Vesicular-arbuscular mycorrhizas of Equisetum species in Norway and the U.S.A.: occurrence and mycotrophy

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Mycol. Res. 97 (6):656-660 (1993) Printed in Great Britain

Vesicular-arbuscular mycorrhizas of Equisetum species in Norway and the U.S.A. : occurrence and mycotrophy

SHIVCHARN S. DHILLION Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.5.A

Typical vesicular-arbuscular mycorrhizal structures occurred in the sporophytes of Equisetum species (11 out of 12 species) collected in Norway and U.S.A. Plants growing in wet (hydric) habitats, sampled in the U.S.A. and Norway, had lower levels of colonization by vesicular-arbuscular mycorrhizal fungi than plants growing in mesic/dry-mesic habitats. Colonization levels ranged from 0 to 46% of the root length. Only E. uariegatum found growing in a wet habitat in Illinois was consistently non-mycorrhizal. Equisetum species appear to be facultatively mycotrophic; the degree of mycotrophy may depend on habitat conditions. The world-wide warming and change from hydric to mesic conditions, during the late Carboniferous period and thereafter, probably contributed to the demise of the Equisetales. The demise of the Equisetales was less likely influenced by the mycotrophic nature of higher plants, fems, cycads and pines.

Vesicular-arbuscular mycorrhizas (VAM) are regularly formed by vascular plants from a wide range of habitats (e.g. Safir, 1987; Brundrett, 1991; Dhillion & Friese, 1993; Dhillion & Zak, 1993), and include members of the Psilotopsida, Lycopsida, Pteridophyta, and Spermatophyta (Boullard, 1959; Stubblefield, Taylor & Trappe, 1987). Gallaud (1905) described the basic mycorrhizal structural components and their variations in different host plants. Since then several features of VAM fungi have sparked speculation about the evolutionary significance of this symbiosis (Malloch, Pirozynski & Raven, 1980). Pirozynski & Malloch (1975) proposed the mycotrophic origin of land plants based on the presence of structures reminiscent of a ubiquitous endophyte in the fossilized rhizomes of the early (Devonian) land plants, Rhynia and Asteroxylon, similar to those that are involved in endotrophic mycorrhizas at the present time. Their proposal was based on the relationships between root system morphology of angiosperms and VAM infection levels. In this respect, Baylis (1976) suggested that fleshy roots (primitive) were highly colonized while fine roots (advanced) were less SO. Boullard (1957, 1959) elegantly demonstrated progressive reduction, in extent and duration, of mycotrophism in the Pteridophyta. The symbiosis among the pteridophytes ranges from obligate and present in both gametophyte and sporophytic generations in Psilotopsida and some Lycopsida and eusporangiate ferns, to facultative mycotrophism in other eusporangiate and most leptosporangiate fems especially Polypodiaceae and Vittariaceae, to absent in Isoetaceae and aquatic Azollaceae, Salviniaceae, Marsileaceae, and Parkeriaceae (Boullard, 1957, 1959; see review by Malloch et al., 1980). These studies also suggested that the dependence

of fern gametophytes on mycotrophism decreased with increase in photosynthetic ability. Although Equiseturn was well represented by its Carboniferous arborescent progenitors, surprisingly, endotrophic symbiosis in it has rarely been reported. In recent years, the mycorrhizal status of Equiseturn species has been somewhat controversial. Ocampo, Martin & Hayman (1980) and Berch & Kendrick (1982) classified the genus as non-mycotrophic and related the demise of the Equisetae and its relatives to their non-mycotrophic nature. In Java, Indonesia, Janse (in Kelly, 1950) stated that jungle-dwelling Equisetae did not harbour any root endophytes, while other workers reported the occurrence of an endophytic 'phycomycete' in E. arvense (Lohman, 1927), and have given accounts of endophytic fungi resembling 'monilioid' hyphae in the stele and cortex of roots (Boullard, 1957; Berch & Kendrick, 1982). Koske ef al. (1985) suggested that these apparently nonmycotrophic species may have been collected from wet habitats, and the lack of VAM colonization may be indicative of the effect of high soil moisture on mycorrhizal development rather than on the mycotrophic potential of Equisetum. In contrast to the above studies, Koske ef a1. (1985) showed that the infection of Equiseturn species by VAM fungi depended upon associated plants and/or soiI moisture conditions, and suggested that the decline of this species was related to the world-wide change from hydric to mesic conditions. Koske et al. (1985), however, based their conclusions on a small sample size, and their conclusions were therefore largely speculative. The validity of observations depends on accurate differentiation of VAM fungi from other root-endophytic fungi. The arbuscule, presumably the primary site of nutrient transfer from fungus to plant, is the morphological and

S. S. Dhillion functional feature most distinctive of all fungi known to form mycorrhizas. The lack of adequate sample size and proper mycorrhizal structure documentation has yielded conflicting information on the mycorrhizal status of Equisetum. The order Equisetales comprises three families, Asterocalamitaceae (fossil), Calamitaceae (fossil) and Equisetaceae (Willis, 1973). Equisetaceae has one genus, Equisetum L., and 23 known species having a worldwide distribution (excluding Australasia). Members are perennial herbs with syrnpodial rhizomes which send up one or two kinds of aerial shoots (reproductive and vegetative shoots). In several species the internodes of the rhizomes are swollen into tubers, which serve for hibernation and vegetative reproduction. The rhizomes produce roots which generally remain in the upper 20 cm of the soil unless the plants are growing in dry regions. This study reports on the mycorrhizal status of twelve members of the Equisetaceae, comprising 52% of all known species (Willis, 1973), in Norway and the U.S.A. and provides evidence for their mycotrophic nature.

MATERIALS A N D METHODS

root segments for clearing, as described above, a 1 g sample of surface-sterilized (Meredith & Anderson, 1992) roots was used as a source of inoculum for the induction of spores (Liberta, Anderson & Dickman, 1983) using sorghum (Sorghum bicolor). The potting soil, root zone soil of the respective species, was autoclaved (twice for 90 min with an interval of 12 h between autoclaving). Induction plants were grown for 6 wk in a growth chamber. Plants were then transferred to a greenhouse and watered (250 ml) daily for 15 wk. Root and root zone soil samples were processed to detect the presence of spores. Soil samples were air dried and stored at 4 OC. Spores were isolated from a 21 cm3 sample employing a wetsieving density gradient procedure (Anderson & Liberta, 1989). Intact, cytoplasm-filled spores were counted and identified using published descriptions (Morton & Benny, 1990; Schenck & Perez, 1990). Since species of Equisetum are known to have large numbers of root hairs, the number of root hairs per cm of root was determined. Three root tips each of three samples of each species (3 root tips/sample and 3 samples/species) were examined for root hair densities. There were no statistically significant differences among species (X fs.D., 20 f6 root hairs cm-l), except for E. kansanum and E. pratense which had significantly (P < 0.001) fewer root hairs per cm of root (9 f3) than did all the other species.

In Norway, sporophyte plants were excavated from regions (valley slopes to a topogenous mire of flat fen type, which represented a range of habitats; forested to aquatic) in Bstradalen valley, Hedrnark province, southern Norway (Table 1).The region belongs to the middle boreal vegetation RESULTS A N D DISCUSSION (see Dahl et al. 1986) and the bedrock is feldspathic quartzite and green schists of the Rsros-nappe complex. The time of .The incidence and proportion of root length colonized by sampling, 9-14 July 1991, in Norway represented the period VAM fungi are presented in Table 1. Members of the of peak biomass production for these plants. In the U.S.A., Equisetaceae were largely found to be capable of forming samples were also collected from a range of habitats, grass- mycorrhizas, colonization levels ranging from 0 to 40%. to tree-dominated areas, in the states of Illinois, Texas and Equisefumfluviafile, E. palusfre and E. variegatum, which occurred in both Norway and the U.S.A., showed similar trends in New Mexico during June 1990, and July 1991 (Table I). Species collected in Norway were E. scirpoides Michx. and colonization (Table I). Favourable habitats for the growth of E. sylvaticum L., and in the U.S.A. were E. arvense L., E. hyemale Equisetum species typically include wet places as standing var. 2 elafurn (Engelm.) Morton Schaffer, E. hyernale var. 1 water in ponds or ditches, marshy areas, wet and mesic pseudohyemale (Farw.) Morton, E. hyemale L. var. afine meadows, moist sandy dunes and moist woods (Koske et al., (Engelm.) A. A. Eaton, E. kansanum J. H., E. laevigafum A. Br. 1985; Table 1).In general, low colonization levels were found and E. prafense Ehrh. Species examined common to both for all species in aquatic habitats collected in Norway and the countries were E. fluviafile L., E. palusfre L. and E. variegatum U.S.A. However, habitats are not limited to wet places, all year Scheich. Plants were classified following Correll & Johnston round, and can include areas which alternate between wet and (1970), Hauke (1978) and Mohlenbrock (1986). dry spells during the year. During dry periods plants are able Plant samples were excavated so as to minimize root loss, to exist via perennating structures below ground awaiting although some fine roots were bound to break. The root more congenial growing conditions. Although plants from system of each plant collected was carefully washed and wet areas have generally been thought to be non-mycorrhizal placed in a glass vial of formalin-acetic acid-alcohol (FAA; there is also ample evidence currently showing the occurrence 10 :35 :10:5 formalin-water-ethanol-acetic acid) and kept for of mycorrhizas in a variety of partially submerged plants (e.g. further processing. Roots in FAA were rinsed in water and Clayton & Bagyaraj, 1984; Tanner & Clayton, 1985; Dhillion, placed for clearing in 10% KOH at room temperature for 7 d. 1992). The data in this study show that roots of several The roots were then stained with trypan blue to assess VAM species occurring in wet habitats (e.g. E. fluviatile and E. colonization and to estimate the total root length, using the variegatum (A) in Norway and E. fluviatile, E. hyemale (A) and gridline intersection method (Giovannetti & Mosse, 1980). E. variegafurn in the U.S.A.; Table 1)either lacked functional Because VAM fungi can only be identified by spores, and mycorrhizal structures (e.g. arbuscules) or showed few hyphae not a11 fungi may be represented by spores at the time of characteristic of VAM fungi. Equisefum arvense, E. hyemale and sampling, an experiment was conducted to induce sporulation E. variegatum in Illinois and E. variegatum in Norway had a to obtain more precise information on the distribution of range of colonization levels, i.e. plants were non-mycorrhizal VAM fungal species. Only E. arvense and E. hyemale were used in the wettest habitats, exhibited low colonization levels in the in the induction experiment. Following the initial removal of moist areas and the highest colonization levels in the dry areas

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Vesicular-arbuscular mycorrhizas of Equisefum Table I. Root colonization and arbuscule formation in Equisetum species collected in Norway and the U.S.A. Species (number of samples examined)

Xf

S.D.

(% RLC)

%VAM samples

Norway E. fluviatile (5) E. palustre (7)

E. scirpoides (5) E. sylvaticum (7) E. variegatum (10) E. variegatum (10) U.S.A. E. arvense (15)

E. arvense (12) E. fluviatile (12) E. hyemale var. 1 (6)

E. hyemale var. 2 (5) E. hyemale (8)

E. hyemale (10) E. hyemale (10)

E. kansanum (8) E. laevigatum (8)

E. pratense (8)

E. variegatum (6) E. variegatum (6)

ARB

HYP

Place of collection

Mire, partially submerged (1p Moist, periodically submerged habitats among pine trees (2) Moist areas surrounding mossdominated areas (2) Far edges of mire, moist-dry (3) (A) Edges of mire, very moist (1) (8)Far edges of mire, moist (2) (A) Buffalo Springs, Texas, wetmoist sandy areas (2) (B) Buffalo Springs, Texas, moist sandy areas (3) Submerged, Evanston, Illinois (1) Open, moist area, Lake Michigan, Chicago, Illinois (2) Moist area, Lake Michigan, Chicago, Illinois (2) (A) Open, marshy wet areas near Lake Michigan, Illinois (1) (B) Open, moist areas, Illinois (2) (C) Opens, shrubs, moist-dry area occasionally very wet, Illinois (3) Open, moist sandy areas, Texas (2) Submerged cottonwood areas, clay loam areas, Albuquerque, New Mexico (1) Among shrubs, moist areas, occasionally very wet, Illinois (2) Jo Daviess County, Illinois, moist sandy areas, near rivers ( 2 ) (A) Wet, sandy-loam, Illinois (1) (B) Moist, sandy-loam, Illinois (2)

RLC. root length colonized, entries show meanf s.D.; %VAM, % of samples examined that were mycorrhizas; ARB, number of samples with arbuscules; HYP, number of samples with VAM hyphae only. " Moisture ranking of sites at time of collection: I, very wet, partially submerged; 2, moist, not submerged; 3, mesic-dry, hard ground. " Samples of this species collected from different habitats (e.g. A, B, ... ). Also see last column of table for brief description of habitat.

(Table I). Such a range in mycotrophy suggests a facultative relationship, and has also been reported for other species examined along a moisture gradient (e.g. Ebbers, Anderson & Liberta, 1987). Other endophytic fungi were present in the roots. Most distinctive structures were spherical clusters of resting spores of Plasmodiophorales-like endophytes and dark sclerotial structures observed in samples collected in west Texas, E. arvense and E. kansanum. These structures were also found to be common in ferns collected in Ontario, Canada (Berch & Kendrick, 1982) and Michigan, U.S.A. (Cooke, personal communication 1990). Norwegian species lacked any non-mycorrhizal endophytic fungi. All specimens, except E. variegafum A, collected in the U.S.A., had coenocytic and knobby hyphae characteristic of VAM fungi (Table I). Vesicles and arbuscules characteristic of VAM fungi were present in roots of all the species of Equisefwn except for E. fluviafile and E. variegatum (A), in Norway, and E. fluviafile, E. hyemale (A),E. laevigatum and E. variegatum, in the U.S.A., which had little or no VAM infection, although a few of these species had roots with coenocytic hyphae (Table 1). In contrast, Berch & Kendrick (1982)

reported 0 % colonization in E. arvense (number of samples examined = 17), E. hyemale var. afhne (3) and E. hyemale var. elatum (1) and E. scirpoides (I), although one sample of E. arvense had 20 % of all segments examined colonized by VAM fungi. Vesicles were found in older root pieces of several species collected in the U.S.A. but none in any of the species from Norway. Arbuscules stained well with trypan blue and were mostly found close to the root epidermis. Unlike hyphae of VAM fungi observed in the roots of grasses and forbs, rarely was there penetration of hyphae deep into the cortical area, i.e. next to the stele. Surprisingly, leaf sheath and branch axils of E. kansanum and E. palusfre in the U.S.A. held a high density of hyphae and spores. These inoculum-rich areas seem to occur in both old and young sections of the plants and probably serve as a ready source of inoculum for new roots (Koske & Gernma, 1990). In this study spores of Glomus aggregafum were not found in the roots of E. hyemale var. pseudohyemale as reported by Koske ef al. (1985), although spores of this species were isolated from root zone and induction experiment soils (see below). Although in this study it was not possible to link spores to

S. S. Dhillion

65 9

Table 2. Spores of VAM species identified in induction experiment and root zone soil of E. arvense and E. hyemale Spores of VAM species Plant species and moisture status of collection site Quisetum arvense Wet sandy areas

Moist sandy areas

Equisetum hyemale Open, marshy wet areas

Open, moist areas

Open, moist-dry areas

Species identified from soil in Species in root zone soil induction experiment

Glomw aggregatum, Gigaspora sp., Glomw sp. I, Glomw sp. 2 Glomw aggregatum, Glomw aggregatum, G ~ g a s ~ o rsp., a Glomw sp. Gigaspora sp., Glomw sp. I. Glomw sp. 2 1, Glomw sp. 2 No spores

No spores

Glomw aggregatum, Gigaspora sp. 1, Glomw fasciculatum complex, Gigaspora sp. 2 Glomw aggregatum, No spores Gigaspora sp. 1, Glomus fasciculatum complex, Gigaspora sp. 2 Glomw aaggregatum, Glomw aggregatum, Gigaspora sp. 1, Gigaspora sp. 1, Glomw fasciculatum complex, Glomw fasciculatum complex, Gigaspora Gigaspora sp. 2 sp. 2

the mycorrhizas, all roots and associated soil samples (root zone) were collected within pure stands of the sampled species, increasing the likelihood of the spores belonging to the Equisetum mycorrhizas. Spores isolated from the induction experiment using E. laevigafum roots were similar in all habitats (Table 2). Although spores were isolated only from the root zone samples of E. hyemale obtained at the driest end of the gradient, and none from the wettest part, the induction experiment revealed that similar VAM spores occupied all sites. It is likely that spodation of these fungal species may be limited to drier sites. The occurrence of VAM fungal species, when considering a range of habitats, should therefore not be based solely on the presence of spores. The low redoxstate of submerged sediments associated with aquatic plants may inhibit germination of spores of VAM fungi (Mortimer, 1942; Mosse et al., 1981) and the development and efficacy of infections (Saif, 1983; Tanner & Clayton, 1985). Mycorrhiza formation may also have been depressed in reduced sediments by high phosphate availability (e.g. Smith, 1980). It is therefore likely that the host species may be more sensitive to changes in environmental conditions across the gradient than its mycobiont associates which appear to occur ubiquitously, as both spores and hyphae (Ebbers ef al., 1987). When E. hyemale and E. laevigatum plants were inoculated with spores isolated from soils of the induction experiment, plants formed mycorrhizas indicating that these fungi were compatible with the Equisetum species tested. The dependence of Equisetum on VAM is not known. There were no differences in root hair densities for all species examined except for E. kansanum and E. pratense which had

significantly lower densities than other species. Since mycorrhizas function as extensions of the root system it is possible that species, e.g. E. kansanum and E. pratense, with fewer root hairs may have higher dependence on VAM than species with more abundant root hairs. Currently experiments are in progress examining the growth dynamics of Equisetum species and their associations with specific VAM fungi. Berch & Kendrick (1982) suggested that the demise of the ancestors of Equisetales, during the Carboniferous, may be due to the Equisetales being non-mycotrophic and to competition with mycorrhiza-forming plants. However, as reported in this study the Equisetales are mycotrophic and their demise may probably be related to climatic changes, going from cool and hydric to warmer and drier conditions. Their reduction may also be related to the evolution of plants with wider-ranging environmental tolerances, through betterdeveloped morphological and physiological features, e.g. deeper dwelling and highly branched root systems, broader leaves (or photosynthesizing organs), better pollination and dispersal mechanisms, and/or enhanced efficiency in the mycorrhizal association. This study indicates that Equisetum species can have a wide range of mycotrophy. The habitat may play an important part in determining the degree of mycotrophy in these plants. Equisetum species of Norway generally showed similar trends to those of the U.S.A. The data collected suggest that Equisetum species are most likely to be facultatively mycotrophic, and their demise may not be related to competition with highly mycotrophic advanced plants, ferns, cycads or pines as suggested by Berch & Kendrick (1982). Although it has been well demonstrated that the Equisetae most frequently occur in wet-moist habitats some members can survive in drier environmental conditions/periods, and may be able to cope effectively with periodically drier conditions via mycorrhiza formation.

I am grateful to James E. Cooke for assistance in the collection and identification of plant species in the U.S.A. Gerd and Erik Olsberg of Tylldalen, Norway, kindly provided assistance in locating sites and collections. I also wish to thank Qishui Zhang, who helped in the processing of samples.

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(Accepted 12 October 1992)

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