Abstract
Fusarium graminearum is the principal cause of fusarium head blight in North America, a disease that has caused severe losses in yield and quality of cereals. In North America, the vast majority of F. graminearum isolates produce 3- or 15-acetyl deoxynivalenol (ADON) in addition to DON. Until recently, 15-ADON isolates predominated, but a rapid shift from 15-ADON to 3-ADON producers in Canada and north central USA has been documented. In order to better understand the effect of this population shift on relative aggressiveness of isolates and mycotoxin accumulation, we tested a total of 58 isolates for 3- and 15-ADON production on two Canadian spring wheat cultivars, ‘Roblin’ (susceptible) and ‘5602 HR’ (moderately resistant). In Experiment 1, three isolates from the Canadian provinces of Manitoba, Saskatchewan and Alberta, each of which produced either 15-ADON or 3-ADON, were tested using spray inoculation. In Experiment 2, 20 isolates which produced 15-ADON and 20 which produced 3-ADON from Manitoba, were tested using point inoculation. There were no significant differences in aggressiveness among isolates based either on geographic origin or mycotoxin type. Analysis of seeds from inoculated heads by gas chromatography/mass spectrometry indicated that the 3-ADON producing isolates had significantly higher DON levels than the 15-ADON isolates in ‘Roblin’ after both spray and point inoculation and in ‘5602HR’ after point inoculation. DON levels following point inoculation by 15-ADON isolates were similar in the two cultivars. The 15-ADON isolates from Alberta produced less DON than 15-ADON isolates from Manitoba and Saskatchewan. Consistently, more ADON was produced by 15-ADON isolates than by 3-ADON isolates. The results of the study suggest that if the percentage of 3-ADON isolates in Canada increases, DON levels in cereals are likely to increase in epidemic years.
Résumé
Fusarium graminearum est la principale cause de la brûlure de l'épi en Amérique du Nord, une maladie qui engendre d'importantes pertes tant sur le plan des rendements que de la qualité chez les céréales. En Amérique du Nord, la plupart des isolats de F. graminearum produisent du 3- ou du 15-acétyl déoxynivalénol (ADON), en plus du déoxynivalénol (DON). Jusqu'à récemment, les isolats produisant du 15-ADON dominaient, mais un remplacement rapide des producteurs de 15-ADON par ceux de 3-ADON s'est opéré et a été documenté au Canada et dans le centre-nord des États-Unis. Afin de mieux comprendre ce remplacement de populations quant à l'agressivité relative des isolats et à l'accumulation de mycotoxines, nous avons testé 58 isolats relativement à la production de 3- et de 15-ADON sur deux cultivars canadiens de blé de printemps : ‘Roblin’ (sensible) et ‘5602 HR’ (moyennement résistant). Au cours de l'Expérience 1, trois isolats provenant des provinces canadiennes du Manitoba, de la Saskatchewan et de l'Alberta, chacun produisant du 3- ou du 15-ADON, ont été testés par pulvérisation. Au cours de l'Expérience 2, 20 isolats qui produisaient du 15-ADON et 20, du 3-ADON provenant du Manitoba ont été testés par injection ponctuelle. Il n'y a pas eu de différence significative quant à l'agressivité chez les isolats, peu importe leur provenance ou le type de mycotoxine produit. L'analyse par couplage GC-SM des semences issues des épis inoculés a montré que les isolats produisant du 3-ADON présentaient des teneurs significativement plus élevées en DON que les isolats produisant du 15-ADON issus de ‘Roblin’, après avoir été inoculés par pulvérisation et injection, et dans le ‘5602 HR’, après inoculation par injection. Les teneurs en DON qui ont fait suite aux inoculations par injection d'isolats produisant du 15-ADON étaient semblables chez les deux cultivars. Les isolats en provenance de l'Alberta, produisant du 15-ADON, ont produit moins de DON que ceux du Manitoba et de la Saskatchewan. Invariablement, de plus grandes quantités d'ADON ont été produites par les isolats 15-ADON que par les isolats 3-ADON. Les résultats de l'étude suggèrent que, si le pourcentage des isolats produisant du 3-ADON augmente au Canada, les teneurs en DON dans les céréales augmenteront probablement durant les années d'épidémies.
Introduction
In North America, Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.) Petch) is the principal cause of fusarium head blight (FHB), a serious disease of small grain cereals that affects all aspects of the grain industry (Gilbert & Tekauz, Citation2000). The disease has caused cumulative losses of billions of dollars since 1993 when the province of Manitoba, Canada and the northern central states of the USA experienced the worst epidemic on record (McMullen et al., Citation1997; Ngange et al., Citation2004).
The fungus produces trichothecene toxins, predominantly deoxynivalenol (DON) and its acetylated derivatives 15-ADON or 3-ADON which may persist and accumulate in the grain. Both 3-ADON and 15-ADON are considered acutely toxic and believed to be occasionally present in crops, animal feed and human food (Pronk et al., Citation2002). 3-ADON affects dividing thymus, spleen and intestine cells and exerts immunotoxic activity (Pronk et al., Citation2002). In addition to these effects, 15-ADON also affects mitotic bone marrow cells and is known to affect animal feed consumption and rate of body weight gain (Pronk et al., Citation2002). Even though a tolerable daily intake (TDI) of 1 μg kg−1 body weight was temporarily established for DON in 1999 (Pronk et al., Citation2002), current data are insufficient to establish a TDI of this toxin. Thus these toxins pose a significant risk to the food supply for both human and animal consumption inhibiting eukaryotic protein synthesis and modifying immune function either by immunosuppression or by immunostimulation (Pronk et al., Citation2002; Pestka & Smolinski, Citation2005).
Until recently, the available data indicated that F. graminearum isolates with a 15-ADON chemotype were the only significant cause of FHB in North America (Abramson et al., Citation1993, Citation2001). However, genetically divergent groups of isolates collected between 1999 and 2000 were identified in Minnesota and North Dakota of which 3-ADON types accounted for 9.4% of the 288 isolates analyzed from these states (Gale et al., Citation2007). A further analysis of 492 isolates of F. graminearum collected in Canada between 1998 and 2004 also documented the presence of increasing numbers of isolates with the 3-ADON chemotype (Ward et al., Citation2008). The latter study demonstrated that groups defined by trichothecene chemotype represented distinct populations and that chemotype frequency in Canada was characterized by a pronounced cline in which the proportion of 3-ADON to 15-ADON isolates decreased from east to west, with the highest percentage (100%) found in Prince Edward Island, and the lowest (5.9%) found in Alberta. Ward et al. (Citation2008) concluded that the 3-ADON-producing F. graminearum population probably resulted from a transcontinental introduction and that this population is rapidly supplanting the original 15-ADON-producing population in parts of North America. Moreover, this study also documented that on average, 3-ADON isolates produced significantly more DON in vitro, were faster growing on agar and produced significantly more conidia than 15-ADON-producing isolates.
Because only preliminary aggressiveness tests using spray-inoculation were reported (Ward et al., Citation2008), the objectives of our work were to expand aggressiveness testing on spring wheat to include point inoculation for comparison with the spray method and to determine if isolates of different provenance and trichothecene chemotype differ in their ability to incite disease and accumulate DON, 3-ADON or 15-ADON in planta.
Materials and methods
Two wheat cultivars, ‘5602 HR’ rated as moderately resistant and ‘Roblin’ rated as susceptible to fusarium head blight (Seed Manitoba, Citation2008), were seeded in 13-cm diameter plastic pots containing Sunshine Mix #4 soil-less mix (Sun Gro Horticulture, Sebo Beach, AB). A slow-release fertilizer (Osmocote, Scotts-Sierra Horticultural Products Co., Marysville, OH) was added at seeding according to manufacturer's instructions, after which plants were fed with a 20:20:20 (N, P, K) (Peters Professional, Scotts-Sierra Horticultural Products Co.) water-soluble fertilizer biweekly.
The isolates of F. graminearum used in the study were collected from wheat seed from the Prairie Provinces in 2004 and deposited in the ARS (NRRL) Culture Collection. provides their NRRL number, chemotype and experiment(s) in which they were used. Isolate histories are available through the ARS Culture Collection website (http://nrrl.ncaur.usda.gov/cgi-bin/usda). For each isolate, molecular chemotypes were predicted with the Luminex assay, a multilocus genotyping assay used to determine species and trichothecene chemotype (Ward et al., Citation2008).
Table 1. Origin, chemotype and experimental use of isolates of Fusarium graminearum.
Monoconidial derived isolates were cultured on potato dextrose agar (PDA, 250 g potatoes boiled, strained and broth made up to 1 L with tap water to which 20 g dextrose and 20 g agar (Difco, Detroit, MI) were added). For macroconidial increase, a five to eight day old colony of each isolate was cut into 1 cm2 pieces, placed in carboxymethyl cellulose broth (CMC, 1.0 g NH4NO3, 1.0 g KH2PO4, 0.5 g MgSO4.7H2O, 1.0 g yeast extract, 15 g CMC, 1 L H2O, 0.2 g streptomycin sulphate [Sigma-AldrichCo., St. Louis, MO]) and incubated under aeration for five to seven days. Inocula were diluted with sterile water and standardized to 5 × 104 macroconidia mL−1 using a hemacytometer. A drop of Tween® 20 (polyoxyethylene 20-sorbitan monolaureate, Fisher Scientific, Fair Lawn, NJ) was added per 100 mL inoculum to reduce water tension. New CMC cultures were prepared every two weeks and fresh inocula were made up from the CMC broth for each inoculation.
For Experiment 1, 18 isolates of F. graminearum, three isolates each that produced 15-ADON or 3-ADON from each of the Canadian provinces of Manitoba, Saskatchewan and Alberta, were tested for relative aggressiveness and toxin production on ‘5602 HR’ and ‘Roblin’. The experimental design was a 3-replicate randomized complete block, where each replicate consisted of a pot containing two or three plants of one cultivar. The experiment was repeated once. The experiments were conducted in a greenhouse with temperature ranges of 20–25 °C with supplemental lighting (16 h light/8 h dark). At anthesis, two to five wheat spikes per plant were spray-inoculated with 2–3 mL of a macroconidial suspension of one of the F. graminearum isolates using a Spraypump (Browne and Co. Ltd., Markham, ON). Inoculated spikes were covered with glassine bags for 48 h to promote a favourable environment for disease development and to prevent cross-contamination among F. graminearum isolates. Plants were kept in the greenhouse until maturity.
For Experiment 2, 20 isolates each from western Canada that produced 3-ADON or 15-ADON were tested for relative aggressiveness and toxin production on the same two hard red spring wheat cultivars. The experimental design was a 3-replicate randomized partial block, with each replicate consisting of a pot containing four plants of one cultivar. The experiment was conducted in growth cabinets with a photoperiod of 16 h light/8 h dark with initial temperatures of 13 °C (light) and 10 °C (dark) until plants were established. Temperatures were increased to 17 °C and 13 °C for light and dark, respectively, until heads emerged, after which temperatures were maintained at 23 °C and 17 °C, for light and dark periods, respectively, for the remainder of the experiment. Ten 15-ADON-producing isolates and 10 that produced 3-ADON were randomly selected for the first trial, and the remainder tested in a second trial. In a third trial, five 15-ADON and five 3-ADON producers were randomly selected to check for uniformity of results across trials. At anthesis, a floret, approximately a third of the way from the tip of the spike on two to three spikes per plant, was point-inoculated by placing 10 μL of a macroconidial suspension of an isolate onto the stigma of the floret using a micropipette. Two or three spikes per plant were inoculated and then covered with glassine bags for 48 h. The average number of spikes tested for each cultivar/isolate was 27.
In both experiments, disease severity was scored at 14 d and 21 d after inoculation (dai) and recorded as percentage infected spikelets. All data were analyzed using SAS Proc Mixed procedure (SAS Institute Inc.). DON and ADON values were square-root transformed. For Experiment 1, province, chemotype and province × chemotype were fixed effects and replicate and isolate (province × chemotype) were set as random effects. A separate analysis of variance was carried out for each cultivar. For Experiment 2, fixed effects were cultivar, chemotype and cultivar × chemotype and random effects were trial and isolate (chemotype).
At maturity, inoculated wheat spikes from both experiments were harvested, hand-threshed, and ground in a Bunn coffee grinder (espresso setting, Bunn® Canada, Bunn Corporation, Aurora, ON). Samples were analyzed for DON, nivalenol, fusarenon-X, 3-ADON, 15-ADON, DAS, HT-2 and T-2 with a limit of quantification (LOQ) of 0.05 ppm for each compound using GC/MS and following the standard Grain Research Laboratory GRL (Canadian Grain Commission) method for Fusarium trichothecene mycotoxins (Tacke & Casper, Citation1996). A slight modification was needed to accommodate the small sample size available for analysis. For each test, the ground sample was extracted with an acetonitrile-water mixture on a reciprocating shaker. The extract was cleaned by elution through a C18-alumina clean-up column and an aliquot was evaporated to dryness and derivatized with a trimethylsilylimidazole-trimethylchlorosilane mix (TMSI-TMCS). The trichothecenes were identified and quantified using a DB-35MS capillary column in an Agilent 6890 Gas Chromatograph with a 5973 Mass Selective Detector. For quality assurance purposes, a normal wheat sample (no detectable DON present), a fortified wheat normal, a certified reference material and an internal DON check sample were routinely included in the same series as the subject samples. A new set of derivatized standards were prepared with every run for calibration purposes (Tacke & Casper, Citation1996).
Results
As expected, FHB severity was lower on the moderately resistant cultivar ‘5602HR’ than on the susceptible cultivar ‘Roblin’. There were no significant differences in aggressiveness among F. graminearum isolates that could be attributed to isolate origin, chemotype or method of inoculation at either 14 or 21 dai ( and ). However, for Experiment 1, there were significant differences in total DON accumulation (mean accumulation over all isolates tested) for cultivar ‘Roblin’ after spray inoculation and significantly more DON was produced by 3-ADON than 15-ADON isolates, 32.6 and 25.8 ppm, respectively (). Levels of the acetyl derivatives were very low compared with the DON values (). Furthermore, the amount of 3-ADON produced by 3-ADON isolates was significantly less than the amount of 15-ADON produced by 15-ADON isolates, 0.80 and 1.42 ppm, respectively. DON production was lower in isolates from Alberta (a and ). On closer examination, the lower DON values for Alberta isolates were from 15-ADON isolates, with a mean value of 17.8 ppm, compared to 28.4 and 32.2 ppm for 15-ADON isolates from Manitoba and Saskatchewan, respectively. The DON values for Alberta 3-ADON isolates were 30.7 ppm compared with 32.7 and 34.4 ppm for 3-ADON isolates from Manitoba and Saskatchewan, respectively. Based on their known trichothecene chemotype, all isolates produced the expected 3-ADON or 15-ADON analogs in planta. In Experiment 2, after point-inoculation, differences between cultivars were smaller, but still significant for certain variables. Total DON (mean of 3- and 15-ADON isolates combined) accumulated in ‘Roblin’ at significantly higher levels than in ‘5602HR’. More DON was also detected in ‘Roblin’ after inoculation with 3-ADON isolates alone than in ‘5602HR’, but there was no difference in levels of DON in the two cultivars after inoculation with 15-ADON producing isolates (b and ). Again, for both cultivars, significantly more 15-ADON was produced by 15-ADON isolates than by 3-ADON isolates.
Table 2. Least squares means for fusarium head blight (FHB) severity on spring wheat cultivars 5602HR (moderately resistant) and Roblin (susceptible) following spray-inoculation with macroconidia of isolates of Fusarium graminearum from Alberta, Manitoba and Saskatchewan producing either 15- or 3-ADON
Table 3. Least squares means for fusarium head blight (FHB) severity on spring wheat cultivars 5602HR (moderately resistant) and Roblin (susceptible) following point-inoculation with macroconidia of isolates of Fusarium graminearum from Manitoba producing either 3- or 15-ADON
Table 4. Type 3 tests of fixed effects for accumulation in wheat cultivars 5602HR and Roblin of deoxynivalenol (DON) and 3- and 15-acetylated DON (3-, 15-ADON, chemotype) following (a) Experiment 1: spray-inoculation with isolates of Fusarium graminearum from Manitoba, Saskatchewan and Alberta and (b) Experiment 2: point- inoculation with 40 Manitoba isolates
Table 5. Least squares means for parts per million (ppm) deoxynivalenol (DON) and acetylated DON (ADON) accumulation in cultivars 5602 HR and Roblin after spray-inoculation with Fusarium graminearum isolates from the Prairie Provinces, producing either 3- or 15-ADON
Table 6. Least squares means for parts per million (ppm) (a) deoxynivalenol (DON) and (b) acetylated DON (ADON) accumulation in cultivars 5602 HR and Roblin after point-inoculation with Fusarium graminearum isolates producing either 3- or 15-ADON
Discussion
There were obvious differences in disease severity levels resulting from either method of inoculation on ‘Roblin’. These two methods of inoculation are used in FHB disease screening to identify two types of resistance: type I – resistance to establishment of initial infection, and type II – resistance to hyphal invasion of the host, or disease spread within the spike (Schroeder & Christenson, Citation1963). Other types of resistance have also been described, including resistance to kernel infection, tolerance to yield reduction, and ability to degrade toxins (Mesterházy, Citation1995), but types I and II were our focus. Spray inoculation more closely mirrors the natural situation, as air-borne spores are deposited randomly on the exterior of wheat spikes (Schmale et al., Citation2005), and this method is used to assess type I resistance. Point inoculation, the act of opening the floret and placing a spore droplet directly onto the stigma, or using a hypodermic syringe to place inoculum directly into the floret, is a highly artificial method which has been used extensively in greenhouse research to measure spread of disease within a spike, or type II resistance (Gilbert & Tekauz, Citation2000). The measure of type II resistance was essential to the study because trichothecenes function in spread but not in initial infection and DON is a known virulence factor (Desjardins et al., Citation1996).
Spray inoculation caused higher levels of disease than point inoculation both at 14 and 21 dai in ‘Roblin’. Spray inoculation incited less disease than point inoculation at 14 dai on ‘5602HR’, but not at 21 dai. As such, ‘5602HR’ appeared to resist initial infection after spray inoculation. However, disease spread within the spike occurred between 14 and 21 dai. The rapid and severe development of disease in ‘Roblin’ is indicative of its lack of resistance to both initial infection and subsequent disease spread within the spike.
The GC/MS results for the fortified and certified reference material samples were within the expected concentration ranges. Samples were analyzed for eight trichothecenes, but DON and 3- and 15-ADON were the only toxins identified in this study. Recent studies report DON-3-glucoside as a toxin of potential importance in barley (Kostelanska et al., Citation2009) and its presence cannot be excluded as a contaminant of wheat. However, available data do not indicate that glucosides make up a large proportion of the total DON in Canadian barley (Kostelanska et al., Citation2009). Significantly more 15-ADON was produced by 15-ADON producing isolates than 3-ADON by 3-ADON producing isolates, but because both acetylated forms were isolated at low levels compared to DON, actual DON levels generated by 3- and 15-ADON-producing isolates are of more critical concern. In this study, the DON levels were not significantly different from each other in the moderately resistant cultivar ‘5602HR’, but 3-ADON isolates produced significantly more DON than 15-ADON isolates in the susceptible cultivar ‘Roblin’. Given that most registered wheat varieties in the Prairie Provinces are rated poor or very poor in resistance to FHB (Seed Manitoba, Citation2008), and that a rapid increase in 3-ADON-producing isolates has been documented (Gale et al., Citation2007; Ward et al., Citation2008), this increase should be a significant concern to the grain industry.
15-ADON isolates from Alberta produced less DON in ‘Roblin’ than those from Manitoba and Saskatchewan, while levels of DON produced by 3-ADON producers from all provinces were not different. Occurrence of FHB in western prairie regions has increased since 1993, when Manitoba experienced the most severe FHB epidemic on record (Gilbert & Tekauz, Citation2000), but levels are still relatively low in Alberta (Clear & Patrick, Citation2009). It is unknown why the 15-ADON isolates from Alberta produced less DON in planta than 15-ADON isolates from the other prairie provinces.
In summary, F. graminearum produced twice as much DON in the susceptible cultivar ‘Roblin’ as moderately resistant cultivar ‘5602HR’ after spray inoculation. Differences in DON accumulation in both cultivars were smaller following point inoculation, but differences were still significant when all isolates were combined, and for 3-ADON isolates alone, but not for 15-ADON isolates. In light of the fact that isolates with the 3-ADON chemotype increased 14-fold between 1998 and 2004 in western Canada (Ward et al., Citation2008), there is a concern regarding potentially high levels of DON in wheat. It appears that while isolates producing either 15-ADON or 3-ADON do not differ in their pathogenicity, the 3-ADON isolates produce on average higher levels of DON, which may exacerbate problems associated with food safety for humans and livestock.
Acknowledgements
The authors gratefully acknowledge Ron Kaethler, Kirsten Slusarenko and Courtney Leclerc for technical support and the Western Grains Research Foundation and Matching Investments Initiative for financial support.
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