JIRCAS Journal No.8: 49 - 59 (2000) 49
Occurrence of an Ephelis Fungus on Ishigaki Island and Observations on its Epiphytic Association with Host Grasses
Michael J. CHRISTENSENa), Keiichi TAKAHASHib),Katsuyuki KOHNOc) Takao TSUKIBOSHid) and Makoto KOBAYASHie)
a) AgResearch Grasslands (Private Bag ll008 Palmerston North, New Zealand) b) Okinawa Subtropical Station, Japan International Research Center for Agricultural Sciences (JIRCAS) (Ishigaki, Okinawa, 907-0002 Japan) Present address : Postharvest Technology Division, National Food Research Institute (NFRI) (Tsukuba, Ibaraki, 305-8642 Japan ) c) Okinawa Subtropical Station, Japan International Research Center for Agricultural Sciences (JIRCAS) (Ishigaki, Okinawa, 907-0002 Japan) d) Department of Environmental Biology, National Institute of Agro-Environmental Sciences (NIAES) (Tsukuba, Ibaraki, 305-8604 Japan) e) Department of Plant Breeding, National Grassland Research Institute (NGRI) (Nishinasuno, Tochigi, 329-2747 Japan)
Received November 8, 1999
Abstract
Distribution of Ephelis-infected grasses on Ishigaki, and the frequency at which infected plants were present, were observed over a three year period. Sites with infected plants were most commonly wet areas that had been undisturbed for many years. The frequency of infection varied widely between grass species. In three of the fifteen grasses found to be hosts to this fungus, infected plants were present at many sites, while in other species, infected plants were present at only one or two sites. Ephelis infection of three of the grasses was found in only one plant. The number of infected tillers at each site was typically low with apparently a random distribution, and no evidence of spread from sites of infection. Infected plants were characterized by stromata that covered the inflorescence with dense mycelial growth, and in most grasses, by hyphae on the surface of the leaf blades. Infected plants typically showed both infected and Ephelis-free tillers. Examination of infected plants of 10 species revealed that the Ephelis fungus was essentially an epiphyte, with hyphae in vegetative tillers being located on the surface of the stem apex, including primordial leaves. Hyphae on the leaf blades were also apparently confined to epiphytic growth. Possible mechanisms for infection, the ecological and economic significance of Ephelis infection on Ishigaki, and areas for future research are outlined.
Key words : Clavicipitaceae, Ephelis, epiphyte, grasses 50 JIRCAS J No.8, 2000
Introduction were the presence and location of hyphae on leaves. The presence or absence of Ephelis stromata on each Fifteen species of grasses growing on Ishigaki reproductive tiller of individual plants was also noted. Island are hosts to a fungus forming a stromata which Hyphal distribution within infected plants was envelop the inflorescence11.12,13l. The black appea noted. Stems of reproductive tillers were slit open, rance of the stromata, and the sterilization of infected some internal tissue removed, placed in a drop of tillers, have given rise to the descriptive name "black aniline blue stain on a microscope slide, covered, choke". Leaves of some species infected with this heated to boiling, and examined using a compound fungus are covered with white hyphal growth. No microscope for the presence of hyphae. Epidermal sexual stage has been observed, but the fungus on all strips were removed from the leaf blades and sheaths the grass species has been identified as belonging to and similarly examined for the presence of the anamorphic genus Ephelis from the presence of intercellular hyphae. The presence of hyphae in stem characteristic elongate narrow conidia13l. Informa apices was assessed by dissecting these from tion from conidial morphology and sequence analysis terminal portions of non-reproductive tillers, staining of the ribosomal DNA ITS region in Ephelis spp. with aniline blue, and examining with a compound obtained from different hosts and locations indicate microscope. strongly that all the grasses are infected with the same species10l. In 1904 an Ephelis species was Results reported on Miscanthus tinctorius and Paspalum thunbergii in Japan and given the name E. japonica 1) Survey ofEphelis-infected grasses in Ishigaki Hennings6l. As the description of conidia and The number of sites where Ephelis-infected plants stromata of E. japonica was similar to that observed were observed varied markedly for each grass in Ephelis-infected grasses in Japan in recent years, species (Table 1). Ephelis infection occurred at many this may be the correct name to use for the Ephelis sites in three grasses; Chrysopogon aciculatus, fungus present in Ishigaki and other locations in Digitaria violascens and Paspalum scrobiculatum, but Japan. This paper reports on the distribution of only in E scrobiculatum were infected plants common Ephelis-infected grasses on Ishigaki Island and the at each site. Ephelis infection was recorded at only frequency at which they were found. Also reported two sites in three of the 15 grasses (Brachiaria are the results of a study that assessed the nature of mutica, Chloris barbata, Imperata cylindrica var. the association between the Ephelis fungus and host koenigii), and the number of infected plants was low. grasses. The remaining nine species were found only at a single site. In three grasses, (Cynodon dactylon, Materials and methods Eriochloa procera, E urvillei), Ephelis infection was observed only on a single plant. Sites with infected 1) Survey of Ephelis-infected grasses in Ishigaki plants were most commonly wet areas that had The presence on Ishigaki Island of grasses with remained undisturbed for many years. Forest was the Ephelis infection stromata on inflorescences and/or least likely habitat where Ephelis-infected grasses white hyphal growth on leaves was recorded over a could be found, with only one species, Leptochloa period of three years. panicea confined to one small site, being located, in spite of intensive surveys conducted over a three 2) Study of the nature of the Ephelis/grass association year period. Stromata were observed on 14 of the During April 2-8 1999, Ephelis-infected plants of 10 species, but not on Cynodon pletostachyrus, a grass species were identified at previously located sites. that does not flower in Ishigaki. Leaf infection was The distribution of the infected plants at each site observed for 13 of the grasses, with infection being was observed, and the plants sampled for particularly conspicuous on B. mutica and C. examination. Appearance of stromata was noted, as pletostachyrus (Table 1). Michael]. CHRISTENSEN et al.: Occurrence of an Ephelis Fungus on Ishigaki Island 51
Table 1. Grass species with Ephelis present on Ishigaki island, their frequency of infection, and symptoms
Grass species Subfamily Tribe
Brachiaria mutica (Forsk.) Stapf (Paragrass) a,c,e) Panicoideae Paniceae (Panicodae)
Chloris barbata Swartz. (Purple top chloris) a,c) Chloridoideae Chlorideae
Chloris divaricata R. Br. a,c) Chloridoideae Andropogoneae
Chrysopogon aciculatus (Retz.) Trin. a,b) Panicoideae Andropogoneae (Andropogonodae)
Cynodon dactylon (L.) Pers. (Bermudagrass) c,d,f) Chloridoideae Chlorideae
Cynodon pletostachyrus (K. Schm.) Pilgera,c,e) Chloridoideae Chlorideae (Giant star grass)
Digitaria eriantha Steud. (Pangola grass) c) Panicoideae Paniceae (Panicodae)
Digitaria violascens Link. a,b) Panicoideae Paniceae (Panicodae)
Eriochloa procera C.H. Hubb.c,d) Panicoideae Paniceae (Panicodae)
Imperata cylindrica (L). Beauv. var. koenigii (Retz). Panicoideae Andropogoneae Durand et Schinz (Needlegrass) a,c,d,f) (Andropogonodae)
Leptochloa panicea (Retz.) Ohwi a,c,d) Chloridoideae Chlorideae
Panicum crus-galli, Beauv. var. praticola Ohwi c) Panicoideae Paniceae (Panicodae)
Panicum repens L. a,c) Panicoideae Paniceae (Panicodae)
Paspalum scrobiculatum G. Forsta,b) Panicoideae Paniceae (Panicodae)
Paspalum urvillei Steud. c,d) Panicoideae Paniceae (Panicodae) a) examined in April 1999 study b) present at many sites c) present at just 1 or 2 sites d) very rare e) leaf blades conspicuously colonized f) leaf blades not colonized 52 JIRCAS ]. No.8, 2000
2) Study of the nature of the Ephelis/grass association On the upper side of the blades they were located Ten species of grasses with Ephelis were identified between the ridges containing the vascular bundles. and examined in the April 1999 survey. Several Numerous elongate narrow conidia were produced by Ephelis-infected plants were examined for each hyphae on the leaf blades. The hyphae appeared species except I. cylindrica, for which only a single confined to the surface of leaves, with no hyphae tiller with a stroma was found. Frequency of infection detected in the mesophyll of the leaf blades or of Ephelis-infected plants at each site, with just two sheaths. Leaves of infected tillers of these two exceptions, was typically low, with perhaps only 1% heavily colonized grasses were conspicuously or less of tillers showing evidence of infection. The shorter than those of Ephelis-free tillers. first exception was an area of mown turf that had been established for over twenty years, and where 5) Stem apex and stem infection many tillers of C. aciculatus were infected with Hyphae were observed on the surface of stem Ephelis. The second exception was with P. apices, including primordial leaves, of all nine of the scrobiculatum where infected tillers were widespread Ephelis-infected grass species for which non at several sites. Distribution of infected plants within reproductive tillers were available for examination. sites was apparently random, with no evidence of Short hyphae apparently growing intercellularly spread from a focal point. within young leaf blades were occasionally observed. Hyphae were not seen in tissues from within hollow 3) Stromata characteristics stems of reproductive tillers, or in tissues removed Stromata in all the grasses were initially from nodes of these tillers. comprised of dense white hyphal growth covering the developing seeds, later becoming black (Plate 1). 6) Distribution of Ephelis infected tillers within plants Hyphal growth was absent below the basal branches Ephelis-infected plants typically had both infected of inflorescences. Numerous narrow, elongate and healthy tillers. In the stoloniferous grasses B. conidia were present on the stromata. No perithecia mutica and C. pletostachyrus, Ephelis infection was were observed. often confined to just a few tillers arising from one part of the branching network of stolons. In non 4) Leaf colonization stoloniferous grasses, infected tillers appeared to be Hyphae were observed on the leaf blades of 8 of randomly distributed. In contrast, all the leaves of the 10 Ephelis-infected grasses examined in the April the infected tillers, with one exception, were 1999 survey. In two grass species, B. mutica and C. colonized by hyphae. The exception was a tiller of B. pletostachyrus, hyphal growth was conspicuous, and mutica where the four youngest leaves were heavily infected plants could be readily detected. Hyphal colonized by epiphytic hyphae, the fifth was sparsely growth on leaves of C. barbata and D. violascens was colonized, while the two oldest leaves had no visible sparse and difficult to detect without the use of a hyphae. stereo dissecting microscope. No hyphae were observed on the leaves of C. divaricata and I. Discussion cylindrica. Hyphal distribution on leaves was noted for the two grasses that were the most conspicuously In this study it was found that the frequency of colonized, B. mutica and C. pletostachyrus (Plate 2). Ephelis infection for each grass species, both in Hyphae were confined almost entirely to the blades; terms of the number of sites located, and the occasionally, sparse hyphae were also present on the frequency at which infected plants were infected sheath, located in close proximity to the ligular zone. within sites varied widely. It is certain that there are Hyphae were never seen on the inner surface of the more sites where Ephelis-infected grasses are sheath adjacent to the stem. Hyphae on blades were present than those found in this survey. However, present as dense, longitudinally orientated masses. the study provides a good indication of the frequency Michael]. CHRISTENSEN et al. : Occurrence of an Ephelis Fungus on Ishigaki Island 53
Pl ate 1. Stroma on Brachiaria 111urica in florescence
Pl ate 2. Hyphae on Cy 11 odo11 plerosrachvrns leaves 54 JIRCAS ]. No.8, 2000
of infection of grasses in Ishigaki. In particular, it m the swollen nodes of these tillers. In Ephelis seems certain that the occurrence of Ephelis infected plants, hyphae were either apparently infection of forest grasses is very rare as these absent in these tissues or at least present at low grasses were subjected to extensive surveys over a concentrations. The efficiency of colonization of period of three years. The most common sites were axillary buds was typically high in Epichloe/ areas of grassland that had remained undisturbed for Neotyphodium associations, with endophyte-free many years, with few infected plants found in tillers being seldom observed. In contrast, healthy recently established paddocks. However, no infected tillers were typically present on the Ephelis-infected plants were detected in close proximity to the sea, plants examined. even in grasslands established for a long period of This distribution of infected grasses within sites time. Within sites, infection was typically very low, provides clues as to how plants become systemically and no sites were found at which the majority of the infected. One possible mechanism for infection is the tillers were infected. penetration of leaves or crowns by hyphae from All of the host grasses were C4 species, and germinating conidia. Such a mechanism is unlikely, as representatives of two sub-families, and three tribes for systemic infection to occur hyphae would have to (Table 1). The general appearance of the host species penetrate through the densely packed layers of leaf varied markedly, ranging from small-leaved plants sheaths and then colonize the stem apex. The sparse that could persist under turf management to tall distribution of infected plants in natural populations broad-leaved types. The relationship of the Ephelis of B. mutica and C. pletostachyrus, species with very fungus to all 10 species of grass examined was high concentrations of conidia being produced on the essentially that of an epiphyte. Hyphae were located surface of leaves, provides evidence that this is not on the surface of the stem apices from where they the mechanism of infection. It is unlikely that can colonize the developing leaves and infection would occur from Ephelis within the soil as inflorescences, and the axillary buds which give rise Clavicipitaceous fungi are in nature obligate to new tillers. The epiphytic nature of the association biotrophs17l. Transmission through the seed appears was also apparent by the dense covering of hyphae to provide the most likely explanation of how plants present on the surface of leaf blades of some grass become systemically infected. Vertical seed species. This epiphytic relationship was similar to transmission has been reported for a symptomless that reported for other Clavicipitaceous fungi which Ephelis sp. found in association with Danthonia have Ephelis conidia, belonging to the genera spicata8l. During the vegetative growth of the plant, Balansia andAtkinsonella 9,171. the fungus inhabits only the external surfaces of the The epiphytic relationship between the Ephelis shoot apex and adjoining rudimentary leaves. How species and the grasses it colonizes on Ishigaki was ever, with the change of the host to the reproductive illustrated by a comparison of the distribution of phase, the fungus becomes incorporated into the hyphae on leaves and reproductive tillers with the developing ovary and ovule, penetrating the micro hyphal distribution of the endophytic Epichloe/ phyle to the megagametophyte wall. Hyphae enter Neotyphodium fungi. In leaves, hyphae of these the embryo sac at early embryogenesis and remain endophytic fungi were typically most concentrated in there during normal embryo development. Since the sheaths, while Ephelis hyphae were almost hyphae, however, invade the surface of the entirely associated with blades. In the blades of embryonic shoot apex before it is enclosed by the Epichloe/Neotyphodium-infected leaves, the hyphae, coleoptile rim, as the case of in Neotyphodium if present, were mainly concentrated in the basal, infected plants, infection of the next gerieration had oldest, region7l. InEphelis-infected plants the hyphae already occurred before seed maturation. Following tended to be uniformly located along the blades. germination, hyphae from the seedling apex may Hyphae were abundant in the stem of reproductive colonize the stem apex of each new tiller. However, tillers of Epichloe/Neotyphodium-infected plants, and vertical seed transmission generally seems unlikely Michael]. CHRISTENSEN et al.: Occurrence of an Ephelis Fungus on Ishigaki Island 55
in grasses infected with the Ephelis species present influence the incidence of Ephelis-infected plants. It in Ishigaki as developing inflorescences of Ephelis is possible that in some grasses Ephelis infection infected grasses in Ishigaki became heavily colonised gives them a competitive advantage. This may result by hyphae, preventing seed development and from enhanced tolerance to insect pests. This dispersal. A possible exception is P. scrobiculatum, hypothesis is supported by insect feeding trials where stromatal development did not always result which have revealed the existence of insect feeding in complete colonisation of inflorescences. This is deterrence in Ephelis-infected grasses in two grass one of the two species of grasses where the hoppers, Oxya yezoensis 14), and Aiolopus thalassinus incidence of Ephelis infection was relatively high at (Takahashi, unpubl. data), and an armyworm, some sites. Mythimna (Pseudaletia) separata (Takahashi, unpubl. Another possible mechanism for new infections data). Further evidence of Ephelis-induced insect with Ephelis is horizontal transmission. With this feeding deterrence is by the higher incidence of a form of transmission, infection might occur by hyphal mealy bug observed on Ephelis-free than on Ephelis penetration of stigmata of neighboring uninfected infected Digitaria eriantha growing in a glass-house. plants, and subsequent growth into developing Over time the incidence of infected perennial plants ovaries and ovules. Hyphae would invade the is likely to increase, even in the absence of fresh developing embryo and colonize the surface of the infection. In long-lived perennial grasses such as C. shoot apex, as in the case of the Ephelis of D. spicata. aciculatus and P. scrobiculatum, infected plants Horizontal infection via stigmata occurs in other increase in size, either from growth of stolons in the Clavicipitaceous fungi forming associations with case of C. aciculatus, or from increases in the number grasses9). With Claviceps species, infection following of tillers as in the case of P. scrobiculatum. Free stigmata penetration is confined to the developing standing plants with enhanced competitive advantage seed and gives rise to fungal sclerotia. Horizontal relative to non-infected plants, would arise from the infection with Balansia obtecta, in contrast, produces death of segments of stolons, and from the viable seeds which give rise to systemically infected fragmentation of the crowns of non- stoloniferous seedlings. A further example is Epichloe typhina, in grasses, increasing the frequency of occurrence of which, as in Ephelis infection of grasses in Ishigaki, infected plants. stromatal development results in sterilization of the Conversely, the low incidence of Ephelis infection host plant2\ The relatively low incidence of Ephelis in most of the host species suggests that infection is infected grasses on Ishigaki indicates that if not beneficial to the host plant or may even have a horizontal transmission does occur it is either a rare detrimental effect on plant survival and persistence. occurrence, or that germination of infected seeds and The growth of epiphytic hyphae on leaf blades may subsequent growth of infected seedlings are cause stress to host plant. Nutrients are required for adversely affected, and few plants become extensive epiphytic hyphal growth to occur on established. A complicating factor is that in other blades, and these hyphae must receive a continuous Clavicipitaceous fungi which form systemic supply of water to prevent desiccation. A possible seedborne infections with grasses following mechanism to ensure a supply of nutrients and water horizontal transmission, hyphae from ascospores and is that the hyphae modify the cuticle and epidermal 2 3 9 not conidia, penetrate into the stigmata • • ). To date, cells of leaves, allowing movement of nutrients and no perithecia have been observed on Ephelis-infected water from the apoplast to the hyphae where they grasses present in Japan. One possibility is that the will be absorbed. Disruption of the cuticle has been dark hyphal masses which develop as stromata age reported in D. spicata plants infected with the may function as sclerotia, and germinate to produce symptomless, seed-transmitted Ephelis species, apothecia and perithecia as occurs with Claviceps spp. associated with hyphal colonization of the internal and B. obtecta. surfaces of ovaries and ovules8). Modification of Factors other than the frequency of infection may epidermal cells and cuticle has also been reported in 56 JIRCAS ]. No.8, 2000
grasses infected with the Clavicipitaceous fungi Ephelis should contribute to the international effort Myriogenospora atramentosa Atk. and B. epichloe to better understand this continuum of association (Weese) Diehl15l; and E. amarillans 16l. Water loss and how these associations may have evolved. associated with increased permeability of the cuticle and epidermal layer may increase the susceptibility Acknowledgement of Ephelis-infected plants to drought, and thus provide an explanation why infected grasses are The authors thank Dr. Melva Philipson and Mr. found mainly in wet areas where the effects of water Wagne Simpson for reviewing the manuscript, and loss will be less damaging. the STA is also acknowledged for providing a travel Infection of grasses with Ephelis appears to have fellowship for M. J. Christensen. little ecological or economic significance in Ishigaki. The typically low incidence of infected plants References indicates a lack of a competitive advantage over non 1) Christensen, M. J. et al. (1993). Taxonomy of infected plants, even though the presence of the Acremonium endophytes of tall fescue fungus may confer protection against some insects. (Festuca arundinacea), meadow fescue (F. It is not known if grasses infected with this Ephelis pratensis), and perennial rye-grass (Lolium species contain alkaloids that are toxic to grazing perenne). Mycol. Res. 97: 1083-1092. animals, as are grasses infected with other 2) Chung, K.-R. and Schardl, C. L. (1997). Sexual Clavicipitaceae fungi. These alkaloids have been cycle and horizontal transmission of the grass extensively studied in C. purpurea 5l, and in symbiont, Epichloe typhina. Mycol. Res. 101: 295- Neotyphodium-infected L. perenne, F. arundinacea, 301. and F. pratensis 1l. Their presence has important 3) Diehl W W (1950). Balansia and the Balansiae implication for the health of grazing animals4l. It in America. Agricultural Monograph. United seems unlikely that even if such alkaloids were States Department of Agriculture, Washington present in Ephelis-infected grasses on Ishigaki the D.C. concentrations would reach harmful levels as the 4) Fletcher, L. R. and Easton, H. S. (1997). The incidence of infected plants is low. evaluation and use of endophytes for pasture The findings of this study point to areas requring improvement. In Neotyphodium/Grass Interac furthur investigations as follows : First, is the Ephelis tions. ed. C. W Bacon and N. S. Hill, New York, fungus transmitted through vertical and/or horizontal Plenum Press, 209-227. transmission? A second area of research is the 5) Flieger, M. et al. (1997). Ergot alkaloids - presence of alkaloids in Ephelis-infected plants. Do sources, structures and analytical methods. all of the grass species contain the same alkaloids? Folia Microbial. 42: 3-30. The findings may contribute to understanding of the 6) Hennings, P. (1904). Fungi fluminense A. CL. E. evolution of biotrophic Clavicipitaceae fungi. It is also ULE Collecti. Hedwigia 43:242-273. possible that infected plants may contain secondary 7) Keogh, R. et al. (1996). Distributions of the metabolites of pharmacological importance. A third fungal endophyte Acremonium lolii, and of the area for research is the effect of epiphytic hyphae on alkaloids lolitrem B and peramine, within the cuticle and epidermal layer of leaves and the perennial ryegrass. N Z. ]. Agric. Res. 39: 121- developing inflorescence. Finally, there exists within 127. the graminicolm.is Clavicipitaceae fungi, a continuum 8) Philipson M. N. and Christey, M. C. (1985). An in the nature of their association with host grasses, epiphytic/endophytic fungal association of ranging from infection being confined entirely to the Danthonia spicata transmitted through the seed resulting in seed death, to associations in which embryo sac. Botanical Gazette 146: 70-81. the fungus grows as a symptomless seed-transmitted 9) Schardl, C. L. and Clay, K. (1997). Evolution of endophyte. More detailed information on the Ishigaki mutualistic endophytes from plant pathogens. Michael J. CHRISTENSEN et al.: Occurrence of an Ephelis Fungus on Ishigaki Island 57
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