the relationship of endophytic fungi to the gametophyte of

The Relationship of Endophytic Fungi to the Gametophyte of the Fern Schizaeapusilla

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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Int. J. Plant Sci. 157(1):53-62. 1996. O 1996 by The University of Chicago. All rights reserved. 1058-5893/96/5701-0007$02.00

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 Raghavanboth 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 torobusta, 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 colonizationwith 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 and its fungal associations.

endophytic fungus is recorded and illustrated as aseptate and the other fungus is illustrated as septate. How-

Schizaea pusilla is a rare and threatened fern re-stricted in North America to acidic bogs of New Jer-

ever, these endophytes remain unidentified and un-

sey, Nova Scotia, and Newfoundland (Montgomery characterized to a further degree. In addition, it is not

and Fairbrothers 1992). The life cycle of S. pusilla is known which endophyte bears the vesicle-like struc-

described in detail by Britton and Taylor (1901). Schi-tures, and the function of these structures is unclear.

zaea pusilla is one of the few ferns that maintains a To further characterize the endophytic fungi of S.

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

Fig. 1 Field-collected gametophyte viewed with stereomicroscope. Subterranean filament cells are typically etiolated or devoid of contents (SbF). Branches of filament cells beneath the substrate produce rhizoidophores (Rp), from which two to three rhizoids (Rh) develop. Rhizoids are often surrounded by brown masses of fungal hyphae (H). Branches of filament cells growing above the substrate (SrF) are bright green and filled with chloroplasts. Solid line = approximate soil level. Bar = 1.0 mm.

COLONIZATION. Spores were surface sterilized with 0.4% (vlv) sodium hypochlorite for 20 min and cultured axenically on a modified Knop's medium (pH 4.5) with 1.2% (wlv) sucrose and 1.2% (wlv) agar (Guiragossian and Koning 1986) under continuous illumination (100 ymol m-> s-I) at 21°C. Axenically grown gametophytes were placed on sterilized Whatman #I filter paper above bog soil samples in 9 cm petri dishes (termed a bog soil culture; fig. 2) and cultured under continuous illumination (2.2 ymol m-2 s-l) for 14 d at 21°C.

ISOLATION OF FUNGAL ENDOPHYTES. Colonized gametophytes were removed from bog soil culture and surface sterilized with a 0.5% (vlv) sodium hypochlorite solution for 5 min (Gerdemann 1955). Sterilized gametophytes were then placed on cornmeal agar (Sigma, 0.8% [wlv]) with 0.05% (wlv) penicillin G (Sigma), 0.05% (wlv) streptomycin sulfate (Sigma), 0.02% (wlv) yeast extract (Difco), and 0.1 % (wlv) dextrose at pH 4.5 and cultured under continuous illumination of 2.2 ymol m-2 s-I at 21°C. A series of hyphal apex cultures (agar blocks containing apical regions of hyphal

samples, homogenized into a paste, and packed into petri dishes above the sandy layer. The top layer of the samples, which was largely organic material, also was homogenized and placed in the petri dishes above the two previous layers and gently packed. A sterilized #I Whatman filter paper disk was placed directly above the soil surface, and axenically grown gametophytes were washed with sterilized distilled H,O onto the filter paper. Bog soil cultures were sealed with Parafilm to avoid desiccation.

growth were excised and placed on new medium) were made on resultant fungal growth. Tip cultures and the resultant fungal isolates were maintained on cornmeal agar with the above antibiotics (pH 4.5) under continuous illumination of 2.2 hmol m-2 s-I at 21°C. Subcultures of fungal isolates were established on Sabouraud Dextrose Agar (SDA) (Difco) and potato dextrose agar for sporulation and screening for con- taminants. Potato dextrose agar (PDA) (0.1 % [wlv] dextrose, 0.02% [wlv] yeast extract, 0.8% [wlv] agar) was prepared by boiling 300 g of cut fresh potato in 1.0 L of distilled water. The potato pieces were removed when cooked and distilled water was added to 1.0 L. Dextrose and yeast extract were added and the solution was brought to pH 4.5.

Sporulation of fungus B isolates was induced by desiccation of colonies. Colonies grown on cornmeal agar were placed in sterilized 100 X 80 mm glass storage dishes and maintained as above until dehydrated. Following the drying period, ca. 16 wk, agar was rehydrated and spores were excised for examination.

RECOLONIZATION. Axenically grown gametophytes were then suspended on sterilized Whatman #I filter paper, supported by sterilized perforated paper (pore size 1.2 mm)

55 S W A T Z E L L ET AL.-ENDOPHYTES

<-- ;F;d; filter

Petri dish with isolated fungus

Fig. 3 Recolonization experiment. Fungal isolate cultures were opened in a laminar flow hood, and sterilized perforated paper was placed above the rim o f the petri dish bottom (diameter = 9 cm). Sterilized #1 Whatman filter paper was placed on top o f perforated paper, and axenically grown gametophytes were washed onto the filter paper with sterilized distilled H,O. Cultures were sealed with Parafilm to prevent contamination and desiccation.

above 9 cm petri dishes containing fungal isolates (fig. 3). Colonized gametophytes, filter paper, and perforated paper were removed after 14 d for examination under light microscopy. Gametophytes were stained intact on filter paper with 1.0% (w/v) lactophenol cotton blue (Larone 1993) for 4 d. Fungal colonization was quantified using a systematic gridline method (Kormanik 1982). Gametophytes were removed from the filter paper for light microscopy following quantification.

Gametophytes collected from bog soil samples, colonized gametophytes grown in bog soil culture, fungal isolates, gametophytes removed from culture with fungal isolates for quantification, and cleared sporophyte roots (according to Koske and Gemma 1989) were examined using differential- interference-contrast (DIC) optics on an Olympus BH-2, or 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 examination and photography.

BOG SOIL ANALYSIS

Soil samples taken from areas near S. pusilla sporophytes in Webb's Mill Bog, Ocean County, New Jersey, were as-

OF S C H I Z A E A G A M E T O P H Y T E S

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 extra-matrical vesicles 10.6 pm in diameter (fig. 4). Roots 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.

COLONIZATION PUSILLA GAMETOPHYTESOF SCHIZAEA BY ENDOPHYTIC FUNGI

Fourteen days following introduction of axenically grown, 6-wk-old gametophytes to bog soil culture, rhizoidophores above the filter paper had changed from light green to gray or brown and contained spherical structures (fig. 7). Fungal hyphae extended upward through the filter paper from the soil and attached to rhizoids growing from rhizoidophores above the filter paper. Gametophytes containing visible vesicular structures within rhizoidophores and/or attached hyphae were then removed from bog soil culture for the fungal isolation and examination by light microscopy.

Rhizoids that developed from rhizoidophores were associated with an aseptate fungus (fig. 8). Hyphae measured 4.5 pm in diameter, had a wall thickness of 0.7 pm, and formed a netlike structure (fig. 8) similar to those described by Britton and Taylor (1901). Hy- phae that had colonized rhizoids formed arbuscular structures within the rhizoids (fig. 9). Colonized rhizoidophores that were disrupted by gently pushing on the cover slip released vesicular structures that measured 11.2 pm in diameter, were intercalary, and were connected by aseptate hyphae 4.3 pm in diameter (fig. 10). Filament cells of gametophytes positioned be-tween colonized rhizoidophores were typically empty of cellular contents with no apparent compromise to the remainder of the host (fig. 1).

Fungal growth from colonized gametophytes on cornmeal agar resulted in two isolates: one septate (fungus A) and one aseptate (fungus B). Hyphae of fungus A on all media were regularly septate, 2.2 pm in diameter, and had a wall thickness of 0.3 pm. Col- onies on cornmeal agar produced pseudothecia (not

56 INTERNATIONAL J O U R N A L O F P L A N T SCIENCES

Table 1

GROSSMORPHOLOGY OF FUNGUS A GROWN ON CORNMEAL AGAR (CA), SABOURAUD DEXTROSE AGAR (SDA), AND POTATO DEXTROSE AGAR (PDA)

Medium Front of colony

Color

Reverse of colony Texture

Colony width (mm)

at 3 mo

CA .. .. ... SDA ... ..

PDA . .. ..

White to tan Variegated dark brown and

rust Dark brown

White Variegated dark brown and

rust Dark brown

Smooth, lacking aerial hyphae Deeply convoluted, lacking aerial hy-

phae Deeply convoluted, lacking aerial hy-

phae

24 36

31

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).

RECOLONIZATION Comparison of calcium, potassium, phosphorus, and OF GAMETOPHYTES BY ISOLATED FUNGI

Both fungus A and fungus B traversed the air space nitrates present in bog soil revealed a deficiency in

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

SWATZELL ET AL.-ENDOPHYTES OF SCHIZAEA GAMETOPHYTES 59


Tabb 2

GROSS MORPHOLOGY OF FUNGUS B GROWN ON CORNMEAL AGAR

(CA), SABOURAUD DEXTROSE AGAR (SDA), AND POTATO DEXTROSE AGAR (PDA)

Color Colony 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.

S W A T Z E L L E T AL.-ENDOPHYTES O F SCHIZAEA G A M E T O P H Y T E S 61

Table 3

COMPARISON SCHIZAEA TO STANDARD ARABLE SOILOF BOG SOIL FROM WHICH PUSILLA WAS COLLECTED IN WEBB'S MILL BOG, NEW JERSEY,

%BSP K Ca ME NO3

Soil type PH (lbla) (lbta) (lbta) (lbta) (lbla) Ca ME K

Bog soil . . . .. . .. .. . . 3.9 6 3 3 <250 <SO <5 6 2 0.4 Standard soil . . .. .. 6.5 60 300 2500 200 28 60 15 3

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 within the gametophyte. Fungus A produced pseu-dothecia in isolated culture and did not produce any of the extramatrical characteristics of the septate fungus described by Britton and Taylor (1901). Therefore, it appeared that the presence of fungus A within the gametophyte was incidental.

Several studies have demonstrated that mycorrhizae recognize a potential host through chemical com-pounds produced by the host (Gianinazzi-Pearson 1984; Smith 1988; Giovannetti et al. 1994; Peterson and Farquhar 1994). A series of recolonization experiments was conducted to clarify these issues. If either fungus A or fungus B were mycorrhizal in nature, fungal isolates would display a positive growth response to the presence of the fern gametophyte without direct contact. Moreover, the presence of each isolate in the host gametophyte and gametophyte response upon contact with each fungal isolate would be helpful in determining the nature of the fungayfern relationship.

Exposure of axenically grown S. pusilla gameto-phytes to isolates of fungus A and fungus B revealed two distinct types of plant/fungal relationships. The random growth pattern of fungus A in the presence of the potential host, the lack of structures, and the death of the host following infection eliminated the possi- bility that fungus A was mycorrhizal. In contrast, fungus B approached the gametophytes directly and no random growth was detected. Moreover, the gametophytes responded by growing downward to contact the fungus in midair. In addition, fungus B produced ar-buscular and vesicular structures within the gametophytes and the colonized gametophyte to remain intact and healthy.

Therefore, fungus B mimics mycorrhizal structure and behavior. Several additional observations suggest that the relationship between S. pusilla gametophytes and this endophytic fungus is an obligatory relationship for the fern. First, nutrient deficiency within the bog soil environment almost necessitates mycorrhizal

dependence. Plants lacking mycorrhizal relationships or a special ability to extract nutrients at the low con- centration of nutrients and availability at the low pH would not survive in the acidic bog environment. Sec- ond, S. pusilla produces specialized structures, shizoid- ophores (for the purpose of harboring the symbiont), and produces these structures even without the presence of the fungus in axenic culture (von Aderkas and Raghavan 1985). Third, the gametophyte has a fila-mentous form and displays negative phototropism (Kiss 1994), features advantageous in contacting a subterranean fungus. Finally, S. pusilla fails to com- plete its life cycle in axenic culture without endophytic fungi (Guiragossian 1985). In contrast, gametophytes grown in bog soil in the field and in the laboratory form antheridia and archegonia and mature to the sporophyte generation (Britton and Taylor 190 1 ; this study).

In conclusion, we have isolated a septate fungus (fungus A) that appears to be present incidentally in the gametophyte of S. pusilla and is nonmycorrhizal. In addition, we have isolated the aseptate fungal endophyte (fungus B) described by Britton and Taylor (1901) and have evidence suggesting this endophyte, fungus B, is mycorrhizal. Furthermore, it seems likely that the relationship between fungus B and S. pusilla is obligatory and that the fern depends on this endophytic fungus to survive in a nutrient-poor environment.

Acknowledgments

Schizaea pusilla and soil samples were collected with permission of the New Jersey Department of Fish, Game, and Wildlife. Many thanks to Chris Beth- man, superintendent, Lebanon State Forest, for his kind assistance. Field collections were made with the assistance of Dr. Helen Kiss. Financial support was provided by an Academic Challenge Grant to the Bot- any Department (Ohio Board of Regents), the Research Challenge Program (Ohio Board of Regents), and the Committee for Faculty Research (Miami University).

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The Relationship of Endophytic Fungi to the Gametophyte of the Fern Schizaea pusillaLucinda J. Swatzell; Martha J. Powell; John Z. KissInternational Journal of Plant Sciences, Vol. 157, No. 1. (Jan., 1996), pp. 53-62.Stable URL:


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Literature Cited

Early Processes Involved in Host Recognition by Arbuscular Mycorrhizal FungiManuela Giovannetti; Cristiana Sbrana; Cable LogiNew Phytologist, Vol. 127, No. 4. (Aug., 1994), pp. 703-709.Stable URL:

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