Agrochemicals: Effect on genetic resistance in yeasts colonizing winter wheat kernels
Graphical abstract
Introduction
Winter wheat is the major cereal crop grown for consumption. Its yield is determined by various factors, including microbial colonies that colonize wheat plants (Salvador et al., 2006). Wheat is susceptible to infections caused by numerous pathogens, mostly Zymoseptoria tritici, Fusarium spp., Blumeria graminis and Oculimacula spp. These pathogens can be controlled with benzimidazole, strobilurin, triazole and morpholine fungicides with various mechanisms of action targeting fungi. The most popular triazole and morpholine fungicides belong to the group of sterol biosynthesis inhibitors (SBIs). Benzimidazoles target a specific site in the β-tubulin subunit of fungal cells (Hahn, 2014). Strobilurins inhibit mitochondrial respiration in fungal cells by binding with the coenzyme ubiquinol in cytochrome b and c1 (enzyme complex III) (Bartlett et al., 2002, Grasso et al., 2006a). Apart from their yield-forming benefits, agrochemicals also exert adverse environmental impacts (Bartlewicz et al., 2016). Broad-spectrum fungicides can affect non-target organisms including humans (Guyton et al., 2015, Bartlewicz et al., 2016). The limited effectiveness of fungicides resulting from the fungicides arising from the emergence of resistant pathogen strains is another important consideration. Strobilurins were applied for the first time in 1996, and within a few years resistant forms of Zymoseptoria tritici, Blumeria graminis and other plant pathogens were reported across Europe (Grasso et al., 2006b, Ishii and Holloman, 2015). The majority of strobilurin-resistant pathogens had point mutations in the cytochrome b (CYTb) gene (Heick et al., 2017).
The mechanism of action responsible for the effect of these crop protection compounds on microorganisms colonizing wheat grain has to be understood to create the best growing conditions for plants and to produce crops of the highest quality. Yeasts, which are investigated less frequently than other saprotrophic fungi such as Cladosporium spp. and Epicoccum spp., play a particularly important role in grain production (Bertelsen et al., 2001). Epiphytic and endophytic yeasts are the predominant fungi colonizing the above-ground parts of cereals (Karlsson et al., 2014). Yeasts are found in both Ascomycota and Basidiomycota, with three subdivisions of Ascomycota containing the bulk of species: (1) Schizosaccharomyces in Taphrinomycotina, (2) Saccharomycotina and (3) Pezizomycotina filamentous fungi (Kurtzman and Robnett, 2013, Fitzpatrick et al., 2006, Kurtzman, 2014). Many yeast species belong to the division Basidiomycota, which additionally complicates their classification (Kurtzman, 2014). Some yeasts are found only in the asexual phase, including the genera Rhodotorula and Cryptococcus (Yurkov et al., 2015). Confounding their discrete classification is the fact that phylogenetic analyses have frequently revealed the polyphyletic nature of yeast genera (Kurtzman et al., 2011). The first studies documenting the ability of yeasts to inhibit the growth of phytopathogens in monocotyledons (Fokkema et al., 1979) were continued and developed by several research teams (Dik et al., 1991, Khan et al., 2004, Schisler et al., 2002, Karlsson et al., 2014), which led to the selection of isolates useful for the protection of cereals (De Curtis et al., 2012). In yeasts that are used for biological protection against plant pathogens, the key mechanisms of action are competition and antibiosis (Wachowska and Borowska, 2014), but they can also influence physiological processes in plants (El-Tarabily and Sivasithamparam, 2006). Yeast abundance increases naturally in stored grain (Druvefors et al., 2002), and it modifies flour strength (Salvador et al., 2006). The application of yeasts during the growing season induces changes also in the content of lipophilic bioactive compounds (including sterols and carotenoids) in wheat grain (Wachowska et al., 2016).
Fungicides eliminate fungal pathogens, but they can also limit the proliferation of saprotrophic fungi, including yeasts. For this reason, the influence of fungicides on non-target fungi has to be well understood (Newton et al., 2010). In some cases the beneficial fungi are less susceptible to fungicides than pathogens (Dawidziuk et al., 2016). A study into species of the genus Fusarium, the causative agents of Fusarium head blight in cereals, demonstrated that the use of fungicides to protect crops against specific pathogens can exacerbate infections caused by other pathogens (Birzele et al., 2002). Such complications can be attributed to the inhibitory effect of fungicides on saprotrophic fungi which compete with pathogens for resources (Henriksen and Elen, 2005). Phyllospheric saprotrophs also accelerate leaf aging, therefore, fungicides can contribute to productivity despite the absence of pathogenic infections (Bertelsen et al., 2001). The effect of fungicides on yeasts colonizing grain has to be thoroughly analyzed to optimize fungicide application. A better understanding of the interactions between fungicides and yeasts is required to facilitate the combination biological control using yeast suspensions with fungicide treatments. The objective of this study was to evaluate the sensitivity of yeasts to three different classes of antifungals that are commonly used in agriculture. These treatments were applied both during the growing season of winter wheat and under in vitro conditions. Isolates of the following yeast species: Aureobasidium pullulans (De Bary) G. Arnaud, Candida albicans (Robin) Berkhout, Candida sake (Saito & Oda) van Uden & H.R. Buckley, Debaryomyces hansenii (Zopf) Lodder & Kreger-van Rij (anamorph: Candida famata (Harrison) S.A Meyer & Yarrow var. famata), Metchnikowia pulcherrima Pitt & M.W. Miller (anamorph: Candida pulcherrima (Lindner) Windisch) and Rhodotorula glutinis (Fresenius) F.C. Harrison, obtained from wheat grain, and the reference isolate of Saccharomyces cerevisiae Meyen ex E.C. Hansen were analyzed in vitro to evaluate their sensitivity to agrochemicals. The isolates obtained from wheat grain were identified based on ITS 1, 5.8S and ITS 2 rDNA sequences along with the morphology of colonies, cells and pseudofilaments. The sequences of selected isolates were deposited in GenBank, and were used to estimate the relatedness between the analyzed species. Selected azoxystrobin-resistant isolates were analyzed to detect point mutations in the CYTb gene (G143A) responsible for resistance to QoIs. The A143 mutation has been found in the strobilurin-producing basidiomycete Mycena galopoda and in plant pathogens, whereas its presence in yeasts has not been previously documented.
Section snippets
Field experiment
The effects of agrochemicals on yeast communities colonizing winter wheat kernels were evaluated under field conditions. A field plot experiment with a randomized block design with four replications was performed in 2010–2012 in Tomaszkowo, Poland (53.71 N, 20.41 E). Winter wheat (Triticum aestivum L., cv. Bogatka) was sown in plots of 25 m2 at 14 g of grain per m2. Seven different plant protection regimes were devised for the plots: four intensive protection plans where plants were treated a
The abundance of yeasts isolated from winter wheat grain after chemical treatment
The abundance of epiphytic yeasts on wheat kernels (3.31 log (CFU+1) on average) was 47-fold higher than the abundance of endophytic yeasts (1.64 log (CFU+1) on average) (Table 4). During six months of storage, yeast abundance increased by 5.6-fold in epiphytes and 12.6-fold in endophytes. Protective treatments did not exert a significant effect on the average abundance of yeasts colonizing the surface of wheat kernels, and the size of endophytic communities was reduced significantly by 85%
Discussion
Yeast communities isolated from winter wheat grain during the three-year field experiment were very abundant, which confirms earlier observations that this group of microorganisms plays a dominant role in this particular ecological niche (Laca et al., 2006, Druvefors et al., 2002). The yeast species identified in the present study were typical of this environment. The predominance of A. pullulans in yeast communities colonizing wheat grain, observed in our experiment, had been previously
Conclusions
Yeasts abundantly colonize winter wheat grain, in particular the surface of kernels. Yeast abundance increases during grain storage. The size of yeast communities on wheat grain to some extent depends on the quality and quantity of the fungicides used in agricultural practices. Agrochemicals exert varied inhibitory effects on yeast isolates. Tebuconazole is four-fold more toxic than azoxystrobin to the majority of tested species, and propiconazole is highly toxic to all C. albicans isolates.
Acknowledgements
The work was partly supported by National Science Centre (Number of project N310 116638). The authors thank to Dr. William Truman, Institute of Plant Genetics, Polish Academy of Sciences for a thorough revision of the manuscript.
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