Control of malting barley Fusarium head blight by bioagents

The routine and prophylactic use of fungicides in cereals leads to increased aggressiveness of Fusarium infections. Cross-resistance to triazole compounds represents a significant health risk to both plants and humans. The application of some widely used fungicides causes increased production of DON. Residual concentrations of hydrophobic triazoles change the chemical profile of malt and cause delayed fermentation with an impact on alcohol content. Increasing legislative restrictions of pesticide applications encourage the search for alternatives, starting with the overview of current state of knowledge on biological protection against Fusarium spp. Despite the fact that bioagents have been researched intensively, including field applications and several registrations, biological preparations for disease control against Fusarium head blight (FHB) of malting barley are not used on a mass scale. Generally, bioagents appear to be quite sensitive to environmental changes and soil variability, which causes problems with the evaluation of their effectiveness under field conditions. For efficient disease control of malting barley, the application based on biopreparations registered against FHB combined with weather prediction system can be recommended. With an emphasis on the occurrence of Fusarium graminearum as a key producer of deoxynivalenol (DON), the prediction system for malting barley should be employed from plant emerging to milk stage. When predicting a high incidence of the pathogen, chemical intervention must be considered. However, repeated application of bioagents in field conditions together with the implementation of bioagents directly into the malting process proved to be a promising way to decrease chemical interventions from the cultivation of malting barley.


Introduction
Despite recent covid issues, the development of mini-and/ or microscale breweries and increasing demand for nonalcoholic beer caused record breaking prices in global markets. While until the 1970s, much more patents and scientific documents were dedicated to the wheat, from the 1970s to the beginning of the 21 st century the ratio of patents and scientific document was similar in both crops, showing an increasing industrial interest in barley, and during the last decade the focus to barley has at least doubled compared to wheat (Giraldo et al., 2019).
The innovations in malting procedures brought new products that caused a shift from homogeneous beer production to increased consumer demand for a larger variety of beer, with craft and micro-brewing becoming increasingly popular (Mellor et al., 2020). Emerging health-oriented lifestyle trends, demographics, stricter legislation, religious prohibitions, and consumer preferences have led to a strong and steady growth of interest in non-alcoholic beers (Salanta et al., 2020). Plus, the potential to exploit the health benefits of whole grain and β-glucans is much higher here (Ehrenbergerova et al., 2008).
Whereas recent climate change in Europe threatens the increasing malting industry which is highly sensitive and vulnerable to malt barley supply (Bindereif et al., 2021), strict requirements to maintain quality remain unchanged (Rani and Bhardwaj, 2021).
Versatile microbiota is inevitably naturally present on cereals, influencing the malting quality parameters (Mastanjevic et al., 2018a). Fusarium spp. contamination of cereals increased in recent years, mainly in barley, wheat, maize, and oats (Piacentini et al., 2019). FHB is an important disease of barley (Hordeum vulgare L.) caused by a complex of toxigenic Fusarium spp. and non-toxigenic Microdochium spp. known to impact significantly upon the yield and several functional parameters of grain related to safety and brewing quality (Nielsen et al., 2014). Published data indicate a high variability according to the type of mycotoxins, the level and extent of fungal contamination and contaminated malt processing technologies (Pinotti et al., 2016).
Germination is the malting step that leads to a significant increase of DON and zearalenone (ZEN) levels (Piacentini et al., 2019). The first step of mashing (45 °C ) has the most significant impact on the transfer of hydrophilic toxins from the grist into the wort (Pascari et al., 2022). Besides toxic metabolites of Fusarium spp. classified as trichothecenes, ZEN, and fumonisins (Ji et al., 2019), aurofusarin and rubrofusarin pigments were identified as being contained in F. graminearum (Mastanjevic et al., 2018b) and found to add to the colour intensity of wort (Cambaza, 2018). As for sensory and physico-chemical stability of beer, another fungal products, hydrophobins, were identified as compounds that cause gushing (Mastanjevic et al., 2017). The presence of toxins produced by F. culmorum, F. graminearum or/and F. poae in barley kernels may negatively influence wort filterability, content of enzymes involved in starch and sugar processes, diastatic power, germination capacity contributing to free amino nitrogen in malt and a reduced growth of Saccharomyces cerevisiae, which leads to a delayed fermentation causing inhibition of ethanol synthesis (Ng et al., 2021a).
Thus, the control of barley grain contamination by fungi such as Fusarium spp., particularly by those producing mycotoxins, secondary metabolites with adverse health effects, is of principal importance (Havlova et al., 2006).
Since the early 1800s, fungicides have repeatedly altered growing methods and farmers' expectations of crop health (Klittich, 2008). Great results were reported for fungicide applications against FHB during last decades (Cendoya et al., 2021;Caldwell et al., 2017;Tateishi et al., 2014). Nowadays, synthetic antifungal compounds are often used routinely and prophylactically. Together with the induced antifungal resistance (Hellin et al., 2018;Deising et al., 2008) this practice decreased economic competitiveness of the crop, as well as biodiversity, and increased the environmental burden of greenhouse gas production (Cech et al., 2022;Lazaro et al., 2021).
Trans-kingdom pathogenicity (Gauthier and Keller, 2013) clearly illustrates the danger associated with the broad-spectrum use of fungicides against Fusarium spp. Vertebrate infections, caused particularly by F. onychomycoses (Uemura et al., 2022;Al-Hatmi et al., 2019), are rare, usually limited to a single organ and tend to respond well to the therapy. By contrast, disseminated fusariosis that affects the immunocompromised hosts, especially hematopoietic stem cell transplant recipients and patients with severe and prolonged neutropenia, is frequently fatal (Nucci and Anaissie, 2007). Many cases of intrinsic resistance to several antifungal drugs, antifungal resistance that developed gradually over the years and emerging issues of acquired resistance have been reported.
Improper use of azoles, especially in agriculture, became a problem in recent decades (Al-Hatmi et al., 2019). Three CYP51 gene paralogues of F. graminearum, FgCYP51B, were identified to be related to azole applications. The CYP51 gene encodes the enzyme primarily responsible for sterol 14α-demethylation, essential for ascospore formation. FgCYP51A is found in many human and agricultural pathogens. This gene is induced by azoles and environmental stress. It encodes sterol 14α-demethylase, can compensate the disruption of FgCYP51B function, and is responsible for intrinsic variation in sensitivity to different azoles. FgCYP51C, a Fusarium-specific CYP51 gene, does not influence sterol 14α-demethylase; it is specifically required for full aggressiveness on host wheat ears.
Due to the treatment with subinhibitory concentrations of azoles, the expression of FgCYP51A was induced up to 30-fold by prochloraz and tebuconazole or 100-fold by epoxiconazole, compared to control (Fan et al., 2013). Some fungicide treatments caused increased levels of mycotoxins (Cendoya et al., 2021;Edwards et al., 2001). A study using dilution series of prothioconazole, azoxystrobin and prothioconazole + fluoxastrobin demonstrated that sub-lethal doses of prothioconazole coincide with an increase in DON production 48 h after the fungicide treatment (Audenaert et al., 2010). Increased DON levels were found for in vitro trials using inoculated wheat plants treated with sub-lethal prothioconazole doses, illustrating the significance of these results from a practical point of view. RT-qPCR showed changes of several factors regulating the biosynthesis of mycotoxins in F. graminearum isolates supplemented with sub-lethal concentrations of azoles compared (Kulik et al., 2012). The mycotoxin analysis revealed higher increase in trichothecene accumulation in most of the tebuconazole-treated samples.
Most of the residua of water-soluble pesticides are eliminated from barley after steeping (Navarro et al., 2015), but hydrophobic residua remain in steeped grain. The impact of fungicidal treatment on malting quality was studied by Havlova et al. (2006). Tebuconazole preparations increased the gushing and higher content of oxalates, pentosans and ß-glucans was recorded versus the control. LC-MS/MS system was employed to examine 89 barley grain samples (Palladino et al., 2021). Residua of fungicidal active ingredients in concentrations under Regulation (EC) No 396/2005 limits were determined in 66 samples, mostly azoxystrobin, carbendazim, chlorothalonil, epoxiconazole, and fluxapyroxad. The influence of sterol biosynthesis inhibiting (SBI) compounds (cyproconazole, diniconazole, epoxiconazole, flutriafol, and tebuconazole; residua) on the fermentation and quality of young ale were studied. Noticeable effect of fungicide residues on the fermentation rate was observed in all cases. From the third day onwards, the fermentation rate was low and at the end of fermentation, statistically significantly different extract and attenuation values were obtained for all samples treated with fungicides. Higher amount of residual sugars, mainly maltose and maltotriose, was found in the beer (Navaro et al., 2011). Trace triadimefon residua influence metabolic activity of S. cerevisiae during fermentation and negatively affect beer sensory qualities (Kong et al., 2016a). In the presence of yeast, triadimefon degradation was faster ( and incorporating of alternative strategies, including biological methods to control the spread of Fusarium spp. pathogens (Uemura et al., 2022).
Achieving a safe, sustainable, fair, climate responsible and affordable food production that respects the principles of sustainability, the environment, biodiversity, and ecosystems while ensuring food security, is an important topic, one of 49 proposals included to the final report of the Conference on the Future of Europe, published on May 9, 2022. Protection and restoration of biological diversity, landscapes and oceans, pollution limitations and adoption of decisive measures to support and guarantee more ecological and climate-oriented agriculture is of utmost importance (Proposal for Regulation EU 2021/2115).

The research highlights and agricultural practice
A thorough understanding of the action mechanisms is needed to maximize consistency and efficacy of biocontrol (Fravel et al., 2003). Trichoderma species are well-studied model fungal organisms used for their biocontrol properties with great potential to alleviate the use of agrochemicals (Rush et al., 2021). The success of Trichoderma spp. as biocontrol agents (BCAs) in the soil ecosystems is based on rapid growth, utilization of various substrates, and resistance to many toxic chemicals, including fungicides (e.g., azoxystrobin, 3,4-dichloroaniline, and trifloxystrobin), herbicides and other organic pollutants (Tyskiewicz et al., 2022). Antibiotic and antimycotic effect of Trichoderma isolates were studied, showing the ability to inhibit DON production by F. graminearum and F. culmorum (Matarese et al., 2012). T. gamsii 6085 was selected in a gene expression study as the best of the genes encoding chitinolytic enzymes associated with mycoparasitism to F. culmorum and F. graminearum. According to the test, it is able to antagonize the pathogens on rice, but not on wheat. Tian et al. (2016) showed that DON could be bio-transformed into its modified form deoxynivalenol-3-glucoside (D3G) by Trichoderma isolates, which effectively suppressed the mycelial growth of F. graminearum. Some Trichoderma isolates biotransform ZEN not into glycosylated forms, but to reduced and sulfated form(s) (Tian et al., 2018). Several growthand defense-related phytohormones were determined in the shoots of plants that were root-colonized by different Trichoderma isolates (Illescas et al., 2021).
Despite the availability of Trichoderma-based preparations against phytopathogenic microbes (Oancea et al., 2017;Oros and Naar, 2017), a highly limited number of in vivo studies investigating their use for biocontrol of cereal crops remains an obstacle to commercialization of Trichoderma fungi. The determination of their effectiveness in the biocontrol of cereal crops under variable weather and climate conditions presents a considerable challenge (Modrzewska et al., 2022).
Pythium oligandrum (Drechsler, 1946) has been extensively studied for the capability to exert biological control (Belonoznikova et al., 2022;Kulisova and Kolouchova, 2021). This complex process includes direct effects through the mycoparasitism in the rhizosphere (Rey et al., 2008;Brožová, 2002) and/or indirect effects mediated by P. oligandrum on the plant, i.e. induction of resistance and growth promotion (Rey et al., 2008). Pellan et al. (2021) performed in vitro bioassay comparisons between F. graminearum and some BCAs including formulated P. oligandrum with use of detached spikelets of wheat. P. oligandrum was able to settle and colonize the lemma awn base palea and quickly produced a large quantity of characteristic oogonia containing oospores with no apparent symptoms on the spikelets (no loss of chlorophyll, necrosis, or desiccation) compared to those inoculated with F. graminearum. The treatment caused a significant level of inhibition with 77% reductions of external colonization of F. graminearum. Further integrative analysis showed that P. oligandrum-based commercially available preparations effectively reduced both vegetative and survival stages of F. graminearum; the recommended commercial use is limited to aerial parts. Recently, formulated P. oligandrum has been registered against ear fusarioses in EU, Sweden, Norway and US for the application on wheat and spring barley. Since DON, the main toxic metabolite of F. culmorum and F. graminearum, is a relatively common natural contaminant in barley, its traces can be detected in many commercial beers (Kostelanska et al., 2009). Results presented by Postulkova et al. (2018) clearly support the hypothesis that P. oligandrum can suppress fungal growth in barley during the malting process with higher efficiency than Geotrichum candidum, except G. candidum suppression of F. oxysporum growth on the artificially contaminated barley. The treatment by P. oligandrum in the steeping stage yielded an optimal suppression of Fusarium contamination (20%) and mycotoxin content (17% DON and 21% D3G) relative to untreated wheat malt (Ng et al., 2021b).
Bacterial isolates from the genus Pseudomonas have been tested for their widespread distribution in soil, ability to colonize the rhizospheres of host plants and produce wide range of compounds antagonistic to serious plant pathogens (Foroutan, 2006). Some Pseudomonas sp. strains can protect barley from pathogenesis by Fusarium spp. fungi, including FHB (Vishnevskaya et al., 2020;Petti et al., 2010;Khan et al., 2006). In the test for potential disease control organisms, two P. fluorescens strains and one P. frederiksbergensis strain significantly reduced both the severity of FHB disease symptoms caused by F. culmorum on wheat and barley and the disease-associated loss in thousand grain weight in glasshouse and field conditions when applied preventively (Khan and Doohan, 2009). In the F. culmorum-inoculated field trials, the treatment with these P. fluorescens strains also significantly reduced the DON levels in wheat and barley grain. Use of a short-lived isotopic tracer to monitor the delivery of photoassimilates into the barley roots infected by F. graminearum showed that Pseudomonas can reduce the pathogen pressure in plants, both by activating plant defense mechanisms and by direct production of antibiotics (Henkes et al., 2011). These effects are hard to distinguish under field conditions, impairing estimations of their relative contributions to the plant health. However, P. chlororaphis strain MA 342 is widely registered for the field foliar applications in cereals including barley against foliar and ear pathogens (Dutilloy et al., 2022;EFSA, 2017).
Within the phylum of gram-positive Actinobacteria, Streptomyces is the largest genus (more than 500 species), famous for its ability to produce diverse assortments of secondary metabolites of which many have antibiotic activities and are used in medicine and agriculture. Indeed, these well-known antibiotic-producing bacteria can exert biocontrol in the soil. Besides the antibiotics, they also produce many other bioactive metabolites (Viaene et al., 2016)  A vast majority of the efforts to control fusariosis of cereals is based on Bacillus strains. Excellent antifungal activities were reported from field conditions evaluations (Mulk et al., 2022;Ntushelo et al., 2019) and several commercial products have been created (Jimenez-Quiros et al., 2022). However, despite the patented applications (Schisler et al., 2003), there is no available Bacillus-based commercial product registered to suppress FHB in barley.
Yeasts are a group of Fusarium biocontrol agents that has recently been attracting increased attention of the scientists. Yeasts produced by organic agriculture show greater antagonistic activity against F. culmorum, F. graminearum and F. poae compared to those isolated from conventional cultivation systems. However, tested in vitro and applied to greenhouse and field grown wheat, neither Cryptococcus carnescens nor C. flavescens were observed to compete for nutrients and inhibit Fusarium spore germination on malting barley (Podgorska-Kryszczuk et al., 2022;Schisler et al., 2014).

Conclusion
Evolutionary factors and the use of fungicides have resulted in more aggressive forms of the FHB pathogens (Fernando et al., 2021). Despite unique modes of action differing from conventional fungicides, registered bioagents have never been used on a mass scale. Most of the recent research studies emphasize the need of further testing in field conditions, which is complicated by a plethora of interfering environmental factors. Even the latest promising preparations based on endophytes isolated from a wild relative of barley Elymus repens (Hoyer et al., 2022), lactic acid bacteria (Byrne et al., 2022) or advanced techniques including the spray-induced gene silencing strategies (Werner et al., 2020) are no exception. From this point, weather is one of the most influencing factors of Fusarium infection and the production of mycotoxins in barley (Janssen et al., 2018;Malachova et al., 2010). It is necessary to emphasize the integration of prediction systems for cereals (Bondalapati et al., 2021;Marzec-Schmidt et al., 2021;Shah et al., 2019;Schoneberg et al., 2018;Musa et al., 2007) and the use of the registered bio-preparations, like P. oligandrum-based products. When the critical rate of pathogen occurrence is exceeded, it is appropriate to consider chemical intervention or the subsequent introduction of bioagents into the malting process.

Acknowledgement
The results were achieved with the financial support of the National Agency for Agricultural Research, Ministry of Agriculture, project no. QK1910197 "The strategy for minimizing the impact of drought on sustainable production and barley malting quality".