Eur J Plant Pathol https://doi.org/10.1007/s10658-018-1506-8 Resistance against Fusarium graminearum and the relationship to β-glucan content in barley grains Charlotte Martin & Torsten Schöneberg & Susanne Vogelgsang & Romina Morisoli & Mario Bertossa & Brigitte Mauch-Mani & Fabio Mascher Accepted: 21 May 2018 The Author(s) 2018 Abstract Fusarium head blight (FHB) caused by Fu- filling, composition and structure. According to our sarium graminearum (FG) is a destructive disease results, we postulate the presence of two distinct resis- impacting barley worldwide. The disease reduces the tance mechanisms in the grain, tolerance to grain filling grain yield and contaminates grains with mycotoxins, despite infection as well as the inhibition of mycotoxin such as the trichothecene deoxynivalenol (DON). Al- accumulation. Differently to wheat, in barley, type IV though the infection mainly affects the grain yield, little resistance (tolerance of the grain to infection) is directly is known about its impact on grain structural and bio- linked with type III resistance (resistance against kernel chemical properties. Yet, such information is instrumen- infection). The resistance against toxin accumulation tal to characterize the facets of resistance in the grains. (named type V resistance in wheat) appeared to be After artificial inoculation of six barley cultivars with independent to all other resistance types. Generally, the FG in a 2 years field test, different levels of symptoms resistance was significantly influenced by the environ- on spikes, of colonisation of grains and of DON content ment and by genotype x environment interactions were observed. The infections caused a reduction in explaining the generally weak stability of resistance in grain weight and an average decrease of 10% of the β- barley. Interestingly, a significant and inverse relation- glucan content in grains, indicating alterations of grain ship between DON contamination and β-glucan content in grains suggests that high β-glucan content in grains contributes to type V resistance. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10658-018-1506-8) contains supplementary material, which is available to authorized users. . . Key words Fusarium head blight deoxynivalenol . . resistance types grain filling health promoting C. Martin F. Mascher (*) Agroscope, Plant Breeding and Genetic Ressources, 1260 Nyon, compound Switzerland e-mail: email@example.com Abbreviation : β-glucan 1,3:1,4-β-D-glucan T. Schöneberg S. Vogelgsang Agroscope, Ecology of Noxious and Beneficial Organisms, 8046 Zürich, Switzerland R. Morisoli M. Bertossa Agroscope, Ecology of Noxious and Beneficial Organisms, Introduction 6593 Cadenazzo, Switzerland Fusarium head blight (FHB) of small grain cereals is B. Mauch-Mani caused by different species of the genus Fusarium.The Agroscope, Plant Protection South of the Alps, University of Neuchâtel, 2000 Neuchâtel, Switzerland disease is known in all cereal producing areas of the world. Eur J Plant Pathol Besides yield losses, infections lead to accumulation of VI) (Mesterházy et al. 1999; Boutigny et al. 2008;Martin different mycotoxins in the grains and grain deformation, et al. 2017). In wheat, all these resistance types are inter- resulting in so called tombstones and reduced process dependent, but independently inherited (Bai et al. 2000). quality (Häller Gärtner et al. 2008;Martinetal. 2017). The wealth of knowledge on FHB in wheat can only be FHB infections can cause significant economic losses partially applied to barley (Bai and Shaner 2004;Berger along the entire value chain (McMullen et al. 1997). In et al. 2014). In wheat, the mycotoxin DON is considered to temperate climates, Fusarium graminearum (FG) is a be an essential virulence factor allowing the pathogen to common pathogen of barley (Ioos et al. 2004; Nielsen invade the rachis and to overcome type II resistance (Maier et al. 2014) and the dominant species in barley, in Swit- et al. 2006). In barley, the fungus was observed to grow zerland (Schöneberg et al. 2016). FHB development starts externally from one spikelet to another, without penetrating after primary infection when spores released from crop the rachis (Jansen et al. 2005); thus, DON appears to be residues, transported by wind and rain, are deposited on redundant in the spread of the pathogen throughout the florets (Bai and Shaner 2004). The infection takes place at spike (Langevin et al. 2004). In barley, resistance evalua- anthesis and is favoured by high humidity conditions and tion should focus on type I resistance, since type II resis- temperatures between 16°C-20°C (Xu 2003; Brennan tance appears to be strong. Concerning grains, differences et al. 2005;Musaet al. 2007). Once established in the in resistance to FHB and DON accumulation between ear, the infection progresses throughout the spike, causing spring barley genotypes have been reported (Buerstmayr water-soaked appearance of the spikelets and progressive et al. 2004). Moreover, significant effects of the genotype blighting. The pathogen interferes with grain development, on yield loss and yield components were described by leading to reduction of filling and changes in composition Chełkowskietal. (2000). Yet, the knowledge about the (McMullen et al. 1997; Bai and Shaner 2004;Oliveira impact of FHB on barley grain structure, chemical com- et al. 2013). During the infection process, position and metabolism are far lower than in wheat grains F. graminearum produces the mycotoxin deoxynivalenol (Foroud and Eudes 2009). Without this information, the (DON) that accumulates in grains. Fodder barley contam- underlying mechanisms of type III, IV, Vand VI resistance inated with DON can result in feed refusal, diarrhea, cannot be accurately characterized. vomiting, andgrowthdepressioninfarmanimal(D'Mello Barley grains contain β-glucan, a soluble fibre which et al. 1999; Dersjant-li et al. 2003). The contamination of accumulates in the cell walls of the endosperm during barley grains for human consumption may also cause maturation; β-glucan represents between 2 to 10% of health problems, since mycotoxins remain in the final the dry mass of the grain (Fincher 1975;Izydorczyk product (Kushiro 2008; Malachova et al. 2012). In Europe, et al. 2000; Zhang et al. 2002; Wilson et al. 2012). High contaminations with DON in cereal products are subject to β-glucan content in grains is recommended for a healthy strict regulations to guarantee food and feed safety. For diet and health benefits as claimed by EFSA and US barley and wheat, maximum limits in unprocessed cereals Food and Drug Administration FDA (European Food -1 for human consumption are set to 1250 μg.kg of DON Safety Organisation 2011;FDA 1997;FDA 2005; set (European Commission Regulation (EC) No Wood 2007). In barley grains, the biosynthesis of β- 1881/2006). glucan depends on the availability of polysaccharides, in The cultivation of resistant varieties is the most sustain- particular sucrose, and on an undisturbed metabolism of able and cost effective way to control yield losses and the cells (Becker et al. 1995). Hence, tracing the content contamination with mycotoxins (Mascher et al. 2005). In in β-glucan of the grain after infection will help to wheat, FHB resistance involves a multitude of resistance understand the impact of the infection on the grain’s mechanisms. Schroeder and Christensen (1963)first ob- biochemical composition and metabolism. β-glucan al- served the resistance against the primary infection (called so possesses antioxidant activity (Kofuji et al. 2012). type I resistance) and the resistance impeding the spread of Antioxidant compounds are a recognized factor of the the infection throughout the spike (called type II resis- plant’s defence system to cope with biotic aggressors tance). Resistance types of the grain include: resistance (Lattanzio et al. 2006;Zhou et al. 2007). To date, the against kernel infection (type III), tolerance to yield loss role of β-glucan against the infection by Fusarium (type IV), resistance against the accumulation of trichothe- pathogens remains to be investigated. cenes mycotoxins in the grain (type V) and also the The aim of the current study was to investigate resis- resistance against the alteration of grain constituents (type tance elements of the barley grain against Eur J Plant Pathol F. graminearum and the accumulation of deoxynivalenol. (2013, Graubünden). Strains were stored in a 1:1 water - For this, we precisely characterized the outcome of Fu- glycerol mix at -80°C. For mass production, strains were sarium infections on grains of barley. The experimental retrieved from deep freezing and cultured on Potato approach was based on the study of six winter barley Dextrose Agar (PDA, BD Difco, Le Pont de Claix, varieties with and without artificial infection at six exper- France) for 1 week at 18°C with 12h/12h UV light and imental field sites with distinct climatic conditions. In the dark. Subsequently, two mycelial discs (5 mm diam.) field, disease incidence and severity were scored as indi- from a well-grown colony were transferred to 100 mL of cators of spike resistance. After harvest, grains were liquid V8-medium in a 250ml Erlenmeyer flask. The examined for fungal infection, thousand kernel weight V8-medium consisted of a 1:5 mix of V8 juice (Camp- (TKW), and DON content, to respectively assess the bell Soup Company, Camden, USA) and distilled water, grain resistance against Fusarium infection (type 3), de- amended with 2 g sodium carbonate per litre as a pH formation (type 4) and toxin contamination (type 5). The corrector (pH =8.5). Cultures were incubated for 7 media β-glucan content was determined to study the impact of days (d) at 24°C on a shaker at 200 rpm in the dark, FG infection on grain composition, structure and func- subsequently filtered through sterile cheesecloth and tioning. By this, the contribution of β-glucaninresis- centrifuged at 4500 rpm for 10 min. The generated pellet tance of barley grains was also investigated. was re-suspended in sterile distilled water and either directly used or stored at -20 °C. Materials and methods Field tests and artificial inoculations Plant material Field tests were conducted at four different locations across Switzerland: Changins (46°24’36^/6°14’06^), Six winter barley genotypes, all registered varieties from Vouvry (46°20’16 ^/6°53’28 ^), Reckenholz different European breeders, have been studied in this (47°16’30^/8°26’45^), and Cadenazzo (46°09’00^/ experiment (Table 1). The set includes the 2 row variety 8°57’00^). Field tests were carried out in 2014 in BCassia^ and the hybrid variety BHobbit^. The variety Changins and Vouvry and in 2014 and 2015 in BWaxyma^, was specially bred for an elevated β-glucan Reckenholz and Cadenazzo. Information about weather content and is recommended for human consumption. conditions of the experimental sites is given in Table 2. The other varieties are generally used as animal feed, are The varieties were planted during the month of Octo- popular in Switzerland and figure on the national rec- ber in 1 m plots with 5 rows and 15 cm interline using a ommended list (Courvoisier et al. 2017). Seedmatic seeding machine (Hege Maschinen, Eging am See, Germany). According to the local habits, seed density -2 Fungal isolates and production of inoculum was 350 seeds m for all varieties. All trials at all sites passed winter without significant losses. At flowering, Infections were carried out with a mix of three rows were well established, dense and without gaps. F. graminearum single conidia isolates from symptom- Artificial inoculations took place when 50 % of the atic barley spikes: FG 13170 (isolated in 2013, canton plants of a plot were at mid anthesis (BBCH 65). Inoc- Fribourg), FG 13192 (2013, Basel-Land) and FG 13269 ulum suspension was prepared from fresh cultures Table 1 Winter barley varieties used in the experiments Varieties Row type Breeder Year of registration Country Cassia 2 KWS 2010 United Kingdom Fridericus 6 KWS 2006 Germany Hobbit (Hybrid) 6 Syngenta 2009 United Kingdom Landi 6 Saatzucht Schmidt 2002 Germany Semper 6 KWS 2009 Germany Waxyma 6 Dieckmann Seeds 2008 Germany Eur J Plant Pathol Table 2 Average temperature, relative humidity, evapotranspiration and sum of precipitation at the six field sites between 15.05. (flowering stage) and 15.07. (grain maturity) in 2014 and between 15.05.and 15.07. in 2015 Temperature (°C) Precipitation (mm) Relative humidity (%) Evapotranspiration (mm) Changins (VD) 2014 17.1 201 68 3.1 Vouvry (VS) 2014 16.9 221 73 2.2 Reckenholz (ZH) 2014 16.8 208 70 2.9 Reckenholz (ZH) 2015 18.1 161 69 3.2 Cadenazzo (TI) 2014 18.9 410 70 2.3 Cadenazzo (TI) 2015 20.8 279 68 2.9 meteoswiss.ch/idaweb Cantons: VD,Vaud; VS, Valais; TI,Ticino; ZH, Zurich 5 -1 adjusted to 2×10 conidia.ml . Immediately before in- Harvest and preparation of grain samples oculation, equal proportions of liquid cultures from each of the three FG strains were mixed and 0.0125% of In Changins, Vouvry and Reckenholz each field test plot Tween® were added. Suspensions were applied with a was individually harvested at full maturity (BBCH 89). hand-sprayer (Spray-Matic 1.25P, Birchmeier) until run- The trials at Cadenazzo could not be harvested, in both off. According to the weather conditions, the inoculated years, and grains could therefore not be analysed. In and control plots were irrigated to maintain humidity on Reckenholz, a plot combine harvester (HEGE 140, the ears for at least 24 h to promote primary infection. A Mähdreschwerke GmbH, Germany) was used. The air- high pressure/low volume overhead mist irrigation sys- flow on the harvester was reduced to recover as much of tem was used in Changins. In Vouvry, Cadenazzo and the kernels as possible. In the other locations, all spikes Reckenholz, water was sprayed manually with a back- were harvested by hand and collected in linen bags. The pack sprayer. The additional water supply was about spikes were threshed with a laboratory thresher -1 600 L ha , at all sites, and did not increase the water (Saatmeister, Kurt Pelz, Germany) and grains were balance significantly (Table 2). To ensure that the plants cleaned using a vertical airflow (Baumann received an adequate quantity of conidia, a second in- Saatzuchtbedarf, Germany) to remove dust and other oculation took place 2 days later. debris. All grains were dried to 14% moisture and stored at 4°C. The grains harvested from one plot are treated as one sample. Sub-samples of 200g were extracted after Disease assessments on spikes mixing each sample 3 times with a riffle divider (Schieritz & Hauenstein AG, Arleheim, Switzerland)- At Cadenazzo, Changins, Reckenholz and Vouvry, disease severity and disease incidence on spikes were determined in both control and inoculated plots. For Analyses of grains this, in each plot, 30 spikes were randomly chosen, labelled, and the total number of spikelets per spike The proportion of grains colonised by the inoculated FG was determined. Disease incidence was scored by strains and other Fusarium spp pathogens was deter- counting the number of infected spikes among the mined for all samples from artificially inoculated plots 30 spikes. Disease severity was recorded by counting using the seed health test procedure described by the number of infected spikelets on each of the 30 Vogelgsang et al. 2008. For this, one hundred grains spikes and expressed as percentage of infected spike- from each sample were surface sterilized and placed on PDA. After one week, the Fusarium species were iden- lets on the total number of spikelets on the 30 spikes. The assessments started with the observation of first tified according to the laboratory manual by Leslie and symptoms, between 7 - 10 d after the last inoculation. Summerell (2006). Subsequently, the progressive blighting of spikelets The presence of naturally occurring Fusarium spe- was scored at 3-d intervals. At least three assessments cies was tested on three samples randomly chosen from were carried out in each plot. non-inoculated plots from each field sites. Eur J Plant Pathol The thousand kernel weight (TKW) (g) was measured TKW due to the infection was calculated for each sam- with a MARVIN optical grain counter (Digital Seed ple from inoculated plot as the difference with the aver- Analyser, GTA Sensorik GmbH, Neubrandenburg, Ger- age TKW of the three samples from non-infected plot of many) and a balance (Mettler PM2000, Mettler-Toledo, the same genotype from the same field test. All calcula- Greifensee, Switzerland). TKW was determined for all tions were conducted on Microsoft® Excel 2013. grain samples, from control and inoculated field plots. Statistical analyses were carried out using the statis- The DON content in whole meal flour from inocu- tical software R (R Core Team 2015). Results of lated plots was determined with the DON ELISA kit AUDPCrel of disease severity in the six environments (Ridascreen® FAST DON, R-Biopharm AG, Darm- were illustrated with a biplot, using the average stadt, Germany), according to the manufactures’ instruc- AUDPCrel of each genotype in each field test (R pack- tions (LOD < 0.2 ppm, LOQ=0.2 ppm). Samples with age BGGEBiplotGUI^, version 1.0-9, Frutos et al. high contamination were diluted 10 times in double 2014). The data of DON content were square-root trans- distilled water. Whole meal flour was obtained by mill- formed to obtain normal distribution. The proportion of -1 ing 10g of sample with a sample mill (1093 Cyclotec grains infected by FG (%), the DON content (mg.kg ) Sample Mill, FOSS, Sweden), using a 1mm screen. and the reduction of TKW (%) were analysed with two- Samples were stored at -20°C until further use. The factor analyses of variances (ANOVA), with the trial site DON content was measured in samples from inoculated as main factor and the varieties as sub-factor. Pearson plots, and in three samples randomly chosen from non- correlations were carried out to test the relationship inoculated plots from each field sites as a control. No between disease incidence, disease severity, the propor- DON was detected in the control samples. tion of infected grains, the reduction of TKW and the The β-glucan content was also determined in whole DON content. To better understand kernel resistance meal flour (see above). For quantification, the Mixed- and the role of β-glucan therein, a principal component linkage β-glucan kit (Megazyme International Ireland analysis (R packages BFactoMineR^, version 1.29, (Le Ltd., Wicklow, Ireland) was used. The streamlined et al. 2008)) was performed with the proportion of method of mixed-linkage β-glucan in barley flour –– grains infected by FG, DON content and reduction of (ICC Standard Method No.166) was adapted to the TKWas Bfactors^ and one variety in one environment as laboratory facilities. As a negative control, the β- Bindividuals^. A hierarchical cluster analysis was car- glucan content was measured in 0.1 g of dry FG myce- ried out after the PCA to class individuals into distinct lium from the strain FG13170. β-glucan content was resistance clusters (R packages Bfactoextra^, version measured in all samples, from inoculated and non- 1.0.4, Kassambara and Mundt 2017).Variation of β- inoculated plots. glucan content (%) in the dataset was analysed with a three-factors ANOVA, using, according to the experi- mental design, the Benvironment^ as a main factor, the Experimental set-up and statistical analysis Btreatment^ indicating inoculation or control treatments Field tests were conducted in a comparable way at all as sub-factor and the different varieties as the smallest locations and included inoculated and non-inoculated source of variation. Then β-glucan content (%) in grains treatments with three replicates arranged in a split–plot from inoculated plots were compared between the three design. The treatments were the main plot whereas the resistance groups defined by the clustering on the PCA. different barley varieties were the sub-plots. All along the study, multiple comparisons were used For each plot, data of disease incidence and severity basedonTukeysHSD (package Bagricolae^,De were integrated by the number of observation days Mendiburu 2015). (number of days between inoculation and the last scor- ing), to obtain AUDPC (area under the disease pressure curve). AUDPC were then divided by the number of Results observation days, and defined relative AUDPC (AUDPCrel) as described elsewhere (Martin et al. Symptoms on spikes 2017). This standardized average daily disease progress value allows the comparison of disease incidence and Symptoms on spikes were scored in all six field tests. disease severity between the trial sites. Variation of AUDPCrel of severity are shown in Fig. 1. Symptoms Eur J Plant Pathol were significantly more severe in Changins and therefore only disease severity is shown here. All data of Reckenholz in 2014 than in all other environments disease severity and incidence are presented in supple- (P<0.05). Only very weak symptoms were observed in mentary materials. In all field tests, none or very low Reckenholz 2015, and in Cadenazzo 2014. Symptoms symptoms were observed on non-inoculated plots. in Cadenazzo 2015 and Vouvry 2014 were moderate. Pooled over all environments, the significantly highest Resistance of grains (P<0.05) disease severities on spikes were measured on BHobbit^ and the lowest on BCassia^ followed by Proportion of grains colonised by Fusarium species BWaxyma^ and BSemper^ (Fig. 1). Results indicate that the varieties performed differently in the different envi- FG was retrieved in all samples from inoculated plots, ronments. While "Hobbit" showed the highest disease attesting to the success of the infection. Natural infections severity in Changins 2014 and Cadenazzo 2015, it was were observed in all samples. In samples from inoculated not the most affected genotype in Reckenholz in 2014. plots, less than 3% of the grains presented Microdochium The varieties "Fridericus" and "Waxyma" showed more nivale and less than 1% were colonised by F.poae or symptoms in Vouvry and Reckenholz in 2014 in com- F.culmorum. In non-inoculated plots, less than 5% of parison with other environments. Yet, disease severity grains were colonized by M.nivale and F.graminearum, on BSemper^ was very similar across the different envi- and sporadically by F.poae and F.culmorum. ronments. Disease incidence and disease severity were The proportion of grains colonized by FG with arti- strongly correlated (Pearson coefficient 0.80, P<0.001), ficial inoculation on the six barley varieties in the four environments is displayed in Fig. 2a. The highest colo- nization was found in Reckenholz in 2015 (75% of grains colonised) followed by Changins 2014, Reckenholz 2014 and Vouvry 2014 (22%). Pooled over all environments, grains from BFridericus^ were signif- icantly more colonised (P< 0.05) than grains of BCassia^ (60% and 38%, respectively) (Fig. 2a). Within the same environment, the proportion of colonised grains between varieties was rather similar, except for Changins in 2014 where BWaxyma^ was only weakly colonised while the 5 other varieties, were strongly colonised (Fig. 2a). The impact of the environment on FG colonisation prevailed over the impact of the geno- type (Table 3). Thousand kernel weight reductions Fig. 1 Genotype main effect plus Genotype x Environment (GGE) biplot showing the average disease severity and its stability A reduction of grain filling due to FG infection, mea- of the six barley genotypes over all six experimental sites. The plot sured by TKW comparisons was observed for all barley has been obtained with R package GGEBiplotGUI with parame- varieties in all environments, and ranged between 2% ters Bscalling=0^ and Bcentering =2^ to illustrate both the six and 20% (Fig. 2b). The lowest reduction was measured barley genotypes and the genotype x environment interactions. The parameter Bsingular value portioning^ was set to 1 (SVP=1) in grains from BCassia^ with an average of 4% over all scaling by the visualising the average points (variety / site) and the environments, whereas for the five other varieties, TKW stability of the genotype. Position of the varieties along the hori- reductions ranged between 8% and 11%. The reduction zontal axis indicates the average disease severity over all environ- of the TKW was also significantly influenced by the ments, with BCassia^ displaying weakest symptoms and BHobbit^ with strongest symptoms. The distance between variety and hor- GxE interactions (P<0.01) (Table 3). In particular, in izontal axis indicates the stability of disease severity. Short dis- Changins 2014, FG infection caused only weak reduc- tances indicate high stability. CH14= Changins in 2014, in 2014, tion of TKW in grains from BCassia^ and BFridericus^ RE14= Reckenholz in 2014, VO14= Vouvry in 2014, CA14= (respectively 2.5% and 6.9% of reduction), but elevated Cadenazzo in 2014, CA15=Cadenazzo in 2015, RE15= Reckenholz in 2015 reductions of up to 14.5% in grains of BHobbit^.In Eur J Plant Pathol Fig. 2 Proportion of Fusarium A a colonised grains (A), reduction of thousand kernel weight (TKW) (B), content of deoxynivalenol a 80 a a (DON) (C) for the six barley va- rieties in four environments. Error a bars represent the standard error ab of the means. Different symbols indicate significantly different varieties in the same environment according to a Tukey-Test with b 30 a α=0.05 Changins2014 Reckenholz2014 Vouvry2014 Reckenholz2015 20 a ab ab a ab ab a ab ab b abc abc bc Changins2014 Reckenholz2014 Vouvry2014 Reckenholz2015 α α ab b β bc cd a a 15 ab 10 a Changins2014 Reckenholz2014 Vouvry2014 Reckenholz2015 Barley variees -1 DON content (mg.kg ) Fusarium colonized grains (%) Reductionof TKW (%) Eur J Plant Pathol Table 3 Compositions of the variances of the proportion of colonised grains, the deoxynivalenol (DON) content and the reduction of thousand kernel weight (TKW) in function of the factors environment, genotype, and genotype x environment interaction -1 Proportion of Fusarium graminearum DON content (mg kg ) Reductions of TKW (%) colonised grains (%) Source of variation Sum of Square Mean Square Sum of Square Mean Square Sum of Square Mean Square Environment 26920 122.9 *** 21.4 7.2 * 335.9 112.0 Error (A) 916 153 4.8 0.8 307.5 51.2 Genotype 4100 819.9 *** 31.1 6.2 *** 287.1 57.4 *** Genotype x Environment 4284 285.6 * 30.4 2.0 *** 399.8 26.7 ** Error (B) 4939 123.5 19.1 0.5 337.3 8.43 Significance level: ***: P<0.001, **: P<0.01, *: P<0.05 contrast, in Reckenholz 2015, no differences between contamination measured in Changins 2014. Overall, varieties were observed (Fig. 2b). the DON content in grains was influenced by the barley genotype as well as by the interaction of the genotype and the environment (Table 3). Accumulation of deoxynivalenol The mycotoxin DON was detected in all samples from Interactions between resistance traits inoculated plots. Contents varied from 5.2 to 49.4 -1 mg.kg . The contamination was significantly lower in Disease incidence and disease severity were highly cor- -1 Reckenholz 2015 (on average 9.4 mg kg )than in all related (P<0.001) (Table 4). A significant (P<0.001) -1 other environments (on average 18.1 mg kg )(Fig. 2c). correlation was also found between the proportion of Over the four environments, the highest average content grains colonised by FG and the reduction of TKW due -1 was detected in grains from BHobbit^ (24.3 mg kg ), to artificial inoculation. However, DON content in while grains from BWaxyma^ were significantly grains was neither related with colonisation nor with -1 (P<0.05) less contaminated (on average 7.9 mg kg ) the reduction of TKW. The severity and the incidence than all other varieties (Fig. 2c). The DON content in of symptoms on the spikes were neither correlated with grains was mainly influenced by the environment and to TKW reduction nor with the proportion of FG colonised a lesser extent by the variety (Table 3). Nevertheless, not grains but with the DON content. all varieties performed similarly in all environments, as The interactions between grain resistance factors indicated by significant GxE interactions (P<0.05) were studied with a Principal Component Analysis (Table 3). Indeed, higher DON contaminations in (PCA) (Fig. 3). Here again, the PCA showed the posi- BCassia^ and in BFridericus^ were observed in grains tive link between the colonisation of grains and the from Reckenholz 2014 than from Changins 2014. The reduction of TKW, whereas the DON content in the opposite was observed for BSemper^ with highest grains was clearly not linked with colonisation and Table 4 Pearson correlation coefficients between observed spikes and grains at four field tests a b c Disease incidence Disease severity ) Proportion of Fusarium DON content graminearum colonised grains in grains Disease severity (% spikelets with symptoms) 0.85*** Proportion of Fusariumgraminearum ns ns colonised grains DON content in grains 0.48*** 0.42*** Ns Reduction of TKW ns ns 0.62*** ns Significant level: ***: P<0.001, ns: not significant a b c d Number of spikes with symptoms; Percent spikelets with symptoms; DON, deoxynivalenol; TKW, thousand kernel weight Eur J Plant Pathol Fig. 3 Biplot representation of a Principal Component Analysis (PCA) of grain resistance against accumulation of DON (deoxynivalenol), grain colonisa- tion (FCG) and reduction of thousand kernel weight (TKW) for six varieties in four environ- ments. Each pair BGenotype:Environment^ is one individual point in the PCA. In- dividuals have been classed into three resistance groups (A-C) using a hierarchical cluster analy- sis. C: Cassia, F: Fridericus, H: Hobbit, L: Landi, S: Semper, W: Waxyma; CH14: Changins 2014, RE14: Reckenholz 2014, RE15: Reckenholz 2015, VO14: Vouvry TKW reductions. The hierarchical cluster analysis iden- measured in grains from Reckenholz in 2014 and 2015 tified three distinct clusters: cluster A includes low DON (4.9% and 4.8%, respectively over all genotypes) com- content, low TKW reductions and low proportion of pared with grains from Vouvry 2014 (4.5%). Content in grains colonised by FG. Cluster B is composed of grains from BCassia^ varied between the different envi- individuals affected by the reduction of TKW and with ronments from 4.4% in Changins 2014 to 5.1% in a high proportion of FG colonised grains but low DON Reckenholz 2014; all other varieties showed stable con- contaminations. Cluster C gathers individuals highly tents across environments (data not shown). It resulted contaminated with DON, along with a wide range of in weak but significant GxE interactions (Table 5). TKW reduction and proportion of FG colonised grains. Artificial inoculations significantly impacted the β- Overall, cluster A consisted mainly of BWaxyma^ and glucan content in grains (P<0.01) (Table 5). By com- BCassia^, regardless of the environment. Cluster B gath- parison, β-glucan contents were generally lower by ered most of the individuals from Reckenholz 2015, and 10% in grains from inoculated plants than in grains from cluster C contained all other remaining variety x envi- control plots (Fig. 4). The decrease was the same in all ronment combinations (Fig. 3). varieties and in all environments (Fig. 4). Impact of the Fusarium infectiononthe β-glucan Link between β-glucan content and grain resistance content in grains traits The comparison of the β-glucan content in infected In our data set, grains contained between 3.1% and 6.8% of β-glucan in dry weight. The highest contents were grains between the three grain resistance groups previ- measured in BWaxyma^ grains from non-inoculated ously defined by hierarchical clustering revealed signif- plots (on average 6.5% over all environments), and the icant differences (P<0.05). While grains in cluster A lowest contents in BLandi^ grains (4.0% over all envi- (resistance to FG infection and DON contamination) ronments). Generally, higher β-glucan content was contained on average 4.8% β-glucan, grains from Eur J Plant Pathol Table 5 Analysis of variance of β-glucan content in grains of 6 The concentration of β-glucan was not linked with any barley varieties, from four different field sites, inoculated or not other grain resistance trait (data not shown). with Fusarium graminearum Source of variation β-glucan content (%) Discussion Sum of Mean Square Square In the current study, the resistance of six barley varieties Environment 3.2 1.1 * against infection with FG was investigated under differ- Error (a) 0.9 0.2 ent field conditions. Besides the symptoms on the Inoculation 7.6 7.6 ** spikes, a particular focus was the symptoms of the Environment x Inoculation 2.3 0.8 grains, the accumulation of the mycotoxin deoxynivalol Error (b) 3.0 0.4 and variations of the β-glucan content. The severity of Genotype 48.8 9.8 *** symptoms on the spike differed between the varieties Genotype x Environment 6.2 0.4 * and between the environments. Generally, varieties with Genotype x Inoculation 1.0 0.2 a higher spike resistance showed reduced accumulation Genotype x Environment x Inoculation 4.0 0.3 of the mycotoxin DON. Negative correlations between spike resistance and toxin accumulation have also been Error (c) 16.3 0.2 reported by other authors (e.g. Tekauz et al. 2000; Choo Significance: ***: P<0.001, **: P<0.01, *: P<0.05 et al. 2004; Berger et al. 2014;Heet al. 2015). Yet, the present results show that DON content is not linked with cluster C (grains accumulating DON) contained signifi- the colonization of the grain by FG. High DON cantly less β-glucan (average of 4.2%) (Fig. 5). The grains of cluster B (elevated proportion of colonized grains and reduced TKW) had an intermediate content (4.5%). A correlation analysis (Pearson correlation coefficient: - 0.29, P<0.01) confirmed the significant inverse relation- ship between DON accumulation and β-glucan content. Grains from non-inoculated plots Grains from inoculated plots b bc bc Cassia Frid eric us Ho bbit Land i Semper Waxyma Fig. 4 Comparison of β-glucan contents in barley grains from Fig. 5 The average β-glucan content in barley grains within the inoculated and non-inoculated plots, pooled over 4 environments. three resistance groups. Group A = grains with a general high Black and grey bars represent average β-glucan content in dry resistance level, Group B = grains susceptible to Fusarium colo- weight of grains. Error bars represent the standard error of the nization and reduction of grain filling, Group C= grains generally means. Different letters indicate significant differences between susceptible, with a high DON accumulation. Different letters varieties over all environments in inoculated and control samples indicate significant differences according to a Tukey test with (P<0.05) α=0.05 β-glucan content (% ofdry weight) Eur J Plant Pathol contamination but a weak grain colonisation was found glucanases and other cell-wall degrading enzymes of in all samples from inoculated plots in Vouvry 2014, Fusarium pathogens (Schwarz et al. 2002;Wang where conditions after anthesis were quite humid. In et al. 2005;Oliveiraetal. 2012a, b). On the other contrast, in Reckenholz in 2015, where elevated tem- hand, the reduced supply of sugars and other nutri- peratures and low precipitations were registered, we ents can lead to a reduced synthesis of β-glucan in found a high proportion of colonised grains but only the developing grain (Fincher 1975; Becker et al. low symptoms on the spikes and low accumulation of 1995; Wilson et al. 2012). In conclusion, our anal- DON in the grains from inoculated plots. In Vouvry in yses demonstrated that FG colonises barley grains 2014, characterized by elevated rainfalls and high hu- and by this synthesizing mycotoxins and causing the midity between flowering and grain maturity, elevated reduction of the grain size and shape as well as DON content was found despite a moderate symptom reducing the content in β-glucan. level on the spike. This supports the important role of The present trials show significant differences of environmental conditions on symptoms development the symptoms of spike and grain between barley and DON accumulation in barley and confirms findings genotypes and environmental conditions. Strong in- of other authors (Tekauz et al. 2000; Bai and Shaner teractions between the genotypes and the environ- 2004;Bernhoft et al. 2012). In practical terms, similar to ments indicate a weak stability of barley resistances wheat and triticale, the absence of symptoms on the traits of spike and grains previously mentioned in spike is not a reliable indicator of low mycotoxin accu- genetic studies (Capettini et al. 2003;DelaPena mulation in the grain (Arseniuk et al. 1999;Mesterházy et al. 1999). In our field analyses, we observed that 2002). Arguably, the different genetic basis for spike the interactions of the genotype with the environ- resistance and for DON accumulation in the grain leads ment determine, to the same extent, the phenotypic to the differential reaction between spike and grain variation of symptoms on the spike, the DON con- (Massman et al. 2011). In the following we aim to tent and the reduction of grain filling. Heritability of accurately phenotype the infection induced symptoms resistance against FG infection is therefore lower on the grains of the six barley varieties. than that in wheat, confirming the findings of other Therefore, we expected to observe typical symptoms authors(Baietal. 2000; Urrea et al. 2002; Capettini of F.graminearum on the grains such as the presence of et al. 2003). Consequently, in practical breeding, the scabby grains, pinkish discolouration, or grain deforma- expected genetic of FHB resistance is rather weak. tion as described by McMullen et al. (1997) and He et al. Nevertheless, among the varieties, we found dif- (2015). None of these symptoms were found in our ferences in resistance against all facets of FHB. The samples, besides the presence of black perithecia that two-row variety BCassia^ showed the highest resis- we attributed to Fusarium. Yet, the colonization of the tance with respect to spike symptoms, FG grain FG inoculum on the grain proves the presence of the colonisation and TKW reduction. BWaxyma^ was pathogen on the grain. It is conceivable, that the elevated the most resistant variety with respect to DON ac- disease pressure and conducive meteorological cumulation and second to BCassia^ in all other cat- conditions allowed the development of the typical egories. In contrast to six-row varieties, two-row symptoms on the grains in these studies. Indeed, spikes may impede the external propagation of the Berger et al. (2014) and He et al. (2015) rated pinkish pathogen from one spikelet to the other (Choo et al. discolouration in grains from intensively irrigated fields 2004;Langevinetal. 2004), Even though in and inoculated with a highly aggressive FG isolate. BCassia^ and BWaxyma^ both spike and grain resis- The FG infection caused significant reduction of tances are present, results of the less resistant geno- the TKW indicating a disruption in grain filling that types show that the two resistance types are acting impacts grain morphology and shape. The quantifi- independently. The degree of colonisation of the cation of β-glucan in the grains revealed a general grain was linked to the reduction of grain filling decrease by 10% in grains from inoculated plots but not linked with accumulation of DON. It is compared to grains from non-inoculated plots, re- known for barley, that the colonisation of the grain gardless the variety and the environment. The lower is independent from the accumulation of trichothe- β-glucan content in infected grains might be attrib- cenes mycotoxins (Langevin et al. 2004; Maier et al. uted on the one hand to the activity of fungal β- 2006). Yet, the degree of abundance of the FG Eur J Plant Pathol Open Access This article is distributed under the terms of the pathogen may impair the grain filling processes. No Creative Commons Attribution 4.0 International License (http:// resistance was found in any of the six barley varie- creativecommons.org/licenses/by/4.0/), which permits unrestrict- ties to prevent β-glucan degradations caused by the ed use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, infection. Overall, our resistance analyses suggest provide a link to the Creative Commons license, and indicate if the existence of two distinct resistance mechanisms changes were made. in the grain, namely: (i) the continuity of grain filling and reduced colonization and (ii) the inhibi- tion of mycotoxin accumulation. Type IV resistance References (tolerance of grains to FHB) seems to be directly linked to type III resistance (resistance to kernel Ames, N. P., & Rhymer, C. R. (2008). Issues Surrounding Health infection). Meanwhile, type V resistance (resistance Claims for Barley 1 – 2. The Journal of Nutrition, 138,1237– against trichothecenes accumulation) appears to be Arseniuk, E., Foremska, E., & Chełkowski, J. (1999). Fusarium independent from all other resistance types. head blight reactions and accumulation of deoxynivalenol Interestingly, varieties with the highest β-glucan con- (DON) and some of its derivatives in kernels of wheat, tent (Fig. 3, cluster A) displayed the lowest DON con- triticale and rye. Journal of Phytopathology, 147(10), 577– tent. Arguably, the nature of interaction between the β- Bai, G. H., Shaner, G., & Ohm, H. (2000). Inheritance of resis- glucan content and the contamination with DON and its tance to Fusarium graminearum in wheat. Theoretical and role in resistance is source of debate. On the one hand, a Applied Genetics, 100,1–8. higher β-glucan content might enhance the resistance Bai, G., & Shaner, G. (2004). Management and resistance in wheat against DON accumulation thus contributing to type V andbarleytoFusariumheadblight. Annual Review of Phytopathology, 42,135–161. https://doi.org/10.1146 resistance by antioxidant activity of β-glucan (Kofuji /annurev.phyto.42.040803.140340. et al. 2012). Indeed, several studies demonstrated the Becker, M., Vincent, C., & Reid, J. S. G. (1995). Biosynthesis of inhibitive potential of natural antioxidant compounds on (1,3)(1,4)-beta-glucan and (1,3)-beta-glucan in barley the production of mycotoxins (Boutigny et al. 2010; (Hordeum vulgare L.). Planta, 10,331–338. Berger, G., Green, A., Khatibi, P., Brooks, W., Rosso, L., Liu, S., Pani et al. 2014;Zhou et al. 2007). On the other hand, et al. (2014). Characterization of Fusarium head blight resis- alower β-glucan content may allow the accumulation of tance and deoxynivalenol accumulation in hulled and hulless DON. Indeed, it has been demonstrated that β-glucan is winter barley. Plant Disease, 98,599–606. able to bind several Fusarium toxins in vitro Bernhoft, A., Torp, M., Clasen, P. E., Løes, A. K., & Kristoffersen, A. B. (2012). Influence of agronomic and climatic factors on (Yiannikouris et al. 2004, 2006). Previous findings rec- Fusarium infestation and mycotoxin contamination of ce- ommend it as a potential detoxifiant of food products reals in Norway. Food Additives & Contaminants: Part A, contaminated with Fusarium toxins (Meca et al. 2012; 29(7), 1129 – 114 0. https://doi.org/10.1080 El-Naggar and Thabbit 2014). In any case, cultivating /19440049.2012.672476. Boutigny, A.-L., Richard-Forget, F., & Barreau, C. (2008). Natural barley varieties with high β-glucan contents can be mechanisms for cereal resistance to the accumulation of recommended to serve two purposes at the same time: Fusarium trichothecenes. European Journal of Plant to reduce the risk of mycotoxin contaminated grains and Pathology, 121(4), 411–423. https://doi.org/10.1007 to further promote the production of health promoting /s10658-007-9266-x. Boutigny, A.-L., Atanasova-Pénichon, V., Benet, M., Barreau, C., food (Ames and Rhymer 2008). & Richard-Forget, F. (2010). Natural phenolic acids from wheat bran inhibit Fusarium culmorum trichothecene bio- Acknowledgments This research was funded by the Swiss Na- synthesis in vitro by repressing Tri gene expression. tional Research Program 69 BHealthy Nutrition and Sustainable European Journal of Plant Pathology, 127(2), 275–286. Food Production^ (contract: SNF 145210 /1). We thank Stefan https://doi.org/10.1007/s10658-010-9592-2. Kellenberger for his highly valuable assistance in field experi- Brennan, J. M., Egan, D., Cooke, B. M., Doohan, F. M., Puri, K. ments, and Florian Combremont and Caroline Litzistorf for their D., Zhong, S., … Forrer, H. R. (2005). Effect of temperature help with laboratory work. on head blight of wheat caused by Fusarium culmorum and F. graminearum. Plant Pathology, 54(2), 156–160. Compliance with ethical standards The manuscript is original. https://doi.org/10.1111/j.1365-3059.2005.01157.x No part of the manuscript has been published before nor is any part Buerstmayr, H., Legzdina, L., Steiner, B., & Lemmens, M. (2004). of it under consideration for publication at another journal. Variation for resistance to Fusarium head blight in spring barley. Euphytica, 137(3), 279–290. Conflict of interest The authors declare they have no conflict of Capettini, F., Rasmusson, D. C., Dill-Macky, R., Schiefelbein, E., interest. 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