The Relationship of Endophytic Fungi to the Gametophyte of the Fern Schizaea Pusilla

The Relationship of Endophytic Fungi to the Gametophyte of the Fern Schizaea pusilla

Lucinda J. Swatzell; Martha J. Powell; John Z. Kiss

International Journal of Plant Sciences, Vol. 157, No. 1. (Jan., 1996), pp. 53-62.

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THE RELATIONSHIP OF ENDOPHYTIC FUNGI TO THE GAMETOPHYTE OF THE FERN SCHIZAEA PUSILLA

LUCINDA J. SWATZELL,' MARTHA J. POWELL,2 AND JOHN Z. KISS Department of Botany, Miami University, Oxford, Ohio 45056

Schizaea pusilla is a rare and threatened fern restricted in North America to acidic bogs of Nova Scotia, Newfound- land, and New Jersey. The gametophyte lives in close association with two endophytic fungi. To characterize the nature of this fern's relationship with these fungi, we introduced axenic gametophytes to bog soil for colonization. Following colonization, the endophytic fungi were isolated and reintroduced to axenic gametophytes. The gametophytes introduced to bog soil were colonized by an aseptate fungus that formed vesicles and arbuscules within the gametophyte. However, culture of colonized gametophytes produced two fungal isolates: an aseptate fungus (fungus B) and a septate fungus (fungus A). Upon reintroduction of fungal isolates to axenically grown gametophytes, the aseptate fungus demonstrated a positive growth response to the presence of the gametophytes and colonized the gametophytes without harm to the host. The septate fungus did not exhibit any specific recognition but contacted the gametophytes randomly, leaving a large percentage of the host nonviable. We propose that the relationship of the septate fungus to the gametophyte of S. pusilla is nonmycorrhizal while the relationship of the aseptate fungus to the gametophyte is mycorrhizal. Further- more, based on lack of nutrient availability in local soils, formation of specialized structures in the gametophyte for harboring fungi, and dependence of the fern on fungal presence for completion of its life cycle, we propose that S. pusilla maintains an obligatory relationship with the aseptate mycorrhizal fungus.

Introduction direction as their initial cells in the spore (von Aderkas and Raghavan 1985). The rhizoid, however, rapidly Symbioses between fungi and lower vascular plants turns toward the substrate (Britton and Taylor 1901) are common and range from obligatory in the Psilo- and the apical protonemal cell orients in a negatively phyta to facultative in leptosporangiate ferns (Boullard phototropic direction by the three- to four-cell stage of 1979). Among leptosporangiate ferns, sporophytes gametophyte development (Kiss 1994). (compared to gametophytes) are more commonly found Gametophytes develop rhizoidophores (fig. I), in association with mycorrhizal fungi (Boullard 1979; which are large, highly vacuolate, specialized struc- Bonfante-Fasolo 1984). However, continuous relation- tures that begin as single spherical cells on the fila- ships between fungal symbionts and ferns throughout mentous gametophyte (von Aderkas and Raghavan both phases have been reported in Schizaeaceae, Glei- 1985). Rhizoidophores develop two to three rhizoids, cheniaceae, and Hymenophyllaceae (Boullard 1979). Of divide longitudinally, and form receptacles for an en- the schizaeaceous ferns, gametophytes of Actinostachys dophytic and purported symbiotic fungus (Britton and spp. (Bierhorst 1968, 1975), Lygodium (Warrington Taylor 1901). Upon colonization by the fungus, rhi- 1972), Schizaea jistulosa, Schizaea melanesica, Schi- zoidophores fill with fungal elements. The rhizoids zaea dichotoma (Bierhorst 1966, 1967, 1968), Schizaea collapse and the purported symbiont is presumed to robusta, Schizaea rupestris (Bierhorst 1971), and Schi- function in absorption of water and nutrients (Britton zaea pusilla (Britton and Taylor 1901) all associate with and Taylor 1901). Britton and Taylor (1901) observed fungal symbionts. Of these Schizaea species, S. mela- structures resembling vesicles in colonized rhizoido- nesica and S. pusilla exhibit continuous relationships phores and noted the continuance of colonization with their endophytic fungi throughout both the game- throughout the gametophyte and sporophyte stages. In tophyte and sporophyte phases instead of facultative re- addition, these workers recorded the presence of a sec- lationships only in the sporophyte stage. Of particular ond endophyte in the gametophytes. 1n their paper, one interest in this article is the gametophyte of S. pusilla endophytic fungus is recorded and illustrated as asep- and its fungal associations. tate and the other fungus is illustrated as septate. How- Schizaea pusilla is a rare and threatened fern re- ever, these endophytes remain unidentified and un- stricted in North America to acidic bogs of New Jer- characterized to a further degree. In addition, it is not sey, Nova Scotia, and Newfoundland (Montgomery known which endophyte bears the vesicle-like struc- and Fairbrothers 1992). The life cycle of S. pusilla is tures, and the function of these structures is unclear. described in detail by Britton and Taylor (1901). Schi- To further characterize the endophytic fungi of S. zaea pusilla is one of the few ferns that maintains a pusilla and to elucidate the nature of their relationship completely filamentous and uniseriate gametophyte to this fern, we examined the endophytes within col- throughout its development (Kiss et al. 1995). In S. onized gametophytes and in isolated cultures. pusilla, following germination of the spore, the first protonemal and rhizoidal cells are oriented in the same Material and methods

'Author for correspondence and reprints. Fax: 513-529-4243. 2Present address: Department of Biology, James Madison Univer- Spores, gametophytes, and sporophytes of Schizaea pus- sity, Harrisonburg, Virginia 22807. illa and soil samples were collected from Webb's Mill Bog, Manuscript received May 1995; revised manuscript received August Ocean County, New Jersey, in September 1993, July 1994, 1995. and September 1994. 54 INTERNATIONAL JOURNAL OF PLANT SCIENCES .. . - Petri dish

. ,--= ../ - axenic * gametophytes

sterile filter paper

/-\ Petri dish with bog soil

Fi. 2 Bog soil culture used in colonization experiments. Bog soil samples approximately 7-8 cm deep were separated into distinct soil layers. The bottom layer, which had a high sand content, was placed into the bottom of a petri dish (diameter = 9 cm). A middle layer with a high clay and organic content was then removed from the samples, homogenized into a paste, and packed into petri dishes Fig. 1 Field-collected gametophyte viewed with stereomicroscope. Subterranean filament cells are typically etiolated or devoid of con- above the sandy layer. The top layer of the samples, which was tents (SbF). Branches of filament cells beneath the substrate produce largely organic material, also was homogenized and placed in the rhizoidophores (Rp), from which two to three rhizoids (Rh) develop. petri dishes above the two previous layers and gently packed. A Rhizoids are often surrounded by brown masses of fungal hyphae (H). sterilized #I Whatman filter paper disk was placed directly above Branches of filament cells growing above the substrate (SrF) are the soil surface, and axenically grown gametophytes were washed with sterilized distilled H,O onto the filter paper. Bog soil cultures bright green and filled with chloroplasts. Solid line = approximate were sealed with Parafilm to avoid desiccation. soil level. Bar = 1.0 mm.

growth were excised and placed on new medium) were made on resultant fungal growth. Tip cultures and the resultant COLONIZATION.Spores were surface sterilized with 0.4% fungal isolates were maintained on cornmeal agar with the (vlv) sodium hypochlorite for 20 min and cultured axenically above antibiotics (pH 4.5) under continuous illumination of on a modified Knop's medium (pH 4.5) with 1.2% (wlv) 2.2 hmol m-2 s-I at 21°C. Subcultures of fungal isolates were sucrose and 1.2% (wlv) agar (Guiragossian and Koning established on Sabouraud Dextrose Agar (SDA) (Difco) and 1986) under continuous illumination (100 ymol m-> s-I) at potato dextrose agar for sporulation and screening for con- 21°C. Axenically grown gametophytes were placed on ster- taminants. Potato dextrose agar (PDA) (0.1 % [wlv] dextrose, ilized Whatman #I filter paper above bog soil samples in 9 0.02% [wlv] yeast extract, 0.8% [wlv] agar) was prepared cm petri dishes (termed a bog soil culture; fig. 2) and cul- by boiling 300 g of cut fresh potato in 1.0 L of distilled tured under continuous illumination (2.2 ymol m-2 s-l) for water. The potato pieces were removed when cooked and 14 d at 21°C. distilled water was added to 1.0 L. Dextrose and yeast extract were added and the solution was brought to pH 4.5. ISOLATIONOF FUNGAL ENDOPHYTES. Colonized gameto- Sporulation of fungus B isolates was induced by desic- phytes were removed from bog soil culture and surface ster- cation of colonies. Colonies grown on cornmeal agar were ilized with a 0.5% (vlv) sodium hypochlorite solution for 5 placed in sterilized 100 X 80 mm glass storage dishes and min (Gerdemann 1955). Sterilized gametophytes were then maintained as above until dehydrated. Following the drying placed on cornmeal agar (Sigma, 0.8% [wlv]) with 0.05% period, ca. 16 wk, agar was rehydrated and spores were ex- (wlv) penicillin G (Sigma), 0.05% (wlv) streptomycin sulfate cised for examination. (Sigma), 0.02% (wlv) yeast extract (Difco), and 0.1 % (wlv) dextrose at pH 4.5 and cultured under continuous illumina- RECOLONIZATION.Axenically grown gametophytes were tion of 2.2 ymol m-2 s-I at 21°C. A series of hyphal apex then suspended on sterilized Whatman #I filter paper, sup- cultures (agar blocks containing apical regions of hyphal ported by sterilized perforated paper (pore size 1.2 mm) SWATZELL ET AL.-ENDOPHYTES OF SCHIZAEA GAMETOPHYTES 55

sessed for calcium, potassium, phosphoms, and nitrate presence and availability by Ohio Agriculture Research and De- velopment Center (Wooster, Ohio), according to Dahnke (1988), and compared to standard results of arable soil used for agriculture. Results

Gametophytes collected from bog soil were associated with hyphae that attached to the rhizoids growing from rhizoidophores. These hyphae produced extramatrical vesicles 10.6 pm in diameter (fig. 4). Roots <-- ;F;d; filter of sporophytes collected from bog soil and cleared for examination were found in association with hyphae that attached to root hairs (fig. 5). Hyphae formed netlike structures around root hairs, similar to the colonization process in the gametophyte rhizoids (see be- low). These hyphae then entered the root from the root hairs and formed oblong vesicles approximately 35 pm X 50 pm (fig. 5) and arbuscules (fig. 6). Hyphae attached to the sporophyte root hairs also produced extramatrical vesicles 1 1.9-16.7 pm in diameter out- side of the root. Petri dish with COLONIZATIONOF SCHIZAEA PUSILLA GAMETOPHYTES BY ENDOPHYTIC FUNGI isolated fungus Fourteen days following introduction of axenically grown, 6-wk-old gametophytes to bog soil culture, rhizoidophores above the filter paper had changed from Fig. 3 Recolonization experiment. Fungal isolate cultures were opened in a laminar flow hood, and sterilized perforated paper was light green to gray or brown and contained spherical placed above the rim of the petri dish bottom (diameter = 9 cm). structures (fig. 7). Fungal hyphae extended upward Sterilized #1 Whatman filter paper was placed on top of perforated through the filter paper from the soil and attached to paper, and axenically grown gametophytes were washed onto the rhizoids growing from rhizoidophores above the filter filter paper with sterilized distilled H,O. Cultures were sealed with paper. Gametophytes containing visible vesicular Parafilm to prevent contamination and desiccation. structures within rhizoidophores and/or attached hyphae were then removed from bog soil culture for the above 9 cm petri dishes containing fungal isolates (fig. 3). fungal isolation and examination by light microscopy. Colonized gametophytes, filter paper, and perforated paper Rhizoids that developed from rhizoidophores were were removed after 14 d for examination under light mi- associated with an aseptate fungus (fig. 8). Hyphae croscopy. Gametophytes were stained intact on filter paper measured 4.5 pm in diameter, had a wall thickness of with 1.0% (w/v) lactophenol cotton blue (Larone 1993) for 4 d. Fungal colonization was quantified using a systematic 0.7 pm, and formed a netlike structure (fig. 8) similar gridline method (Kormanik 1982). Gametophytes were re- to those described by Britton and Taylor (1901). Hy- moved from the filter paper for light microscopy following phae that had colonized rhizoids formed arbuscular quantification. structures within the rhizoids (fig. 9). Colonized rhizoidophores that were disrupted by gently pushing on the cover slip released vesicular structures that mea- Gametophytes collected from bog soil samples, colonized sured 11.2 pm in diameter, were intercalary, and were gametophytes grown in bog soil culture, fungal isolates, ga- connected by aseptate hyphae 4.3 pm in diameter (fig. metophytes removed from culture with fungal isolates for 10). Filament cells of gametophytes positioned be- quantification, and cleared sporophyte roots (according to tween colonized rhizoidophores were typically empty Koske and Gemma 1989) were examined using differential- of cellular contents with no apparent compromise to interference-contrast (DIC) optics on an Olympus BH-2, or the remainder of the host (fig. 1). with an Olympus stereo microscope, and photographed with Kodak T-MAX 100, Kodak Technical Pan (ASA 50), and/ or Ektachrome Tungsten 160T film. Fungal isolates were stained with lactophenol cotton blue for 24 h prior to ex- Fungal growth from colonized gametophytes on amination and photography. cornmeal agar resulted in two isolates: one septate (fungus A) and one aseptate (fungus B). Hyphae of BOG SOIL ANALYSIS fungus A on all media were regularly septate, 2.2 pm Soil samples taken from areas near S. pusilla sporophytes in diameter, and had a wall thickness of 0.3 pm. Col- in Webb's Mill Bog, Ocean County, New Jersey, were as- onies on cornmeal agar produced pseudothecia (not 56 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Table 1

GROSSMORPHOLOGY OF FUNGUS A GROWN ON CORNMEAL AGAR (CA), SABOURAUDDEXTROSE AGAR(SDA), AND POTATO DEXTROSE AGAR (PDA) Color Colony width (mm) Medium Front of colony Reverse of colony Texture at 3 mo CA ...... White to tan White Smooth, lacking aerial hyphae 24 SDA ..... Variegated dark brown and Variegated dark brown and Deeply convoluted, lacking aerial hy- 36 rust rust phae PDA ..... Dark brown Dark brown Deeply convoluted, lacking aerial hy- 31 phae

shown) 340-700 pm in diameter. Gross morphology Fungus B approached the gametophytes directly, as (table 1; fig. 11,A, D, E) of fungus A varied in color evidenced by the lack of staining on the filter paper and texture with different media. Fungus A grown on (fig. 13B). Moreover, gametophytes in these cultures SDA produced a rust-colored exudate that seeped from grew downward through the filter paper and support the base of the colonies. paper to form rhizoidophores and contacted the fungus Hyphae of fungus B on all media were aseptate and in midair. Fungus B colonized 99.4% (n = 177) of the produced occasional septa at branch points. Hyphae population samples, and all remained viable. One ga- were 2.8 pm in diameter with a wall thickness of 0.5 metophyte, which lacked rhizoidophores and was only pm. Gross morphology (table 2; fig. 11,B, C, F) of at the six-cell stage, was not viable. Colonized game- fungus B varied in texture with different media. Col- tophytes in fungus B cultures contained vesicular onies grown on cornmeal agar and subjected to des- structures and arbuscules (not shown). iccation produced spores ranging from 35 pm to 65 pm in diameter with an outer wall containing regularly spaced alveoli (fig. 12).

RECOLONIZATIONOF GAMETOPHYTES BY ISOLATED FUNGI Comparison of calcium, potassium, phosphorus, and nitrates present in bog soil revealed a deficiency in Both fungus A and fungus B traversed the air space between the agar medium and the filter paper beneath these nutrients in the natural growth substrate of Schi- the gametophytes. Fungus A approached the filter pa- zaea pusilla gametophytes (table 3). For example, cal- per, attached at the nearest section of the paper or cium is present at 10% (pounds per acre) expected for along the sides of the petri dishes, fanned outward, and normal arable land. Nitrates present were immeasura- encountered the gametophytes randomly (fig. 13A ). ble, less than 5 lbla, and phosphorus was measured at No vesicles or arbuscules were visible. Fungus A left only 6 lbla. Moreover, at the cation exchange capacity 10.3% (n = 195) of the population samples nonviable and pH of bog soil in this area (which ranges from 3.3 (i.e., fewer than three uncompromised chlorophyllous to 4.9 [Guiragossian 1985; this study]), nutrients avail- cells remaining in the entire gametophyte). able for plant growth are almost nonexistent.

Figs. 4-6 Fig. 4, Field-collected gametophyte viewed with differential-interference-contrast optics (DIC). An aseptate hypha (H) attaches to the rhizoid (Rh) of a young gametophyte. An extramatrical vesicle (V)is borne on a hyphal branch. F = filament cell. SC = spore coat of Schizaea pusilla. Bar = 100 pm. Fig. 5, Cleared sporophyte root from field-collected plant viewed with DIC optics. Fungal colonization extends into the sporophyte stage and the colonizing fungus produces vesicles (V) within the colonized roots. H = hypha. Bar = 100 pm. Fig. 6, Arbuscules (arrows) within cleared sporophyte root from field-collected plant viewed with brightfield optics. Hyphae (H) attach to the root hair (RH), grow through the root hair into the root cortex, and produce arbuscules (arrows). Bar = 50 pm.

Figs. 7-9 Fig. 7, Rhizoidophore of a bog soil cultured gametophyte from colonization experiment viewed with DIC optics. Colonizing fungus formed vesicular structures (arrow) within the rhizoidophore (Rp). The vesicles are connected by aseptate hyphae (arrowhead). Bar = 40 ym. Fig. 8, Fungal hyphae attached to a rhizoid in bog soil culture from colonization experiment viewed with DIC optics. Aseptate hypha (H)formed a netlike structure around the rhizoid (Rh) of a rhizoidophore (Rp). Filament cells (F) containing numerous chloroplasts showed no evidence of fungal disturbance. SC = spore coat of Schizaea pusilla. Bar = 40 ym. Fig. 9, Arbuscular structures within a rhizoid from bog soil culture viewed with brightfield optics. Early within the colonization process, rhizoids (Rh), which were stained with lactophenol cotton blue, contain arbuscular structures (A). Bar = 10 ym.

Figs. 10, 11 Fig. 10, Disrupted rhizoidophore viewed with DIC optics. Gametophytes removed from bog soil culture from colonization experiment and subjected to a gentle squash procedure beneath a cover slip spilled the contents of their rhizoidophores (Rp). Fungal vesicles (V) were intercalary and connected by thick-walled hyphae (H). F = filament cell. Bar = 40 pm. Fig. 11, Fungal isolates. Morphology and color of the two isolates, fungus A and B, varied with culture medium. A, Fungus A on Sabouraud's Dextrose Agar (SDA); B, Fungus B on cornmeal agar; C, Fungus B on PDA; D, Fungus A on cornmeal agar; E, Fungus A on potato dextrose agar (PDA); F, Fungus B on SDA. Bar = 5 cm. SWATZELL ET AL.-ENDOPHYTES OF SCHIZAEA GAMETOPHYTES 57 58 INTERNATIONAL JOURNAL OF PLANT SCIENCES SWATZELL ET AL.-ENDOPHYTES OF SCHIZAEA GAMETOPHYTES 59 60 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Tabb 2

GROSSMORPHOLOGY OF FUNGUS B GROWN ON CORNMEAL AGAR (CA), SABOURAUDDEXTROSE AGAR (SDA), AND POTATO DEXTROSE AGAR (PDA) Colony Color width Medi- Front of Reverse of (mm) at um colony colony Texture 3 mo CA .. . . White to White to Smooth, lacking ae- 24 ivory ivory rial hyphae SDA .. White to White to Coarse, hairy, aerial 24 ivory ivory hyphae 1-3 mrn high PDA .. White to White to Coarse, hairy, aerial 24 ivory ivory hyphae 1-3 mm high

Initial colonization from bog soil culture revealed an aseptate fungus (4.5 pm in diameter) that attached to and grew through the rhizoid to enter the rhizoidophore. No septate hyphae were observable within the gametophyte following initial colonization. How- ever, following sterilization of colonized gametophytes and subsequent isolation procedures, two fungal isolates (one was septate while the other was aseptate) were obtained. To establish the identity of the isolates and characterize their respective relationships with Schizaea pusilla, it was necessary to assign each fungal element present in the gametophyte to the appro- priate fungal isolate. Fungus A produced narrow, thin-walled, septate hyphae, while fungus B produced thick-walled, aseptate hyphae. Fungus A, while not apparent following initial bog soil colonization, was indeed present in some form within the gametophytes. However, based on size (hyphae between vesicular structures were 4.3 p,m in diameter) and the lack of septa in the colonizing fungus, fungus B was the one that formed the vesicular structures within the rhizoidophores. Following the colonization experiments, however, the relationship of fungus B or fungus A to S. pusilla gametophytes was still unclear. No evidence as yet suggested whether the vesicular structures of fungus B were true vesicles of a mycorrhizal fungus or the haus- toria of a parasite. Furthermore, fungus A did not pro-

$s. 12,13 Fig. 12, Spore produced in culture by fungus B isolate viewed with DIC optics. Spores are golden and are pocketed at regular intervals with hexagonal alveoli. Bar = 20 pm. Fig. 13, Demonstration of recognition in recolonization experiment. Filter paper was removed from recolonization cultures with gametophytes intact and stained with lactophenol cotton blue. A, Fungus A approached filter paper at the nearest vantage point (i.e., a sag or wrin- kle in the filter paper) and along the sides of the petri dishes. Above the filter paper, stained hyphae (arrows) fan out randomly. B, Lack of staining on the filter paper suggests that fungus B approached the gametophytes (arrowheads) directly. Bar = 5 cm. SWATZELL ET AL.-ENDOPHYTES OF SCHIZAEA GAMETOPHYTES 61

Table 3

COMPARISONOF BOG SOIL FROM WHICH SCHIZAEAPUSILLA WAS COLLECTED IN WEBB'SMILL BOG, NEWJERSEY, TO STANDARD ARABLE SOIL %BS P K Ca ME NO3 Soil type PH (lbla) (lbta) (lbta) (lbta) (lbla) Ca ME K Bog soil ...... 3.9 6 3 3 <250

Note. In comparison with typical results from analysis of soil used for agriculture, phosphorus, potassium, calcium, magnesium, and nitrates are present in low amounts (lbla = pounds per acre) in bog soil. Base saturation (%BS), the percentage of these nutrients available to the fern at the pH and cation exchange capacity, is minimal. Soil analysis was performed by the Research Extension Laboratory, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691. duce specialized structures that defined its presence dependence. Plants lacking mycorrhizal relationships within the gametophyte. Fungus A produced pseu- or a special ability to extract nutrients at the low con- dothecia in isolated culture and did not produce any centration of nutrients and availability at the low pH of the extramatrical characteristics of the septate fun- would not survive in the acidic bog environment. Sec- gus described by Britton and Taylor (1901). Therefore, ond, S. pusilla produces specialized structures, shizoid- it appeared that the presence of fungus A within the ophores (for the purpose of harboring the symbiont), gametophyte was incidental. and produces these structures even without the pres- Several studies have demonstrated that mycorrhizae ence of the fungus in axenic culture (von Aderkas and recognize a potential host through chemical com- Raghavan 1985). Third, the gametophyte has a fila- pounds produced by the host (Gianinazzi-Pearson mentous form and displays negative phototropism 1984; Smith 1988; Giovannetti et al. 1994; Peterson (Kiss 1994), features advantageous in contacting a and Farquhar 1994). A series of recolonization exper- subterranean fungus. Finally, S. pusilla fails to com- iments was conducted to clarify these issues. If either plete its life cycle in axenic culture without endophytic fungus A or fungus B were mycorrhizal in nature, fun- fungi (Guiragossian 1985). In contrast, gametophytes gal isolates would display a positive growth response grown in bog soil in the field and in the laboratory to the presence of the fern gametophyte without direct form antheridia and archegonia and mature to the spo- contact. Moreover, the presence of each isolate in the rophyte generation (Britton and Taylor 1901 ; this host gametophyte and gametophyte response upon study). contact with each fungal isolate would be helpful in In conclusion, we have isolated a septate fungus (fun- determining the nature of the fungayfern relationship. gus A) that appears to be present incidentally in the Exposure of axenically grown S. pusilla gameto- gametophyte of S. pusilla and is nonmycorrhizal. In ad- phytes to isolates of fungus A and fungus B revealed dition, we have isolated the aseptate fungal endophyte two distinct types of plant/fungal relationships. The (fungus B) described by Britton and Taylor (1901) and random growth pattern of fungus A in the presence of have evidence suggesting this endophyte, fungus B, is the potential host, the lack of structures, and the death mycorrhizal. Furthermore, it seems likely that the re- of the host following infection eliminated the possi- lationship between fungus B and S. pusilla is obligatory bility that fungus A was mycorrhizal. In contrast, fun- and that the fern depends on this endophytic fungus to gus B approached the gametophytes directly and no survive in a nutrient-poor environment. random growth was detected. Moreover, the gametophytes responded by growing downward to contact the Acknowledgments fungus in midair. In addition, fungus B produced ar- Schizaea pusilla and soil samples were collected buscular and vesicular structures within the gameto- with permission of the New Jersey Department of phytes and the colonized gametophyte to remain intact Fish, Game, and Wildlife. Many thanks to Chris Beth- and healthy. man, superintendent, Lebanon State Forest, for his Therefore, fungus B mimics mycorrhizal structure kind assistance. Field collections were made with the and behavior. Several additional observations suggest assistance of Dr. Helen Kiss. Financial support was that the relationship between S. pusilla gametophytes provided by an Academic Challenge Grant to the Bot- and this endophytic fungus is an obligatory relation- any Department (Ohio Board of Regents), the Research ship for the fern. First, nutrient deficiency within the Challenge Program (Ohio Board of Regents), and the bog soil environment almost necessitates mycorrhizal Committee for Faculty Research (Miami University).

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You have printed the following article: The Relationship of Endophytic Fungi to the Gametophyte of the Fern Schizaea pusilla Lucinda J. Swatzell; Martha J. Powell; John Z. Kiss International Journal of Plant Sciences, Vol. 157, No. 1. (Jan., 1996), pp. 53-62. Stable URL: http://links.jstor.org/sici?sici=1058-5893%28199601%29157%3A1%3C53%3ATROEFT%3E2.0.CO%3B2-0

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Literature Cited

Early Processes Involved in Host Recognition by Arbuscular Mycorrhizal Fungi Manuela Giovannetti; Cristiana Sbrana; Cable Logi New Phytologist, Vol. 127, No. 4. (Aug., 1994), pp. 703-709. Stable URL: http://links.jstor.org/sici?sici=0028-646X%28199408%29127%3A4%3C703%3AEPIIHR%3E2.0.CO%3B2-0