toward human pathogens, these genomes are still unparalleled resources for revolutionizing the current knowledge of fungi, and they have thus far revealed new insights into evolution, sexual reproduction, gene families, gene acquisition, gene structure, and gene regulation. Genomics offers the chance to carry out science more rapidly and powerfully than ever before by pinpointing the gene “parts” of a fungus and opening a window on how those parts interact. Genome sequencing will undoubtedly fuel improvements in bioremediation using fungi, industrial-scale fermentation processes, medical diagnosis, and disease therapies.

Because fungal genomes are relatively small compared to animal genomes, genome sequencing for these species would be cheap and easy, and technological advances are making genome sequencing less expensive and more facile every year. The tremendous diversity already discovered among the limited number of fungal genomes sequenced to date highlights the importance of seizing the opportunity of cheaper sequencing and conducting more comprehensive sampling of the rest of the fungal kingdom. Sequencing of fungal genomes has become “democratized” in a way, since the capability is now more accessible to labs with limited budgets. Where one genome sequence exists, additional individuals of the same species can be “resequenced” at 25X coverage by high-throughput methods for less than $10,000. Within five years, it will be possible to sequence a new fungal genome with similar coverage for only a few hundred dollars.

Data generation is no longer the primary limitation in genomics; the main challenge lies in the analysis and annotation of genomes and ultimately on the extraction of meaningful information. Falling prices for sequencing will make it possible to assess genome variation among multiple isolates and eventually turn genome sequencing into an assay. However, without a central database with a dedicated staff, these data will not be available to all researchers, and their potential will not be achieved.

FUNGI TO TARGET FOR GENOME SEQUENCING

There are three areas where additional fungal genomic sequence is desperately needed: unrepresented phyla, close relatives of socially-important fungi, and population samples of these same fungi. Regarding unrepresented or underrepresented phyla, among the fungal genome sequences available now are representative species from four of the eight fungal phyla, including Ascomycota, Basidiomycota, Zygomycota, and Chytridiomycota, but most of the sequences originate from the ascomycetes, the most highly populated of the fungal phyla. The available zygomycete sequences originate from only three species (Phycomyces blakesleeanus, Mucor circinelloides, and Rhizopus oryzae) and the chytridiomycete sequences originate from only one species, Batrachochytrium dendrobatidis. The highest priority is for a second Chytridiomycota, the first Blastocladiomycota, the first Entomophthoromycotina, the first Zoopagomycotina, and the first Kickxellomycotina. Considering the significance of the many genomics-fueled discoveries outlined above and the paucity of fungal genome sequences available, sequencing more fungal genomes from a wider diversity of species is a high priority.

To better apply comparative, evolutionary genomics to pathogenic fungi, it is essential to have genome sequence from nonpathogenic species that are so closely related that nucleotide substitution has not yet saturated. For example, with Coccidioides species, the relative Uncinocarpus is too distant. What is needed are sequences from Chrysosporium.

Finally, to fully apply comparative, evolutionary genomics to socially-important fungi, population samples are needed. Here, 20 to 30 resequenced genomes per population would allow biologists to extract the most information about adaptive mutations that are most likely to be important to pathogenicity.

In all cases, whether the genomes are representatives of undersampled phyla, extremely close relatives of pathogens, or individuals in pathogen populations, the data must be made available thought a central, curated database.

The genomes that will benefit the most researchers should be prioritized for sequencing by assembling concerned scientists to work together to compile a proposal with defined rationales for the fungi of greatest interest. It is important to have representative sequences from each of the eight fungal phyla, but it would also be very useful to have selected groups of fungi that are particularly well sampled so that scientists may begin to understand the forces that drive evolution. Additional human fungal pathogens should also be sequenced, including, for example, Sporothrix schenckii, which is the only representative of the dimorphic fungal pathogens not currently in the process of being sequenced. This fungus is not only the causative agent of disease in humans, but is also related to plant pathogens of the Ceratocystis species.

Nonculturable fungi are another priority for genome sequencing. The ability to culture microorganisms is important, not only for diagnostic purposes in medicine but also to facilitate laboratory studies. Currently, a large proportion of microorganisms are nonculturable using traditional approaches, but in recent work researchers have successfully applied the knowledge gained from genome sequences to extrapolate culture conditions. For example, the genomes of two species of Malassezia associated with eczema and dandruff were recently sequenced, revealing that a gene that encodes a key enzyme for lipid biosynthesis is missing, a deficit that explains the strains' requirements for lipids when grown in the lab. Researchers have taken a similar approach with bacteria that previously could be grown only in the presence of their host; by sequencing these genomes they were able to extrapolate the appropriate conditions for cultivation. This strategy could be applied to nonculturable fungi like Pneumocystis species, which can currently only be grown in the lungs of infected animals.

Studies on basal, early diverging fungi and related organisms have much to teach us about the nature of the fungal family tree and about our own evolution from a unicellular ancestor. Genome sequences from key representative species can be used to determine whether microsporidia are fungi or the closest sister group of species to the fungal kingdom, thereby defining the earliest group of species that define the fungal kingdom. The microsporidia are a highly successful group of animal and plant pathogens with at least 1200 known species, of which at least 13 infect humans. They are obligate intracellular pathogens that cannot be grown freely in culture and have dramatically reduced genomes (as small as 2 to 3 MB, compared to 9 to 10 MB for the smallest free living fungi) that have undergone dramatically accelerated evolution. Further understanding of their relationship to fungi will be critical to understanding their pathogenic lifestyle and development of novel therapeutics.

The question of the origins and relationship of species to the fungal kingdom is of interest not only because it addresses fundamental questions about what is a fungus and what is not, but because it also has implications for the history of human evolution. Recent advances in molecular phylogeny have revealed that the animal and fungal kingdoms descended from a common ancestor (thought to have been an aquatic, unicellular, motile organism with a posterior flagellum) roughly one billion years ago, indicating that animals and fungi share a common history and are more closely related to each other than previously thought. This area is currently the focus of a genome initiative addressing the origins of the evolution of multicellularity, with a focus on the genomes of 10 organisms, including three basal fungi (Allomyces macrogynus, Spizellomyces punctatus, and Mortierella verticillata), a species in a sister clade to the fungi (Nuclearia simplex, a member of the Nucleariidae, a sister group to the fungi), and representatives of the sister clade to the metazoa (including species in the Choanoflagellates (such as Monosiga brevicollis) and also the Ichthyosporea and Capsaspora).

TRAINING NEEDS

There are a few identifiable weaknesses in mycology training today. Improving connections between mycologists and bioinformatic specialists and boosting training in classical mycology, a field that is currently lagging behind other areas of expertise, are of particular concern.

Mycology is overwhelmed with data, and the field is in search of ways to use it effectively. There is a great need to connect mycologists with bioinformatic specialists to improve education and training in mycology. Students and scientists alike need training in how to use databases and exploit the available resources in fungal genomics. With these tools in hand, mycologists will be prepared to capitalize on the promise of genomics and begin to uncover the function of the vast number of genes that have yet to be characterized. The summer course in Molecular Mycology at the Marine Biological Laboratory in Woods Hole, Massachusetts, offers one opportunity to train scientists in mycology and pathogenesis that could extend to fungal bioinformatics.

There is a clear need to train more fungal physiologists and classical mycologists to maintain a pool of expertise in these fields. With respect to education and training, fungal physiology and classical mycology are losing ground to other, more recent branches of molecular mycology. Mycology must retain its knowledge base in classical mycology, lest scientists be forced to rely solely on molecular typing to identify environmental and clinical fungi, a technique that can give results that are inconsistent with biology. The field also needs to train experts in systematics who know fungi and can maintain culture collections.

Lastly, mycology is badly in need of support for training. In many of the funding agencies, there are sizeable gaps between the funding afforded to fungal biology and that afforded to other, comparable branches of biology.