Impact of selected antagonistic fungi on Fusarium species – toxigenic cereal pathogens

Fusarium-ear blight is a destructive disease in various cereal-growing regions and leads to significant yield and quality losses for farmers and to contamination of cereal grains with mycotoxins, mainly deoxynivalenol and derivatives, zearalenone and moniliformin. Fusarium pathogens grow well and produce significant inoculum on crop resiudues. Reduction of mycotoxins production and pathogen sporulation may be influenced by saprophytic fungi, exhibiting antagonistic effect. Dual culture bioassays were used to examine the impact of 92 isolates (belonging to 29 fungal species) against three toxigenic species, i.e. Fusarium avenaceum (Corda) saccardo, F. culmorum (w.G.smith) saccardo and F. graminearum schwabe. Both F.culmorum and F. graminearum isolates produce trichothecene mycotoxins and mycohormone zearalenone and are considered to be the most important cereal pathogens worldwide. infection with those pathogens leads to accumulation of mycotoxins: deoxynivalenol (DoN) and zearalenone (ZEA) in grains. Fusarium avenaceum isolates are producers of moniliformin (MoN) and enniatins. isolates of Trichoderma sp. were found to be the most effective ones to control the growth of examined Fusarium species. the response of Fusarium isolates to antagonistic activity of Trichoderma isolates varied and also the isolates of Trichoderma differed in their antagonistic activity against Fusarium isolates. the production of MoN by two isolates of F. avenaceum in dual culture on rice was reduced by 95% to 100% by T. atroviride isolate AN 35. the same antagonist reduced the amount of moniliformin from 100 μg/g to 6.5 μg/g when inoculated to rice culture contaminated with MoN, which suggests the possible decomposition of this mycotoxin.

iNtRoDUCtioN Fusarium species are cosmopolitan necrotrophic pathogens of cereals, pulse crops and many other plants, important in Agricultural and forest landscape.Fusa rium ear (head) blight (FHB=scab) has been known for more than 100 years and is one of the important diseases of wheat and other small grain cereals. in Europe it is caused mostly by F. graminearum, F. culmorum, F. avenaceum and F. poae.other Fusarium species are less important due to their lower incidence and aggressiveness (Arseniuk et al. 1999;Bai, shaner 1994;parry et al. 1995;Chełkowski 1998;wakuliński, Chełkowski 1993;Bottalico, perrone 2002).FHB leads to significant loss of grain yield and quality.several fungal secondary metabolites, e.g.deoxynivalenol (DoN), nivalenol (NiV), zearalenone (ZEA) and moniliformin (MoN) and also their derivatives may contaminate cereal grains (McMullen et al. 1997;Jones, Mirocha 1999;Bottalico 1998;Chełkowski 1998).the disease has been re-emerging in many cereal-growing regions worldwide (parry et al. 1995;McMullen et al. 1997;Jones, Mirocha 1999;Bottalico, perrone 2002).
Fusarium culmorum and F. graminearum survive saprophytically on plant resi-aprophytically on plant resi-on plant residues after harvest and serve as a source of inoculum for the subsequent year.Both species colonize particularly frequently maize stalks and F. graminearum produces significant amounts of ascospores and conidia (sutton 1982).Fusarium avenaceum is a cosmopolitan species and one of the most important ones in agriculture and the forest environment in the moderate climatic zone (Nelson et al. 1983;Kwaśna et al. 1991;Leslie, summerell 2006).
simultaneous co-occurrence of the three mycotoxins: monilformin, deoxynivalenol and nivalenol was found in high percentage of positive samples (33% and 35%), during 1998 and 1999 epidemics of FHB in wheat in poland (tomczak et al. 2002).
there are few species among soil microorganisms antagonistic to Fusarium, able to reduce their population in soil and debris.several fungal species were examined for the ability to reduce the inoculum potential of Fusarium pathogens, mainly by reduction of biomass in plant residues colonized by Fusarium (łacicowa, pięta 1985;Ligitt et al. 1997;Luongo et al. 2005;Lutz et al. 2003;Dawson et al. 2004).several antagonists were found to reduce infection of ears and accumulation of DoN in wheat grains (Ligitt et al. 1997;Dawson et al. 2004).Clonostachys, Gliocladium and Trichoderma species reduced the colonization of wheat and maize by pathogenic Fusarium species and suppressed the sporulation of the latter (woo et al. 2005;Luongo et al. 2005).there is only scarce information on fungal antagonists of toxigenic Fusarium species and their ability to reduce the production of mycotoxins in solid substrates.Trichoderma species have been examined for more than 50 years and are known as highly effective in biological control of a wide range of plant pathogens of soil origin.they are also known to produce over 120 secondary metabolites, in-they are also known to produce over 120 secondary metabolites, including antifungal metabolites (Kubicek, Harman 1998;woo et al. 2005).Cooney et al. (2001) showed that 6-pentyl-alpha-pyrone (6pAp), which is a metabolite of T. harzianum (tHF2/3), can reduce the production of deoxynivalenol by F. graminearum on agar medium by 66 to 81%. the authors developed an agar bioassay technique to examine FusariumTrichoderma interaction on their secondary metabolites level.
T. harzianum type 4 and named T. aggressivum has been found recently to be com-has been found recently to be competitor of commercial mushroom Agaricus bisporus, known to be the cause of green mold and significant losses to mahrooms producers (savoie, Mata 2003).
the aim of this paper was to examine the effect of 29 fungal species isolates (including Trichoderma) originated from soil, compost and cereals, on the growth of three toxigenic Fusarium species in dual cultures and a mutual interaction between the antagonists and Fusarium isolates in bioassays, and to examine the reduction of moniliformin production in dual cultures of F. avenaceum with selected effective Trichoderma antagonists.

MAtERiAL AND MEtHoDs
Fungal isolates.isolates of tested fungi originated from culture collections of the institute of plant Genetics, polish Academy of sciences, poznań, poland (KF and AN), the institute of science of Food production, Bari, italy (itEM) and the Department of Forest pathology, Agricultural University, poznań, poland (tab.1).Fungal isolates originating from soil, compost and cereal grains were maintained on a synthetic low nutrient agar sNA (Nirenberg 1981;Kwaśna et al.1991).identification and nomenclature of Trichoderma and Gliocladium isolates was followed according to Gams and Bissett 1998.
Dual culture bioassays.A modified bioassay of Mańka (1974) was applied to examine growth reduction of three toxigenic species isolates: F. avenaceum, F. cul morum and F. graminearum.A Fusarium isolate and a tested fungus (an antagonist) were inoculated onto 9 cm petri dishes at a distance of 1cm on potato dextrose agar (pDA).As a control each fungus was cultured separately.Agar plates were then incubated at 25˚C at diffused daylight.inhibition of Fusarium growth by the antagonist was evaluated using a modified Mańka (1974) scale -8 to +8 presented in figure 1, where 0 indicated no inhibition and +8 a total inhibition, with the Fusarium mycelium in >95% overgrown by the antagonist.Growth rate of each control culture was measured after 2-7 days.At least two replications were performed.
to examine the reduction of Fusarium toxin production in solid substrates 50 g of commercial rice were soaked with 15 ml of distilled water in a 300 ml Erlenmayer flask overnight before sterilization run for 30 min.at 121˚C.then it was inoculated with Fusarium and the tested fungus (antagonist).inoculation was made with four 4mm diameter discs of each fungus cut from the edge of 1-week-old culture on pDA and 2 ml of sterile water.Cultures were incubated at 25˚C and shaken each day to support a uniform growth of mycelium.in the control each fungus was cultured isolates), 15AcDoN chemotype (most of F. graminearum isolates) and NiV chemotype (several isolates of both species).
Competitive abilities of candidate fungi against toxigenic Fusarium isolates were examined in dual culture bioassays on agar and solid substrate bioassay.Fusa rium avenaceum, F. culmorum and F. graminearum produce aurofusarin and other carmine-red pigments on pDA medium (Vesonder, Goliński 1989).Trichoderma isolates produce a green pigment of polyphenolic nature.Colony pigmentation is a useful marker when studying the interaction between Fusarium isolates and their antagonists.Fusarium isolates growth in dual cultures with competitive isolates were visually reduced after 4 days.A week after inoculation growth inhibition and mycoparasitism of Trichoderma isolates were observed -the plate was green and overgrown with Trichoderma with significant production of abundant conidia in pustules over mycelium of Fusarium isolates, with only a small spot of the plate remaining red.Fusarium growth and red pigment production were strongly inhibited by isolates of T. atroviride, T. harzianum, T. hamatum, T. longibrachiatum and T. koningii.
Isolates of F. graminearum (KF 844 and KF 2870) were grown simultaneously with 92 antagonistic isolates and exhibited different susceptibility to the presence of the antagonists: isolate KF 2870 was significantly less resistant and isolate KF 844 exhibited a higher level of resistance against the same antagonistic mycoparasite (Fig. 2A, B). the antibiosis effect was not found in any of the candidate antagonists in dual culture on pDA.
the interaction between eight isolates of Trichoderma and six isolates of Fusa rium is presented in tables 2 and 3. thus, on one hand the same Trichoderma isolate exhibited different antagonism against various Fusarium isolates, and on the other hand antagonistic isolates exhibited various aggressiveness against the same isolate of Fusarium.
Trichoderma isolates are able to produce antibiotics -inhibitors of other fungi (such as 6pAp), and several enzymes, which hydrolyse fungal structures -conidia and mycelia -and macromolecules -chitin, cellulose, hemicellulose, beta glucan, xylem and proteins (łacicowa, pięta 1985;Kubicek, Harman 1998;Cooney et al. 2001).this ability allows Trichoderma isolates to utilise the mycelium of Fusarium ta b l e 2 Growth inhibition of four isolates of F. graminearum and F. culmorum, belonging to three chemotypes (3AcDoN, 15AcDoN and NiV) by eight isolates of Trichoderma in dual cultures on pDA medium after 7 days at 25˚C (in -8 to +8 scale) as a source of nutrients.our biotest experiments confirmed the mycoparasitism of Trichoderma species over all three species and chemotypes of toxigenic Fusarium.three types of interaction between Trichoderma species and plant pathogens have been recognized: antibiosis, competition for nutrients and hyperparasitism (woo et al. 2006).No antibiosis was observed in our experiments.Results of our previous paper (Buśko et al.2008) correspond well with the finding of Cooney et al. (2001) on the impact of T. harzianum isolates on a DoN-producing F. graminearum in the agar medium bioassay.the same authors proved the inhibition of DoN production by a Trichoderma metabolite 6pAp, by as much as 80%. the mechanism of DoN content reduction in F. graminearum cultures remains unsolved.it was shown that DoN when added to an agar medium inoculated with Trichoderma was not metabolised by the fungus (Cooney et al. 2001).
A question arises whether isolates of Trichoderma, growing on the mycelium of toxigenic Fusarium species, are also able to transform or degrade such mycotoxins as DoN, NiV, MoN, ZEA, and others -in total 19 mycotoxins identified in grain samples (Chełkowski 1998;Bottalico 2002).Until now, the ability to decompose DoN has been found very rarely among microorganisms.only one mixed culture among 1285 microbial cultures, isolated from farmland soil, cereal grains and others sources, transformed DoN into two products that can be separated chromatographically (mainly 3-keto-4-deoxynivalenol; Voelkl et al. 2004).
in our previuos laboratory experiments F. culmorum and F. graminearum cultures grown on rice produced five trichothecenes: fusarenone X (up to 21 mg/kg), nivalenol (up to 3.7 mg/kg), deoxynivalenol (up to 310 mg/kg), 3Ac-DoN (up to 228 mg/ kg) and 15Ac-DoN (up to 184 mg/kg).production of five trichothecene mycotoxins DoN, 3AcDoN, 15AcDoN, nivalenol and fusarenone X was reduced by over 95% in dual culture bioassay by Trichoderma isolates AN 22 and AN 35 (Buśko et al. 2008).DoN concentration in the bioassay in microcosms was reduced by 45% and fungal biomass was reduced by 15% in studies of Noef et al. 2006.Consequently, mean DoN production per biomass was significantly lower -36% in dual culture with T. atroviride than in solitary culture of F. graminearum and the above mentioned authors did not find any evidence for the role of DoN production in F. graminearum defence against T. atroviride.ta b l e 3 Growth inhibition of two isolates of Fusarium avenaceum in dual culture by eight isolates Trichoderma (in -8 to +8 scale) on pDA medium after 7 days at 25˚C A significant reduction of wheat head infection by F. graminearum and DoN accumulation in kernels was found by Dawson et al. (2004) when ears were preinoculated by fungal antagonists, such as T. harzianum, Clonostachys rosea and F. equiseti.
Recently three major compounds exhibiting antifungal activity were identified to be produced by t22 and t39 isolates of T. harzianum, that are already used as active agents in a variety of commercial biopesticides (Vinale et al. 2006).over 50 commercial biopesticides were found effective in biological control of soil and residue borne pathogens (woo et al. 2005).
Fusarium avenaceum isolates produced moniliformin up to 100 mg/kg (tab.4). the isolate T. atroviride AN35 was found the most effective among the examined accessions against toxigenic Fusarium isolates used in this study.this antagonist was able to reduce moniliformin production in dual culture bioassay on rice by 95-100%, depending on the applied F. avenaceum isolate.the same isolate reduced by 93% the amount of MoN in a rice culture of F. avenaceum itEM 3411 (KF 2603) -from 100 μg/g to 6.5 μg/g (tab.4).
the application of Trichoderma competitors may reduce growth of Fusarium species through competition in crop debris and mycotoxin production as well.However, the development of the formulation of biofungicides for practical control of diseases remains a very important task (Kubicek, Harman 1998;woo et al. 2006).
Both in the literature and in our experiments presented in this paper significant interaction between toxigenic Fusarium species and Trichoderma competitotors was found to be of complex character, with importance of several characters such as growth rate, enzymes and secondary metabolites production (Harman 2006).
it can be concluded that competitive Trichoderma isolates are candidate fungi for biological control of toxigenic Fusarium species aggressive to cereals (such as F. culmorum, F. graminearum and F. avenaceum) and in reducing their inoculum, as well as preventing mycotoxin accumulation in plant tissues and crop residues in field.
ta b l e 1 Growth rate of 92 candidate antagonistic isolates from soil, compost and cereals and Fusarium isolates on pDA medium in mm/day at 25˚C ranged from 9 to 20 mm/day (tab.1).three chemotypes were identified using specific primers for pCR amplification of DNA among F. culmorum and F. graminear um isolates used in our experiments: 3-AcDoN chemotypes (mostly F. culmorum two F. graminearum isolates KF 844 and KF 2870 in bioassay on pDA medium after 7 days at 25˚C (in -8 to +8 scale of inhibition).
. there is high similarity ta b l e 4 Reduction of moniliformin produced by two isolates F. avenaceum in dual culture bioassay and decomposition of MoN by Trichoderma harzianum AN 35 isolate on rice after 21 days at 25˚Cof Trichoderma isolates morphology under laboratory conditions and there are many confusions in the literature, concerning identification of species.Actual list of over 40 species and their descriptions are available on website: http://nt.ars-grin.gov/taxadescriptions/keys/trichodermaindex.cfm.