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Introduction

Fusarium head blight (FHB) is an economically important disease of barley in Canada and the United States (McMullen et al. 1997; Tekauz et al. 2000). The disease reduces yield and results in poor seed quality, which has a negative impact on the market value of the grain and on seed vigour (Horsley et al. 2006). In addition, the disease causes kernel contamination with mycotoxins, such as deoxynivalenol (DON) and nivalenol (Campbell et al. 2002; Müeller et al. 1997), which are harmful to humans and livestock. Deoxynivalenol is a known inhibitor of protein synthesis and has an interactive and additive effect with other mycotoxins (Miller et al. 2001). Grain contamination with DON is a food and feed concern in many countries around the world (Campbell et al. 2002; Placinta et al. 1999). Furthermore, the malting and brewing industries reject grain with even minute amounts of DON because of quality issues with the resulting malt and beer (Beattie et al. 1998; Horsley et al. 2006).

Although Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.) Petch.] is considered the most important causal agent of FHB and DON production in small grain cereals in Canada and the United States, several other Fusarium spp. are often isolated from infested barley kernels (Clear et al. 1996; Salas et al. 1999; Sturz and Johnston 1985; Tekauz et al. 2000; Xue and Chen 2010). These Fusarium spp. have been proven to be pathogenic on barley and play a role in the etiology of FHB in barley. Xue et al. (2006a) compared the pathogenicity of eight Fusarium spp. isolated from kernels of small grain cereals and demonstrated that F. crookwellense Burgess, Nelson & Toussoun, F. culmorum (W.G. Sm.) Sacc. and F. graminearum were highly pathogenic; F. avenaceum (Fr.) Sacc. was intermediate; and F. acuminatum Ellis & Everh., F. equiseti (Corda) Sacc., F. poae (Peck) Wollenw., and F. sporotrichoides Sherb. were weakly pathogenic in causing FHB in barley. In Europe, F. graminearum, F. culmorum or, more rarely, F. avenaceum are the prevalent species found on barley depending on the country, with F. culmorum being more abundant in northern countries (Bottalico and Perrone 2002; Nielsen et al. 2011).

It is known that harvest date affects the incidence of seed-borne Fusarium spp. in wheat in Ontario. Xue et al. (2004) demonstrated that the incidence of total Fusarium spp. increased about twofold, from 9.5% in seed harvested early to 19.8% following delayed harvest. In addition, wheat cells can metabolize DON, and this was consistent with field observations of a decline in DON concentration (Miller and Arnison 1986). However, Langseth and Stabbetorp (1996) found that delayed harvest generally reduced DON levels in barley, although this trend depended on year and location. The objective of this study was to compare the effect of five harvest dates on seed-borne Fusarium spp. and DON concentration in barley in Ontario. This information could be useful in determining whether an early harvest could be used as a management tool to reduce grain contamination with Fusarium pathogens and DON in barley.

Materials and Methods

Field experiments were conducted at three locations in Ontario: Emo Agricultural Research Station (48°38’N 93°52’W) and New Liskeard Agricultural Research Station (47°28’N 79°30’W), both in northern Ontario, and Agriculture and Agri-Food Canada’s (AAFC) Eastern Cereal and Oilseed Research Centre (ECORC) in Ottawa (45°23’N 75°42’W), eastern Ontario, during 2004-2005. The soil type was clay loam at Emo, clay at New Liskeard, and sandy loam in 2004 and loam in 2005 in Ottawa. All sites were under conventional tillage with a 3-year rotation of canola/cereal (wheat, barley or oat)/cereal at Emo and New Liskeard, and soybean/corn/cereal in Ottawa. Three common barley cultivars, AC Vision, Brucefield, and OAC Baxter, similar in maturity but differing in susceptibility to FHB, were used at each location for both years. The experiments were arranged in a split-plot design with four replications. The three cultivars were the main plots and the five harvest dates were the subplots. Plots were seeded on May 7 and 17 in Emo, June 3 and May 26 in New Liskeard, and April 30 and May 16 in Ottawa in 2004 and 2005, respectively, at a rate of 400 seeds m‑2. Plots were 10 rows wide, 5.0 m long with 15 cm row spacing in Emo, 8 rows wide, 5.0 m long with 18 cm row spacing in New Liskeard, and 6 rows wide, 6.0 m long with 18 cm row spacing in Ottawa. The plots were fertilized according to soil test recommendations and treated with applications of appropriate herbicides for weed control according to standard management practices (OMAFRA 2002).

The five harvest dates were “very early harvest” (VEH), “early harvest” (EH), “normal harvest” (NH), “late harvest” (LH), and “delayed harvest” (DH), corresponding to plants at Zadoks’ growth stages of 91, 92, 93 early stage, 93 late stage, and 94, respectively (Zadoks et al. 1974). The VEH began when the grain reached approximately 25% moisture (w/w) on August 12 and 8 in Emo, September 14 and August 24 in New Liskeard, and August 4 and July 27 in Ottawa, in 2004 and 2005, respectively (Table 1). At this point, the grain was mature and the glumes were no longer green. The four subsequent harvest dates followed at approximately 1-wk intervals. Plants were at the hard kernel stage with less than 18% moisture at EH, at the loosening kernel stage in daytime at both NH and LH, and overripe with the straw collapsing at DH. Harvest of the two middle rows was done using a rice binder, the grain was air dried and then threshed using a stationary threshing machine at Emo. Small plot combines were used to harvest 1.0 m long rows in the middle of each plot in New Liskeard (8 rows) and Ottawa (6 rows). Grain was air dried to approximately 12% moisture (w/w) at low temperatures ranging from 13 to 17°C. A sample of approximately 500 g of seed was taken from each plot for the isolation of Fusarium spp. The samples were stored at 5°C until processed.

Table 1

Harvest dates for barley grown in Emo, New Liskeard, and Ottawa, Ontario, in 2004 and 2005.

Harvest dates for barley grown in Emo, New Liskeard, and Ottawa, Ontario, in 2004 and 2005.
a

VEH = very early harvest; EH = early harvest; NH = normal harvest; LH = late harvest; DH = delayed harvest.

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Approximately 3 mo after harvest, a random subsample of 100 kernels from each plot was taken to determine the level of infection by Fusarium spp. The kernels were surface-disinfected for 1.5 min with 0.5% NaOCl, rinsed three times with sterile distilled water, and drained on sterile filter paper. Five kernels were placed in each Petri dish containing modified potato dextrose agar (mPDA) (10 g L‑1 dextrose, which is 50% of the label rate) amended with 20 ppm streptomycin sulfate. Dishes were placed under mixed lightings consisting of fluorescent UV tubes and artificial daylight, on a 12-h light/dark cycle for 7-14 d at 22-25°C. Fusarium spp. were identified directly from mPDA or, if necessary, subcultured onto carnation leaf agar (CLA) medium for their identification. The fungi were identified by microscopic examination of macro- and/or microconidia, using the taxonomic keys for Fusarium spp. described by Nelson et al. (1983) and the illustrated morphological manuals by Samson et al. (2000) and Watanabe (1994).

Analyses for DON concentration were done at the Mycotoxin Research Laboratory, ECORC, AAFC, using a 30-g seed subsample from each plot. Samples were ground to a fine powder in a Retsch Ultra-Centrifugal Mill Type ZM-l (Brinkman Instruments Inc., Rexdale, ON) with a 0.75 mm wire mesh. A ground sample of 1.00 g was used for DON analysis. The concentration of DON was determined by the competitive direct enzyme-linked immunosorbent assay (CD-ELISA) procedure using monoclonal antibodies (MABs) as described by Sinha et al. (1995).

Logarithmic transformation of DON values and square root transformation of incidence of Fusarium spp. were used in the analysis of variance to stabilize variance and to respect the assumption of homogeneity of model residuals. Data were back-transformed to the original scale for presentation, and treatment means were separated by Fisher’s least significant difference (LSD) test at a probability level of P< 0.05 transformed data. Pearson’s correlation coefficients were calculated to examine relationships between harvest date and incidence of Fusarium spp. and DON concentration. Analyses were performed using Proc GLM in SAS/STAT® (SAS Institute Inc., Cary, NC).

Results

Eight Fusarium spp. were recovered from 4,007 of the 36,000 seeds tested in 2004 and 2005 (Table 2). Among the eight species of Fusarium identified, F. equiseti, F. sporotrichioides and F. poae predominated and were isolated from 4.4%, 3.3%, and 1.6% of the kernels, representing 39.3%, 29.4%, and 14.2% of the pathogen population, respectively. Fusarium graminearum and F. avenaceum were recovered from approximately 1% of the kernels and composed 8.3% and 6.6% of the population, respectively. Other species, including F. acuminatum, F. culmorum, F. semitectum and an unknown one, collectively occurred only on 0.2% of the kernels and represented 2% of the population.

Table 2

Frequency of Fusarium spp. isolated from kernels of barley grown in northeastern Ontario in 2004 and 2005.

Frequency of Fusarium spp. isolated from kernels of barley grown in northeastern Ontario in 2004 and 2005.
a

Number of infected seeds out of 36,000 tested.

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Analysis of variance indicated that harvest date had a significant effect on the incidence of two of the five most common species and on all Fusarium spp., while the type of cultivar affected the incidence of three of the five Fusarium spp., all Fusarium spp. and DON concentration (Table 3). Location had a significant effect on the incidence of the five most common species, all Fusarium spp., and DON concentration. The year and location × year interaction had a significant effect on the incidence of four of the five most common species, all Fusarium spp., and DON concentration. Other significant effects observed were for the harvesting date × location interaction on the incidence of F. equiseti, F. sporotrichioides, and all Fusarium spp.; the cultivar × location interaction on the incidence of F. sporotrichioides and DON concentration; and the harvest date × year interaction on the incidence of F. equiseti and F. sporotrichioides. The interactions of harvest date × cultivar, harvest date × cultivar × location, cultivar × year, harvest date × cultivar × year, harvest date × location × year, cultivar × location × year, and harvest date × cultivar × location × year were not significant for any species nor for DON concentration (Table 3).

Table 3

Mean squares from the analysis of variance for the effects of harvest date, cultivar, location, year and their interaction on the incidence of the most common seed-borne Fusarium spp. and deoxynivalenol (DON) content in barley in northeastern Ontario in 2004 and 2005.

Mean squares from the analysis of variance for the effects of harvest date, cultivar, location, year and their interaction on the incidence of the most common seed-borne Fusarium spp. and deoxynivalenol (DON) content in barley in northeastern Ontario in 2004 and 2005.
a

Fa = F. avenaceum; Fe = F. equiseti; Fg = F. graminearum; Fp = F. poae; Fs = F. sporotrichioides.

*, **

Significant at P<0.05 and P<0.01 levels, respectively.

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Of the five harvest dates, VEH had the lowest incidence of F. sporotrichioides and total Fusarium spp. (Table 4). The incidence of F. sporotrichioides and total Fusarium spp. at EH and NH was also significantly lower than at the later harvest dates (LH and DH). The incidence of F. avenaceum was not significantly different among LH, NH, EH and VEH. DON concentration in the harvested grain ranged from 0.20 to 0.28 ppm and did not differ statistically among the five harvest dates (Table 4).

Table 4

Effect of harvest date and barley cultivar on the incidence of the five most common seed-borne Fusarium spp. and deoxynivalenol (DON) in grains of barley grown in northeastern Ontario in 2004 and 2005.

Effect of harvest date and barley cultivar on the incidence of the five most common seed-borne Fusarium spp. and deoxynivalenol (DON) in grains of barley grown in northeastern Ontario in 2004 and 2005.
*

VEH = very early harvest; EH = early harvest; NH = normal harvest; LH = late harvest; DH = delayed harvest.

**

Fa = F. avenaceum; Fe = F. equiseti; Fg = F. graminearum; Fp = F. poae; Fs = F. sporotrichioides.

†

Means followed by the same letter in a column under each factor are not significantly different at P = 0.05 (LSD).

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Among the three cultivars used, ‘OAC Baxter’ had the lowest levels of seed-borne Fusarium spp. (7.8%) and DON concentration (0.19 ppm); it was significantly different from ‘Brucefield’ for both parameters, and from ‘AC Vision’ for all Fusarium spp. (Table 4). The three cultivars ranked the same in their level of seed infection by F. avenaceum,F. graminearum, and F. sporotrichioides, and were not significantly different for seed-borne F. equiseti and F. poae.

Among the three locations over the 2 yr, the incidence of the five most common species, all Fusarium spp., and DON concentration was significantly higher at the Ottawa site than at Emo and New Liskeard (Table 4). For a single yr over the three locations, the incidence of F. equiseti, F. poae, F. sporotrichioides, and all Fusarium spp. was significantly lower in 2004 than 2005. However, the incidence of F. graminearum and DON concentration was significantly higher in 2004 than in 2005 (Table 4).

Correlation analysis revealed that the incidence of F. sporotrichioides and all Fusarium spp. increased significantly with delayed harvest (Table 5). The incidence of F. avenaceum correlated positively with that of F. sporotrichioides, and that of F. sporotrichioides with all Fusarium spp. The incidence of F. equiseti,F. graminearum, F. poae, and DON concentration did not correlate with harvesting date.

Table 5

Correlation coefficients relating harvest date and incidence of the five most common seed-borne Fusarium spp. and deoxynivalenol (DON) concentration in barley in northeastern Ontario in 2004 and 2005.

Correlation coefficients relating harvest date and incidence of the five most common seed-borne Fusarium spp. and deoxynivalenol (DON) concentration in barley in northeastern Ontario in 2004 and 2005.
a

Fa = F. avenaceum; Fe = F. equiseti; Fg = F. graminearum; Fp = F. poae; Fs = F. sporotrichioides; Total = Sum of all Fusarium spp.

*, **

Significant at P < 0.05 and P < 0.01 levels, respectively. The correlation coefficients were calculated using treatment means with three degrees of freedom (n = 5).

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Discussion

This study demonstrated that F. equiseti and F. sporotrichioides were the two predominant seed-borne Fusarium spp. in barley in northeastern Ontario and that the incidence of F. sporotrichioides and all Fusarium spp. increased with a delayed harvest date (Tables 2 and 4). The five most common Fusarium spp. observed in the present study are the same as those recorded for causing FHB on barley in central and eastern Ontario in 2004 and 2005 (Xue et al. 2005, 2006b). Fusariumgraminearum was recovered from only less than 1% of the seed and ranked as the 4th most common species following F. equiseti, F. sporotrichioides, and F. poae in the present study. The relatively low incidence of F. graminearum recorded in the present research was likely related to the cool weather prevailing in June, July and August at the testing locations in 2004 and the hot and dry weather conditions for the same period in 2005. Weather conditions across the province during growing seasons in both 2004 and 2005 were less favourable to the endemic of F. graminearum, but likely favoured the presence of F. equiseti, F. sporotrichioides, and F. poae. As a result, both seasons were considered non-epidemical years for FHB in Ontario (Xue et al. 2005, 2006b). The results suggest that although F. graminearum is the principal cause of FHB in barley in Canada, it might be less prevalent than other etiological components of FHB under lighter disease pressure in a non-epidemic year in Ontario.

Deoxynivalenol is the major mycotoxin in FHB-infected kernels and is a food and feed safety concern for barley in eastern Canada (Choo et al. 2004). The DON concentration found in the present study was low and below the Canadian legislative limit of 1.0 mg kg‑1 for swine, young calves and lactating dairy animals (FAO 2003), likely as a result of the low incidence of F. graminearum, the most important DON-producing species. Although the concentration of DON and the incidence of F. graminearum were not affected by the harvest date, they were significantly affected by cultivar, location, and year (Tables 3 and 4). For instance, a significantly higher level of DON and seed-borne infection by F. graminearum was found on ‘Brucefield’ than on the other cultivars; in Ottawa than in the other locations; and in 2004 than in 2005 (Table 4). These results are in agreement with Langseth and Stabbetorp (1996) who conducted a similar experiment in Norway and reported that the effects of year, location, and variety were more important than the effect of harvest date for DON concentration. The results of the present study are also similar to those reported by Xue et al. (2004) for spring wheat in eastern Ontario, where a delayed harvest had no effect on DON concentration in the harvested grain.

The three barley cultivars used in the present study showed differences in the incidence of F. graminearum and DON concentration in kernels, but the genotypic variability was not affected significantly by date of harvest, location and year, either individually or by a combination of these factors, except for the difference in DON concentration between cultivars, which was affected by location (Table 3). The varietal differences observed were similar to the reaction of these cultivars to FHB as observed in cultivar performance trials. ‘AC Vision’ and ‘OAC Baxter’ appear to be moderately resistant while ‘Brucefield’ appears to be susceptible (Xue, unpublished data). The results confirmed that genotypic variation in the levels of seed-borne F. graminearum and DON occurs in harvested grain, and that breeding for resistance should be possible.

Current research on FHB management in barley in Canada has been focussing mainly on the control of F. graminearum and DON, recognized as the primary causal species of FHB and mycotoxin, respectively (Tekauz et al. 2000; Choo et al. 2004). Little attention has been given to the presence of other Fusarium spp. and mycotoxins. Results of the present research indicate that F. equiseti and F. sporotrichioides were the predominant Fusarium spp. in barley in non- epidemical years for FHB in Ontario. Both species are weak FHB pathogens of barley (Xue et al. 2006a) and produce low or non-detectable levels of DON (Desjardins 2006). However, these species as a group are important producers of other mycotoxins, including beauvericin, fusarochromanone, moniliformin, zearalenone, T-2, HT-2, and T-2 tetraol, which are more toxic than DON (Desjardins 2006; Hestbjerg et al. 2002; Salas et al. 1999; Thrane et al. 2004). The presence of the different mycotoxins, though at low levels, could pose chronic adverse health effects in humans and livestock through contaminated barley grain feed or products. However, only DON concentration was determined in this study and the possible presence of other Fusarium mycotoxins and their concentration in the harvested grain as affected by harvest date were not examined. The potential role of the weakly pathogenic species F. equiseti and F. sporotrichioides as competitors of F. graminearum on the barley spike and their possible role in reducing the production of DON by F. graminearum may be worthy of future studies. Additional research is also needed to define the geographic distribution and importance of F. equiseti and F. sporotrichioides as contaminants of barley grain in Ontario and the possible negative effects if multiple mycotoxins contaminate grain.