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Biocontrol Science and Technology

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Potential of fungal antagonists for

biocontrol of Fusarium spp. in wheat
and maize through competition in crop
debris
a a a
Laura Luongo , Massimo Galli , Luciana Corazza , Ellis Meekes
b b b b
, Lia De Haas , Carin Lombaers Van Der Plas & Jürgen Köhl
a
Plant Pathology Research Institute (Istituto Sperimentale per la
Patologia Vegetale) , Roma, Italy
b
Plant Research International , Wageningen, The Netherlands
Published online: 18 Jan 2007.

To cite this article: Laura Luongo , Massimo Galli , Luciana Corazza , Ellis Meekes , Lia De Haas ,
Carin Lombaers Van Der Plas & Jürgen Köhl (2005) Potential of fungal antagonists for biocontrol
of Fusarium spp. in wheat and maize through competition in crop debris, Biocontrol Science and
Technology, 15:3, 229-242, DOI: 10.1080/09583150400016852

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 Biocontrol Science and Technology, May 2005; 15(3): 229
/242

Potential of fungal antagonists for biocontrol of

Fusarium spp. in wheat and maize through
competition in crop debris

LAURA LUONGO1, MASSIMO GALLI1, LUCIANA CORAZZA1,

ELLIS MEEKES2, LIA DE HAAS2, CARIN LOMBAERS VAN DER
PLAS2, & JÜRGEN KÖHL2
1
Plant Pathology Research Institute (Istituto Sperimentale per la Patologia Vegetale), Roma,
Italy, and 2Plant Research International, Wageningen, The Netherlands
Downloaded by [Aston University] at 15:49 09 January 2014

(Received 22 March 2004; returned 4 May 2004; accepted 10 August 2004)

Abstract
Pathogenic Fusarium spp. cause head blight in wheat or ear rot in maize leading to yield losses
and also a reduction in quality due to mycotoxin contamination of the grain. Infected crop
residues are the main inoculum source for epidemics. Saprophytic fungi, obtained from cereal
tissues or necrotic tissues of other crops, were screened for their ability to colonise wheat straw
and maize stalks and to suppress sporulation of pathogenic Fusarium spp. Results of bio-assays
conducted under controlled conditions were variable among Fusarium spp. and host substrates
for most antagonists tested, such as yeasts, Trichoderma spp. and non-pathogenic Fusarium spp.
Isolates of Clonostachys rosea consistently suppressed sporulation of F. culmorum and
F. graminearum on wheat straw, and of F. culmorum , F. graminearum , F. proliferatum and F.
verticillioides on maize stalks. Isolates of C. rosea , C. cladosporioides and F. equiseti were applied to
pieces of maize stalks or flowering ears in preliminary experiments conducted under field
conditions. The colonisation of stalk pieces by pathogenic Fusarium spp. was assessed after 9
months. Colonisation of stalk pieces by pathogenic Fusarium spp. was significantly reduced at
several sampling dates. However, results obtained with the antagonists were not consistent for
all sampling dates and between experiments.

Keywords: Competitive substrate colonisation, Fusarium culmorum, F. graminearum,

F. proliferatum, F. verticillioides, saprophyte, antagonist selection, maize ear rot

Introduction
Head blight of small grain cereals, especially wheat, and ear rot of maize can be caused
by various Fusarium spp. (Bottalico & Logrieco 1988; Leslie et al. 1990; Parry et al.
1995). Infections result in significant yield losses and also a reduction in quality
because mycotoxins such as deoxynivalenol, nivalenol, zearalenone or moniliformin
are produced by the pathogens in the grain (Chelkowski 1989; Yoshizawa 1991).
F. graminearum , followed by F. culmorum were the most dominant species on
wheat grain in The Netherlands in 2000 and 2001 (Waalwijk et al. 2003). Ear rot

Correspondence: Jürgen Köhl, Plant Research International, P.O. Box 16, 6700 AA Wageningen, The
Netherlands. Tel: 31 317 476017. Fax: 31 317 423110. E-mail: jurgen.kohl@wur.nl

ISSN 0958-3157 print/ISSN 1360-0478 online # 2005 Taylor & Francis Group Ltd
DOI: 10.1080/09583150400016852
 230 L. Luongo et al.

in maize for grain production is caused mainly by F. graminearum , F. proliferatum ,

F. verticillioides and F. culmorum (Vigier et al. 2001; Moretti et al. 2002).
Wheat and maize are most susceptible to ear infection during anthesis (Ooka &
Kommedhal 1977; Parry et al. 1995). There is no further polycyclic development
and spread of the disease within the crop from infected ears to other ears.
Consequently, inoculum sources are a direct threat for only a short time during
the growing season, and disease incidence and severity are closely related to the
amount of primary inoculum present (Sutton 1982). Inoculum sources can be
found within and outside the crop. Most pathogenic Fusarium spp. produce conidia
as inoculum which are mainly splash-dispersed over short distances of a few
centimetres to metres (Jenkinson & Parry 1994; Parry et al. 1995; Fernando et al.
1997, 2000). An exception is the teleomorph G. zeae of F. graminearum which
produces ascospores in perithecia on crop residues. Such ascospores are mainly
wind-spread and can travel distances of several metres, but a few may also be
transported for several kilometres (Fernando et al. 1997; Maldonado-Ramirez &
Bergstrom 2000). Thus, ear infections of wheat and maize are mainly caused by
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spores originating from the same field and, in part, by G. zeae, originating from
neighbouring fields.
Crop residues such as maize stalks are decomposed slowly and can therefore
be present in subsequent crops. Cotton and Munkvold (1998) demonstrated that
F. moniliforme , F. proliferatum and F. subglutinans survive in maize stubble on soil
surfaces for at least 630 days under North American conditions. In many studies,
residues of previously infected crops have been found to be the main sources of spores
infecting ears of wheat and maize (Sutton 1982; Shaner 2003). Monoculture, reduced
tillage or no-tillage systems strongly favour disease development because of the
presence of stubble resulting in high inoculum pressure (Dill-Macky & Jones 2000;
Gilbert & Tekauz 2000).
A general rule for reducing the risk of ear infection of wheat or maize by pathogenic
Fusarium spp. is to limit residues of infected crops in susceptible crop fields.
Monoculture of wheat or maize should be avoided and maize, as a potential source
of Fusarium spp., should not be grown in rotation with wheat. If this cannot be
avoided, stubble should be ploughed in carefully so that no stubble is left on the soil
surface. Protection of soil from erosion and the urge to save energy and costs may
force farmers to apply reduced- or no-tillage systems. New methods are needed to
enhance decomposition of wheat and maize stubble on soil surfaces in such cropping
systems.
Antagonistic fungi applied to crop debris may reduce survival and multiplication of
necrotrophic pathogens present in the residues of diseased crops and enhance
decomposition (Köhl & Fokkema 1998). Early colonisation of crop residues by
antagonists may also prevent saprophytic colonisation of such substrates by soil- or
air-borne inoculum of pathogenic Fusarium spp. after harvest as observed by Cotton
and Munkvold (1998).
The objective of our study was to select potential antagonists that suppress
saprophytic colonisation and sporulation of toxigenic Fusarium spp. on residues of
wheat and maize crops. Saprophytic fungi isolated from various necrotic plant
tissues were tested in bioassays to compare their efficacy in reducing sporulation
of several Fusarium spp. on wheat and maize stubble. In preliminary field tests,
selected antagonists were applied to pieces of maize stalks and flowering maize ears to
 Potential of fungal antagonists for biocontrol 231

study their effect on colonisation by Fusarium spp. A similar set of antagonists has
been screened by Dawson et al. (2002a,b) for use on wheat ears during flowering
to prevent infection.

Materials and methods

Fungal pathogens and antagonists
Fusarium culmorum (W:G. Smith) Sacc. (isolate 807) and F. graminearum Schwabe
(isolate 820), used for antagonist screening on wheat straw, were obtained from
infected wheat grains sampled in The Netherlands in 1998 and 1999. F. culmorum
(mcISPaVe1463) and F. graminearum (mcISPaVe1460) were obtained from wheat
residues in Italy in 1998; F. proliferatum (Matsushima) Nirenberg (mcISPaVe1519)
and F. verticillioides (Sacc.) Nirenberg (mcISPaVe1172) were obtained from maize
residues in Italy in 1998 and 1999.
A total of 135 candidate antagonists were included in the study, of which the
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majority were isolated from straw, stubble, seed surfaces, phyllosphere or roots of
cereal crops. A few isolates were from necrotic tissues of other crops. Fifty-nine
isolates originated from The Netherlands (Plant Research International), 52 from the
United Kingdom (Bateman and Dawson, Rothamsted Research) and 24 from Italy
(Istituto Sperimentale per la Patologia Vegetale).
Pathogenic Fusarium spp. were grown on Spezieller Nährstoffarmer Agar (SNA)
(Nirenberg 1976) for 14 days at 188C with 12 h blacklight (350 nm)/day. Candidate
antagonists were grown on oatmeal agar or potato dextrose agar (PDA, Oxoid) at
188C for 14 days in the dark, except non-pathogenic Fusarium spp. which were grown
with 12 h blacklight/day. Slow-growing fungi were incubated for 28 days. Forty
candidate antagonists, belonging to Chaetomium globosum Kunze:Fries, Epicoccum
spp. and several non-pathogenic Fusarium spp., sporulated poorly on agar and were
excluded from further studies. The remaining isolates belonged to the following
species (with number of isolates tested on wheat straw/maize stubble): Acremonium
strictum Gams (1/1), Aspergillus repens de Bary (3/0), Aureobasidium pullulans (de
Bary) Arnaud (6/2), Botrytis cinerea Pers.: Fries (1/0), Chaetomium globosum (1/1),
Chaetomium sp. (0/3), Cladosporium cladosporioides (Fr.) de Vries (6/2), C. herbarum
(Pers.: Fries) Link (2/1), Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert &
W. Gams (syn. Gliocladium roseum ) (11/10), Clonostachys rosea f. catenulata (Gilman
& Abbott) Schroers (syn. G. catenulatum ) (1/0), Cryptococcus albidus (Saito) Skinner
(1/0), C. laurentii (Kufferath) Skinner (2/0), Epicoccum nigrum Link (3/3), Fusarium
aquaeductuum Lagerheim (1/0), F. equiseti (Corda) Saccardo (9/10), F. flocciferum
Corda (3/1), F. oxysporum (5/2), F. poae (Peck) Wollenweber (1/0), F. sambucinum
Fuckel (1/0), F. solani (Mar.) Sacc. (1/1), Gliocladium nigrovirens van Beyma (1/0),
Idriella bolleyi (Sprague) von Arx (9/5), Penicillium brevicompactum Dierckx (1/1),
P. commune Thom (1/1), P. echinulatum Fassatiova (1/1), P. waksmanii Zaleski (1/0),
Scopulariopsis brevicaulis (Sacc.) Bainier (1/1), Sporobolomyces sp. (1/0), Trichoderma
aureoviride Rifai (1/1), T. harzianum Rifai (3/3), T. koningii Oudemans (2/1),
T. polysporum (Link: Pers.) Rifai (1/0), T. pseudokoningii Rifai (1/1), T. strictipilis
Bissett (1/0), T. viride Pers.: Fries (9/7), Trichothecium roseum (Pers.: Fries) Link (1/1),
and Ulocladium atrum Preuss (1/0).
 232 L. Luongo et al.

Bioassay under controlled conditions

A bioassay based on wheat straw inoculated with F. culmorum 807 or F. graminearum
820 and candidate antagonists was developed. Straw of winter wheat cv. Tambor,
which had not been sprayed with pesticides during the growing season, was cut into
4-cm pieces each with a central intact node. The pieces were sealed in plastic bags and
gamma-irradiated (4 Mrad). Before use in bioassays, sterile straw pieces were placed
in Erlenmeyer flasks (250 ml) containing sterile tap water and soaked for 6 h. Three
water-soaked pieces were transferred to a sterile moist chamber consisting of a Petri
dish (9 cm diameter) containing one sterile 1.5-mm thick filter paper (8.5 cm
diameter) with a sterile filter paper (8 cm diameter) on top of it, moistened with 10 ml
of sterile tap water. Spore suspensions were sprayed under sterile conditions using
atomisers at approximately 5 ml cm 2. Conidial suspensions of F. culmorum or
F. graminearum (1 /104 conidia ml 1) were applied first. After 6 h incubation at 158C
in the dark, the straw pieces were treated with sterile tap water containing 0.01%
Tween 80 (control) or spores of candidate antagonists. Petri dishes sealed with
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parafilm were further incubated (completely randomised) at 158C with 12 h

blacklight/day. For both F. culmorum and F. graminearum, three replicate Petri dishes
were used for each treatment with a candidate antagonist. For the water control
treatment, six replicates were used. In 10 of the 11 bioassays, C. rosea 016 was
included as standard for comparison of the different bioassays.
The number of conidia of F. culmorum and F. graminearum produced on the straw
pieces after 21 days was counted using a microscope for bioassays with most candidate
antagonists except in assays employing non-pathogenic Fusarium spp. For these, spore
numbers were estimated from the number of colony forming units (CFUs) after
plating on agar, since not all pathogenic Fusarium spp. could be distinguished
microscopically from non-pathogenic Fusarium spp. by their spores. For microscopic
counting, straw pieces from each Petri dish were placed in an Erlenmeyer flask
(100 ml) containing 10 ml of a washing liquid (20% ethanol in tap water containing
0.01% Tween 80). Flasks were shaken on a reciprocal shaker for 10 min, and the
concentration of conidia in the suspension was determined microscopically for
F. culmorum and F. graminearum using a haemacytometer. For counting after plating,
spore suspensions were plated using a spiral plater on PDA (10 g l 1) with Triton
X-100 (Sigma) (2 ml l1) added. Plates were incubated for 5 days at 308C. Under
these conditions, F. graminearum and F. culmorum produced very small pink and dark
red colonies, respectively. Colonies of F. aquaeductuum , F. equiseti, F. flocciferum ,
F. oxysporum, F. poae , F. sambucinum and F. solani were orange, white, yellow, white/
purple, salmon/yellow/brown, white and grey/white, respectively, and could easily be
distinguished from those of F. culmorum or F. graminearum .
Bioassays on maize stubble were subsequently carried out with the same set of
candidate antagonists except that the 34 isolates with weak antagonism against
Fusarium spp. on wheat were excluded. An additional eight isolates, belonging to
Chaetomium sp. or F. equiseti , were included. Experiments with maize stubble were
conducted similarly to those on wheat stubble. Four 4-cm pieces of gamma-irradiated
maize stubble were placed in moist chambers consisting of Petri dishes (20 cm
diameter), each with a sterile filter paper moistened with 25 ml sterile water. A total
of 10 experiments were conducted, each with eight to 10 antagonists tested against
F. culmorum mcISPaVe1463, F. graminearum mcISPaVe1460, F. proliferatum
mcISPaVe1519 and F. verticillioides mcISPaVe1172. After 21 days incubation, conidia
 Potential of fungal antagonists for biocontrol 233

were washed off the maize stubble and the numbers of conidia of pathogenic Fusarium
spp. produced on maize stubble were determined microscopically. Data were
processed as described for experiments with wheat stubble.

Bioassays under field conditions

Maize stalks. Five 6-cm stalk pieces were sewed on a small plastic strip (2.5 cm
distance between pieces) and gamma-irradiated (4 Mrad). Water-soaked stalk pieces
were first sprayed with conidial suspensions (1/106 conidia ml 1) of C. rosea 016,
C. rosea 1457, C. cladosporioides 761 or F. equiseti 1168 at approximately 5 ml cm 2.
After 30 min, the stalks were sprayed with conidial suspensions (1/104 conidia ml1)
of F. verticillioides mcISPaVe1172, F. proliferatum mcISPaVe1519 or F. graminearum
mcISPaVe1460. Four replicates of the following pathogen/antagonist combinations
were tested: F. verticillioides mcISPaVe1172/C. rosea 016, F. v./C. rosea 1457, F. v./C.
cladosporioides 761, F. v./F. equiseti 1168; F. proliferatum mcISPaVe1519/C. rosea 016,
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F. p./C. rosea 1457, F. p./C. cladosporioides 761, F. p./F. equiseti 1168; F. graminearum
mcISPaVe1460/C. rosea 016, F. g ./C. rosea 1457, F. g ./C. cladosporioides 761, and
F. g./F. equiseti 1168. Stalk pieces treated with the Fusarium spp. without antagonists
served as controls. For each pathogen/antagonist combination, each of four replicate
plots was inoculated with four plastic strips of five stalk pieces. After inoculation,
strips were placed on field soil in a randomised block design with approximately 20 cm
between strips. Two experiments were carried out on an experimental farm at Tor
Mancina (Rome) from September to June in 2001
/2002 and 2002
/2003, respec-
tively. During these experiments, stalk pieces were sampled at 2-month intervals. Two
similar experiments with the antagonists C. rosea 1457 and C. cladosporioides 761 were
conducted on an experimental farm at Wageningen from December to April in 2001
/
2002 and 2002
/2003, respectively, with one sampling date at the end of the
experiment.
Sampled stalk pieces were surface sterilised (30 s in a solution of ethanol (10%) and
sodium hypochlorite (8%), then rinsed for 60 s in sterile distilled water) and incubated
for 7
/10 days separately in Petri plates (20 cm diameter) containing modified PDA
(MPDA: 20 g l1 potato dextrose agar, streptomycin sulphate 160 mg l 1,
nitroaniline 6.5 mg l 1 and neomycin 60 mg l 1) at 258C. Colonies of F. verticillioides,
F. proliferatum or F. graminearum were identified based upon colony morphology and
microscopic examination of coniophores and conidia. The percentage of stalk pieces
producing colonies of the applied Fusarium species was recorded/replicate.

Maize ears. Conidial suspensions (1 /106 conidia ml 1; approximately 10 ml/ear) of

C. rosea 016, C. rosea 1457, C. cladosporioides 761 or F. equiseti 1168 were sprayed
using a hand-held pressure sprayer on the silk (stigmas) of tagged ears at the blooming
stage in July in a maize crop at an experimental farm in Tor Mancina (Rome). After
inoculation, each treated ear was covered with a polyethylene bag for 3 days. After 3
days, conidial suspensions of F. verticillioides , F. proliferatum or F. graminearum were
sprayed (1/104 conidia ml 1). The pathogen/antagonist combinations were the
following: F. verticillioides /C. rosea 016, F. v./C. rosea 1457, F. v./C. cladosporioides ,
F. v./F. equiseti ; F. proliferatum /C. rosea 016, F. p./C. rosea 1457, F. p./C. cladosporioides ,
F. p./F. equiseti ; F. graminearum /C. rosea 016, F. g ./C. rosea 1457, F. g./C.
cladosporioides , F. g./F. equiseti . Ears only treated with Fusarium spp. without
 234 L. Luongo et al.

antagonists served as controls. After inoculation, each cob was covered again with a
polyethylene bag for 3 days. Each antagonist
/pathogen combination was applied in
four replicates on 15 ears/replicate on plants located in micro-plots arranged in
randomised blocks. Ears were collected at the end of October 2001 and 2002,
respectively, and the kernels were removed from the cobs. A sample of 100 maize
seeds/replicate of each treatment was surface sterilised (30 s in a solution of ethanol
(10%) and sodium hypochlorite (8%), then rinsed for 60 s in sterile distilled water)
and incubated at 258C for 7
/10 days on MPDA plates (20 cm diameter; 25 kernels/
plate). Colonies of F. verticillioides , F. proliferatum or F. graminearum were identified
based upon colony morphology and microscopic examination of conidiophores and
conidia. The percentage of maize kernels producing colonies of the applied Fusarium
species was recorded/replicate.
During field experiments at Tor Mancina (Rome), air temperature, the soil
temperature at 5 cm of depth, rainfall and RH were recorded by a Delta-T Logger
DL2e (Delta-T Devices Ltd, Cambridge, UK) placed in the field.
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Statistical analysis
The numbers of conidia of F. culmorum or F. graminearum obtained/replicate, each
consisting of three straw pieces, were log10-transformed and analysed by analysis of
variance (ANOVA), using GENSTAT 5 version 4.1 (Genstat Committee, Algorithm
Group Inc.), with pathogen species and applications of water or candidate antagonists
as independent variables. Least significant difference (LSD)-tests (a /0.05) were
carried out for separation of means. The efficacy of the antagonists was calculated
from the back-transformed mean log10-numbers of Fusarium conidia of antagonist
and water treatments.
Percentages of stalks or ears producing colonies of Fusarium spp. were analysed
after arcsin transformation by ANOVA followed by LSD-tests (a /0.05).

Results
Bioassays on wheat straw
The number of conidia (transformed to log10), produced by F. culmorum (807) (per
three straw pieces) in the control treatments was 6.82 averaged over all the
experiments. For F. graminearum (820), the number of conidia/three straw pieces
(transformed to log10) in the various control treatments averaged 6.24. There was
considerable variation between replicates of the various treatments, resulting in LSD
values (a /0.05) for the log10-transformed numbers for the different experiments
between 0.3 and 1.0 for comparisons between control treatments and antagonist
treatments. Results of the bio-assays for the standard isolate C. rosea 016 were very
consistent in all experiments. Sporulation of F. culmorum on C. rosea (016)-treated
straw pieces was significantly reduced by 85
/99%, and that of F. graminearum by 91
/
100% in 10 repeated experiments (Table I). In the few cases that other antagonists
were tested twice, results were also similar in repeated experiments.
Statistically significant reductions of more than 80% in sporulation of F. culmorum
or F. graminearum were found for 19 and 23 antagonists, respectively, and for 15
antagonists against both Fusarium spp., out of the 95 isolates tested. Amongst the
fungal species included in the screening, isolates of C. rosea , a few isolates of F. equiseti
 Potential of fungal antagonists for biocontrol 235
Table I. Effect of isolates of C. rosea on reduction of conidial production by Fusarium culmorum (F.c.) and
F. graminearum (F.g.) on wheat straw and F. culmorum , F. graminearum , F. proliferatum (F.p.) and
F. verticillioides (F.v.) on maize stubble pieces.

Efficacy (%)a

Wheat straw Maize stalks

Isolate no. F.c.807 F.g.820 F.c.1463 F.g.1460 F.p.1519 F.v.1172

W4 91*b/ 99* 83*/65 6 B/0 85* B/0

W7 96*/100* 89*/98* 72* 74* 70* 52*
W15 98* 95* 50* 7 82* 80*
1316 100* 98* 81* 90* 37* 76*
1355 100* 100* 62* 19 43* 98*
016c 93 (85*
/99*) 96 (91*
/100*) 86* 67* 58* 80*
GR1 1456 99* 94* 62* 75* 69* 49*
GNL 1457 99* 96* 91* 96* 97* 98*
H1 99* 98* 82* 76* 97* 92*
H2 51 17 23* 36* 6 92*
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J83 95* 98*
/
/
/
/

a
Calculated from back-transformed numbers of conidia/CFUs produced by F. culmorum , F. graminearum ,
F. proliferatum and F. verticillioides . bStatistically significant reduction (LSD; a /0.05) of conidial numbers
(after log10-transformation) produced by F. culmorum , F. graminearum , F. proliferatum or F. verticillioides by
antagonist treatment compared to water treatment (control). cAverage (range) for 10 experiments.

and single isolates of Chaetomium globosum and Epicoccum nigrum were highly
effective (Figure 1). Isolates of non-pathogenic Fusarium spp. tended to show
moderate antagonism. Yeasts, and yeast-like Aureobasidium pullulans, as well as
common saprophytes of straw, such as Idriella bolleyi and Penicillium spp., were
identified as weak competitors against both pathogenic Fusarium spp. under the
conditions of the screening experiment. The six isolates of C. cladosporioides reduced
sporulation of F. culmorum (807) by only 20% but that of F. graminearum (820) by
80%. Isolates of T. harzianum and T. viride were weaker antagonists than C. rosea or
Fusarium spp. against both F. culmorum and F. graminearum .

Bio-assays on maize stubble

In bio-assays on maize stubble, the number of conidia (transformed to log10)
produced by F. culmorum (1463), F. graminearum (1460), F. proliferatum (1519) and
F. verticillioides (1172) (per four stubble pieces) in the control treatments averaged
4.95, 5.15, 6.02 and 5.97 for the different experiments. Variation between replicates
for the various treatments was less than for experiments with wheat straw pieces
resulting in LSD values (a /0.05) between 0.02 and 0.14 for comparisons between
control treatments and antagonist treatments. Although isolates with weak antagonism
against F. culmorum (807) and F. graminearum (820) on wheat straw were excluded
from experiments with maize stubble, antagonists were generally less effective on
maize stubble against the four pathogenic Fusarium spp. In many cases, no significant
reductions of sporulation of Fusarium spp. were observed. Statistically significant
reductions of more than 80% of sporulation of F. culmorum (1463), F. graminearum
(1460), F. proliferatum (1519) or F. verticillioides (1172) were found for 9, 11, 12 and
 236 L. Luongo et al.

100

Reduction in sporulation [%]

80

60

40

20

Idriella bolleyi
Aspergillus

cladosporioides
pullulans

Cladosporium
Aureobasidium

Clonostacys

equiseti
Epicoccum

Fusarium

Fusarium

Trichoderma
nigrum

harzianum

Trichoderma
repens

Fusarium
oxysporum

viride
rosea

flocciferum
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Figure 1. Reduction in sporulation of F. culmorum ( ) and F. graminearum ( ) by antagonist species

(reduction [%] calculated in relation to water treatment [ /100%]). Median, average and standard
deviation grouped/species, when three isolates or more/antagonist species were tested on pieces of wheat
straw.

21 antagonists, respectively, out of the 61 isolates tested. Only one isolate, belonging
to C. rosea , reached such a control level against all four Fusarium spp. Overall,
antagonism against F. culmorum (1463) and F. graminearum (1460) tended to be
weaker than against F. proliferatum (1519) and F. verticillioides (1172) (Figure 2). The
strongest antagonists against all Fusarium spp. tested were isolates of C. rosea . Isolates
of T. viride were moderately effective against F. proliferatum and F. verticillioides but
not effective against F. culmorum (1463) or F. graminearum (1460). Isolates of
T. harzianum were most effective against F. graminearum (1460).

Correlation between antagonism against different Fusarium spp. and on different substrates
No correlations were found when the efficacies of the antagonists on wheat straw
against F. culmorum (807) or F. graminearum (820) were compared with the efficacies
of the same antagonists in the other series of experiments using maize stubble
as substrate against other isolates of F. culmorum (1463) or F. graminearum (1460)
(R2 /0.0137 for F. culmorum and R2 /0.0043 for F. graminearum ; GENSTAT
5 version 4.1). A weak correlation was found when the reduction percentages of
F. culmorum and F. graminearum were compared separately for experiments with wheat
straw or maize stubble (R2 /0.2448 for wheat straw and R2 /0.1449 for maize
stubble). However, six of the 10 isolates of C. rosea significantly (LSD-test; a /0.05)
reduced sporulation of all six isolates of pathogenic Fusarium spp. when tested on
wheat straw or on maize stubble (Table I).

Bioassays under field conditions

Maize stalks. The percentage of maize stalks producing colonies of F. verticillioides ,
F. proliferatum or F. graminearum was higher in the first experiment (2001
/2002)
 Potential of fungal antagonists for biocontrol 237

100

Reduction in sporulation [%]

80

60

40

20

0
Clonostachys

equiseti

Idriella bolleyi

viride
Trichoderma

Trichoderma
rosea

Epicoccum
nigrum

Fusarium

harzianum
Downloaded by [Aston University] at 15:49 09 January 2014

Figure 2. Reduction in sporulation of F. culmorum ( ), F. graminearum ( ), F. proliferatum ( ) and

F. verticillioides ( ) by antagonist species (reduction [%] compared to water control [/100%]). Median,
average and standard deviation grouped/species, when three isolates or more/antagonist species were tested
on pieces of maize stubble.

conducted at Tor Mancina (Rome) than in the second (2002/2003), and also
varied between sampling dates within the experiment (Figure 3). At various
sampling dates, significantly (LSD-test; a /0.05) fewer Fusarium colonies were
found for antagonist-treated pieces. Most consistent reduction of Fusarium
colonisation was found for C. rosea 016. No antagonist treatments stimulated
Fusarium . No statistically significant treatment effects on Fusarium colonisation
were found for stalk pieces exposed to field conditions at Wageningen (Figure 4),
but Fusarium colonisation tended to be less on pieces treated with C. rosea 1457 or
C. cladosporioides 761.

Maize ears. Ears inoculated with F. verticillioides or F. proliferatum during flowering

yielded kernels of which approximately 60% were colonised by the inoculated
pathogen for both experiments. After inoculation with F. graminearum 20% of the
kernels were colonised by the pathogen in the first experiment. No F. graminearum
was found in the second experiment. In the first experiment, ear treatments with
C. rosea 016 and C. cladosporioides 761 reduced colonisation of kernels with both
F. verticillioides and F. proliferatum by approximately 50%. In the second experi-
ment, C. rosea 1457 and C. cladosporioides 761 significantly reduced kernel
colonisation by F. verticillioides. F. proliferatum colonisation was reduced by
treatments with C. cladosporioides and F. equiseti . Three of the four antagonists
were also effective against F. graminearum (Figure 5).

Discussion
In our screening programme, the effects of many antagonists were inconsistent when
tested under controlled conditions on wheat or maize stubble against several
pathogenic Fusarium spp. No significant correlation was found for their efficacy on
different substrates or against the different Fusarium spp.
 238 L. Luongo et al.

c
2001-2002 2002-2003
70 70
bc F. verticillioides
60 60 F. verticillioides
b
b
50 50

40 b 40
a
a a a
30 a b b b 30
aa ab ab a
a a a b a
20 a a a a 20
a a a b a
a a a
10 10 a a a a a a a
a a
a a a a a a
0 0
Percentage stalk pieces with Fusarium (%)

Sep Nov Jan Mar May Jul Sep Nov Jan Mar

50 b F. proliferatum 50 F. proliferatum
45 a ab 45
ab
40 b 40
a a
35 bbb 35
bb
30 30 a
b b b b a
25 b a 25 a
a
20 ab 20 a
ab aa
15 ab ab a a 15 ab ab a b
a
a 10 ab a a
10 a a aa a
a a
5 5 a aa
Downloaded by [Aston University] at 15:49 09 January 2014

0 0
Sep Nov Jan Mar May Jul Sep Nov Jan Mar
c
35
c
F. graminearum 35 F. graminearum
30 30
a
25 a 25
bc aaa b
20 b 20

15 15

10 10
a a
5 a
5 a
a
0 0
Sep Nov Jan Mar May Jul Sep Nov Jan Mar

Clonostachys rosea (016) NL

Clonostachys rosea (1457) I
Cladosporium cladosporioides (761) NL
Fusarium equiseti (1168) I
Control

Figure 3. Percentage of maize stalk pieces producing colonies of F. verticillioides , F. proliferatum or

F. graminearum after inoculation with conidial suspensions (1/106 ml 1) of antagonists or water as control
followed by inoculation with conidial suspensions (1 /104 ml 1) of the assessed Fusarium sp. Inoculated
stalk pieces were exposed to field conditions from September until July and samples were assessed at
2-month intervals. Bars belonging to the same sampling date and assessed Fusarium sp. with a common
letter do not differ significantly (LSD-test; a/0.05). Experiments were carried in 2001
/2002 and 2002
/
2003 in a field in Tor Mancina (Rome).

Yeast and yeast-like A. pullulans , potential antagonists which prevent infections by

spores of necrotrophic pathogens (Fokkema 1971) on substrates that include wheat
flowers (Schisler et al. 2002), did not reduce Fusarium sporulation on necrotic tissue.
Yeasts may colonise the surface of necrotic tissue but do not invade its interior,
and thus are unable to compete with Fusarium spp. during substrate colonisation.
Non-pathogenic Fusarium spp., especially some isolates of F. equiseti , showed strong
antagonism against pathogenic Fusarium spp. Such isolates may have ecological
characteristics, e.g., in substrate utilisation and temperature requirements, which are
very similar to those of pathogenic Fusarium spp. Due to the expected niche
adaptation to necrotic cereal tissues, strong competitive ability during substrate
 Potential of fungal antagonists for biocontrol 239

80 2001-2002
2001-2002

70

60

50

40

Percentage stalks with Fusarium spp. (%)

a
30 a
a a a
20
a a a a
10

0
F. v. F. p. F. g.

80 a 2002-2003
2002-2003
70

60

50
Downloaded by [Aston University] at 15:49 09 January 2014

40 a
a a
30 a

20 a
a
10 a a

0
F. v. F. p. F. g.
Clonostachys rosea (1457) I
Cladosporium cladosporioides (761) NL
Control

Figure 4. Percentage of maize stalk pieces producing colonies of F. verticillioides , F. proliferatum or

F. graminearum after inoculation with conidial suspensions (1/106 ml 1) of antagonists or water as control
followed by inoculation with conidial suspensions (1 /104 ml 1) of the assessed Fusarium sp. Inoculated
stalk pieces were exposed to field conditions from December until sampling in April. Bars belonging to the
same Fusarium sp. with a common letter do not differ significantly (LSD-test; a/0.05). Experiments were
carried in 2001
/2002 and 2002
/2003 in a field in Wageningen.

colonisation was expected at the beginning of the study. However, results obtained in
the various bio-assays were not consistent among the different pathogenic Fusarium
spp. tested and on tissues of wheat or maize. Ulocladium atrum 385, an antagonist
specifically selected for competitive substrate colonisation to outcompete Botrytis spp.
by nutrient competition during colonisation of necrotic host tissues (Köhl et al. 2003),
did not suppress Fusarium spp. on wheat straw. Evidently, Fusarium spp. are able to
utilise cereal residues better than U. atrum, which was originally isolated from a
necrotic onion leaf. Surprisingly, isolates of T. harzianum and T. viride had only
moderate effects on sporulation of Fusarium spp., and results against the various
Fusarium spp. varied on maize stubble. Isolates of Trichoderma spp. are antagonistic
against a broad range of pathogens, including F. pseudograminearum on wheat
straw (Wong 2002). However, reports on antagonism of Trichodema spp. against
Fusarium spp. are relatively rare. Although closely related to Trichoderma , isolates of
Clonostachys spp., especially of C. rosea , were consistently found to be superior
antagonists, suppressing sporulation of the four pathogenic Fusarium spp. included in
the study regardless of host tissue.
 240 L. Luongo et al.

90 2001

80

70 cc
c
c
bc b
60

ab
ab
50
ab
ab
ab
40 aa

30 aa
a c
c
Percentage kernels with Fusarium (%)

20 bc
bc
ab
10 a
a a
a
0
F. v. F. p. F. g.
Downloaded by [Aston University] at 15:49 09 January 2014

2002
c
90
bc bc
80 c
c
bc b
70
ab
60 a

50
a
40

30

20

10

0
F. v. F. p. F. g.
Clonostachys rosea (016) NL
Clonostachys rosea (1457) I
Cladosporium cladosporioides (761) NL
Fusarium equiseti (1168) I
Control

Figure 5. Percentage kernels colonised by F. verticillioides , F. proliferatum or F. graminearum at harvest. Ears

were inoculated in the field during flowering with conidial suspensions of antagonists (1 /106 ml 1) or
water as control, followed by inoculation with conidial suspensions (1 /104 ml 1) of the assessed Fusarium
sp. Ears were covered with plastic bags for 3 days after inoculation. Bars for the same Fusarium sp. and year
with a common letter do not differ significantly (LSD-test; a /0.05).

Two isolates of C. rosea , and an isolate of C. cladosporioides and F. equiseti , which

showed superior antagonism in the screening programme were selected for pre-
liminary experiments under field conditions in maize. Results of experiments with
pieces of maize stalks gave variable results. The level of Fusarium colonisation differed
between the two years, possibly because of different micro-climatic conditions, e.g.,
F. graminearum was only found after periods with frequent rainfall. All antagonists
tested were able to suppress Fusarium colonisation at certain sampling periods. Effects
of the single application of antagonist in late summer could still be detected several
 Potential of fungal antagonists for biocontrol 241

months later, e.g., the percentage stalk pieces colonised by F. proliferatum was
significantly reduced from 35% in the control treatment to below 5% for stalk pieces
treated with C. rosea 016 (experiment 2001
/2002 in Rome). However, the effect of
the antagonists was not consistent among the different experiments or sampling dates
within experiments. This may be due to the differential effect of micro-climatic
conditions on the antagonistic
/pathogen interaction. More knowledge on the
ecological demands of the antagonists is necessary for further exploitation. Improved
methods for quantification of Fusarium mycelium present in the host tissue, e.g., by
real-time PCR (Waalwjik et al. 2004), could be used in detailed studies on substrate
colonisation under field conditions. Such experiments should also include treatments
of host tissues already colonised by pathogenic Fusarium spp. since they are able to
infect and colonise host tissue before it senesces. C. rosea has been described as an
endophyte of various plants (Sutton et al. 1997). The possible endophytic colonisation
of maize and wheat tissues by C. rosea isolates found antagonistic against Fusarium
spp. should be investigated. Such an early colonisation of host tissue may favour the
antagonist during competitive colonisation of the tissue during senescence.
Downloaded by [Aston University] at 15:49 09 January 2014

The application of antagonists on flowering maize ears to prevent colonisation by

pathogenic Fusarium spp. gave promising results in the preliminary field experiments.
Further experiments carried out under disease conducive conditions are needed
without artificial stimulation of fungal colonisation as used in the preliminary
experiments. During such experiments, monitoring of mycotoxin contents will be
necessary to study the possible effect of antagonists on mycotoxin production in
kernels by pathogenic Fusarium spp.
The results of the screening programme followed by preliminary experiments
conducted under field conditions demonstrated the potential of several antagonists to
control pathogenic Fusarium spp. in wheat and maize when targeted at crop residues
after harvest, and at flowering ears.

Acknowledgements
This study was financed by the European Commission (QLK1-1999-00996,
ControlMycotoxFood), and the Dutch Ministry of Agriculture, Nature Management,
and Fisheries.

References
Bottalico A, Logrieco A. 1988. Osservazioni sulla fusariosi del mais in Basilicata. II. Influenza di alcuni
fattori colturali. Informatore Fitopatologico 38:55
/58.
Chelkowski J. 1989. Mycotoxins associated with corn cob fusariosis. In: Chelkowski J, editor. Fusarium -
mycotoxins, taxonomy and pathogenicity. Amsterdam: Elsevier. pp 53
/62.
Cotton TK, Munkvold GP. 1998. Survival of Fusarium moniliforme , F. proliferatum , and F. subglutinans in
maize stalk residue. Phytopathology 88:550
/55.
Dawson WAJM, Bateman GL, Jestoi M, Rizzo A. 2002a. Biological control of ear blight of wheat caused by
Fusarium culmorum . Journal of Applied Genetics 43A:217
/222.
Dawson WAJM, Bateman GL, Köhl J, de Haas BH, Lombaers-van der Plas CH, Corazza L, Luongo L,
Galli M, Jestoi M, Rizzo A. 2002b. Controlling infection of cereal grain by toxigenic Fusarium spp. using
fungal competitors. Proceedings of the BCPC Conference 2002
/ Pests & Diseases. pp 347
/352.
Dill-Macky R, Jones RK. 2000. The effect of previous crop residues and tillage on Fusarium head blight of
wheat. Plant Disease 84:71
/76.
Fernando WGD, Paulitz TC, Seaman WL, Dutilleul P, Miller J. 1997. Head blight gradients caused by
Gibberella zeae from area sources of inoculum in wheat field plots. Phytopathology 87:414
/421.
 242 L. Luongo et al.
Fernando WGD, Miller JD, Seaman WL, Seifert K, Paulitz TC. 2000. Daily and seasonal dynamics of
airborne spores of Fusarium graminearum and other Fusarium species sampled over wheat plots.
Canadian Journal of Botany 78:497
/505.
Fokkema NJ. 1971. The effect of pollen in the phyllosphere of rye on colonisation by saprophytic fungi and
on infection by Helminthosporium sativum and other leaf pathogens. Netherlands Journal of Plant
Pathology 77(Suppl. 1):1
/60.
Gilbert J, Tekauz A. 2000. Review: Recent developments in research on fusarium head blight of wheat in
Canada. Canadian Journal of Plant Pathology 22:1
/8.
Jenkinson P, Parry DW. 1994. Splash dispersal of conidia of Fusarium culmorum and Fusarium avenaceum .
Mycological Research 98:506
/510.
Köhl J, Fokkema NJ. 1998. Biological control of necrotrophic foliar fungal pathogens. In: Boland GJ,
Kuykendall LV, editors. Plant-microbe interactions and biological control. New York: Marcel Dekker. pp
49
/88.
Köhl J, Molhoek WML, Goossen-van de Geijn HM, Lombaers-van der Plas CH. 2003. Potential of
Ulocladium atrum for biocontrol of onion leaf spot through suppression of sporulation of Botrytis spp.
BioControl 48:349
/359.
Leslie J F, Pearson CAS, Nelson PE, Toussoun TA. 1990. Fusarium spp. from corn, sorghum, and soybean
fields in the central and eastern United States. Phytopathology 80:343
/350.
Maldonado-Ramirez SL, Bergstrom GC. 2000. Temporal patterns of ascospore dicharge by Gibberella zeae
Downloaded by [Aston University] at 15:49 09 January 2014

from colonized corn stalks under natural conditions. In: Proceedings of the 2000 National Fusarium
Head Blight Forum, Cincinatti, USA. pp 159
/162.
Moretti A, Corazza L, Balmas V, Santori A, Ritieni A. 2002. Funghi tossigeni e micotossine: filiera
cerealicola. Informatore Fitopatologico 12:17
/22.
Nirenberg H. 1976. Untersuchungen über die morphologische und biologische Differenzierung in der
Fusarium -Sektion Liseola. Mitteilungen der biologischen Bundesanstalt für Land- und Forstwirtschaft
169:1
/117.
Ooka JJ, Kommedhal T. 1977. Wind and rain dispersal of Fusarium moniliforme in corn fields.
Phytopathology 67:1023
/1026.
Parry DW, Jenkinson P, McLeod L. 1995. Fusarium ear blight (scab) in small grain cereals
/ a review. Plant
Pathology 44:207
/238.
Schisler DA, Khan NL, Boehm MJ, Slininger PJ. 2002. Greenhouse and field evaluation of biological
control of fusarium head blight on durum wheat. Plant Disease 86:1350
/1356.
Shaner G. 2003. Epidemiology of Fusarium head blight of small grain cereals in North America. In: Leonard
KJ, Bushnell WR, editors. Fusarium head blight of wheat and barley. St. Paul, USA: The American
Phytopathological Society. pp 84
/119.
Sutton JC. 1982. Epidemiology of wheat head blight and maize ear blight caused by Fusarium graminearum .
Canadian Journal of Plant Pathology 4:195
/209.
Sutton JC, Li D-W, Peng G, Yu H, Zhang P, Valdebenito-Sanhueza RM. 1997. Gliocladium roseum : A
versatile adversary of Botrytis cinerea in crops. Plant Disease 81:316
/328.
Yoshizawa T. 1991. Natural occurrence of mycotoxins in small grain cereals (wheat, barley, rye, oats,
sorghum, millet, rice). In: Smith JE, Henderson RS, editors. Mycotoxins and animal foods. Boca Raton,
FL: CRC Press. pp 301
/324.
Vigier B, Reid LM, Dwyer LM, Stewart DW, Sinha RC, Arnason JT, Butler G. 2001. Maize resistance to
gibberella ear rot: symptoms, deoxynivalenol and yield. Canadian Journal of Plant Pathology 23:99
/105.
Waalwijk C, Kastelein P, de Vries I, Kerényi Z, van der Lee T, Hesselink T, Köhl J, Kema G. 2003. Major
changes in Fusarium spp. in wheat in The Netherlands. European Journal of Plant Pathology 109:743
/
754.
Waalwijk C, Heide R van der, Vries I de, Lee T van der, Schoen C, Costrel-Decorainville G, Haeuser-Hahn
I, Kastelein P, Köhl J, Lonnet P, Demarquet T, Kema GHJ. 2004. Quantitative detection of Fusarium
species in wheat using TaqMan. European Journal of Plant Pathology 110:481
/494.
Wong PTW, Mead JA, Croft MC. 2002. Effect of temperature, moisture, soil type and Trichoderma species
on the survival of Fusarium pseudograminearum in wheat straw. Australasian Plant Pathology 31:253
/257.