[ 57 ]

Trans. Br. mycol. Soc. 66 (1) 57-64 (1976) Printed in Great Britain

COLONY INTERACTIONS AND HYPHAL INTERFERENCE BETWEEN

SEPTORIA NODORUM AND PHYLLOPLANE FUNGI

By A. M. SKIDMORE* AND C. H. DICKINSON

Department of Plant Biology, The University of N ewcastle upon Tyne

Interactions between dual cultures of S eptoria nodorum and several other cereal phylloplane
fungi were studied in vitro to determine their antagonistic ability and their tolerance of
antagonism. No fungi tested were outstandingly successful antagonists and interference
reactions between opposed hyphae rarely occurred. Septaria caused the hyphae of some
test fungi to fail to plasmolyse and others to burst. In experiments designed to examine volatile
and non-volatile metabolites growth of Septaria was rarely affected by colonies of the test
fungi.
In a previous paper (Dickinson & Skidmore, 1976) paired combinations, 3'5 ern apart on potato
interactions between spores and germ-tubes of dextrose agar (PDA) (Oxoid, pH 5'6) in four
Septoria and several cereal phylloplane fungi were replicate 9 cm Petri dishes. Controls were single
examined, Such interactions probably account for and dual inoculated cultures of the same fungus.
the majority of interfungal contacts on the leaves Duplicate experiments were incubated in darkness
of temperate crops. In favourable circumstances at 15° and 25 °C, in polythene bags to prevent
extensive vegetative development of phylloplane drying out. Interactions were assessed using a key
organisms occurs and pathogens may also grow based on the observations ofPorter (1924) and Dick-
extensively on the leaf surface, when there may inson & Boardman (1971). Porter recognized five
be numerous hyphal contacts. separate modes of interacting colony growth :
Fungal colonies growing in proximity may effect (A) Mutually intermingling growth where both
significant changes in one another or they may fungi grew into one another without any macro-
require intimate hyphal contact. Changes include scopic signs of interaction.
trophic stimulation, morphogenetic alterations and (Bi) Intermingling growth where the fungus
various degrees of growth inhibition. Interactions being observed is growing into the opposed fungus
between hyphae appear to be significant in deter- either above or below or above and below its
mining the pattern of colonisation of dung colony, and its corollary.
(Ikediugwu & Webster, 1970a, b), competition (13 ii) Intermingling growth where the fungus
between species of Trichoderma and other fungi under observation has ceased growth and is being
(D ennis & Web ster, 1971a, b, c) and the control of overgrown by another colony.
Heterobasidion annosum by Peniophora gigantea (C) Slight inhibition where the fungi approached
(Ikediugwu, Dennis & Webster, 1970). However, each other until almost in contact and a narrow
little is known of the ability of foliar pathogens to demarcation line, c. 1-1. mm, between the two
compete in this way. colonies was clearly visible.
(D) Mutual inhibition at a distance of > 2 mm.
The interacting fungi were assigned values on
MATERIALS AND METHODS a 0-5 scale for each type of interaction (Fig. 1).
Between the two extremes of no inhibition and
The fungi used in these experiments, together with considerable mutual inhibition the assignment of
Torula herbarum and Drechslera graminea, and the intermediate values, especially the status of inter-
culture methods used were described by Skidmore mingling growth patterns and growth around other
& Dick inson (1973) and Dickinson & Skidmore colonies, was difficult. Growth around another
(1976). fungus was placed at a lower point on the scale (3)
Colony interactions than slight mutual inhibition (4), since in the former
instance the antagonized fungus was able to with-
The influence of the fungi on each other in cul- stand inhibitors produced by the antagonist.
ture was examined by inoculating all possible In evaluating interactions, each fungus was as-
* Present address: ICI Plant Protection Division, sessed for its ability to inhibit growth of another
Jealott's Hill Res. Stn, Bracknell, Berks. fungus, when it was termed active, and for its
 Septoria nodorum
A. Mutually intermingling growth
( ;>
C. Mutual slight inhibition
~~
Fig. 1. Interactions observed between adjacent fungal colonies on agar media.
Based on the observations of Porter (1924).
response to antagonism, i.e. its status as a passive
species.
Assessments were made when the fungi had
achieved an equilibrium after which there was no
further alteration in the growth pattern. In general,
observations were made after 21 days at 15° and
after 14 days at 25°.
Hyphal interference
Hyphal interference between these fungi was in-
vestigated using modifications of the methods
described by Ikediugwu & Webster (1970a, b).
Pieces of cellophane were placed on PDA in Petri
dishes (Dickinson & Skidmore, 1976), and left
overnight to allow excess water to evaporate. Using
four replicate dishes per treatment, opposing
fungal species were then inoculated approximately
2 cm apart on the diameter of the dishes. The two
colonies met or came into close proximity within
1-3 days, depending on the species and the tem-
perature of incubation. Microscopic examination
of the colonies at 15° was made in a controlled
temperature room. The colonies incubated at
25° were examined at laboratory ambient tem-
perature (c. 23-25°). Sheets of Perspex were
cut to accommodate the microscope objectives
B. Overgrowth by antagonist
II
or
~
o~
D. Mutual inhibition at a distance
~ ~
and thereby maintain a high humidity over the
fungi. More detailed observations were made on
small squares of cellophane cut from the areas of
intermingling growth. These squares were mounted
on microscope slides and a plasmolysing solution
of 70 % (w jv) glycerine in water introduced be-
neath the coverslip to detect changes in per-
meability.
Non-volatile metabolites
The effectof accumulations of non-volatile meta-
bolites on the growth of Septaria was examined
using the method of Gibbs (1967). Petri dishes con-
taining cellophane on PDA, as before, were inocu-
lated centrally with 5 mm disks cut from the edge
of actively growing cultures of the test fungi. Four
replicates were used per treatment. Duplicate
experiments were incubated at 25° dark and
15-25 ° light /dark (Dickinson & Skidmore 1976).
After 4 days, growth of the inoculated fungus was
measured and the cellophane removed with the
adhering colony. D. sorokiniana, A. alternata and
S. botryosum penetrated the cellophane and grew
into the medium if incubated for longer
periods. After the cellophane had been removed a
5 mm disk of S. nodorum, cut from the edge of a
growing colony, was placed in the central position.
 A. M. Skidmore and C. H. Dickinson 59
The dishes were replaced in their original incuba-
tion conditions and the extension growth of S. no-
dorum recorded for up to 14 days.

Volatile metabolites
The methods used to examine the possible pro-
duction.of volatile metabolites were essentially those
of Martin (1963) and Dennis & Webster (1971b).
Four replicate Petri dishes ofPDA were inoculated
centrally with 5 mm disks of each fungus, put in
polythene bags, and incubated at either 25° dark
or 15-25° light/dark. After 6 days the Petri dish
lids were replaced by cultures of S. nodorum on
PDA, fixed with Sellotape and re-incubated. Con-
trols consisted of S. nodorum over PDA dishes.
Growth of Septoria was measured at regular
intervals.
RESULTS
Colony interactions
All of the interactions observed between the ten
test fungi are given in Table 1. In general more
antagonistic interactions, i.e. those rated 5 (Fig. 1),
were observed at 15° than at 25° where there
were many interactions involving some form of
intermingling growth.
Septaria was more antagonistic at 25° than at
15° but was variable. It tended to show greatest
activity against slower growing species.
At 15° Aureobasidium and D. sorokiniana were
involved in mutually inhibitory interactions (i.e.
rated 4 or 5) with all of the other fungi and were
° less active at 25°. Both excreted conspicuous
<:l
I:l:i~
.,
.l:
13
{
'"
N
°
'"
'"
amounts of pigmented compounds into the agar
at 15° but much less so at 25° as judged by optical
density measurements.
The two Cladosporium species behaved in a very
similar manner in both incubation regimes, being
most active against slow growing fungi. Stemphy-
lium and Alternaria exhibited similar activity at
both temperatures with the striking exception of
their reaction to Torula. At 15° Stemphylium
was inhibitory (i.e, rated 4) to Torula whereas
Alternaria and Torula colonies produced inter-
mingling growth. At 25° intermingling growth
occurred between Stemphylium and Torula, whereas
Alternaria (rated 3) hyphae overgrew those of
Torula (rated 2). D. graminea did not show uniform
activity against the test fungi but it always pro-
duced orange-coloured crystals in the agar around
the hyphae adjacent to the opposed fungus. These
crystals were almost certainly anthroquinones, as
both helminthosporin and catenarin have been
shown to be produced by this species (Miller,
1961).
60 Septoria nodorum

5
 A. M. Skidmore and C. H. Dickinson 61

Table 2. Hyphal interference reactions observed on cellophane when all possible combinations
of ten leaf fungi were examined at 15° and 25°
(The types of reaction observed in the cytoplasm are given as v = vacuolation, g = granulation,
np = no plasmolysis and b = hyphal bursting.)
Incubation
temperature Type of
(0C) Active species Passive species reaction
15 Aureobasidium pullulans v. Botrytis cinerea v
Drechslera graminea v. Septoria nodorum v
Septoria nodorum v. Botrytis cinerea b
Septaria nodorum v. Drechslera graminea np
Stemphylium botryosum v. Torula herbarum b
Torula herbarum v. Botrytis cinerea b
25 Drechslera graminea v, Cladosporium herbarum np
Drechslera graminea v. Cladosporium macrocarpum np
Septoria nodorum v. Alternaria alternata np
Septoria nodorum v. Botrytis cinerea b
Septaria nodorum v. Drechslera graminea np
Stemphylium botryosum v. Torula herbarum b

to interact in all of the ways recognized in Fig. 1,

Hyphal interference
One of the results of hyphal interference is an
alteration in the permeability of antagonized cells.
Ikediugwu & Webster (1970a) used a number of
techniques to detect permeability changes in the
hyphae, including plasmolysing solutions of either
70 % (w/v) glycerine or 65 % (w/v) glucose in water
and vital stains. The stains were incorporated into
the medium on which the fungi were growing.
Uptake of the stain only occurred into those cells
whose permeability was unimpaired. In the present
study preliminary experiments showed that each
of several vital stains affected the radial growth of
the fungi on PDA, even at low concentrations,
which invalidated their use.
Ikediugwu & Webster (1970a) showed that the
growth rate of antagonized hyphae slowed con-
siderably on contact. A similar phenomenon was
noticed in this study but no attempt was made to
quantify these observations.
Of the 100 possible hyphal interference reactions
only six were observed at each temperature
(Table 2). They occurred with combinations of
fungi whose colonies had previously been shown
except for mutual inhibition at a considerable
distance.
The occurrence of hyphal interference in two
instances where it had previously been shown that
the colonies did not come into contact may be
explained by the fact that the inocula in this in-
stance were placed close together and it was
probable that diffusible inhibitory metabolities
had not accumulated before the hyphae met.
Positive interference reactions were also obtained
with two combinations which had previously
been recorded as having mutually intermingling
hyphae. At 15° both fungi in the Septaria/D.
graminea combination caused mutual vacuolation
and the cessation of growth in about 25 % of in-
stances where hyphae were seen to come into con-
tact and thus the overall appearance of the colonies
(Table 1) was ofcontinuing growth by both fungi.
At 25° only the D. graminea hyphae became
granular and failed to plasmolyse. The Septaria/
Alternaria interaction at 25° was of a similar
nature with the Alternaria hyphae becoming
granular and failing to plasmolyse (Fig. 2).
The remaining positive combinations concerned

Figs. 2-5 . Hyphal interference reactions amongst some phylloplane fungi from barley. Figs. 2 and 3
taken of colonies on cellophane mounted on microscope slides, flooded with 70 % (w/v) glycerine and
illuminated by phase contrast. Figs. 3 and 4 taken of colonies directly observed on agar dishes.
Fig. 2. Septaria (arrowed) versus Alternaria. Hyphae of the latter have granular contents and did not
plasmolyse in 70 % (w/v) glycerine.
Fig. 3. Septaria (arrowed) versus Botrytis. The hyphal tip cel1s of Botrytis in contact with or near to
those of Septoria have ruptured.
Figs. 4, 5. Torula (narrow hyphae, arrowed) versus Botrytis. On the surface of the cellophane Botrytis
hyphae appear to be coiling around those of Torula and some of the Botrytis hyphal tips have ruptured
(Fig. 4). Aerial hyphae of Torula appear to be coiling around those of Botrytis (Fig. 5).
 62 Septoria nodorum
intermingling hyphae where one or other fungus more, 1976) the data given in the present paper
appeared to cease growth. It is obvious, however, would appear to be analogous to the period of
that the capacity for hyphal interference was not active fungal growth during leaf senescence and
restricted to the apparently active fungus as in following the death of the host when breakdown of
four instances species which had ceased extension host resistance and the release of cell contents
growth were capable for causing hyphal interference enhances the nutrient status of the substrate. Few
in the opposed hyphae. workers have explored the interactions which may
The types of interference observed were vacuo- occur during the early stages of the colonisation of
lation, granulation of the cytoplasm, failure to senescent aerial, herbaceous tissues although there
plasmolyse and hyphal bursting, which was most are 'succession' studies (Hudson, 1968; Jensen,
spectacular in combinations of Septaria or Torula 1974). Saprophytic growth of necrotrophic patho-
with Botrytis (Figs. 3-5). Torula was itself an- gens may determine the extent of their reproduc-
tagonized by Stemphylium at both 15° and 25° with tion or survival during adverse conditions.
a proportion of hyphae bursting on contact. Saprophytes also may depend on successful growth
In some instances positive reactions occurred on newly dead tissues to enable them to sporulate
on only relatively few occasions, but in other casesprofusely.
up to 50 % of hyphal tips burst (Septoria/Botrytis) The group of fungi examined here included
and 70 % of hyphae failed to plasmolyse (Septoriaj commonly occurring cereal phylloplane saprophytes
D. graminea). and pathogens together with Botrytis and Torula
which are respectively primary and secondary
Non-volatile metabolites colonizers of newly dead herbaceous tissues (Web-
The growth of Septoria on agar which had pre- ster & Dix, 1960; Hudson, 1968). This group of
viously supported the growth of other test fungi fungi did not appear, in general, to cause or suffer
was comparable to growth on uncolonized media pronounced interactions between vegetative hyphae
except in one case. Previous growth of B. cinerea and the majority tolerated extensive vegetative de-
at 25° resulted in a marked reduction in growth velopment of adjacent species.
of S. nodorum. This may perhaps be explained by Of the reactions observed, the most variable
the nutrient impoverishment of the medium by were those involving colony versus hyphal inter-
B. cinerea, since it had grown considerably prior ference reactions and further studies are needed to
to its removal, or by the pH of the medium becom- determine why small numbers of hyphae may be
ing unfavourable for growth of S. nodorum. affected whilst the majority appear to grow on
unhindered. Dennis & Webster (1971C) suggest
that in the case of Trichoderma hyphal interference
Volatile metabolites
only occurred when two hyphae came into contact
The growth of Septoria colonies was not markedly but even if this were the case then as two colonies
affected when it was inoculated above actively grew into one another it would seem likely that there
growing colonies of most of the test fungi. Com- were many more contacts than there were hyphae
parison of the results at 25° in darkness and at affected in these studies. The infrequency of such
15-25° in the light-dark regime showed that the hyphal interference reactions amongst phylloplane
latter conditions resulted in a longer period of fungi, as compared to those between dung fungi
uniform growth of the Septaria colonies, compared (Ikediugwu & Webster, 1970a, b) and stump
with the controls. Alternaria depressed, and colonizers (Ikediugwu et al. 1970) is perhaps not
Aureobasidium enhanced the growth rate of surprising when the apparent successions on dung
Septoria at 25°. When the dishes were opened (Webster, 1970) and on wood (Kaarik, 1974) are
at the conclusion of the experiment, there were compared with the microbial activity on leaves.
noticeable aromas associated with all of them. The bursting reactions observed may be a necro-
trophic type of hyperparasitic interaction (Barnett
DISCUSSION
& Binder, 1973) where the antagonizing fungus
is obtaining nutrients from the dead host cells.
The experiments described here show that dif- Bartnicki-Garcia & Lippman (1972) using a number
ferent types of interaction are possible between of different fungi have shown that similar pheno-
paired fungi on agar media. Similar interactions mena, such as increase in vesicle formation, hyphal
have been shown by numerous workers (van den swelling and bursting, are influenced by tempera-
Heuvel, 1970; Dickinson & Boardman, 1971; ture, pH, ionic strength and character of the
Fokkema, 1973). bathing solution. These factors are thought to
By comparison with an earlier account of spore cause an imbalance between cell wall synthesis and
interactions on leaf surfaces (Dickinson & Skid- cell wall lysis by affecting cell wall synthetase and
 A. M. Skidmore and C. H. Dickinson
cell wall splitting enzymes and the discharge of
vesicles containing autolytic enzymes.
The inhibitions and stimulations caused by vola-
tile compounds were of minor importance com-
pared to those described by Dennis & Webster
(1971 b) and no attempt was made to isolate them.
The use of Sellotape in this method is open to
criticism, as volatile materials may be produced
from the adhesive film. However, any effect would
have also been seen on the controls.
Many studies have been made of the activity of
Botrytis as a facultative pathogen causing a 'grey
mould' disease of glasshouse crops, soft fruits and
stored vegetable products. Less is known of its
saprophytic activities, though it can be commonly
isolated from leaves (Barnes, 1971; Pugh & Buck-
ley, 1971 ) and was regarded by Hudson (1968) as a
primary saprophytic colonizer of senescing tissues.
I t is more aggressive than some of the common
phylloplane inhabitants (Dickinson & Skidmore,
1976). Torula has a somewhat different physiology
from the other test fungi and the re sults presented
here help to explain its success as a secondary
colonizer of herbaceous tissues (W ebster & Dix,
1960).
The relatively harmonious growth of the phyllo-
plane inhabitants in vitro suggests that the habitat
on leaves may be unable to support more aggressive
species, whose demands for nutrients and space
would probably be considerable (Dickinson &
Skidmore, 1976), though it would be unwise to
extrapolate too far from results obtained in vitro
to any in vivo situation.
A. M. Skidmore gratefully acknowledges the
provision of a SRC research studentship which
enabled these studies to be carried out.
REFERENCES
BARNES, G. (1971). Inhibition of Erysiphe polygoni on
clover leaf surfaces by saprophyte spores. In Ecology
of leaf surface micro-organsims (ed. T. F. Preece and
C. H. Dickinson), pp. 557-565. London: Academic
Press.
BARNETT, H. L. & BINDER, F. L. (1973). The fungal
host-parasite relationship. Annual Review of Phyto-
pathology 11, 273-292.
BARTNICKI-GARCIA, S. & LIPPMAN, E. (1972). The
bursting tendency of hyphal tips of fungi: presump-
tive evidence for a delicate balance between wall
synthesis and wall lysis in apical growth . Journal 0/
General Microbiology 73, 487-500.
DENNIS, C. & WEBSTER, J. (1971 a). Antagonistic pro-
perties Of species groups of Trichoderma . 1 . Produc-
tion of non-volatile antibiotics. Transactions of the
British Mycological Society 57, 25-39.
DENNIS, C. & WEBSTER, J. (1971 b). Antagonistic pro-
perties of species groups of Trichoderma. 2. Produc-
tion of volatile antibiotics. Transactions of the
British Mycological Society 57, 41-48.
DENNIS, C. & WEBSTER, J. (1971C). Antagonistic pro-
perties of species groups of Trichoderma. 3. Hyphal
interaction. Transactions of the British Mycological
Society 57, 363-369.
DICKINSON, C. H. & BOARDMAN, F. (1971). Physiologi-
cal studies of some fungi isolated from peat. Transac-
tions of the British Mycological Society 55, 293-305.
DICKINSON, C. H. & SKIDMORE, A. M. (1976). Interac-
tions between germinating spores of S eptaria
nodorum and phylloplane fungi. Transactions of the
British My cological Society 66,45-56.
FOKKEMA, N. J. (1973). The role of saprophytic fungi
in antagonism against Drechslera sorokiniana (Hel-
minthosporium sativum ) on agar plates and on rye
leaves with pollen. Physiological Plant Pathology 3,
195-205.
GIBBS, J. N. (1967). A study of the epiphytic growth
habit of Fomes annosus. Annals of Botany 31,755-774.
HEUVEL, J. VAN DEN (1970). Antagonistic effects of
epiphytic microorganisms on infection of dwarf bean
leaves by Alternaria zinniae . Mededelingen uit het
Phytopathologisdi Laboratorium 'Willie Commelin
Scholten' 84, 1-84.
HUDSON, H. J. (1968). The ecology of fungi on plant
remains above the soil. New Phytologist 67, 837-874.
IKEDIUGWU, F. E. O. & WEBSTER, J. (1970a). Antagon-
ism between Coprinus heptemerus and other copro-
philous fungi. Transactions of the British Mycological
Society 54, 181-204·
IKEDIUGWU, F. E. O. & WEBSTER, J. (1970 b). Hyphal
interference in a range of coprophilous fungi.
Transactions of the British Mycological Society 54,
2°5-210.
IKEDIUGWU, F. E. 0 ., DENNIS, C. & WEBSTER, J.
(1970). Hyphal interference by Peniophora gigantea
against Het erobasidion annosum. Tran sactions of the
British Mycological Society 54, 307-309.
JENSEN, V. (1974). Decomposition of angiosperm tree
leaf litter. In Biology of Plant Litter Decomposition
(ed. C. H. Dickinson and G. J. F. Pugh ), pp . 69-
104. London: Academic Press.
KAARIK, A. A. (1974). Decomposition of wood. In
B iology of Plant Litter Decomposition (ed. C. H.
Dickinson and G. J. F. Pugh ), pp. 129-174. London:
Academic Press.
MARTIN, C. M. (1963). Studies on the nature and
effects of volatile fungal metabolites. Ph .D . thesis ,
University of Glasgow.
MILLER, M. W. (1961). The Pfizer handbook of micro-
bial metabolites. New York : McGraw-Hill.
PORTER, C. L. (1924). Concerning the characters of
certain fungi as exhibited by their growth in the
presence of other fungi. American Journal of Botany
11,168-188.
PuGH, G. J. F. & BUCKLEY, N. G. (1971). The leaf
surface as a substrate for colonisation by fungi. In
Ecology of leaf surface micro-organisms (ed. T. F.
Preece and C. H. Dickinson), pp. 431-445. London:
Academic Press.
 Septoria nodorum
SKIDMORE, A. M . & DICKINSON, C. H. (1973). Effect of WEBSTER, J. & Drx, N . J. (1960). Succession offungi on
phylloplane fungi on the senescence of excised decaying cocksfoot culms . 3. A comparison of the
barley leaves. Transactions of the British Mycological sporulation and growth of some primary sap ro-
Society 60, 107-116. phytes on stem, leaf blade and leaf sheath. Tran sac-
WEBSTER, J. (1970). Coprophilous fungi. Transactions tions of the British My cological Society 43, 85-99.
of the British Mycological Society 54, 161-180.

(A ccepted for publication 12 June 1975)