305 Mycoi. Res. 96 ( 4 ) :305-308 (1992) Printed in Great Britain Endophytic aquatic hyphomycetes of roots of spruce, birch and maple K. R. SRIDHAR' AND F. B A R L O C H E R ~ , ~ Department of Biosciences, Mangalore University, Mangalagangofri 574 199, Mangalore, India Department of Biology, Mount Allison University, Sackville, N.B., EOA 3C0, Canada Aquatic hyphomycetes were isolated from living root tissues of spruce, birch and maple in a woodland stream of Nova Scotia by plating and aeration techniques. Of the 12 species recorded, Anguillospora filiformis and Heliscw lugdunensis were most common. Fungi were more common on bark than on decorticated segments. Aerated root segments yielded three species that were not recorded by the plating method. Anguillospora filifomis, Articulospora anfipodea, Cylindrocarpon aquaticum, Mycocentrospora clavafa, Mycocentrospora sp., Tetrabrachium elegans and Varicosporiumgiganteum are recorded for the first time as root endophytes. Riparian plants extend part of their roots into streams and rivers. Since aquatic hyphomycetes are the major fungal colonizers of plant detritus in running waters, it seems likely that they would also infect healthy or decaying aquatic roots. This was recently confirmed by Fisher, Petrini & Webster (1991) who examined the bark and xylem of aquatic roots of Alnus glutinosa (L.) Gaertner. In the present study, living aquatic roots of three additional tree species were examined for the presence of aquatic hyphomycetes. Five trees of white spruce (Picea glauca (Moench) Voss), mountain maple (Acer spicatum Lam.) and white birch (Befula papyrifera Marsh.) were chosen from the bank of the Boss Brook, a softwater stream in Fenwick, N.S., Canada (Barlocher, 1987). From each tree, 20-25 cm long and 0.7-1.0 cm thick sections were cut from roots exposed to flowing water at a depth of 0.5 to 1 m. The roots were 8-12 yr old. Samples were taken in January (spruce), February (birch) and March (maple) 1991. The material was transferred to the laboratory in polythene bags and processed within 5 h. The root sections were thoroughly rinsed in running distilled water and cut into 1 cm segments. The bark was separated from each segment. Bark and decorticated segments (referred to as xylem throughout this paper) were surface sterilized by immersion in 96% ethanol ( I min), sodium hyphochlorite (6% available chlorine; 3 min) and 96% ethanol (0.5 min). Immediately after surface sterilization, the segments were rinsed in sterile distilled water and placed individually on Petri plates containing 1% malt extract agar (MEA) supplemented with penicillin/streptomycin solution (Sigma 0906; 400 IU penicillin G ml-I, 0.4 mg streptomycin ml-I). The plates were incubated at 20 OC for 7-21 d depending on the growth of fungi. Isolation was made by transfer of mycelial fragments to MEA plates without antibiotics and incubation at 20'. After To whom reprint requests should be sent 20 2-3 wk gowth, small sections of the colony were suspended in sterile distilled water, aerated for up to 48 h and scanned for conidiophores and conidia of aquatic hyphomycetes. To determine potential spore production of endophytic aquatic hyphomycetes, surface-sterilized bark and xylem segments (I cm long) were separately suspended in sterile distilled water, and aerated for 96 h. The water was filtered through Millipore filters (8 ~ m )which , were stained with acid fuchsin and scanned for conidia. The dry weight was determined for each sample after subjecting the root material to 100' for 24 h. Twelve, five and seven species of aquatic hyphomycetes were recorded as root endophytes of spruce, birch and maple respectively (Tables 1, 2). Of the 50 segments plated out, 18-92% yielded at least one species, &24% yielded two species, and 0-4 % yielded three species (Table 3). In all cases, aquatic hyphomycetes were more common on bark than on xylem segments. This confirms observations by Fisher et al. (1991). In view of the relative scarcity of aquatic hyphomycetes on conifer needles (Barlocher, 1982), it is of interest that Picea roots harbour a range of species comparable to that of deciduous trees. In addition to aquatic hyphomycetes, terrestrial fungi and sterile mycelia were isolated (Table I), though their numbers were much lower than in the alder roots studied by Fisher ef a/. (1991). Out of a total of nine taxa isolated by plating, Anguillospora jiliformis Greathead, Heliscus lugdunensis Sacc. & Theny and Tetrabrachium elegans Nawawi & Kuthubutheen were common to all three tree species (Table I). Their frequency of occurrence ranged from 4-32 % for bark and &14 % for xylem (Table 4). Varicosporium elodeae Kegel and Varicosporium giganteum Crane were isolated exclusively from spruce roots. Articulospora tetracladia Ingold, Cylindrocarpon aquaticum (Nils.) Marvanovi & Descals and Mycocentrospora sp. were confined to spruce and maple, whereas Mycocentrospora clavata Iqbal Endophytic aquatic hyphomycetes 3 06 Table 1. Endophytic aquatic hyphomycetes in roots of P. glauca, B. papyrifera and A . spicatum. Number of isolations from 50 segments each of bark and xylem: percentage in parentheses, B, bark; X, xylem; Cultures that did not produce sigmoid or tetraradiate spores upon aeration were classified as terrestrial isolates P. glauca B. papyrifera A . spicaium Anguillospora filiforrnis Articulospora tetracladia Cylindrocarpon aquaticum Heliscus lugdunesis Mycocentrospora clavata Mycocentrospora sp. Tetrabrachiurn elegans Varicosporium elodeae Varicosporium giganteum Total isolations Terrestrial isolates Table 2. Conidia produced per gram dry weight of aquatic roots of P ghuca, B. papyrifera and A. sprcatum, after 96 h of aeration, Average, with range in parentheses P. glauca Anguillospora filiformis Bark Xylem Articulospora antipodea Bark Xylem A . atra Bark Xylem A. tetracladia Bark Xylem Ciavariopsis aquatica Bark Xylem Cylindrocarpon aquaticum Bark Xylem Heliscus lugdunensis Bark Xylem Mycocentrosporn clavata Bark Xylem Tetrabrachiurn elegans Bark Xylem Varicosporium elodeae Bark Xylem Varicosporium giganteum Bark Xylem B. papyrifera A . spicatum 41 (0-114) 32 (0-1 17) 0 0 35 (0-133) 13 (0-67) 0 0 0 0 9 (0-44) 10 (0-50) 0 0 0 0 40 (&loo) 64 ( 5 2 4 0 ) 0 4 (&la) 0 0 7 (0-33) 6 (0-17) 0 0 29 (0-101) 122 (0-467) 8 (0-29) 268 (0-1002) 879 (14-4205) 0 0 0 0 55 (0-201) 30 (0-114) 0 1 (0-7) 0 11 (0-57) 56 (0-257) 3 (0-9) 3 (0-14) 0 0 0 33 (0-89) 0 34 (0-171) 0 11 (0-29) 9 (0-31) 0 0 7 (0-36) 0 0 0 0 0 23 (0-80) 0 0 0 0 K. R. Sridhar and F. Barlocher 307 aquatic hyphomycetes, Fisher ef al. (1991) isolated 12 species from living aquatic roots of Alnus glutinosa. Five of their C, three species isolates (Articulospora atra, A. tetracladia, Clavariopsis aquatica, Heliscus lugdunensis and Varicosporiurn elodeae) were also common in the present study. Anguillospora filiformis, ArficuloP. glauca spora anfipodea, Cylindrocarpon aquaticum, Mycocentrospora Bark 82 6 0 clavata, Mycocenfrospora sp., Tetrabrachium elegans and VaricoXylem 38 0 0 sporium giganteurn are recorded for the first time as root B papyrifera endophytes (Table 5). Thus, it appears that aquatic roots of Bark 36 18 0 Xylem 18 0 0 riparian trees are indeed commonly colonized by aquatic A. sprcatum hyphomycetes. In addition, these fungi are occasionally found Bark 92 24 4 on soil root surfaces, associated with root rot, and as Xylem 32 2 0 endophytes in soil roots (Table 5). The results suggest that roots of different species may be colonised by different fungal species, but the numbers of Table 4. Frequency of occurrence (in %; 50 segments) of three common samples studied so far is too small to state this with any aquatic hyphornycetes from roots of spruce, birch and maple degree of confidence. Other potential factors that may Tefrabrachium Anguillospora Heliscus influence numbers and types of endophytic fungi include elegans filiformis lugdunensis geographic location, stream type, the age of the root and the season. In any case, it seems clear that aquatic roots provide P. glauca a stationary refuge for some aquatic hyphomycetes. This, Bark 4 22 8 Xylem 0 10 0 together with the ability to survive in terrestrial situations B. papyrifera (Bandoni, 1981; Park, 1974; Sanders & Webster, 1978; Bark 20 8 6 Sridhar & Kaveriappa, 1987) may help them maintain their Xylem 4 8 2 presence in a given stream reach despite the unidirectional A. spicatum flow of water. The greater incidence of fungi in the bark Bark 32 20 16 Xylem 14 6 2 suggests that this is where the primary invasion takes place; subsequently, interior tissues may be invaded. The role of root-associated aquatic hyphomycetes in plant nutrition, root was confined to spruce and birch (Table I). Two sterile white absorption efficiency and senescence or susceptibility to m~celialcolonies were isolated from spruce bark. diseases remains to be investigated. Aeration of surface sterilized bark and xylem yielded 11 One of the isolates of Heliscw lugdunensis from spruce bark aquatic hyphomycetes (Table 2). Three of these, namely produced the teleomorph after 40 d when subcultured colonies Arficulospora antipodea Roldin, Arficulospora afra Descals and were exposed to continuous light. According to Fisher, Anson Clavariopsis aquafica de Wildeman were not recorded by the & Petrini (1986), endophytic fungi of healthy plant parts often plating method. All three species were confined to spruce bark develop both the sexual and asexual reproductive state in a and xylem. Possibly, these fungi may have been unable to moist chamber after the death of the plant tissue. Since compete successfully with terrestrial endophytes on MEA considerable numbers of aquatic hyphomycetes are established medium. O n the other hand, Mycocentrospora sp., which was as endqphytes in aquatic roots, these may serve as a helpful isolated by plating out spruce and maple segments, was not tool to establish teleomorph-anamorph connections. recorded in the aerated samples. Two species, Articulospom Rough estimates of the visible root biomass in the Boss fetracladia and Heliscw lugdunensis were recorded in all aerated Brook suggests that it equals or exceeds that of dead branches tree species. and twigs. Though spore production from living roots ( < I For all three tree species, aeration yielded more fungal mg-'; Table 2) is considerably lower than from dead twigs species than plating, despite the lower number of replicates. In (100-150 mg-'; Barlocher, 1981) or from dead leaves (up to contrast to studies with dead leaves, the aeration period 6000 mg-'; Barlocher, 1982), there is considerable turnover of necessary to induce spore production was considerably longer root biomass, especially in the smaller roots (Waid, 1974). The (4 versus 2 d; Barlocher, 1982). This indicates that mycelial amount of dead root material that becomes available during g o w t h was necessary before sporulation could occur (e.g. one growing season must therefore be substantial. In addition from inside the root to the surface) or, the mycelium was to shedding particulate matter, roots excrete many water dormant and became activated only upon the death of the soluble or mucilaginous organic compounds. The potential root. A similar delay was found in the plated segments; bark contribution of these various root products to the nutrition of required 4-5 d of incubation and xylem 7-10 d to produce aquatic hyphomycetes, as well as of other stream organisms, visible mycelium on MEA medium. Loose conidia, filtered has so far been neglected by ecologists. from the stream, never survived the surface sterilization sequence (unpublished observations). It therefore seems safe One of us (K.R.S.) is thankful to Mangalore University for to concIude that the conidia produced from root segments granting a study leave during the tenure of this investigation. were indeed derived from mycelial structures inside the root. The financial support of the Natural Sciences and Engineering In the only other study designed to recover endophytic Research Council of Canada is gratefully acknowledged. Table 3. Frequency of occurrence of aquatic hyphomycetes (in %; 50 segments). A, at least one species isolated from a segment; B, two species; Endophytic aquatic hyphomycetes Table 5. Aquatic hyphomycetes reported from roots. Habitat: A, aquatic, T, terrestrial. Root part examined: B, bark; S, surface; X, xylem (root without bark); 7, undefined Anguillospora fii$ormls A . longissima (Sacc. & Syd.) Ingold Articulospora antipodes A . atra A . tetracladia Campylospora parvula Kuzuha Clathrosphaerina zalewskii van Beverwijk Clavariopsis aquatica Cylindrocarpon aquaticum Filosporella sp. Heliscus lugdunensis Lunulospora curvula Ingold Mycocentrospora acerina (Hartig) Deighton Mycocentrospora clavata Mycocentrospora sp. 1 Mycocentrospora sp. 2 Pseudoanguillospora sp. Tetrabrachium elegans Tetracladium marchalianum de Wildeman 7 . setigerum (Grove) Ingold Tricladitcm chaetocladium Ingold T , s~lendens Ingold Plant Habitat Root part A . spicatum 3.papyrifera P. glauca Fragaria sp. A A A B,X B,X B,X T ? 12 12 12 6 P. glauca Alnus glutinosa A A B,X B 12 4 P. glauca A . glutinosa A . spicatum B. papyrifera P. glauca A . glutinosa Alnus sp. Fagus sylvatica L. A T T T Daucw sp. T ? A . spicatum B. papyrifera P. glauca A . glutinosa A . spicatum P. glauca A . ghtinosa A . spicatum B. papyrifera P. glauca Fragaria sp. A A A A A A A A A 1 A B,X B,X B,X B B,X B,X B B,X B,X B,X T ? T A S S B T A A B B B 2.3 4 4 A . glutinosa A B B,X Fagus sylvatica T T Gent~anasp. Fragaria sp. A . glutinosa A . glutinosa T Varicosporium giganteum Varicosporium sp. Plant Habitat Root part Ref. P. glauca A B,X 12 Arctostaphylos uva-ursi (L.) Sprengel T ? 11 References: I, Bant (1963); 2, Fisher & Petrini (1989); 3, Fisher & Petrini (1990); 4, Fisher et al. (1991); 5, Gourley (1969); 6, Nemec (1969); 7, Parkinson & Thomas (1969); 8,Taylor & Parkinson (1965); 9,Waid (1954); 10, Watanabe (1975); 11,Widler & Miiller (1984); 12, this study. REFERENCES A A Phaseolus sp. Phaseolus vulgaris L. 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