Efficiency of various fungicide treatments on the occurrence of Fusarium spp. associated with spring barley (Hordeum vulgare L.

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1 Efficiency of various fungicide treatments on the occurrence of Fusarium spp. associated with spring barley (Hordeum vulgare L.) grains MARTIN KMOCH 1, IVANA SAFRANKOVA 1, ALEXANDRA MALACHOVA 1,2, PAVLINA SMUTNA 1, LUCIE JANECKOVA 1, JAROSLAVA EHRENBERGEROVA 1, KATERINA VACULOVA 3, RADIM CERKAL 1 1 Department of Crop Science, Breeding and Plant Medicine Mendel University in Brno Zemedelska 1, Brno CZECH REPUBLIC 2 Department IFA-Tulln University of Natural Resources and Life Sciences, Vienna Konrad Lorenz str. 20, 3430 Tulln AUSTRIA 3 Agrotest Fyto, Ltd. Havlickova 2787/1, Kromeriz CZECH REPUBLIC martin.kmoch@mendelu.cz, cerkal@mendelu.cz Abstract: - A spectrum of fungi was identified in ten spring barley varieties treated by various fungicides. The presence of the Fusarium spp. was determined and the effect of a fungicide treatment and a variety on the grain infection was evaluated. A microbiological method was used for fungi identification. In selected isolates, the identification of Fusarium spp. was verified by the PCR method. A total of 4182 microorganisms from 12 genera was isolated; a large proportion was represented by Alternaria and Cladosporium genus; the occurrence of Aspergillus, Penicillium, Rhizopus, and Mucor was rare. The ratio of the Fusarium spp. was approx. 30%. The Fusarium genus was represented by the following species: F. poae (41.9%), F. graminearum (25.1%), F. avenaceum (14.7%), F. tricinctum (10.0%), F. sporotrichioides (3.8%), F. verticillioides (1.9%), F. culmorum (1.6%), and F. oxysporum (1.0%). Varieties Gladys and Sebastian were most infected by fungi of the Fusarium spp.; Bojos and Radegast were the least infected varieties. The occurrence of pathogens of the Fusarium spp. was reduced by % by the combination of fungicides Hutton (active substances prothioconazole, spiroxamine, tebuconazole; application in the BBCH 39 growth stage) + Zantara (bixafen, tebuconazole; BBCH 65). Treatment by Hutton, Zantara, and Prosaro 250 EC had an influence on the percentage of F. graminearum in the microflora and F. verticillioides was completely eliminated from the pathogen spectrum. Key-Words: - PCR, FHB, scab, ear blight, fungicide treatment, active ingredients, variety 1 Introduction Fusarium fungi are the most common pathogens of cereals worldwide. Although 70 species have been already described in the latest laboratory manual [1], only a relatively small number is important with regard to food spoilage in its broad concept [2]. In Europe, Fusarium graminearum, F. culmorum, F. poae, and F. avenaceum are the most common fungal species that cause a disease called Fusarium head blight (FHB) which can result in considerable economic losses due to lower yields and decline of grain quality [3]. Xu et al. [4] describe the differences regarding occurrence of FHB pathogens among individual countries e.g. in Hungary and Italy, there is a regular occurrence of F. graminearum and F. poae, and a rare occurrence of F. culmorum. In the Czech Republic, the thermophilic F. graminearum is becoming predominant. Natural infections by fungi of the Fusarium spp. are mostly weak; however, they may cause considerable yield losses once conditions suitable for epidemic occurrence set in. Secondary, toxic metabolites of Fusarium fungi mycotoxins ISBN:

2 may pose a serious health risk not only for humans but also for farm animals. Apart from Fusarium genus, other fungi and microorganisms can also be found on the grains [5, 6]. Common companions of Fusarium spp. are fungi of the Alternaria and Cladosporium genus, in some cases also the originators of leaf spotting. Most Fusarium spp. infecting barley can produce one or more mycotoxins, but the presence/absence of mycotoxins in the grains do not necessarily correlate with the infection level [7, 8]. Fungi of the Fusarium genus produce mainly trichothecenes (especially deoxynivalenol and nivalenol) and their derivates, T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and neosolaniol [9, 10]. F. graminearum and F. culmorum are considered to be main producers of deoxynivalenol [10]. These two species are also known as the most important producers of zearalenone and its derivates. F. avenaceum is responsible for trichothecene production as well as for formation of enniatins and moniliformin [11]. Climate changes contribute to spread of so far not common Fusarium spp. Consequently, the spectrum and levels of mycotoxins in various commodities are changing annually [12]. Probably due to climate conditions changes, chemotypes F. culmorum responsible for NIV production [13] or F. sporotrichioides and F. poae synthetised HT-2 and T-2 toxins [14]. The occurrence of pathogens may be controlled by application of fungicides. Their effectiveness depends on the active ingredients, the manner and term of application, the meteorological conditions, the virulence of the pathogenic strain and the fungi species involved. Generally, fungicides with triazole chemistry (tebuconazole, metconazole, and bromuconazole) seem to be the most effective. Depending on a cultivar s resistance, the aggressiveness of toxinogenic fungi, and other factors mentioned above, efficacy exceeding 70% might be achieved [15, 16]. Unfortunately, individual species of the Fusarium genus show a specific sensitivity to active ingredients. For example, triazoles selectively control F. culmorum and F. avenaceum and reduce levels of DON, but show little control of M. nivale; strobilurins selectively control F. avenaceum [17]. In this work, we have therefore focused on: i) the identification of the fungal spectrum in grains of spring barley treated by various fungicides, in different growth stages and inoculated by F. culmorum; ii) screening for occurrence of particular species of the Fusarium genus on grains; iii) establishment of the effect of the fungicide treatment and the variety on the grain infection by the Fusarium spp. 2 Material and Methods 2.1 Characterization of growing locality, experimental design and barley grain samples The total of 160 barley samples (2011 harvest) were analyzed (10 variety x 4 variants of fungicide treatment x 2 inoculation variants x 2 replications). A set of 10 different spring barley varieties recommended for beer production was studied (Aksamit, Bojos, Gladys, Kangoo, Malz, Prestige, Sebastian, Radegast, Tocada, and Xanadu). The barley varieties were planted on approximately 3600 m 2 plots located in Žabčice (16 37 E, N). The experimental station is situated in the south of Moravia (Czech Republic) in a maize production region at the average level above sea of 178 m. The region s dominating soil type is middle heavy to heavy gleyic fluvisols FLq and FLg. The locality is considered to be one of the warmest districts in the Czech Republic [18]. To evaluate the impact of fungicide treatment on Fusarium spp. occurrence, various fungicides and inoculation by F. culmorum were applied. An overview of the fungicides and their active ingredients used are displayed in Table 1; the inoculation by F. culmorum took place at the beginning of flowering (BBCH 61 64). The concentration of the inoculum was adjusted to the value of conidia/ml suspense (approx. 10 million of conidia/m 2 ). 2.2 Identification of pathogens A microbiological method was used for the identification of fungi of particular genera and for the species identification. The identification was verified for selected isolates (F. avenaceum, F. graminearum, F. poae, and F. culmorum) by polymerase chain reaction (PCR). Cultivation of grains (3x10 grains) disinfected on their surface by sodium hypochlorite (5%) was carried out at the temperature of C and 12/12 hour light regimen on the potato-dextrose agar; pathogens identification took place on 4 th to 7 th day of the cultivation. A roll-tube method was employed for the evaluation of the Fusarium fungi infection of grains. 100 grains disinfected on the surface by sodium hypochlorite (5%) were fixed by starch glue between sheets of filtration paper. First, the paper was rolled up and then placed vertically into a container with water solution (50 ppm iprodione) in such a way so that gradual rise of the solution to the germinating grains occurred. After 6 days of ISBN:

3 incubation (temperature of C, 12/12 hour light regimen), the number of Fusarium fungi colonies growing out of individual grains was evaluated. Mycelium samples were taken from the infected grains in order to grow a pure culture and to identify species of the Fusarium genus according to the morphological characteristics of macroconidia and microconidia [1]. An optical microscope (Olympus BX41, magnification ) and stereomicroscope (Olympus SZX12) were used for identification. PCR method was used for the confirmation of Fusarium spp. indentified by the roll-tube method. For detection of the Fusarium spp., DNA from 100 mg of fungal mycelium was isolated by DNeasy Plant Minikit (Qiagene, Germany). DNA quality and quantity were established by the spectrophotometer (NanoDrop 2000c). DNA purity was measured as the ratio of the absorbance at 260 and 280 nm, while the ratio of 260/280 fluctuated around 8 (pure DNA has A260/A280 approx 1.8). Table 1 Fungicides used in spring barley fields Treatment Fungicide Application rate BBCH Active ingredient(s) Control - H2+Z3 Hutton Zantara 0.8 l ha l ha prothioconazole, spiroxamine, tebuconazole bixafen, tebuconazole H2+P3 Hutton Prosaro 250 EC 0.8 l ha l ha prothioconazole, spiroxamine, tebuconazole prothioconazole, tebuconazole H1+P3 Hutton Prosaro 250 EC 0.8 l ha l ha prothioconazole, spiroxamine, tebuconazole prothioconazole, tebuconazole The following primers were used for DNA amplification: F. avenaceum JIAf/r [19], F. graminearum Fg16F/R [20], F. poae Fp82F/R [21], F. culmorum OPT18F470/OPT18R470 [22]. Taq PCR Core Kit (Qiagene, Germany) was used for PCR-reactions. The reactions took place in the volume of 25 µl. Reaction conditions (thermocycler ApolloTM ATC401) and the concentrations of reagencies (puffers, nucleotides, primers, Taqpolymerase) were adjusted according to the above cited publications. A positive (standard) and negative (without a template) controls were included into reactions. DNA fragments separation was carried out on the horizontal electrophoresis (power source Consort EV265, tub Cleaver MSMAXI0) on 1 1.5% tris-acetate-edta-agarose gel. Visualization of the amplified DNA after coloring by 1% ethidium bromide was conducted on UVtransilluminator (Foto/UV 21). 3 Results and Discussion 3.1 Pathogen spectrum on barley grains In the total of 160 barley grain samples, 4182 microorganisms from 12 genera (Alternaria, Aspergillus, Bipolaris, Cladosporium, Drechslera, Epicocum, Fusarium, Mucor, Nigrospora, Penicillium, Rhizopus, Stemphylium) were isolated; yeast cells were seldom found. The saprophytic species of the Alternaria and Cladosporium genus were the most frequently represented species. On the other hand, the occurrence of Aspergillus and Penicillium was rare as well as the Rhizopus and Mucor genera. The proportion of the Fusarium spp. was 30.4% and a higher number of fungi (16%) was isolated from naturally infected stands. 3.2 Infection of barley grains by fungi of Fusarium spp. the method of roll-tubes and PCR Natural infection of spring barley grains by Fusarium fungi was very weak in However, a distinct varietal sensitivity to infection was observed. Within the experimental sample set, the most infected varieties were Gladys >Sebastian >Aksamit >Kangoo >Malz, the less infected ones were Bojos< Radegast< Xanadu< Tocada< Prestige (data not shown). Inoculation of barley stands by F. culmorum in the BBCH growth stage caused only a very small change of the pathogen spectrum the presence of F. culmorum on grains increased by approx. 1% max. (data not shown). The results show that application of the Hutton (BBCH 39) and Zantara fungicides (BBCH 65) had the highest suppression effect. Bixafen is one of the active substances contained in Zantara. This fungicide combination managed to reduce the occurrence of Fusarium spp. by 41.8% (non-inoculated variants) and 31.4% (variants that were inoculated by F. culmorum). A distinct fungicide effect did not occur after application of fungicides Hutton and Prosaro ISBN:

4 250 EC that contained active ingredients prothioconazole, tebuconazole, and spiroxamine; the occurrence of Fusarium spp. was lower only by 4 18% in comparison to the non-treated control variant (see Fig. 1). Chemical control of cereals against Fusarium spp. has been the subject of a large number of studies. Most of them concluded that the fungicide effectiveness dependence on the timing and frequency of application as well as the active ingredient [23, 24]. According to Chala et al. [25] and Mesterhazy et al. [15], a treatment at the flowering stage is most effective in terms of decreasing the infection by Fusarium spp. High sensitivity in artificially inoculated wheat stands was proved in case of tebuconazole [23, 15]; the effect of propiconazole did not differ from the one of tebuconazole. The suppression of pathogens of the Fusarium spp. by triazols is very dependent on the application dose [26]. Fig. 1 Spring barley grain infection by Fusarium spp. (in %), Žabčice, CR, 2011 Legend: H fungicide Hutton, Z Zantara, P Prosaro 250 EC; 1 growth stage BBCH 25 30, 2 BBCH 39, 3 BBCH 65. When identifying Fusarium spp. pathogens from the non-treated variants (non-inoculated), the presence of the following species was established: F. poae (41.9%), F. graminearum (25.1%), F. avenaceum (14.7%), F. tricinctum (10.0%), F. sporotrichioides (3.8%), F. verticillioides (1.9%), F. culmorum (1.6%), and F. oxysporum (1.0%; Fig. 2A). At the beginning of this millennium, Hysek et al. [27] published that F. culmorum and F. poae were the most frequently isolated species found in barley in the Czech Republic. Currently, the thermophilic F. graminearum species is pre-dominant. Together with F. avenaceum, it is the most frequent species in the neighboring Germany as well as Netherlands [28]. Simpson et al. [17] propose that F. graminearum is sensitive to active ingredients from the group of triazoles that were used in our experiments. A positive influence of a fungicide treatment containing active ingredients prothioconazole, spiroxamine, tebuconazole (Hutton), bixafen, tebuconazole (Zantara) and prothioconazole, tebuconazole (Prosaro 250 EC) resulted in a lower occurrence of grains infected by F. graminearum and the absence of F. verticillioides (see Fig. 2A D). A distinct decrease of wheat infection by F. graminearum and F. culmorum as a result of treatment by fungicides containing the active ingredient tebuconazole was confirmed also by Ioos et al. [24]. However, the effectiveness of the treatment differed among years and each fungus of the Fusarium spp. responded to the fungicide treatment in different way. Based on in vitro experiments, Jones [29] published sensitivity of F. graminearum to benomyl, tebuconazole, and mancozeb. 4 Conclusion Agricultural practices use several effective approaches to reduce the Fusarium infection as well as the following mycotoxin production. A selection of the suitable variety for respective locality and environmental conditions and the use of a fungicide treatment can be one of the used approaches. However, a reliable fungicide treatment completely eliminating the Fusarium spp. infection has not been developed so far. Therefore, the combination of more fungicide preparations together with the optimal timing of application seems to be the most effective way. The experiment results proved that the suitable combination of active ingredients of some fungicides (Hutton + Zantara) together with the optimal term of application (BBCH 39 and 65) can significantly eliminate the occurrence of Fusarium spp. pathogens (by up to 40%), even in case of inoculation by F. culmorum. Combination of active ingredients can also result in changes of the spectrum and representation of pathogens as was observed in all variants treated by fungicides in cases of F. graminearum or F. verticillioides. Varieties Gladys and Sebastian were the most susceptible to the infection by fungi of the Fusarium spp. out of all the ten tested spring barley varieties. On the other hand, Bojos and Radegast were the least infected varieties. Unfortunately, the commercial situation regarding the commodity of barley in the Czech Republic does not make it feasible to respect biological advantages of some varieties, hence the issue of fungicide protection of barley will need more attention in the future. ISBN:

5 Fig. 2 Representation of the Fusarium spp. (in %) in spring barley grains (non-inoculated variants), Žabčice, CR, 2011 A) Untreated control B) Hutton (BBCH 39) + Zantara (BBCH 65) C) Hutton (39) + Prosaro 250 EC (BBCH 65) D) Hutton (25 30) + Prosaro 250 EC (BBCH 65) Acknowledgement The research was financially supported by the project No. QI111B044 Comprehensive strategy for decreasing a negative impact of Fusarium spp. toxicogenic fungi infection in cereals and their derivated products. Preparation and implementation of this article is supported also by resources of the project CZ.1.07/2.3.00/ The excellence of Ph.D. studies at FA MENDELU for the following scientific European career. References: [1] Leslie JF, Summerell BA, Bullock S, The Fusarium Laboratory Manual, Blackwell Publishing, [2] Pitt JI, Hocking AS, Fungi and Food Spoilage (3 rd edition), Springer Science+business Media, [3] Bottalico A, Perrone G, Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe, European Journal of Plant Pathology, Vol.108, No.7, 2002, pp [4] Xu XM, et al., Predominance and association of pathogenic fungi causing Fusarium ear blight in wheat in four European countries, European Journal of Plant Pathology, Vol.112, No.2, 2005, pp [5] Laitila A, et al., Indigenous microbial community of barley greatly influences grain germination and malt quality, Journal of the Institute of Brewing, Vol.113, No.1, 2007, pp [6] Olstorpe M, Schnurer J, Passoth V, Microbial changes during storage of moistcrimped cereal barley grain under Swedish farm conditions, Animal Feed Science and Technology, Vol.156, No.1 2, 2010, pp [7] Edwards SG, Influence of agricultural practices on fusarium infection of cereals and subsequent contamination of grain by trichothecene mycotoxins, Toxicology Letters, Vol.153, No.1, 2004, pp [8] Malachova A, et al., Fusarium mycotoxins in various barley cultivars and their transfer into malt, Journal of the Science of Food and Agriculture, Vol.90, No.14, 2010, pp [9] Nicholson P, et al., Molecular tools to study epidemiology and toxicology of fusarium head blight of cereals, European Journal of Plant Pathology, Vol.109, No.7, 2003, pp ISBN:

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