NATURAL TOXINS 1:147-149 (1992)
INT~DUCTION
Fungal Endophytes of Plants: Biological and Chemical Diversity Keith Clay Department of Biology, Indiana University, Bloomington, Indiana
This issue of Natural Toxins highlights a poorly investigated but extremely common source of toxins of biological origin. Endophytic fungi exist asymptomatically within the aerial tissues of most, if not all, plants. These symbioses represent a diverse assemblage of fungi and hosts with a wide range of functional interactions. Historically, the interior of healthy plant leaves has been assumed to be sterile but recent research indicates that this perspective should be replaced; plant leaves are proving to be virtual microbial ecosystems on the same scale as plant root systems or digestive systems of ruminants. The simple act of surface sterilizing leaves and plating them on nutrient agar reveals a diversity of fungi within, existing in a state of innocuous or beneficial symbiosis. This should not be surprising given that plant leaves probably provide the most abundant living substrate for heterotrophic organisms in terrestrial ecosystems. It is their chemical diversity, and not their biologically diversity, that is largely responsible for the mounting interest in fungal endophytes. Toxins produced by endophytes independently or in association with their host plants cause significant agricultural losses from livestock poisonings while at the same time, they represent highly desirable traits to incorporate into other plants for enhanced resistance to pests. An increasing number of studies in natural habitats suggest that endophytes influence a range of plant population, community, and ecosystem processes through their toxic effects on herbivorous animals. While there is considerable recent research on fungal endophyte infections of trees, shrubs, and herbaceous plants other than grasses [Carroll, 19881, most research has focused on the clavicipitaceous endophytes of grasses [Clay, 19891. These endophytes are important in the livestock and turfgrass industries in both a positive and negative sense. The fungi are closely related to the more notorious Claviceps species and produce many of the same alkaloid toxins. However, unlike Claviceps, the endophytes are perennial and systemic within the aboveground tissues of their hosts. Many are also hereditary and seed borne; they represent one of the most highly specialized and coadapted symbioses that have not 0
1992 Wilw-Liss, Inc
achieved organellar status. Much current research is designed to move endophytes from one host to another and to alter their toxin-producing abilities. Recent papers by Siege1 et al. 119901and Hill et al. [1991] illustrate attempts to understand the production of alkaloids and manipulate their expression in grasdendophyte symbioses. The work of Schardl and his colleagues (described herein) indicate how molecular techniques may be used to directly alter toxin production by endophytes. Three papers in this issue consider various aspects of grass/endophyte associations, reflecting the economic importance of their toxins and the concomitant research efforts. Leuchtmann provides an overview of the systematics and host relations of clavicipitaceous endophytes of grasses. There are a number of well defined sexual species in addition to a larger complex of asexual forms whose distributions are related to the C-3 or C-4 photosynthetic mechanisms of their hosts. Photosynthetic mechanism is also correlated with the preponderance of contagious (by spores) versus maternal (through seeds) transmission of the fungi. The recent interest in applications of grass endophytes in plant protection and biotechnology has led to attempts to inoculate endophytes into alien hosts. Leuchtmann’s review of the mixed results from these studies suggests that grasslendophyte compatibility relationships are variable among taxa and probably have a complex genetic basis. He also points out several fascinating examples of endophytes of grasses which are functionally similar to the clavicipitaceous endophytes but are phylogenetically unrelated. These cases strongly suggest that a variety of fungal groups have undergone convergent evolution to fill a common niche in plants as a source of natural toxins. The alkaloid toxins from endophyte-infected pasture grasses cause widespread economic losses in the livestock industry. The same or related toxins are responsible for insect resistance of infected grasses as well. Some toxins are clearly fungal in origin, based on their production in pure fungal cultures, while others are of unknown origin,
Received June 1, 1992; accepted for publication August 20,1992
148
CLAY
being found only in infected grasses but not uninfected grasses or pure cultures. Endophyte infections are also common in wild grasses but they have been poorly studied from a chemical perspective. Probably fewer than 1‘!4 of infected grasses have been examined chemically. In one exception, a new type of ergot alkaloid was discovered from endophyte-infected sandbur grass (Cenchrus echinatus) [Powell et al., 19901. Detailed surveys of wild endophyte-infected grasses will undoubtably reveal a tremendous diversity of novel alkaloid toxins. In this issue, Powell and Petroski review the chemistry of endophyte alkaloids including analytical techniques, the major infected pasture grasses, and the primary groups of endophyte alkaloids. Increased knowledge of chemistry is essential for solving the livestock toxicity problems and for biotechnological manipulation of the grass/endophyte symbiosis. Endophytes are probably one of the most common and widespread forms of biocontrol but they also present significant in grasses. The enhanced fitness and pest resistance of many important endophyte-infected grasses provide a strong rationale for research to eliminate the mammalian toxicity of infected tall fescue, perennial ryegrass, and others. Molecular genetic techniques are powerful tools for the genetic manipulation of endophytes to eliminate or modify genes critical for the production of alkaloid toxins, or for the introduction of foreign DNA sequences coding for desirable metabolites. They are also useful for unraveling the phylogenetic history of endophytes and their host relations. Here Schardl and Tsai review much of their recent research on molecular studies of endophytes with a focus on mating studies, phylogenetic analysis, construction of clone libraries, and transformation systems necessary for modifying or disrupting alkaloid biosysnthetic pathways. Their innovative phylogenetic analyses show, for the first time, that the production of certain ergot alkaloids appears to be restricted to specific endophyte lineages. While clavicipitaceous endophytes of grasses have received the lion’s share of attention, they are by no means the only type of endophyte. The diversity of endophyte symbioses between other plants and other fungi is extraordinarily high. Few, if any, plants may be free of endophytes. There might easily be more than one million species of endophytic fungi [Hawksworth, 19911, surpassing the diversity of virtually all other forms of life on Earth. Many of these endophytes are also characterized by the production of chemical toxins with biological activity against animals and plant pathogens. Petrini, Sieber, Toti, and Viret provide an overview of biodiversity of endophytes from a wide range of hosts. Most endophytes that have thus far been isolated are ascomycetes or conidial forms lacking a sexual state. There is good evidence that adaptation of endophytes to their hosts occurs at several levels. There is specificity for
tissue type and tissue age within a host and there is specificity for particular host species in mixed plant communities. While many endophyte taxa can be isolated from a given host, only a few are typically dominant. Extensive sampling may be required to “capture” most endophytes. Further, they point out the importance of the type of isolation process and nutrient media utilized; varying these factors can produce distinct assemblages of endophytes from a single host. We may be seeing only a small percentage of the actual endophytes present, some of which may be unculturable under any circumstances. The enzymatic capabilities of endophytes and their production of secondary metabolites appears to be related to their lifestyle. Most are capable of degrading cuticle, epidermis, cell walls, and intercellular materials and utilizing these components as energy sources. Most endophytes investigated also produce a large array of metabolites including hormones like auxins and cytokinins which can affect plant growth, antibacterial and antifungal compounds, and toxins with effects on animals that consume infected plant tissues. As with the clavicipitaceous endophytes, they have potential applications as biocontrol agents, mycoherbicides, and genetic vectors but they also have drawbacks as well. Given the preponderance of endophytic symbioses in nature, the likely possibility arises that they may mediate interactions among plants and herbivores via toxin production by the fungus. Microbes on and in plant leaves have generally been ignored in plant-herbivore studies despite the fact that there are several well-documented examples of herbivores being affected by plant resident fungi. In the final contribution on endophytes, Hammon and Faeth consider the potential role of endophytes at the population, community, and ecosystem levels. They suggest that most endophytes are probably not mutualistic, or beneficial to their hosts, and outline conditions where mutualistic symbioses are to be expected. It may be hard to separate distinguish the biologically significant, mutualistic endophytes from other commensalistic, or neutral, endophytes when many species co-occur within a single host plant. Major theoretical issues in plant-herbivore interactions could be influenced by endophyte infections. For example, the presence of endophytes could provide host plants novel chemical attributes so that from the perspective of an insect herbivore, host plants may essentially be a new, unpalatable species. Long recognized seasonal trends in herbivory, where young tissues are preferentially consumed over older tissues, could reflect the buildup of endophyte infections over time. Endophyte infections could also affect plant-insect interactions in more complex, indirect ways. Taper et al. [I9861 describe a fascinating interaction between gall-forming cynipid wasps, their host oak trees, and an unidentified, leafinhabiting fungus. The wasps prefer to oviposit on trees
149
FUNGAL ENDOPHYTES OF PLANTS
and leaves with higher tannin levels even though tannins inhibit larval growth. This apparent maladaptation in fact may not be maladaptive when one considers that the fungus, which attacks developing galls, is inhibited by increasing tannin concentrations. Although the data base is relatively meager at this point, Hammon and Faeth present a strong case that endophytes potentially have a pervasive role in plant / herbivore interactions. Anyone with an interest in natural toxins should be aware of the biological and chemical diversity of fungal endophytes infecting plants. Their role in natural and agricultural plant communities is largely a function of their ability to produce a wide array of biological toxins with activity against organisms that feed on their hosts. Taken together, the following interdisciplinary compilation of papers provides a broad and up-to-date overview of the state of endophyte research and identifies the important areas for future research by scientists of diverse background and expertise.
REFERENCES Carroll G (1988): Fungal endophytes in stems and leaves: From latent pathogen to mutualistic symbiont. Ecology 69:2-9. Clay K (1989): Clavicipitaceous endophytes of grasses: Their potential as biocontrol agents. Mycol Res 92:l-12. Hawksworth D L (1991): The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol Res 95:641-655. Hill NS, Parrott WA, Pope D D (1991): Ergopeptine alkaloid production by endophytes in a common tall fescue genotype. Crop Sci 3 1 :1545- 1547. Powell RG, Plattner RD. Y a k s SG, Clay K, Leuchtmann A (1990): Ergobalansine, a new ergot-type peptide alkaloid isolated from Cmchrus echinufus (sandbur grass) infected with Bulimia obtecfa. and produced in liquid cultures of B. obferfuand Bnlunsiu cyperi. J Nat Prod 53:1272-1279. Siege1 MR, Latch GCM, Bush LP, Fannin FF, Rowan DD, Tapper BA, Bacon CW, Johnson MC (1990): Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response. J Chem Ecol 16:3301-33 15. Taper ML. Zimmermann EM, Case TJ (1986): Sources of mortality for a cynipid gall-wasp (Dryocosmus thtbiosus (Hymenoptera:Cynipidae)): The importance of tannin/ fungus interaction. Oecologia 68:437-445.
INT~DUCTION
Fungal Endophytes of Plants: Biological and Chemical Diversity Keith Clay Department of Biology, Indiana University, Bloomington, Indiana
This issue of Natural Toxins highlights a poorly investigated but extremely common source of toxins of biological origin. Endophytic fungi exist asymptomatically within the aerial tissues of most, if not all, plants. These symbioses represent a diverse assemblage of fungi and hosts with a wide range of functional interactions. Historically, the interior of healthy plant leaves has been assumed to be sterile but recent research indicates that this perspective should be replaced; plant leaves are proving to be virtual microbial ecosystems on the same scale as plant root systems or digestive systems of ruminants. The simple act of surface sterilizing leaves and plating them on nutrient agar reveals a diversity of fungi within, existing in a state of innocuous or beneficial symbiosis. This should not be surprising given that plant leaves probably provide the most abundant living substrate for heterotrophic organisms in terrestrial ecosystems. It is their chemical diversity, and not their biologically diversity, that is largely responsible for the mounting interest in fungal endophytes. Toxins produced by endophytes independently or in association with their host plants cause significant agricultural losses from livestock poisonings while at the same time, they represent highly desirable traits to incorporate into other plants for enhanced resistance to pests. An increasing number of studies in natural habitats suggest that endophytes influence a range of plant population, community, and ecosystem processes through their toxic effects on herbivorous animals. While there is considerable recent research on fungal endophyte infections of trees, shrubs, and herbaceous plants other than grasses [Carroll, 19881, most research has focused on the clavicipitaceous endophytes of grasses [Clay, 19891. These endophytes are important in the livestock and turfgrass industries in both a positive and negative sense. The fungi are closely related to the more notorious Claviceps species and produce many of the same alkaloid toxins. However, unlike Claviceps, the endophytes are perennial and systemic within the aboveground tissues of their hosts. Many are also hereditary and seed borne; they represent one of the most highly specialized and coadapted symbioses that have not 0
1992 Wilw-Liss, Inc
achieved organellar status. Much current research is designed to move endophytes from one host to another and to alter their toxin-producing abilities. Recent papers by Siege1 et al. 119901and Hill et al. [1991] illustrate attempts to understand the production of alkaloids and manipulate their expression in grasdendophyte symbioses. The work of Schardl and his colleagues (described herein) indicate how molecular techniques may be used to directly alter toxin production by endophytes. Three papers in this issue consider various aspects of grass/endophyte associations, reflecting the economic importance of their toxins and the concomitant research efforts. Leuchtmann provides an overview of the systematics and host relations of clavicipitaceous endophytes of grasses. There are a number of well defined sexual species in addition to a larger complex of asexual forms whose distributions are related to the C-3 or C-4 photosynthetic mechanisms of their hosts. Photosynthetic mechanism is also correlated with the preponderance of contagious (by spores) versus maternal (through seeds) transmission of the fungi. The recent interest in applications of grass endophytes in plant protection and biotechnology has led to attempts to inoculate endophytes into alien hosts. Leuchtmann’s review of the mixed results from these studies suggests that grasslendophyte compatibility relationships are variable among taxa and probably have a complex genetic basis. He also points out several fascinating examples of endophytes of grasses which are functionally similar to the clavicipitaceous endophytes but are phylogenetically unrelated. These cases strongly suggest that a variety of fungal groups have undergone convergent evolution to fill a common niche in plants as a source of natural toxins. The alkaloid toxins from endophyte-infected pasture grasses cause widespread economic losses in the livestock industry. The same or related toxins are responsible for insect resistance of infected grasses as well. Some toxins are clearly fungal in origin, based on their production in pure fungal cultures, while others are of unknown origin,
Received June 1, 1992; accepted for publication August 20,1992
148
CLAY
being found only in infected grasses but not uninfected grasses or pure cultures. Endophyte infections are also common in wild grasses but they have been poorly studied from a chemical perspective. Probably fewer than 1‘!4 of infected grasses have been examined chemically. In one exception, a new type of ergot alkaloid was discovered from endophyte-infected sandbur grass (Cenchrus echinatus) [Powell et al., 19901. Detailed surveys of wild endophyte-infected grasses will undoubtably reveal a tremendous diversity of novel alkaloid toxins. In this issue, Powell and Petroski review the chemistry of endophyte alkaloids including analytical techniques, the major infected pasture grasses, and the primary groups of endophyte alkaloids. Increased knowledge of chemistry is essential for solving the livestock toxicity problems and for biotechnological manipulation of the grass/endophyte symbiosis. Endophytes are probably one of the most common and widespread forms of biocontrol but they also present significant in grasses. The enhanced fitness and pest resistance of many important endophyte-infected grasses provide a strong rationale for research to eliminate the mammalian toxicity of infected tall fescue, perennial ryegrass, and others. Molecular genetic techniques are powerful tools for the genetic manipulation of endophytes to eliminate or modify genes critical for the production of alkaloid toxins, or for the introduction of foreign DNA sequences coding for desirable metabolites. They are also useful for unraveling the phylogenetic history of endophytes and their host relations. Here Schardl and Tsai review much of their recent research on molecular studies of endophytes with a focus on mating studies, phylogenetic analysis, construction of clone libraries, and transformation systems necessary for modifying or disrupting alkaloid biosysnthetic pathways. Their innovative phylogenetic analyses show, for the first time, that the production of certain ergot alkaloids appears to be restricted to specific endophyte lineages. While clavicipitaceous endophytes of grasses have received the lion’s share of attention, they are by no means the only type of endophyte. The diversity of endophyte symbioses between other plants and other fungi is extraordinarily high. Few, if any, plants may be free of endophytes. There might easily be more than one million species of endophytic fungi [Hawksworth, 19911, surpassing the diversity of virtually all other forms of life on Earth. Many of these endophytes are also characterized by the production of chemical toxins with biological activity against animals and plant pathogens. Petrini, Sieber, Toti, and Viret provide an overview of biodiversity of endophytes from a wide range of hosts. Most endophytes that have thus far been isolated are ascomycetes or conidial forms lacking a sexual state. There is good evidence that adaptation of endophytes to their hosts occurs at several levels. There is specificity for
tissue type and tissue age within a host and there is specificity for particular host species in mixed plant communities. While many endophyte taxa can be isolated from a given host, only a few are typically dominant. Extensive sampling may be required to “capture” most endophytes. Further, they point out the importance of the type of isolation process and nutrient media utilized; varying these factors can produce distinct assemblages of endophytes from a single host. We may be seeing only a small percentage of the actual endophytes present, some of which may be unculturable under any circumstances. The enzymatic capabilities of endophytes and their production of secondary metabolites appears to be related to their lifestyle. Most are capable of degrading cuticle, epidermis, cell walls, and intercellular materials and utilizing these components as energy sources. Most endophytes investigated also produce a large array of metabolites including hormones like auxins and cytokinins which can affect plant growth, antibacterial and antifungal compounds, and toxins with effects on animals that consume infected plant tissues. As with the clavicipitaceous endophytes, they have potential applications as biocontrol agents, mycoherbicides, and genetic vectors but they also have drawbacks as well. Given the preponderance of endophytic symbioses in nature, the likely possibility arises that they may mediate interactions among plants and herbivores via toxin production by the fungus. Microbes on and in plant leaves have generally been ignored in plant-herbivore studies despite the fact that there are several well-documented examples of herbivores being affected by plant resident fungi. In the final contribution on endophytes, Hammon and Faeth consider the potential role of endophytes at the population, community, and ecosystem levels. They suggest that most endophytes are probably not mutualistic, or beneficial to their hosts, and outline conditions where mutualistic symbioses are to be expected. It may be hard to separate distinguish the biologically significant, mutualistic endophytes from other commensalistic, or neutral, endophytes when many species co-occur within a single host plant. Major theoretical issues in plant-herbivore interactions could be influenced by endophyte infections. For example, the presence of endophytes could provide host plants novel chemical attributes so that from the perspective of an insect herbivore, host plants may essentially be a new, unpalatable species. Long recognized seasonal trends in herbivory, where young tissues are preferentially consumed over older tissues, could reflect the buildup of endophyte infections over time. Endophyte infections could also affect plant-insect interactions in more complex, indirect ways. Taper et al. [I9861 describe a fascinating interaction between gall-forming cynipid wasps, their host oak trees, and an unidentified, leafinhabiting fungus. The wasps prefer to oviposit on trees
149
FUNGAL ENDOPHYTES OF PLANTS
and leaves with higher tannin levels even though tannins inhibit larval growth. This apparent maladaptation in fact may not be maladaptive when one considers that the fungus, which attacks developing galls, is inhibited by increasing tannin concentrations. Although the data base is relatively meager at this point, Hammon and Faeth present a strong case that endophytes potentially have a pervasive role in plant / herbivore interactions. Anyone with an interest in natural toxins should be aware of the biological and chemical diversity of fungal endophytes infecting plants. Their role in natural and agricultural plant communities is largely a function of their ability to produce a wide array of biological toxins with activity against organisms that feed on their hosts. Taken together, the following interdisciplinary compilation of papers provides a broad and up-to-date overview of the state of endophyte research and identifies the important areas for future research by scientists of diverse background and expertise.
REFERENCES Carroll G (1988): Fungal endophytes in stems and leaves: From latent pathogen to mutualistic symbiont. Ecology 69:2-9. Clay K (1989): Clavicipitaceous endophytes of grasses: Their potential as biocontrol agents. Mycol Res 92:l-12. Hawksworth D L (1991): The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol Res 95:641-655. Hill NS, Parrott WA, Pope D D (1991): Ergopeptine alkaloid production by endophytes in a common tall fescue genotype. Crop Sci 3 1 :1545- 1547. Powell RG, Plattner RD. Y a k s SG, Clay K, Leuchtmann A (1990): Ergobalansine, a new ergot-type peptide alkaloid isolated from Cmchrus echinufus (sandbur grass) infected with Bulimia obtecfa. and produced in liquid cultures of B. obferfuand Bnlunsiu cyperi. J Nat Prod 53:1272-1279. Siege1 MR, Latch GCM, Bush LP, Fannin FF, Rowan DD, Tapper BA, Bacon CW, Johnson MC (1990): Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response. J Chem Ecol 16:3301-33 15. Taper ML. Zimmermann EM, Case TJ (1986): Sources of mortality for a cynipid gall-wasp (Dryocosmus thtbiosus (Hymenoptera:Cynipidae)): The importance of tannin/ fungus interaction. Oecologia 68:437-445.
