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.
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A
A
Phaseolus sp.
Phaseolus
vulgaris L.
P. glauca
Fragaria sp.
(Accepted 15 October 1991)
A
A
A
A
A . glutinosa
P. glauca
A . spicatum
P. glauca
A . glutinosa
A . glutinosa
A. spicatum
8. papyrifera
P. glauca
A . glutinosa
Tumularia aquatica (Ingold) A . glutinosa
Descals & Marvanova
Varicosporium elodeae
Ref
Table 5. contd
12
12
12
4
12
12
4
12
12
12
6
10
I0
4
S
4
4
9
T
T
S
S
7
8
A
T
B,X
?
12
5
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