New Phytol. (1993), 124, 93-100 Root colonization of Lupinus latifolius Agardh. and Pinus contorta Dougl. by Phialocephala fortinii Wang & Wilcox BY T . E. O ' D E L L ^ * , H . B. MASSICOTTE^f AND J. M. ^Department of Botany & Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA ^Department of Forest Science, Oregon State University, Corvallis, Oregon 97331, USA {Received 10 September 1992; accepted 19 January 1993) SUMMARY Root colonization patterns were studied after Phialocephala fortinii w as inoculated on Lupinus tatifolius (broadleafed lupin), a nitrogen-fixing legume, and Pinus contorta (lodgepole pine). The fungus colonized epidermal and cortical cells in the root hair zone on ultimate pine roots, as well as cortical and epidermal cells of primary roots of both hosts. Fungal colonization was inter- and intracellular with sclerotia forming in cells of both hosts. Labyrinthine tissue, a type of fungal differentiation that occurs in the Hartig net of ectomycorrhizas, formed sporadically on pine roots. Similar colonization has been observed on conifers and many other plants, but this report is the first showing that a single fungus can form such structures on both pine and lupin. Key words: Lupinus latifolius, Pinus contorta, Phialocephala fortinii. Mycelium radicis atrovirens, septate root endophytes. INTRODUCTION Fungi colonizing roots include well-known pathogens and mutualists as well as frequently observed types with unknown functions. Researchers may call intracellular fungal colonization that does not fit recognized categories of mycorrhizal or pathogenic infection 'neutral' (Smith & Smith, 1990), 'endophytic' (Currah, Siegler & Hambleton, 1987; Stoyke &Currah, 1991), 'pseudomycorrhizal'(Melin, 1923; Kowalski, 1973; Wilcox & Wang, 1987), 'weakly pathogenic' (Egger & Paden, 1986; Wilcox & Wang, 1987), or 'dark-septate' (Haselwandter & Read, 1980; Currah & Van Dyk, 1986). Such colonization occurs in roots of Lupinus sp., other legumes, conifers, and many vascular plants (Melin, 1923; Peyronel, 1924; Thomas, 1943; Haselwandter & Read, 1982; Currah & Van Dyk, 1986; Cazares, 1992; Fischer, 1992; O'Dell & Trappe, 1992). In most cases the fungi responsible have not been cultured or identified. We will refer to the more or less symptomless, intracellular fungal colonization of * Current address: Department of Botany KB-15, University of Washington, Seattle, WA, 98195, USA, t Current address: Department of Forest Sciences, 193-2357 Main Mall, University of British Columbia, Vancouver, B V6T 1Z4, Canada. roots collectively as being caused by septate endophytes (SF); some are hyaline, hence 'dark-septate' is inappropriate. SF are reported to increase the growth of some host plants (Haselwandter & Read, 1982; Wilcox & Wang, 1987), but for the most part a growth response is unknown. Although they are widespread and of potential ecological importance, SF are generally poorly documented (Harley & Smith, 1983, p. 359; Smith & Smith, 1990). The structure of the root-fungal interface can evidence the nature of a symbiosis (Bracker & Littlefield, 1973; Smith & Smith, 1990). For example, many root pathogens degrade host tissues and colonize the vascular cylinder while most types of mycorrhizal fungi form complex structures presumably involved in nutrient exchange (e.g. arbuscules in vesicular-arbuscular mycorrhizas; labyrinthine tissues in ectomycorrhizas). Phialocephala fortinii Wang & Wilcox is one fungus known to form septate endophytic colonization of several hosts. The type specimen of P. fortinii is an isolate from Pinus sylvestris roots. It is one of several fungal taxa known from many coniferous hosts referred to collectively as Mycelium radicis atrovirens Melin (Wang & Wilcox, 1985). In dual culture studies, P. fortinii killed seedlings of 94 T. E. O'Dell, H. B. Massicotte andJ. M. Trappe Pinus resinosa and Picea rubens within seven months at pH 5-7; at pH 3-0, the seedlings survived but were stunted, with fungal colonization of stelar tissues and some Hartig-net formation (Wilcox & Wang, 1987). The association was called pseudomycorrhizal, meaning it was caused by 'weak pathogens with some morphological traits of ectomycorrhizas' (Wilcox & Wang, 1987). P. fortinii was isolated from Arctostaphylos uva-ursi, Cassiope mertensiana, Luetkea pectinata and Vaccinium scoparium by Stoyke & Currah (1991) who also studied its colonization of Menziesia ferruginea roots in dual culture. Stoyke & Currah (1991) described P. fortinii as an endophyte of these hosts, it formed extensive hyphal wefts on the root surface and ' intracortical sclerotia of compact, darkly pigmented and irregularly lobed, thick-walled hyphae'. Though P. fortinii was frequently isolated from ericaceous hosts, Stoyke & Currah (1991) clearly distinguish its 'endophytic' colonization from ericoid mycorrhizas by its formation of intracellular sclerotia rather than hyphal coils. P. fortinii has also been isolated from several orchid species (Currah et al, 1987; Currah, Hambleton & Smreciu, 1988) but colonization of these hosts has not been characterized. In a survey of field-collected lupin roots, O'Dell & Trappe (1992) found septate endophytes in 7 of 12 species and 19 of 44 collections examined. Some of these lupins grew in conifer forests. A fungal isolate from roots of Lupinus latifolius Agardh. growing under Pinus contorta Dougl. produced Phialocephala fortinii conidiophores in culture, the first report of its occurrence on a legume. We examined the structures formed by P. fortinii inoculated on lupin and pine under controlled conditions to characterize structures formed by their interactions. MATERIALS AND METHODS Plant material Seeds of Lupinus latifolius were collected from a naturally occurring population in the GifTordPinchot National Forest, Lewis County, Washington. A voucher collection from this population is in the Oregon State University herbarium. Seeds were stored at 0 °C until use. Seeds of Pinus contorta were obtained from the U S D A Forest Service. The pine seeds were wetted in Tween 80 (J. T . Baker Co.), rinsed with distilled water, disinfected in 5 % HgOa Figure 1. Lupinus latifolius seedlings in growth pouch, inoculated with agar plug (arrow) oi Phialocephala fortinii, showing tap rot and adventitious roots. Nodules are present along the roots. Figure 2. Conidiophore of P. fortinii in pure culture with typical 'broom'-shaped 3F" conidiophore (bar, 10/<m). Figure 3. Portion of primary Figures 1-3. Phialocephala fortinii on root systems and in root of L. latifolius with intracellular colonization by P. fortinii. pure culture Phialocephala colonization of lupin and pine overnight and germinated on 1 % malt extract agar. Lupin seed was treated similarly with the addition of a 30 min. scarification in concentrated HCl after wetting. Germinated seeds lacking contaminating bacteria and fungi had seed coats removed and were transferred to growth pouches (Northrup King, Minneapolis, M N ) containing 10 ml of sterile, distilled water one week after germination. Fungi Lupin seedlings from a population in the Ochoco National Forest, Wheeler Co., Oregon known from previous sampling to be colonized by SF were excavated along with field soil and grown in a greenhouse for 3 months before isolating rootcolonizing fungi and nodulating bacteria. Nodules and root segments were washed in Tween 80, surface sterilized in 0-1 % HgClj and crushed with forceps on yeast extract-mannitol agar (YFM) (10-0 g mannitol, 0-4 g yeast extract, 0-1 g NaCl, 0-2 g MgSO4, 0-5 g K2HPO4 per litre). Slow-growing fungal colonies were subcultured on to YFM, M M N (modified Melin-Norkrans medium; Molina & Palmer, 1982) or 0'5 % malt extract agar (MFA). Fungal cultures were maintained on MFA or M M N agar slants. Inoculum of P. fortinii was prepared by growing fungal cultures for 1 month on Petri dishes of M M N agar, collecting 10 mm diameter plugs from the growing margin of the fungal colony, and placing these colonized plugs on water agar Petri plates for one week before transferring them to growth pouches containing lupin or pine seedlings. Bacteria Bacterial cultures were obtained by the same method as the fungi. Slow-growing bacteria colonies were streaked to purity on YFM and single colonies subcultured on YFM agar slants and frozen for later use. Those isolates tested formed nodules, able to reduce acetylene, on roots of Lupinus latifolius and accordingly can be designated Bradyrhizobium sp. {Lupinus). Lupinus seedlings were inoculated by pipetting about 0-5 ml (10^ colony-forming units) of a diluted broth culture directly on the roots of seedlings after 1 wk in the growth pouch. 95 bacterial inoculation received sterile YFM. A single agar plug colonized by 1 of 3 fungal isolates, or uncolonized for controls, was placed below the radicle of each seedling. Plants were grown under artificial light in a 20 °C water bath for 8 wk before clearing and staining to assess fungal colonization. Growth pouch. Ten to 15 seedlings of each species were grown at 20 °C in growth pouches under fluorescent light with a 9/15 h day/night cycle, watered with distilled water as necessary, and fertilized with 5 ml of half-strength modified MelinNorkrans nutrient solution (Marx & Bryan, 1975) monthly during the course of the study. Lupins were inoculated with Bradyrhizobium sp. {Lupinus) after 1 wk; both plant species were inoculated with P. fortinii after 4—5 wk in the growth pouch (Fig. 1), or were left uninoculated as controls. Lupin roots were harvested 12 wk after inoculation; pine roots were harvested 24 wk after inoculation. External morphology, light microscopy and clearing of roots The external morphology of root systems was examined periodically with a dissecting microscope to monitor fungal colonization. Colonized root segments of five individuals of each species were processed for microscopy. Root segments for sectioning were cut with a razor blade, placed in 2-5 °o glutaraldehyde in 0-1 M H F P F S buffer for 4 h, then rinsed four times in cold buffer, dehydrated in a graded ethanol series over 3 d, infiltrated with LR White acrylic resin (London Resin Company) and embedded in gelatin capsules. Fmbedded roots were sectioned with glass knives on a microtome and stained with 0-1 % Toluidine blue O in l ' 0 % sodium borate. For clearing, roots were placed in 3-0% KOH overnight in a 70 °C water bath, then rinsed in distilled water and mounted in polyvinyl alcohol. Photography Photomicrographs were recorded on Kodak T M A X 100 film with a green filter to enhance contrast and developed in T M A X developer (Kodak Co.) following the manufacturer's instructions. Inoculation and growing conditions Gnotobiotic culture. Pure culture synthesis tubes containing one piece of Whatman No. 3 filter paper, 13x18 cm, and 50 ml modified Murashige & Skoog (1962) woody plant medium (minus N, minus carbohydrate, one-fourth strength P) were autoclaved, planted with one seedling of L. latifolius, and inoculated with 1x10^ colony-forming units of Bradyrhizobium sp. {Lupinus) in YFM. Controls for RESULTS Isolation of fungi About 15 isolates, from at least three taxa (judging from colony morphology), were obtained from lupin roots. When inoculated onto lupin roots in axenic culture, the pattern of colonization produced by one of these resembled that of field-collected roots. After 15-18 months' storage at 5 °C this fungal culture 96 T. E. O'Dell, H. B. Massicotte andj. M. Trappe f 9' Figures 4-9. For legend see opnosite. Phialocephala colonization of lupin and pine 97 formed conidiophores characteristic oi Phialocephala fortinii (Fig. 2) and it is the subject of this study. The other isolates remained sterile and colonized only the surface of lupin roots under these conditions. fungus did not penetrate the vascular tissues. Occasional patches of intercellular labyrinthine fungal tissue (similar to Hartig net tissue) formed at the surface of primary roots of pine (Fig. 8) but were not observed on lupin. Colonization of lateral pine roots tended to occur in the proximal portion of the roots. The basal portion of these roots often had a sporadic mantle and fungal colonization of wounds produced by the emergence of higher order lateral roots was consistently observed (Fig. 12). Growth pouch In growth pouches, root branching differed considerably between the two plant species. Lupinus latifolius grew with a tap root, and only a fewadventitious and lateral roots emerged, forming a loosely racemose root system (Fig. 1). All lupin plants inoculated with Bradyrhizobium sp. {Lupinus) formed nodules (Figs 1, 5, 6) that were absent from those not inoculated. Older lupin and pine roots had a zone of cavitation (collapsed cells) within the cortex (Figs 10, 11, 13). Pinus contorta had a tap root with first, second- and third-order lateral branching; dichotomy was rarely seen. P. fortinii grew evenly on to the pouch from agar plugs. Occasionally the fungus grew within fibres of the pouch forming sclerotium-like, irregularly swollen cells similar to those described in roots (see below). Loose wefts of hyphae grew along the root surface of both lupin and pine with some branching, surface patches of sclerotia, and inter- and intracellular colonization of the outer cortex (Figs 3-7, 11-13). The cortical colonization resulted in localized areas of strong colonization and sclerotium development scattered along primary and secondary roots of both hosts (Figs 3-7). Primary and secondary roots and nodules of L. latifolius were colonized by inter- and intracellular hyphae of P. fortinii (Figs 3-7). Colonization was restricted to the epidermis and outer cortex of roots and nodules (Figs 4—6, 11). Root hairs and other epidermal cells contains both cylindrical hyphae and sclerotia. The sclerotia were composed of thickwalled, irregularly lobed and compacted cells which sometimes formed sheets several cells thick. Similar intercellular sheets of sclerotia were observed in the cavitation zone (Fig. 13). Colonization was never observed in the endodermis or vascular cylinder of either host. Colonization of Pinus contorta occurred on first order and second order lateral roots and resembled that of L. latifolius. Sclerotia formed inter- and intracellularly in epidermal and cortical cells (Figs 7, 13); root hairs were often colonized (Fig. 9). The DISCUSSION Colonization of L. latifolius and P. contorta by P. fortinii was strikingly similar. All the structures occurring on lupin, plus some additions, were present on pine. Inter- and intracellular hyphae and intracellular sclerotia commonly occurred on primary and secondary roots of both hosts. In addition, P. fortinii formed labyrinthine tissue on pine, on which it also colonized proximal portions of ultimate lateral roots, where tissues are disrupted during lateral root emergence. Labyrinthine tissue is characterized by the digitate growth typical of ectomycorrhizal Hartig nets in contrast to the more sclerotium-like intracellular growth (observed on both hosts in this study) of other root-inhabiting fungi such as Gaeumannomyces graminis var. tritici (Deacon, 1981). The colonization of P. contorta by P. fortinii described here is virtually identical to P. fortinii colonization of Cassiope mertensiana, Luetkea pectinata and Menziesia ferruginea (Stoyke & Currah, 1991). It also resembles the colonization of P. contorta by Geopyxis carbonaria and Trichophaea hemisphaerioides, both of which form complex intracellular structures (sclerotia?) in the root cortex but fail to penetrate the vascular cylinder (Fgger & Paden, 1986). Another striking similarity is with the formation of a rudimentary Hartig net by G. carbonaria on roots of P. contorta. Clearly, P. fortinii can grow intracellularly in roots of a wide variety of plants without obvious harm to the host. No adverse reaction to P. fortinii, extensive degradation of host tissue, or colonization of vascular tissues was observed for either host, although such symptoms of pathogenicity were reported on Pinus resinosa by Wilcox & Wang (1987). After 4 months in growth pouches and extensive colonization by P. Figures 4-9. KOH-cleared roots of Pinus contorta and Lupinus latifolius colonized by Phiatocephata fortinii Figure 4 Cleared root of L. latifolius showing colonization by P. fortinii. Intracellular sclerotia in epidermal cells of primary root (arrow) and extraradical hyphae (bar, 50 fim). Figures 5, 6. Cleared root nodule of L. tatifolius colonized by P. fortinii. Figure 5 (inset) Nodule and primary root with associated extraradical hyphae. Figure 6 Intracellular sclerotia (arrow) in epidermal cells of root nodule (bar, 100/<m). Figure 7. Cleared primary root of P contorta colonized by P. fortinii. Intracellular sclerotia in root epidermal cells (arrow) and intercellular hyphae (double arrow), (bar, 100 //m). Figure 8. Cleared primary root of P. contorta showing labyrinthine tissue (arrow) of P. fortimi (Bar, 10 /im). Figure 9. Transverse hand section of P. contorta root showing colonization of root hairs by P. fortinii (bar, 30 fim). ANP 124 98 T. E. O'Dell, H. B. Massicotte and jf. M. Trappe 10 M * Figures 10-13. For legend see opposite. Phialocephala colonization of lupin and pine fortinii the plants and root systems of both species appeared healthy. T h e formation of similar fungal structures in roots of both hosts without significant adverse reaction indicates a neutral, commensal or mutualistic association. Colonization of proximal portions of lateral roots of pine by P. fortinii is quite different from the colonization of distal portions of roots typical of ectomycorrhizal fungi on compatible hosts (e.g. Massicotte, Peterson & Ashford, 1987). Therefore, P. fortinii appears to occupy a rhizoplane niche distinct from that used by ectomycorrhizal fungi. Simultaneous colonization of root systems by P. fortinii and typical ectomycorrhizal fungi may occur, as evidenced by the isolation of P. fortinii and dark sterile fungi [many of which may be P. fortinii (Stoyke & Currah, 1991)] from ectomycorrhizas (Levisohn, 1954; Trappe, 1962; Summerbell, 1989; Wang & Wilcox, 1985). The development of a cavitation zone on lupin and pine roots is not well understood. Since control roots (Fig. 10) as well as those colonized by P. fortinii exhibited this feature, it is not caused by P. fortinii, but is either part of a normal secondary root development or an artifact of the growing conditions. One possible cause is the relative dryness maintained in the growth pouches, required to reduce bacterial contamination of lupin seed. Drought causes cavitation in roots of Agave sp. (North & Nobel, 1991). The separation of colonized outer cortex from the inner cortex by a cavitation zone makes direct physiological interaction between these hosts and P. fortinii unlikely. This, together with lack of growth response of L. latifolius to inoculation with P. fortinii (O'Dell, 1992), indicates that P. fortinii is a commensal saprotroph of L. latifolius roots under these conditions (growth pouch and greenhouse). This does not preclude a beneficial association with other hosts, or with Lupinus under other conditions. Stoyke & Currah (1991) believed that a dark septate fungus, that was found by Haselwandter & Read (1982) to increase growth and phosphorus concentration of two Carex species, was P. fortinii. Though P. fortinii has been isolated from roots of members of the Pinaceae, Rosaceae, Orchidaceae, and Leguminosae, its colonization of roots and effects on host growth have received only scant study (Wang & Wilcox, 1985; Currah et al, 1987; Stoyke & Currah, 1991; O'Dell, 1992). The wide range of hosts and habitats where P. fortinii occurs indicates 99 the potential for significant ecological functions awaiting discovery. Gallaud (1905) first described SE colonization on Allium sphaerocephalum L. and Ruscus aculeatus L. Peyronel (1922) documented it on Triticum aestivum L. and then reported SE on 135 species of angiosperms (Peyronel, 1924). Although convinced by his observations of fungal cultures and field-collected roots that several different fungal taxa were represented, for the sake of simplicity, Peyronel referred to all of them as 'the Rhizoctonia'. Colonization by 'the Rhizoctonia', typically involved simple and branched hyphae that sometimes produced 'more short, branched, clavate...barrel-shaped segments, morphologically similar to conidia of Oidium, or better yet, Monilia' (Peyronel, 1924). These swollen hyphae sometimes aggregate and coil into bunches of thick-walled cells which Peyronel called ' stromatic nodules'. Similar structures were observed on roots of members of the Pinaceae by Melin (1924), who called them ' pseudomycorrhizas' to indicate what he judged to be their parasitic rather than mutualistic behaviour. Melin called pseudomycorrhizal fungi Mycelium radicis atrovirens (M.r.a.)., a name that has since been applied to many sterile dark fungi isolated from ectomycorrhizas. Strains of M.r.a. vary in the structures that they form on ectomycorrhizal hosts. Some form classic ectomycorrhizas, others 'pseudomycorrhizas', still others are characterized as pathogenic (Kowalski, 1973; Wilcox & Wang, 1987). T h e M.r.a. complex includes at least two form-species based on conidial morphology. Some isolates of M.r.a. were discovered by Richard & Fortin (1973) to form Phialocephala dimorphospora Kendrick conidiophores after extended exposure to low temperature. Wang & Wilcox (1985) described P. fortinii from an isolate obtained from roots of Pinus sylvestris. Phialocephala dimorphospora formed 'pseudomycorrhizas' on roots of Pinus resinosa Ait. and increased host growth at low pH (3-5), whereas Phialocephala fortinii was pseudomycorrhizal or pathogenic (based on degradation of host tissues and colonization of vascular tissues) on the same host (Wilcox & W^ang, 1987). Since most experiments regarding M.r.a. have used unidentified isolates, it is hardly surprising that confusion remains as to whether these organisms are parasitic, commensal or mutualistic. Recent improvements in molecular biology techniques make it possible to identify sterile cultures Figures 10-13. Sections of LR White-embedded roots of Pinus contorta and Lupinus laiifolius Figure 10. Transverse section of uncolonized L. latifolius root, eavitation zone (*) within cortex (bar, 20 fim). Figure 11 Longitudinal section of L. latifolius root showing sclerotium (arrow) and intracellular hypha of P. fortinii Cavitation zone (*) is obvious within the cortex (bar, \Q fim). Figure 12. Longitudinal section of P contorta short root colonized by P. fortinii showing basipetal colonization of wound caused by secondary root emergence (arrow) as well as cavitation zone (*). (bar, 10//m. Figure 13. Transverse section of P. contorta short root colonized by P. fortinii. Sclerotia (arrows) are present, as well as single hyphae (double arrows) (bar, 10/tm). 7 2 100 T. E. O'Dell, H. B. Massicotte andJ. M. Trappe rAcademie Polonaise des Sciences Serie des Sciences Biologique 21: 767-770. Levisohn L 1954. Aberrant root infections of pine and spruce seedlings. New Phytoiogist 53: 284-290. Marx DH, Bryan WC. 1975. Growth and ectomycorrhizal development of loblolly pine seedlings in fumigated soil infested with fungal symbiont Pisolithus tinctorius. Forest Science 21: 245-254. ACKNOWLEDGEMENTS Massicotte HB, Peterson RL, Ashford AE. 1987. Ontogeny of Eucalyptus pilularis-Pisolithus tinctorius ectomycorrhizae. I. The research was funded by the Indo-US Science and Light microscopy and scanning electron microscopy. Canadian Technology Initiative and National Science Foundation Journal of Botany 65: 1927-1939. Grant BSR 8717427. Sheri Shenk and Al Soeldner Melin E. 1923. Experimentelle Untersuchungen uber die Konstiprovided helpful guidance with microtechnique. The tution und oekologie den Mykorrhizen von Pinus silvestris und Picea abies. (Experimental studies on the constitution and USDA Forest Service Pacific Northwest Research Station ecology of the mycorrhizae of Pinus silvestris and Picea abies.) provided facilities for this research. Dr Peter Bottomley Mykologische Untersuchungen und Berichte 2: 73-330. provided facilities for bacteria isolations. Melin E. 1924. Zur Kenntnis der Mykorrhizapilze von Pinus montana Mill. (Towards knowledge of the mycorrhizal fungi of Pinus montana Mill.) Botanska Notiser 1924: 69-92. Molina R, Palmer JG. 1982. 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