Potential of fungal antagonists for biocontrol of Fusarium spp. in wheat and maize through competition in crop debris

Potential of fungal antagonists for biocontrol ofFusarium spp. in wheat and maize throughcompetition in crop debris

LAURA LUONGO1, MASSIMO GALLI1, LUCIANA CORAZZA1,

ELLIS MEEKES2, LIA DE HAAS2, CARIN LOMBAERS VAN DER

PLAS2, & JURGEN KOHL2

1Plant Pathology Research Institute (Istituto Sperimentale per la Patologia Vegetale), Roma,

Italy, and 2Plant Research International, Wageningen, The Netherlands

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

AbstractPathogenic Fusarium spp. cause head blight in wheat or ear rot in maize leading to yield lossesand also a reduction in quality due to mycotoxin contamination of the grain. Infected cropresidues are the main inoculum source for epidemics. Saprophytic fungi, obtained from cerealtissues or necrotic tissues of other crops, were screened for their ability to colonise wheat strawand maize stalks and to suppress sporulation of pathogenic Fusarium spp. Results of bio-assaysconducted under controlled conditions were variable among Fusarium spp. and host substratesfor most antagonists tested, such as yeasts, Trichoderma spp. and non-pathogenic Fusarium spp.Isolates of Clonostachys rosea consistently suppressed sporulation of F. culmorum andF. 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 topieces of maize stalks or flowering ears in preliminary experiments conducted under fieldconditions. The colonisation of stalk pieces by pathogenic Fusarium spp. was assessed after 9months. Colonisation of stalk pieces by pathogenic Fusarium spp. was significantly reduced atseveral sampling dates. However, results obtained with the antagonists were not consistent forall 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: Jurgen Kohl, Plant Research International, P.O. Box 16, 6700 AA Wageningen, The

Netherlands. Tel: 31 317 476017. Fax: 31 317 423110. E-mail: [email protected]

ISSN 0958-3157 print/ISSN 1360-0478 online # 2005 Taylor & Francis Group Ltd

DOI: 10.1080/09583150400016852

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

https://www.researchgate.net/publication/226467418_Major_Changes_in_Fusarium_spp_in_Wheat_in_the_Netherlands?el=1_x_8&enrichId=rgreq-c77965ff-0dee-4bfb-b678-a55cfdf5fded&enrichSource=Y292ZXJQYWdlOzIzMzY3MDgxNjtBUzoxNzI3NDY0Nzg3MjcxNzBAMTQxODE5NzM3ODkyNA==

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

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 (Kohl & 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

230 L. Luongo et al.

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

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 Nahrstoffarmer 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).

Potential of fungal antagonists for biocontrol 231

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 158Cin 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

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 l�1) 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


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 ml�1)

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,

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 l�1 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


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.

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


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

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*

J83 95* 98* �/ �/ �/ �/

aCalculated 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.


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)

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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.


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.

Idrie

lla b

olle

yi

Clo

nost

achy

sro

sea

Epi

cocc

umni

grum

Fus

ariu

meq

uise

ti

Tric

hode

rma

harz

ianu

m

Tric

hode

rma

virid

e

0

02

04

06

08

001

Red

uctio

n in

spo

rula

tion

[%]

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.


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

2001-2002 2002-2003

Clonostachys rosea (016) NL

Clonostachys rosea (1457) I

Cladosporium cladosporioides (761) NL

Fusarium equiseti (1168) I

Control





Control





Control

0

10

20

30

40

50

60

70

0

5

10

15

20

25

30

35

40

45

50

0

5

10

15

20

25

30

35

Sep Nov MarJan May Jul

Sep Nov Jan Mar May Jul

Sep Nov Jan Mar May Jul

0

5

10

15

20

25

30

35

40

45

50

0

5

10

15

20

25

30

35

Sep Nov Mar

Sep Nov Jan Mar

Sep Nov Jan Mar

F. verticillioides

Jan

F. verticillioides

F. proliferatum F. proliferatum

F. graminearum F. graminearum

a

a

0

10

20

30

40

50

60

70

b

bc

c

a

aa

a a

a

aa

a

b

a

ababa

b

a aa

a

a

a

bbb b

a aa

a aa

aa

aa

a a a a

a

a

a aab

a

a

aa

a

a

a

a aa

a

a

a

ab

ab

abab

b

b bb

bab

bab

b b

b b

b

ab

ab aba

a

a

a aa

a

a

a a

a

aa

a

a

b

b

a

aa a

a

b

bc

c

c

aa a

a

ba

htiw

seceipkla ts egat necre

Pmui rasu

F)

%(

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).


https://www.researchgate.net/publication/43263163_Greenhouse_and_Field_Evaluation_of_Biological_Control_of_Fusarium_Head_Blight_on_Durum_Wheat?el=1_x_8&enrichId=rgreq-c77965ff-0dee-4bfb-b678-a55cfdf5fded&enrichSource=Y292ZXJQYWdlOzIzMzY3MDgxNjtBUzoxNzI3NDY0Nzg3MjcxNzBAMTQxODE5NzM3ODkyNA==

https://www.researchgate.net/publication/35089596_The_effect_of_pollen_in_the_phyllosphere_of_rye_on_colonization_by_saprophytic_fungi_and_on_infection_by_Helminthosporiu_sativum_and_other_leaf_pathogens_Neth_J_PI_Path?el=1_x_8&enrichId=rgreq-c77965ff-0dee-4bfb-b678-a55cfdf5fded&enrichSource=Y292ZXJQYWdlOzIzMzY3MDgxNjtBUzoxNzI3NDY0Nzg3MjcxNzBAMTQxODE5NzM3ODkyNA==

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 (Kohl 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.

hti

w sklats eg at

necreP

mu iras

uF

)%(

.p

ps

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



Control

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

2001-2002

2002-2003

a

aa

a a

a

a a

a

0

10

20

30

40

50

60

70

80

a

a

a

aa a

a a

a

0

10

20

30

40

50

60

70

80

2001-2002

2002-2003

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.


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

htiw

slenrekegatnecreP

muirasuF)

%(

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

Clonostachys rosea (016) NLClonostachys rosea (1457) I



Control

a

c

ab

bc c

a

ab

a

ab

b

bc

a a

ab

c

0

10

20

30

40

50

60

70

80

90

bc

ab

a

c cbc bc

b

a

c

0

10

20

30

40

50

60

70

80

90

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

a

c

ab

bc c

a

ab

a

ab

b

bc

a a

ab

c

2001

2002

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).


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.

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.

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Documents

Potential of fungal antagonists for biocontrol of Fusarium spp. in wheat and maize through competition in crop debris