New Pltytoi.

(1996), 132, 119-122

Survival and spread of the endophyte

Stagonospora pteridiicola in Pteridium
aquihnum, other ferns and some flowering
plants

BY P. J. F I S H E R
Department of Biological Sciences, Hatherly Laboratories., University of Exeter,
Exeter EX4 4PS, UK
{Received 18 May 1995; accepted 26 September 1995)

SUMM.^RY
Pteridium aquilinum (L.) Kuhn was sampled for colonization by Stagonospora pteridiicola in Great Britain,
Hungary and Australia. British samples gave the highest incidence of f)8",, during September and 12"o at the
beginning of the growing season. Hungarian samples showed a similar frequency. The fungus was not found in
Australian bracken. Five field-collected fern species other than bracken did not contain the fungus in May when
bracken already had a colonization frequency of \2"i, in the pinnules. Sampling after the bracken had died in
November demonstrated that the fungus had continued growth as a saprobe. Glasshouse-grown bracken sprayed
with a spore suspension showed 96 "o colonization after 21 d, whereas four fern species andfi\'eflowering plants,
similarly treated, gave colonization frequencies of 0-3 "o. Other glasshouse-grown bracken, similarly sprayed,
showed that colonization declined over 5 months from 75 ",, to 40'^,j and that the fungus showed little spread into
fresh unsprayed growth on these plants. The possible species specificity oi the futigus is discussed.

Key words: Endophytes, Fteridium aguUmum, Stagonospora pteridiicola, spray inoculation, species specificity.

autumn plants. Aureobasidtum pulhdans. Cylindro-

INTRODUCTION
Bracken [Pteridium aquilinum (L.) Kuhn] is a
widespread Australasian plant whose typical form
ranges from Britain and Sweden to the Caucasus,
and throughout most of Africa. Variants extend to
South-east Asia and North America (Willis, 197S),
Bracken has extensively colonized heathland, moor-
land, woodland and some pastures across Britain
where it has often hecome dominant (Macleod,
1982; Scragg, 1982). Godfrey (1974) characterized
hriefly the phylloplane mycoflora of P. aquilinum,
and more extensive investigations have been carried
out by Frankland (1966, 1969, 1976) who isolated
species of Phoma and Stagonospora from apparently
healthy petioles. This suggested that at least some of
the fungi recorded from ferns might coexist within
the tissues of their host as symptomless endophytes.
Petrini, Fisher & Petrini (1993) undertook the first
detailed study of the endophyte community in
bracken growing on two sites on Dartmoor, Devon
UK, Endophytes were isolated from the pinnules,
leaf veins, rachis and rhizomes of healthy spring and
carpon destrnrtans and Phoma sp. dominated the
endophyte community in the spring, and four
species: A. pultulans, Ramichloridium schulzeri,
Stagonospora sp. and Sordarta fimicola dominated in
the autumn. The autumn sampling also showed that
c. 40'\, of all samples from the rachis were infected
with a Stagonospora sp. This species was previously
unknown and was described by Fisher & Puni-
thalingam (1993) as Stagonospora pteridiicola. There
are no records of it having been isolated prev'iously
from any other plants in the field.
The present investigation w as undertaken to study
the frequency of S. pteridiicoto in its endophytic and
saprobic states in naturally-occurring bracken at
various times of the year at two sites in Devon, and
one in each of Cumbria (Britain), Hungary and
Victoria (Australia) and to test other indigenous fern
species for the endophyte. In addition, artificial
inoculations were conducted with glasshouse-grown
bracken plants, other fern species and flowering
plants to test infectivity, fungal spread and species-
specificity of S. pteridiicola.
120 P. y. Fisher

ethanol for 30 s, after Petrini etal. (1993). The pieces

MATERIALS AND METHODS were then placed in groups of five into Petri dishes
S. pteridiicola was isolated during July and Sep- containing 1-5 "^ Oxoid* malt extract agar (MEA)
tenriber 1994 from healthy plants of P. aquilinum supplemented with 250 mg T^ terramycin to sup-
growing at a site in Stoke Woods near Exeter, press bacterial growth. All plates were incubated at
Devon. UK (Grid Ref, SX9I5 958) and at a site on 20 + 2 °C for 5-14 d depending upon the growth rate
Haldon Hills near Exeter (Grid Ref. SX9O7825). of the en:ierging fungi. Isolation was by transfer of
Samples were also taken during September from the mycelium to 2"o MEA plates without antibiotics. S.
Zemple'n mountain near the Slo\'akian border of pteridiicola could be recognized 3 ^ d after plating of
north-east Hungary, and during November from the leaf fragments. Viability of" the spore suspension
Diamond Creek, a stream passing through grassland used for spraying the plants was tested by spreading
with scattered trees, near Eltham, Victoria, Aust- 0-1 ml onto MEA plates (1-5 "a) and examining the
ralia. Dead, brown fronds were collected after a plates under a dissecting microscope ( x 250) after
number of frosts during November from the Stoke 24 h.
Woods and Haldon sites as well as from Murthwaite. A one-way analysis of variance was performed on
Fell End, Ravenstonedale Cumbria (Grid Ref. the raw frequencies of isolation from both field and
SD34718987). November samples were collected to glasshouse material. Multiple comparisons were
assess the frequency of 6\ pteridiicola in its saprobic made using Fisher's least significant difference
phase. At each sampling, 24 pinnules {c. 1 cm'^) were (LSD) test. All computations were performed on the
removed from each of five fronds derived from statistical package Systat 5.2 (W'ilkinson. 1989).
different plants, taken to the laborator\' in paper bags
and processed within 24 h after collection for all RESULTS
British plants, and 5 d after collection for all other
plants. At Stoke Woods there was a statistically significant
increase in frequency of coionization of S. pteri-
In addition, fronds and pinnules offiveeach of the
diicola in September compared with July when the
following ferns were similarly sampled from Slapton
fungus was growing as an endophyte. This was
Ley Nature Reserve, Devon, during May: Dryopteris
followed by an insignificant decrease in November
borreri Newman, D. dilatata (Hoffm. A. Gray.), D.
when the fungus had entered a saprobic phase in the
filix-mas (L.) Schott, Phyllitis scolopendrium (L.)
brown dead leaves of bracken (Table 1, Fig. 1).
Newman, Polystichum setiferum (Forsskal) More ex
W^oynar, and P. aquilinum, and processed as de-
scribed above to test for colonization by 5. pteri-
diicola in the field. For the glasshouse studies (a), five
bracken plants were chosen that had reached a height
of c. 30 cm when grown from fragments of rhizomes
obtained from, the Stoke Woods site and planted in
soil taken from this site. These plants were sprayed
with 5 ml of a spore suspension (2xlO^mr^),
covered with a polythene bag for 24 h to maintain
high relative humidity during spore germination,
then sampled after 21 d in the manner described
above. An equal number of plants was left unsprayed
but otherwise treated similarly to serve as controls.
These plants were re-sampled after 5 months when
a distinction was made between old sprayed plant
material and new growth which had not been
sprayed. For glasshouse studies (b), the ferns: D.
borreri, P. scolopendrium, P. setiferum, Polypodium
vulgareL., atidP. aquilinum and the flowering plants: HJ HS HN SJ SS
Cucurbita pepo L., Raphanus sativus L., Zea mays L.. Samples
Glycine hispida Maxin. and Hibiscus esculentus L. Figure 1. Box-plot display of actual infection frequencies
were also infected by spraying and treated in the out of 24 for Stagonospora pteridiicola in Pteridium
same way as plants in glasshouse studies (a) except aquilinum in the field. The codes refer to the origin of the
that, for the flowering plants and P. scolopendrium, plant material (first letter) and sampling date (second
leaves or fronds w ere divided into 24 parts of similar letter). C: Cumhria; H: Haldon; S: Stoke Woods; U:
Hungar\; J: July; S: September; N : November. The
size to the pinnules described earlier and ail plants upper, median and lower horizontal lines of tbe boxes
were sampled only once after 21 d. All plant material represent the 75th, median and the 25tb percentiles
was surface sterilized by immersion sequence, 75"'o respectively of the colonization density. The ends of the
ethanol for 1 min, 20 "o NaClO for 3 mm and 75% 'whiskers' (vertical bars) denote the outermost values.
The symbol * denotes outliers.
 Endophyte in Pteridium aquilinum 121

Table 1. Fisher's Least-Significant-Difference Test Matrix of pairwise comparison probabilities

CN HJ HN HS SJ SN SS US
HJ 1 0(1
HN 0-03 (102 1-00
HS 0.17 0 26 000 1-00
SJ 042 0 30 0-17 0-03 1(X)
SN 013 0-20 O-(HI 0^87 0 02 1 00
SS 0-02 0 03 0(K) 0^26 0-00 0 33 100
us 0'5/ 0-42 O'll 0'06 0-81 0-04 0-(X) 0-00

The codes refer to the origin of the plant material (first letter) and sampling date (second letter). C: Cumbria;
H: Haldon; S: Stoke Woods; U: Hungary; J: July; S: September; N: November. The boxed values are those where
0

Different results were recorded from the plants

sampled at Haldon, situated ahout 10 miles from
Stoke Woods, where the increase in infection
frequency was not significant from July to Sep-
tember but the decrease in November was significant
(Table 1, Fig, 1). When the Hungarian data were
compared with those for Stoke Woods and Haldon in
September, the Hungarian plants showed a lower
colonization frequency, hut only with the Stoke
Woods data was there a significant difference (Table
1, Fig. 1). A comparison between infection fre-
quencies for the Stoke Woods, Haldon and Raven-
stonedale collections for November samplings, when
the fungus had entered a saprobic phase, shows a
significantly lower infection frequency for the
Haldon site compared with the other two (Table 1,
Fig. 1). Such differences will depend on a number of
unknown variables, such as frequency of the fungus S5 N5
in the pinnules during its endophytic stage and the Plant
type of competition encountered by the fungus by Figure 2. Box-plot display of actual infection frequencies
other micro-organisms after plant death. The fungus out of 24 for Stagonospora pteridiicola in Pteridium
was not detected in the Australian bracken which aquilinum in the glasshouse experiments. SI : spray
was sampled during early spring and summer. infected plants after 21 d, C: unsprayed controls, S5: SI
plants after 5 months, N5: unsprayed new growth on S5
The ferns D. borreri, D. dilatata, D.fi/ix-mas, P. plants.
scolopendrium and P. setiferum, sampled in mid-May
at Slapton Ley, showed no colonization by 5.
pteridiicola but P. aquilinum collected from the same suspension of 5 . pteridiicola, Raphanus sativus had
site had a mean colonization frequency of 12 '\) in the an infection frequency of less thans 2 "o- The other
pinnules. species remained uninfected.
Glasshouse plants from study (a), sprayed with the When the conidiai suspension used for spraying
spore suspension and tested after 21 d, showed 75 ''o the plants was applied to 1-5 "o MEA test plates,
colonization compared with controls which them- lOO'^'fi spore germination was obtained after 24 h.
selves showed an indigenous presence of the fungus
of 10*^0 (Fig. 2). The same sprayed plants, tested
again after S months, showed a population of the DiscrssiON
fungus declining from 75°,, to 42"o, but new The field data for infection frequencies during the
unsprayed growth on these plants remained largely growing season of 5, pteridiicola in Britain and
uninfected (Fig. 2) indicating that, at least under Hungar\^ are similar to those described by Petrini et
glasshouse conditions, the fungus showed little al. (1993), witb colonization increasing to a maxi-
spread. The four fern species, D. borreri, P. scolo- mum during Septetnber, although tbis varied be-
pendrium, P. setiferum and P. viilgare. sprayed with a tween sites. W'hat bad not been show'n previously is
spore suspension in the glasshouse study (b), gave that tbe fungus persists well as a saprobe in dead
infection frequencies of 1-3 °o whereas the sprayed bracken fronds. In tbis respect the fungus can be
bracken control gave 96"n colonization. Of the five classed as an endophytic saprobe, defined by Fisher
flowering plants which were sprayed with the spore & Petrini (1992) as a fungus that begins its life cycle
122 P. J. Fisher
as a typical endophyte in often quite young plants
without causing disease symptoms but becomes a
saprobe after the plant has died.
The field survey at Slapton Ley during May, when
zero infection was recorded for S. pteridiicola in the
ferns D.borreri, D. dilatata, D.filix~mas, P.scolo-
pendrium, and P.setiferum and 12"(, in bracken,
suggests that the fungus might colonize bracken
more readily than it does the other ferns. It would be
important to repeat sampling at other times of the
year to support this evidence, which suggests species
specificity m the field.
The glasshouse studies undertaken here further
support the suggestion that the fungus more readily
infects bracken than it does other plants. When
susceptibilit>' of four artificially-infected ferns and
five species of flowering plants was compared with
that of similarly-treated bracken plants, the bracken
showed a relatively high degree of susceptibility, and
the other ferns and fiowering plants show ed virtually
none. Howe\er, these glasshouse experiments were
carried out with non-axenic bracken, which raises
the possibility that the bigb infection frequency in
inoculated bracken might ha\'e been the result of
stimulated latent infections. Host tissue and organ
specificity have been demonstrated for wheat endo-
phytes (Sieber, 1985) and for endophytes of Norway
spruce (Sieber, 1988, 1989).
Of particular interest are those bracken plants
infected in the glasshouse then allowed to continue
their grow^th for a further 5 months before infection
frequencies were determined in the old and new
growth. The declining incidence of the fungus in the
old growth and its near absence in any new growth
suggest that the physiology of the endophyte is non-
aggressive. It also implies an infection process which
allows the fungus to enter the plant and remain there
in an inactive state until the plant dies. A question
which remains to be answered is how comparatively
young plants sampled in the field during July, which
might have reached a height of 2 m during the first
5 wk of the growing season, already show a high
degree of infection of the pinnules when detailed
examination under a dissecting microscope fails to
reveal any spore-producing conidiomata. Set against
this, the evidence from the glasshouse study suggests
that there is little spread from infection loci, set up
by germinating spores on the pinnule surface, to
other parts of the plant. Two possible explanations
remain, which might well operate concurrently.
Firstly, the fungus has been shown to be present in
the rhizomes (Petrini et al., 1993), which readily
overwinter and give rise to new shoots in the spring,
and these might caro' the infection to the top of the
young plants systemically. Secondly, established
stands of bracken normally leave a thick covering of
bracken detritus in the autumn after the plants have
died, and this might give rise to numerous infection
sites through w hich the young fronds have to emerge
in the spring. Rain splash probably also plays an
important role in the movement and deposition ol"
the spores even when only a few conidiomata have
ripened. Although no exact counts have been taken
of the number of spores produced by one conidioma,
it was relatively simple in the laboratory to produce
a spore suspension of 10* mP ^ from 1-3 conidiomata.
Since the spores are hydrophilic and readily wet-
table, and their viability approaches 100 "^'o, infection
in the field by conidia deposited on pinnule surfaces
probably forms an important part of the infection
process at certain times of the year.
ACKNOWLEDGEMENTS
We would like to thank Mariann Marschall for collecting
and sending the bracken samples from Hungary and Jenny
Hoffman for doing the same in Australia. With thanks also
to Dr. (). Petrini for critically reading the manuscript.
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