Sensitivity of western Canadian Pyrenophora teres f. teres and P. teres f. maculata isolates to propiconazole and pyraclostrobin

Abstract

Pyrenophora teres f. teres (Ptt) and Pyrenophora teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can be managed in western Canada with fungicides containing propiconazole and pyraclostrobin. Given the potential for development of fungicide resistance, the sensitivity of a collection of Ptt and Ptm isolates to propiconazole and pyraclostrobin was evaluated using microtitre plate bioassays. The concentration of propiconazole needed to inhibit fungal growth by 50% (EC₅₀) was 1.5 mg L⁻¹ for Ptt and 2.3 mg L⁻¹ for Ptm, while the EC₅₀ of pyraclostrobin was 0.015 mg L⁻¹ for Ptt and 0.024 mg L⁻¹ for Ptm. Subsequently, 39 Ptt and 27 Ptm isolates were screened with discriminatory doses of 5 mg propiconazole L⁻¹ and 0.15 mg pyraclostrobin L⁻¹. Inhibition of growth as a result of propiconazole was 12–95% for Ptt and 48–92% for Ptm; growth inhibition as a result of pyraclostrobin was 40–100% and 24–100%, respectively. Two Ptt isolates were insensitive to propiconazole, while one Ptm isolate was insensitive to pyraclostrobin. The latter also showed decreased sensitivity to propiconazole. The identification of net blotch isolates insensitive to these fungicides emphasizes the need for farmers to employ integrated crop management strategies to avoid fungicide resistance build-up.

Résumé

Pyrenophora teres f. teres (Ptt) et Pyrenophora teres f. maculata (Ptm), les agents causaux respectifs des formes réticulée et helminthosporienne de la tache réticulée de l’orge, peuvent être maîtrisés dans l’Ouest canadien avec des fongicides à base de propiconazole et de pyraclostrobine. Compte tenu de la capacité de ces agents à développer une résistance aux fongicides, la sensibilité d’une collection d’isolats de Ptt et de Ptm au propiconazole et à la pyraclostrobine a été évaluée par essais biologiques au moyen de microtitrage sur plaque. La concentration de propiconazole requise pour inhiber de 50% la prolifération fongique (EC₅₀) était de 1.5 mg L⁻¹ pour Ptt et de 2.3 mg L⁻¹ pour Ptm, tandis que l’EC₅₀ pour la pyraclostrobine était de 0.015 mg L⁻¹ pour Ptt et de 0.024 mg/l⁻¹ pour Ptm. Par la suite, 39 isolats de Ptt et 27 de Ptm ont été criblés avec des doses discriminatoires de 5 mg L⁻¹ de propiconazole et de 0.15 mg L⁻¹ de pyraclostrobine. L’inhibition de la prolifération engendrée par le propiconazole était de 12 à 95% chez Ptt et de 48 à 92% chez Ptm; l’inhibition de la prolifération engendrée par la pyraclostrobine était de 40 à 100% et de 24 à 100%, respectivement. Deux isolats de Ptt étaient insensibles au propiconazole, tandis qu’un isolat de Ptm l’était à la pyraclostrobine. Ce dernier a également affiché une sensibilité réduite au propiconazole. L’identification des isolats de tache réticulée insensibles à ces fongicides souligne l’importance pour les producteurs d’utiliser des stratégies de gestion intégrée des cultures afin d’entraver le développement de la résistance aux fongicides.

Keywords:

• barley
• net blotch
• propiconazole
• pyraclostrobin

Mots clés:

• Orge
• propiconazole
• pyraclostrobine
• tache réticulée

Introduction

The necrotrophic fungus Pyrenophora teres Drechs. (anamorph: Drechslera teres [Sacc.] Shoem.) has two morphologically identical but genetically distinct forms: P. teres f. teres (Ptt) and P. teres f. maculata (Ptm), which incite the net form of net blotch (NFNB) and spot form of net blotch (SFNB) of barley (Hordeum vulgare L.), respectively (McDonald 1963; Smedegard-Petersen 1978). These are among the most economically important foliar diseases across the major barley growing regions of the world, including western Canada with 2.5 million hectares planted to barley in 2016 (Tekauz 1990; Steffenson 1997; Liu et al. 2011; Statistics Canada 2016). Yield losses of 10–40% are typical in severe cases of NFNB, although Ptt has the potential to cause total yield loss (Steffenson et al. 1991; Steffenson 1997; Murray & Brennan 2010). Likewise, yield losses of up to 44% were reported for SFNB (Jayasena et al. 2007). Crop rotation, fungicide application and the deployment of resistant cultivars can be used to manage net blotch of barley in western Canada (van den Berg & Rossnagel 1991; Turkington et al. 2004, 2005, 2006, 2011, 2012, 2015). However, many western Canadian farmers have adopted a canola-cereal-canola rotation, which is not sufficient to manage barley leaf spot diseases such as net blotch (Mathre 1997; Tekauz 2003; Turkington et al. 2011, 2015). Moreover, many two and six row barley cultivars, especially malting types, are susceptible to net blotch (Alberta Government 2016a). As a consequence, many farmers routinely use fungicides as their main tool for managing barley leaf diseases (Turkington et al. 2011, 2015; Poole & Arnaudin 2014). Most fungicides are registered for use on barley from stem elongation to head emergence, but also are registered for tank mixing with herbicides and may be applied at earlier crop growth stages (Turkington et al. 2015; Alberta Government 2016a). When weather conditions are conducive to disease development and the barley cultivar is susceptible, many farmers will follow an application of fungicide that is tank-mixed with herbicide(s) with a second fungicide-only application between flag leaf and head emergence (Turkington et al. 2015; Alberta Government 2016a).

Both Ptt and Ptm are stubble-borne pathogens, producing asexual conidia and sexual pseudothecia, which produce ascospores on overwintered infected crop debris (Piening 1961, 1968; van den Berg & Rossnagel 1991; Peever & Milgroom 1994; Duczek et al. 1999). Based on a 1:1 ratio of the two mating types for Ptt and Ptm in western Canada (Akhavan et al. 2015), it is likely that both forms of the net blotch pathogen undergo regular cycles of sexual reproduction in this region. Moreover, Ptt and Ptm populations in western Canada are genetically diverse, with almost 90% of the population consisting of distinct haplotypes (Akhavan et al. 2016b). These diverse Ptt and Ptm populations with mixed reproduction and an outcrossing mating system fall into the category of pathogens that have a greater likelihood of quickly adapting to fungicides with a single mode of action (McDonald & Linde 2002), ultimately resulting in a reduction or loss of fungicide effectiveness.

The fungicides propiconazole and pyraclostrobin are commonly applied to manage net blotch on barley in western Canada (Alberta Government 2016b). Propiconazole has been used on various crops including barley since the early 1990s as the foliar fungicide Tilt (propiconazole, 250 g L⁻¹; Syngenta Canada Inc.) (Xue et al. 1994; Thomas 1997). Propiconazole is a triazole belonging to the demethylation inhibitors (DMIs) group. This group of fungicides inhibits 14α–sterol demethylase, an enzyme used by fungi to synthesize ergosterol, which in turn is needed for fungal cell membrane development (Dahl et al. 1987; reviewed in Parker et al. 2014). Excessive use of DMIs has been reported to select for insensitivity in cereal pathogens such as Ptt (Mair et al. 2016), Blumeria graminis DC Speer f. sp. hordei Marchal (Délye et al. 1998), Zymoseptoria tritici (Desm.) Quaedvlieg & Crous [formerly known as Mycosphaerella graminicola (Fuckel) Schroeter] (Leroux et al. 2007; Bean et al. 2009; Cools & Fraaije 2013), and Fusarium graminearum Schwabe [teleomorph: Gibberella zeae (Schwein.) Petch] (Talas & McDonald 2015), increasing the level of insensitivity within these pathogen populations (Kuck et al. 2012). Insensitivity to fungicides usually results from point mutations in the gene(s) encoding the proteins targeted by specific fungicides. Alterations in the CYP51 gene in plant pathogens were reported to be one of the major mechanisms resulting in reduced sensitivity towards DMIs (reviewed in Parker et al. 2014). Mair et al. (2016) reported the occurrence of insensitivity among Ptt isolates collected from Australia against multiple DMI compounds. This insensitivity was correlated with both a mutation in the Cyp51A gene and overexpression of both the Cyp51A and Cyp51B genes, which are paralogues of the Cyp51 gene. Rallos and Baudoin (2016) also reported that CYP51 over-expression and target-site mutation contributed to insensitivity in Erysiphe necator.

Pyraclostrobin is classified within the strobilurin or quinone outside inhibitors (QoI) group of fungicides, and is the active ingredient in the fungicide Headline 250 EC (BASF Canada, Mississauga, ON, Canada). Products containing pyraclostrobin have been registered and utilized by western Canadian farmers for cereal leaf spot management since the early 2000s (BASF 2003; Government of Canada: Health Canada Pest Management Regulatory Agency 2011). Marzani (2011) investigated the effectiveness of several QoI fungicides on Ptt isolate growth and reported that pyraclostrobin was the most active QoI, resulting in high growth inhibition. Strobilurins have a single site of activity and prevent mitochondrial respiration in fungi by binding to the Qo site of cytochrome bc1, interfering with electron transfer in complex III (Sauter et al. 1999; Bartlett et al. 2002; Grasso et al. 2006). The Fungicide Resistance Action Committee (FRAC 2016) has labelled strobilurins as compounds at a high risk of selecting for insensitivity in pathogen populations.

In the current study, a microtitre bioassay was used to assess the level of fungicide sensitivity. This method is more efficient relative to other more conventional techniques, such as the agar radial growth bioassay (Tremblay et al. 2003). The microtitre bioassay is an approved method of assessing fungicide sensitivity for P. teres (FRAC 2006a, 2009) and other species of Pyrenophora (FRAC 2006b) to DMI, QoI and succinate dehydrogenase inhibitor (SDHI) fungicides. It involves measurement of the optical density of fungal tissues in the presence of different concentrations of chemicals in a 96-well microplate, followed by comparison of the growth of the same isolate in non-amended controls (Pijls et al. 1994; Tremblay et al. 2003). For DMIs, discriminatory doses from near the half-maximal effective concentration (EC₅₀) value (Smith et al. 1991; Peever & Milgroom 1992, 1993) to 100 times greater than the baseline EC₅₀ (Villani & Cox 2011) have been employed. The inhibition of fungal growth for a single fungicide concentration close to the EC₅₀ value was shown to be an appropriate response to measure with DMI fungicides (Smith et al. 1991) and was also shown in P. teres to be highly correlated with EC₅₀ values determined with a series of concentrations (Peever & Milgroom 1992, 1993). However, Romero & Sutton (1997) suggested that a discriminatory dose slightly higher than both the mean EC₅₀ value and the highest detected EC₅₀ is more useful for monitoring sensitivity to DMI fungicides. For QoIs, discriminatory doses from near the EC₅₀ value (Mondal et al. 2005) to almost 70 times greater than the baseline EC₅₀ (Fraser et al. 2016) have been typically employed.

Information on the sensitivity of local isolates of P. teres to fungicides used commercially is required in the formulation of integrated net blotch management strategies. Although both propiconazole and pyraclostrobin fungicides have been applied extensively in barley fields in western Canada, no information is available on the fungicide sensitivity of P. teres populations to these compounds. The objectives of the current study were to: (i) determine the EC₅₀ of propiconazole (DMI) and pyraclostrobin (QoI) for a set of Ptt and Ptm isolates from western Canada; (ii) test the null hypothesis of a lack of significant differences between the EC₅₀ values obtained for Ptt and Ptm; (iii) quantify the degree of sensitivity to propiconazole and pyraclostrobin in a representative collection of Ptt and Ptm isolates from commercial barley crops across western Canada; and (iv) test the null hypothesis of a lack of significant differences in propiconazole and pyraclostrobin sensitivity among Ptt and Ptm isolates from the three western Canadian provinces of Alberta, Saskatchewan and Manitoba.

Materials and methods

Fungal isolation and preparation of inoculum

A total of 39 Ptt and 27 Ptm isolates collected from commercial fields and research plots across western Canada (Alberta, Saskatchewan and Manitoba) were studied. These included 36 Ptt and 21 Ptm isolates representing different clades identified in earlier genetic analyses (Akhavan et al. 2016b), plus an additional three isolates of Ptt and six isolates of Ptm from another collection made in 2012. All 39 Ptt and 27 Ptm isolates also were previously characterized for their virulence on a selection of barley genotypes and differentials (Akhavan et al. 2016a). The collection included 17 Ptt isolates from Alberta, 12 from Saskatchewan, and 10 from Manitoba, as well as eight Ptm isolates from Alberta, 10 from Saskatchewan, and nine from Manitoba (Supplementary Table 1). Two previously collected isolates of Ptt (WRS858 and WRS102) and one of Ptm (WRS857), which were obtained from Dr A. Tekauz (Agriculture and Agri-Food Canada, Winnipeg, MB), were also included for comparison in experiments to test the sensitivity of Ptt and Ptm isolates to propiconazole and pyraclostrobin. These three isolates were collected prior to registration of both propiconazole and pyraclostrobin in Canada. Isolate WRS102 was originally collected from Saskatchewan (Metcalfe et al. 1970), while WRS858 and WRS857 were collected from Manitoba (Tekauz & Mills 1974; Ho et al. 1996).

Table 1. The effective concentration of propiconazole (mg L⁻¹) required to inhibit fungal growth by 50% (EC₅₀) in eight isolates each of Pyrenophora teres f. teres (Ptt) and P. teres f. maculata (Ptm).

Ptt	Experiment 1	Experiment 2	Average	Ptm	Experiment 1	Experiment 2	Average
MB15	0.842	0.780	0.811	MB22	1.182	0.993	1.088
SK26	1.011	0.763	0.887	SK88	1.956	2.289	2.123
ABV01	1.236	0.786	1.011	SK69	1.367	3.052	2.210
SK24	1.340	0.876	1.108	AB82	2.064	2.453	2.259
ABV28	1.383	1.622	1.503	MB23	2.705	1.821	2.263
MB10	1.862	1.717	1.790	AB61	1.726	2.375	2.051
AB01	2.334	2.194	2.264	MBV37	2.116	2.809	2.463
AB38	3.043	2.935	2.989	SKV12	4.322	3.052	3.687
Average	1.632	1.459	1.545	Average	2.180	2.356	2.268

These isolates were randomly selected from across western Canada to establish a baseline sensitivity to propiconazole. A paired t-test indicated that there was no significant difference between EC₅₀ values calculated in each of the two independent experiments conducted for Ptt (P = 0.07) and Ptm (P = 0.61). Therefore, the two EC₅₀ values for each isolate were averaged. A log10-transformed fungicide concentration was used to linearize fungal growth inhibition (as a proportion of the control) to fit a regression line to estimate the EC₅₀ using probit analysis.

To obtain single-spore isolates, segments (10 mm × 5 mm) were cut from barley leaves with symptoms of NFNB or SFNB, and soaked in 50% ethanol for 15 s and in 2% sodium hypochlorite for 30 s, flushed with sterile water and placed on moistened filter paper in 9 cm-diameter plastic Petri dishes (Tekauz 1990). To enhance fungal sporulation on the leaves, the Petri dishes were incubated at 20 ± 0.5°C in a growth chamber with a 12 h photoperiod under a mixture of common fluorescent lamp light and a black light blue fluorescent lamp emitting near ultraviolet light at 368 nm. Following 3–5 days of incubation, single conidia of P. teres were transferred onto 10% V-8 juice agar (V-8 juice, 100 mL; CaCO₃ 3 g; Difco agar 20 g; distilled water 900 mL) amended with 50 mg L⁻¹ kanamycin, and incubated as above to form new colonies (Tekauz 1990). Single-spore isolates were preserved at −80°C in an ultra-low temperature freezer, either as 0.5 cm-diameter mycelial plugs in 50% sterile glycerol or as conidial suspensions in 25% sterile glycerol. Identification of the isolates as P. teres was based on the morphological features of the conidiophores and conidia and species-specific PCR analysis. The form of P. teres (Ptt or Ptm) was confirmed by form-specific PCR as described by Akhavan et al. (2015). To validate the results of the form-specific PCR, isolates were inoculated onto the barley cultivar ‘Steptoe’ as described previously by Tekauz (1990).

Conidial suspensions were produced as per the technique developed by Lamari & Bernier (1989) for Pyrenophora tritici-repentis (Died.) Drechs., as modified by Aboukhaddour et al. (2013). Briefly, the isolates were removed from a −80°C ultra-low temperature freezer, and the mycelial plugs were transferred onto fresh 10% V-8 juice agar supplemented with 50 mg L⁻¹ kanamycin in 9 cm-diameter Petri dishes, with one plug in the centre of each Petri dish. Alternatively, for those isolates preserved as a frozen conidial suspension, approximately 100 µL of thawed spore suspension was transferred onto the same medium. The Petri dishes were incubated in darkness at room temperature for 5–7 days, until the colonies were 4–5 cm in diameter. Approximately 10 mL of sterile distilled water was then added to each Petri dish, and the fungal mycelium was flattened with the bottom of a sterile glass tube. The water was decanted and the Petri dishes were incubated overnight under fluorescent light at room temperature, followed by 24 h incubation in darkness at 15°C to encourage sporulation. The spore suspensions were prepared by adding 4–5 mL of sterile distilled water to the fungal colonies, and then gently dislodging the conidia from the conidiophores with the help of a sterile inoculation loop or an artist’s brush.

Calculation of the mean EC₅₀

Independent experiments were run to determine EC₅₀(s) for propiconazole and pyraclostrobin. Experiments were conducted using a completely randomized design (CRD) with three replicate wells assigned for each treatment. There were three replicate control wells per isolate that were not amended with fungicide. All experiments were repeated.

Fungal spore concentrations were estimated using a Fuchs Rosenthal Counting Chamber (Hausser Scientific, Blue Bell, PA) and manually adjusted to 1 × 10⁴ conidia per mL for both Ptt and Ptm. An aliquot of 100 µL of the spore suspension, containing approximately 1000 spores, was transferred into each well of a 96-well microtitre plate (Thermo Scientific, Waltham, MA, Catalog # 167008), which contained 100 µL of the fungicide solution prepared in 2× concentrated potato dextrose broth (PDB; Difco Laboratories, Detroit, MI). The combined solution was mixed thoroughly. Kanamycin (100 mg L⁻¹) and streptomycin (20 mg L⁻¹) were added to the concentrated media to avoid bacterial contamination. Technical grade propiconazole and pyraclostrobin fungicides were purchased from Sigma-Aldrich Corporation (USA) and were diluted to achieve end concentrations. The EC₅₀ of propiconazole was determined based on eight different concentrations of the fungicide (0, 0.25, 0.5, 1, 2, 3, 4 and 5 mg L⁻¹) and eight isolates each of Ptt and Ptm. These isolates were randomly selected from the full collection of 39 Ptt and 27 Ptm isolates. The EC₅₀ value of pyraclostrobin was determined based on eight different concentrations of the fungicide (0, 0.0015, 0.0075, 0.015, 0.075, 0.15, 0.45 and 1.05 mg L⁻¹) and 12 isolates each of Ptt and Ptm, with four isolates of each form randomly selected from each province.

The endpoint optical density (OD) of samples in the 96-well microtitre plates was measured at 405 nm, on a Molecular Devices SpectraMax M3 microtitre plate reader (Sunnyvale, CA), immediately after filling the plates. Plates were then incubated in darkness at 20°C for 7 days. Following incubation, the OD was measured and final values for absorbance were obtained by subtracting the values of the initial reading from those of the final reading. Subsequently, for each isolate-concentration replicate, growth inhibition was calculated using the equation: 1 – [(absorption for each treatment/absorption of non-amended control)], and then presented as a percentage.

Since some fungi exhibit in vitro insensitivity to the QoIs based on an alternative respiratory pathway using alternative oxidase 1 (AOX), a preliminary assessment was conducted to determine if the addition of salicylhydroxamic acid (SHAM), which blocks this pathway (Wood & Hollomon 2003; Miguez et al. 2004), was required. The pyraclostrobin sensitivity of three isolates each of Ptt and Ptm was assessed in the presence or absence of 100 mg L⁻¹ SHAM (99%; Sigma-Aldrich, St. Louis, MO). Although a paired t-test indicated no significant difference for the results obtained in the presence or absence of this chemical (results not shown), all pyraclostrobin bioassays were conducted in the presence of 100 mg L⁻¹ SHAM to avoid any potential alternative respiration, which could confound sensitivity assessments.

Evaluation of the sensitivity of Ptt and Ptm isolates

As in the case for the EC₅₀ assessment, independent experiments were performed for each fungicide and for each pathogen form, employing a completely randomized design (CRD) with three replicate wells per isolate × concentration combination. All experiments were repeated. There were three replicate control wells per isolate with no fungicide amendment. Each of the 39 Ptt and 27 Ptm isolates was tested using the microtitre bioassay procedure, described previously, at a discriminatory dose of 5.0 mg L⁻¹ for propiconazole, and 0.15 mg L⁻¹ for pyraclostrobin. For both fungicides, these concentrations provided overall growth inhibitions of almost 90% for Ptt and 86% for Ptm when averaged over the representative isolates used in the calculation of mean EC₅₀ values (Fig. 1). The dose response curve was not, however, used directly to calculate the average EC₅₀ values, so as to not violate the assumptions of normality and homogeneity of variance required to pool the data from individual isolates. Therefore, average EC₅₀ values were estimated as described in the data analysis section below. The discriminatory dose of propiconazole (5 mg L⁻¹) was 2.6× greater than the average EC₅₀ values calculated for the Ptt and Ptm isolates, and was 1.4 times greater than the highest calculated EC₅₀ value among the 16 examined P. teres isolates which was 3.69 mg L⁻¹ for Ptm isolate SKV12. The discriminatory dose of pyraclostrobin (0.15 mg L⁻¹) was 7.7× greater than the average EC₅₀ values calculated for Ptt and Ptm, and was 2.5 times greater than the highest calculated EC₅₀ value among the 24 examined P. teres isolates which was 0.061 mg L⁻¹ for Ptm isolate MBV37.

Fig. 1 Dose response curve of the mean fungal growth inhibition of representative Pyrenophora teres f. teres and P. teres f. maculata isolates from western Canada in response to propiconazole (a) and pyraclostrobin (b). Eight and 12 isolates each of Ptt and Ptm, respectively, were employed in the experiments with propiconazole (16 isolates in total) and pyraclostrobin (24 isolates in total). Three replicate wells were included for each combination of isolate and concentration. Experiments were conducted twice, and the mean fungal growth inhibitions were averaged. Growth inhibition is relative to non-amended control treatments.

Fungal isolates were grouped as sensitive to propiconazole and pyraclostrobin if growth was reduced by >70%, intermediate if growth was reduced by 30–70%, and insensitive if growth was reduced <30% (Thaher 2011; Bowness et al. 2016). For each isolate replicate, per cent growth inhibition was calculated as described above and averaged over the three replicates and the two experiments. All isolates for which reduced sensitivity was observed were examined further by treatment with higher discriminatory doses of each fungicide. In the case of propiconazole, all isolates with growth inhibition values of <50% at 5.0 mg L⁻¹ were also evaluated against discriminatory doses of 10 mg L⁻¹, 20 mg L⁻¹ and 100 mg L⁻¹ of the fungicide. In the case of pyraclostrobin, isolates with growth inhibition values of <50% at 0.15 mg L⁻¹ were further tested against a new discriminatory dose of 0.45 mg L⁻¹.

Data analysis

For each isolate, the EC₅₀ value was independently estimated by probit analysis (Finney 1971; Stammler et al. 2012) with SPSS statistical software (IBM SPSS Statistics for Windows, Version 23.0, IBM Corporation, Armonk, NY). The maximum likelihood procedure was used for linear regression analysis to fit the regression of the response versus the concentration (Marzani 2011). Briefly, a log₁₀-transformed fungicide concentration was used to linearize fungal growth inhibition (as a proportion of the control) and a regression line was fitted. The resulting linear regression equation was used to calculate the fungicide concentration at which fungal growth was inhibited by 50% (EC₅₀) for each isolate. Paired t-tests (PROC TTEST) were performed with SAS 9.3 (SAS Institute Inc., Cary, NC) to determine if the EC₅₀ values calculated for a set of isolates in an experiment were significantly (P < 0.05) different from the EC₅₀ values calculated for the same set when the entire test was repeated. Since there was no significant difference between the estimated values, the final EC₅₀ values of the eight Ptt and eight Ptm isolates (for propiconazole) and 12 Ptt and 12 Ptm isolates (for pyraclostrobin) were calculated by averaging the arithmetic means of the calculated EC₅₀ values from the two experiments. For analysis of the fungal growth inhibition data, the assumptions of normality and variance homogeneity of the residuals were tested using Shapiro–Wilk and Kolmogorov–Smirnov tests, and Leaven’s test, respectively (PROC UNIVARIATE) in SAS 9.3 (SAS Institute). The growth inhibition data were then subjected to a one-way analysis of variance (PROC GLM) to test the null hypothesis of a lack of significant difference in the propiconazole and pyraclostrobin sensitivities of Ptt and Ptm isolates from Alberta, Saskatchewan and Manitoba.

Results

Determination of EC₅₀ of propiconazole for Ptt and Ptm isolates

The mean EC₅₀ of propiconazole for the Ptt isolates was 1.5 mg L⁻¹ and ranged from 0.8 mg L⁻¹ for isolate MB15 to 3.0 mg L⁻¹ for isolate AB38. Similarly, the mean EC₅₀ of propiconazole for the Ptm isolates was 2.3 mg L⁻¹, and ranged from 1.1 mg L⁻¹ for isolate MB22 to 3.7 mg L⁻¹ for isolate SKV12 (Table 1). A two-tailed t-test indicated that the difference between the EC₅₀ values obtained for Ptt and Ptm was just above the P = 0.05 level of significance (P = 0.07); therefore, we failed to reject the null hypothesis of a lack of significant difference between the propiconazole EC₅₀ values obtained for Ptt and Ptm.

Assessing propiconazole sensitivity of Ptt and Ptm isolates

No significant differences were observed in the propiconazole sensitivity of Ptt or Ptm isolates collected from each of the western Canadian provinces of Alberta, Saskatchewan or Manitoba (P = 0.25 for Ptt and P = 0.15 for Ptm). Growth inhibition values in response to propiconazole (5 mg L⁻¹) among the 39 Ptt isolates ranged from 12.4% for isolate AB48 to 95.2% for isolate AB28. A mean growth inhibition of 78.7% was obtained for all of the Ptt isolates combined. Two isolates, AB48 and AB11, collected from the Lacombe and Bentley areas in central Alberta, had a growth inhibition of <30%. Another four isolates had growth inhibition values of 30–70%, while the growth inhibition of the remaining 33 isolates was 70–100% (Table 2, Fig. 2). Growth inhibition for the Ptt reference isolates WRS102 and WRS858 was 84.8% and 92.0%, respectively. The isolates AB48 and AB11 were designated as insensitive to the fungicide, based on the low growth inhibition observed, and also were assessed at discriminatory doses of 10 mg L⁻¹, 20 mg L⁻¹ and 100 mg L⁻¹ propiconazole. Both isolates had a growth inhibition of <50% at the 10 mg L⁻¹ dose, but growth inhibition at 20 mg L⁻¹ was >50%. Neither AB48 nor AB11 grew at a dose of 100 mg L⁻¹ propiconazole.

Table 2. Propiconazole sensitivity of Pyrenophora teres f. teres (Ptt) and P. teres f. maculata (Ptm) isolates collected in 2009–2012 from Alberta, Saskatchewan and Manitoba, Canada.

Ptt	Per cent growth inhibition	Sensitivity	Ptm	Per cent growth inhibition	Sensitivity
AB28	95.2	Sensitive	ABV14	92.1	Sensitive
MB10	93.3	Sensitive	MB22	91.7	Sensitive
MB14	93.3	Sensitive	AB61	91.6	Sensitive
ABV01	91.4	Sensitive	SK100	90.5	Sensitive
SK16	90.7	Sensitive	SK87	89.8	Sensitive
SK41	90.2	Sensitive	AB57	89.2	Sensitive
MB02	90.0	Sensitive	AB68	86.1	Sensitive
SK53	89.3	Sensitive	AB82	82.6	Sensitive
ABV28	89.1	Sensitive	MB16	81.8	Sensitive
AB53	88.3	Sensitive	AB58	80.9	Sensitive
SK03	88.1	Sensitive	MB32	80.6	Sensitive
AB01	87.9	Sensitive	MB24	79.3	Sensitive
MB05	87.6	Sensitive	SK73	77.2	Sensitive
ABV18	87.1	Sensitive	SK88	75.5	Sensitive
AB04	86.1	Sensitive	SKV08	74.2	Sensitive
AB35	86.0	Sensitive	SKV10	72.4	Sensitive
SK05	85.9	Sensitive	SKV12	72.3	Sensitive
SK26	85.8	Sensitive	AB74	72.0	Sensitive
AB06	85.6	Sensitive	MB23	71.4	Sensitive
MB04	85.6	Sensitive	SK69	70.4	Sensitive
SK24	84.9	Sensitive	MB21	67.3	Intermediate
SK52	81.3	Sensitive	AB79	66.1	Intermediate
MB11	79.4	Sensitive	MBV25	64.7	Intermediate
SK08	78.9	Sensitive	MBV37	58.7	Intermediate
MB01	78.5	Sensitive	MB26	49.2	Intermediate
AB51	78.0	Sensitive	SK64	48.4	Intermediate
MB06	77.7	Sensitive	SK60	48.2	Intermediate
MB03	76.4	Sensitive
SK33	76.3	Sensitive
AB38	74.7	Sensitive
AB34	72.7	Sensitive
SK01	71.4	Sensitive
SK07	70.2	Sensitive
AB12	68.6	Intermediate
MB15	64.2	Intermediate
AB16	63.2	Intermediate
AB32	62.6	Intermediate
AB11	22.1	Insensitive
AB48	12.4	Insensitive

Sensitivity to propiconazole was assessed based on the reduction in fungal growth in a microtitre plate bioassay at a discriminatory dose of 5 mg L⁻¹. The endpoint optical density (OD) of samples in the 96-well microtitre plates was measured at 405 nm immediately after filling the plates. Plates were then incubated in darkness at 20°C for 7 days. Following incubation, the OD was measured and final values for absorbance were obtained by subtracting the values of the initial reading from those of the final reading. Subsequently, for each of the three isolate replicates, growth inhibition was calculated using the equation: 1 – [(absorption for the treatment /absorption of non-amended control)], and then averaged. The experiments were then repeated and the mean of the two growth inhibition values for each isolate was presented as a percentage.

Fig. 2 Frequency distribution of inhibition of fungal growth in 39 Pyrenophora teres f. teres and 27 P. teres f. maculata isolates from western Canada in response to 5 mg L⁻¹ propiconazole in potato dextrose broth. Inhibition of fungal growth is expressed as a percentage relative to a control treatment in which no propiconazole was included.

For the Ptm isolates, growth inhibition in response to propiconazole (5 mg L⁻¹) ranged from 48.2% for isolate SK60 to 92.1% for isolate ABV14. The mean growth inhibition was 75.0% over all of the Ptm isolates examined. No isolates had growth inhibition values of <30%, while growth inhibition ranged from 30–70% for seven isolates and from 70–100% for the remaining 20 (Table 2, Fig. 2). Growth inhibition for the Ptm reference isolate WRS857 was 73.2%. Three isolates with growth inhibition values of 48–49% (MB26, SK64 and SK60) also were evaluated at discriminatory doses of 10 mg L⁻¹, 20 mg L⁻¹ and 100 mg L⁻¹ propiconazole. Only SK64 from Dollard, Saskatchewan, had a growth inhibition of <50% at 10 mg L⁻¹ or 20 mg L⁻¹ propiconazole, but did not grow at a dose of 100 mg L⁻¹.

Determination of EC₅₀ of pyraclostrobin for Ptt and Ptm isolates

The mean EC₅₀ of pyraclostrobin for the Ptt isolates was 0.015 mg L⁻¹, and ranged from 0.002 mg L⁻¹ for isolate MB03 to 0.059 mg L⁻¹ for isolate SK24. In the case of the Ptm isolates, the mean EC₅₀ of pyraclostrobin was 0.024 mg L⁻¹, ranging from 0.008 mg L⁻¹ for isolate SK69 to 0.061 mg L⁻¹ for isolate MBV37 (Table 3). A two-tailed t-test indicated that the difference between the EC₅₀ values obtained for Ptt and Ptm was just above the P = 0.05 level of significance (P = 0.06). Therefore, as was the case for propiconazole, we failed to reject the null hypothesis of a lack of significant difference between the pyraclostrobin EC₅₀ values obtained for Ptt and Ptm.

Table 3. The effective concentration of pyraclostrobin (mg L⁻¹) required to inhibit fungal growth by 50% (EC₅₀) in 12 isolates each of Pyrenophora teres f. teres and P. teres f. maculata.

Ptt	Experiment 1	Experiment 2	Average	Ptm	Experiment 1	Experiment 2	Average
MB03	0.002	0.003	0.002	SK69	0.004	0.012	0.008
MB10	0.003	0.001	0.002	AB74	0.009	0.01	0.009
MB01	0.007	0.001	0.004	AB58	0.005	0.014	0.01
AB38	0.002	0.006	0.004	MBV25	0.011	0.013	0.012
SK01	0.006	0.009	0.008	AB82	0.007	0.024	0.016
ABV28	0.01	0.007	0.009	MB23	0.013	0.019	0.016
SK53	0.009	0.01	0.01	MB22	0.023	0.026	0.024
MB15	0.002	0.018	0.01	SKV12	0.036	0.015	0.026
AB01	0.014	0.012	0.013	AB61	0.04	0.026	0.033
ABV01	0.021	0.026	0.024	SK88	0.026	0.042	0.034
SK26	0.026	0.043	0.034	SK60	0.025	0.062	0.043
SK24	0.056	0.062	0.059	MBV37	0.059	0.063	0.061
Average	0.013	0.017	0.015	Average	0.021	0.027	0.024

Four isolates from each of the three provinces of Alberta, Saskatchewan and Manitoba, Canada, were selected randomly to establish the baseline sensitivity to pyraclostrobin. A paired t-test indicated that there was no significant difference between EC₅₀ values calculated in each of two independent experiments conducted for Ptt (P = 0.13) and Ptm (P = 0.20). Therefore, the two EC₅₀ values for each isolate were averaged. A log10-transformed fungicide concentration was used to linearize fungal growth inhibition (as a proportion of the control) to fit a regression line to estimate the EC₅₀ using probit analysis.

Assessing pyraclostrobin sensitivity of Ptt and Ptm isolates

No significant differences were detected in the pyraclostrobin sensitivity of Ptt or Ptm originating from Alberta, Saskatchewan or Manitoba (P = 0.11 for Ptt, and P = 0.20 for Ptm). Growth inhibition values in response to pyraclostrobin (0.15 mg L⁻¹) for the 39 Ptt isolates ranged from 40.5% for isolate ABV01 to 100% for isolates ABV28, SK01 and MB05. No isolates had a growth inhibition of <30%, four had a growth inhibition of 30–70%, and 35 had a growth inhibition of 70–100%. The mean growth inhibition for all of the Ptt isolates was 89%. The Ptt reference isolates WRS102 and WRS858 had growth inhibition values of 84% and 99%, respectively. The sensitivity of two isolates with growth inhibition values of 40.5% and 44.9% (ABV01 and SK24, respectively) also was evaluated at a discriminatory dose of 0.45 mg L⁻¹ pyraclostrobin, but growth of both was inhibited by >50% at this concentration of fungicide.

In the case of the 27 Ptm isolates, growth inhibition ranged from 23.8% for isolate SK64 to 100% for isolate AB79. One isolate (SK64) had a growth inhibition value of <30%, five isolates had a growth inhibition of 30–70%, and 21 isolates had a growth inhibition of 70–100% (Table 4, Fig. 3). The mean growth inhibition over all of the Ptm isolates was 84.6%. Growth of the Ptm reference isolate WRS857 was completely inhibited (100%). Isolate SK64 was designated as insensitive to pyraclostrobin based on its low growth inhibition, and this isolate, along with three others (SK100, MB16 and MB22) that had growth inhibition values of 38–49%, were evaluated under a higher discriminatory dose of 0.45 mg L⁻¹ pyraclostrobin. At this concentration, all of the isolates were inhibited by >50%. Isolate SK64 also showed decreased sensitivity to propiconazole (see above).

Table 4. Pyraclostrobin sensitivity of Pyrenophora teres f. teres (Ptt) and P. teres f. maculata (Ptm) collected in 2009–2012 from Alberta, Saskatchewan and Manitoba, Canada.

Ptt	Per cent growth inhibition	Sensitivity	Ptm	Per cent growth inhibition	Sensitivity
ABV28	100	Sensitive	AB79	100	Sensitive
SK01	100	Sensitive	SKV10	99.9	Sensitive
MB05	100	Sensitive	SKV12	99.6	Sensitive
MB04	99.8	Sensitive	MB32	99.2	Sensitive
AB53	99.5	Sensitive	AB58	99.2	Sensitive
MB06	99.4	Sensitive	MB24	98.6	Sensitive
MB15	99.2	Sensitive	AB68	98.1	Sensitive
AB51	99.1	Sensitive	ABV14	97.9	Sensitive
MB10	98.4	Sensitive	AB74	97.0	Sensitive
SK08	98.3	Sensitive	SK69	96.0	Sensitive
MB02	98.3	Sensitive	SK88	95.8	Sensitive
AB34	97.9	Sensitive	MBV25	95.8	Sensitive
AB38	97.6	Sensitive	AB61	95.7	Sensitive
AB04	97.4	Sensitive	SK60	95.0	Sensitive
SK41	97.2	Sensitive	MB26	94.5	Sensitive
SK03	96.8	Sensitive	MB23	94.2	Sensitive
SK05	96.6	Sensitive	MB21	93.7	Sensitive
MB11	96.1	Sensitive	MBV37	92.6	Sensitive
AB16	96.0	Sensitive	SK73	89.2	Sensitive
SK33	94.9	Sensitive	AB57	85.5	Sensitive
MB01	94.7	Sensitive	AB82	83.5	Sensitive
MB14	94.5	Sensitive	SK87	64.3	Intermediate
MB03	93.2	Sensitive	SKV08	58.8	Intermediate
AB01	91.9	Sensitive	MB16	49.6	Intermediate
AB48	90.3	Sensitive	MB22	48.8	Intermediate
SK53	90.3	Sensitive	SK100	38.3	Intermediate
AB12	89.9	Sensitive	SK64	23.8		Insensitive
AB35	86.1	Sensitive
SK52	85.4	Sensitive
SK26	85.4	Sensitive
AB06	85.3	Sensitive
ABV18	84.4	Sensitive
AB11	81.1	Sensitive
SK07	75.3	Sensitive
AB32	72.3	Sensitive
AB28	63.2	Intermediate
SK16	61.7	Intermediate
SK24	44.9	Intermediate
ABV01	40.5	Intermediate

Sensitivity to pyraclostrobin fungicide was assessed based on the reduction in fungal growth in a microtitre plate bioassay at a discriminatory dose of 0.15 mg L⁻¹. The endpoint optical density (OD) of samples in the 96-well microtitre plates was measured at 405 nm immediately after filling the plates. Plates were then incubated in darkness at 20°C for 7 days. Following incubation, the OD was measured and final values for absorbance were obtained by subtracting the values of the initial reading from those of the final reading. Subsequently, for each of the three isolate replicates, growth inhibition was calculated using the equation: 1 – [(absorption for the treatment /absorption of non-amended control)], and then averaged. The experiments were then repeated and the mean of the two growth inhibition values for each isolate was presented as a percentage.

Fig. 3 Frequency distribution of inhibition of fungal growth in 39 Pyrenophora teres f. teres and 27 P. teres f. maculata isolates from western Canada in response to 0.15 mg L⁻¹ pyraclostrobin in potato dextrose broth. Inhibition of fungal growth is expressed as a percentage relative to a control treatment in which no pyraclostrobin was included. Experiments were performed in the presence of 100 mg L⁻¹ salicylhydroxamic acid (SHAM).

Discussion

Propiconazole and pyraclostrobin are commonly used to manage net blotch of barley and other cereal leaf diseases in western Canada. To evaluate the sensitivity of Ptt and Ptm to these fungicides, the first step was to establish an EC₅₀ for each compound. In fungicide sensitivity screening, isolates with no history of exposure to the fungicidal compounds are typically used to calculate a baseline EC₅₀. However, since there were no P. teres isolates collected before the registration of propiconazole or pyraclostrobin, randomly selected Ptt and Ptm isolates from across western Canada were used to estimate the baseline EC₅₀ of these fungicides in this study. The EC₅₀ values calculated based on the sensitivity of these isolates, which may have been exposed to propiconazole or pyraclostrobin in the past, could be higher than the EC₅₀ values obtained for isolates that had never been exposed to these products. Nonetheless, this study provides important information on the current fungicide sensitivity of P. teres populations in western Canada, serving as a baseline against which to compare future assessments. To our knowledge, this is the first evaluation of the sensitivity of Ptt and Ptm to propiconazole and pyraclostrobin in this important barley growing region.

Ascospores and conidia are the most important primary inocula in the disease cycle of both Ptt and Ptm in western Canada, and are responsible for primary infection of barley tissues (Piening 1961, 1968; van den Berg & Rossnagel 1991; Duczek et al. 1999). Conidia are also the main inoculum source for most secondary infections. Therefore, conidia were used in this study to calculate the EC₅₀ values, and also to assess the sensitivity of a collection of P. teres isolates to propiconazole and pyraclostrobin.

The EC₅₀ of propiconazole for the Ptt isolates ranged from 0.8 to 3.0 mg L⁻¹ with a mean value of 1.5 mg L⁻¹, while the EC₅₀ for the Ptm isolates ranged from 1.1 to 3.7 mg L⁻¹ with a mean of 2.3 mg L⁻¹ (Table 1). Using an agar radial growth procedure, Campbell & Crous (2002) calculated mean EC₅₀ values of 1.2 and 0.4 mg L⁻¹ propiconazole for two Ptt populations (with a range of 0.004–7.693 mg L⁻¹ for individual isolates), and of 3.0 and 1.4 mg L⁻¹ for two Ptm populations (with a range of 0.138–5.772 mg L⁻¹) collected from four separate fields in South Africa. The wider range of EC₅₀ values reported by Campbell & Crous (2002) suggests the occurrence of more variability in the propiconazole sensitivity of South African versus Canadian isolates of P. teres. Direct comparisons are difficult to make, however, since the EC₅₀ values in the current study were determined in a microtitre plate bioassay using conidial suspensions, as opposed to the mycelial growth that was measured by Campbell & Crous (2002).

In the case of pyraclostrobin, the EC₅₀ values for individual Ptt isolates ranged from 0.002 to 0.059 mg L⁻¹ with a mean of 0.015 mg L⁻¹. The EC₅₀ values for Ptm isolates ranged from 0.008 to 0.061 mg L⁻¹ pyraclostrobin with a mean of 0.024 mg L⁻¹ (Table 3). Marzani (2011) calculated a mean EC₅₀ of 0.1–0.22 mg L⁻¹ pyraclostrobin for wild-type Ptt isolates, and of 0.28–0.69 mg L⁻¹ for mutant Ptt isolates with increased pyraclostrobin insensitivity. Marzani et al. (2013) observed similar EC₅₀ values for some of the mutant and wild-type isolates, suggesting that isolates were highly sensitive to pyraclostrobin. While the EC₅₀ values reported by Marzani (2011) were substantially higher than those reported in this study, those estimates were based on a modified radial growth bioassay compared with the microtitre procedure employed in the current study, making direct comparisons difficult.

Only two of the 39 Ptt isolates, AB48 and AB11, both of which were collected in central Alberta, had a growth inhibition of <30% and thus were regarded as propiconazole insensitive when tested at a discriminatory dose of 5 mg L⁻¹ propiconazole. These two isolates were closely related genetically, as revealed previously by simple sequence repeat marker analysis (Akhavan et al. 2016a). The growth inhibition of both isolates remained <50% even when the discriminatory dose was doubled to 10 mg L⁻¹ propiconazole. While no Ptm isolates were designated as insensitive to propiconazole, the growth inhibition of isolates SK60 and SK64 was only 48.2% and 48.4%, respectively. Further testing with a discriminatory dose of 10 mg L⁻¹ propiconazole showed that inhibition of the growth of SK64 remained <50%. When isolates AB48, AB11 and SK64 were evaluated at an even higher discriminatory dose of 20 mg L⁻¹ propiconazole, only SK64 had a growth inhibition of <50%. On the basis of these results, isolate SK64 appears to be the most insensitive to propiconazole among all Ptt and Ptm isolates evaluated. None of the isolates grew on 100 mg L⁻¹ propiconazole, a concentration almost 53× greater than the mean EC₅₀ values calculated for Ptt and Ptm. This suggests that it is unlikely that any of the P. teres isolates examined possess extreme or qualitative insensitivity to propiconazole. Alterations in the CYP51 gene in many plant pathogens (reviewed by Parker et al. 2014), including Ptt (Mair et al. 2016), have been reported to be the main mechanism for increased insensitivity towards DMIs. Azole insensitivity is increasing in pathogen populations as a result of the selection pressure imposed by the excessive application of triazole fungicides (Parker et al. 2014). Interestingly, isolate SK60 was previously reported to be one of the two Ptm isolates virulent on barley breeding line CI 9214, which is classified as highly resistant to SFNB (Akhavan et al. 2016a).

None of the Ptt isolates out of the 39 examined had a growth inhibition of <30% when tested at a discriminatory dose of 0.15 mg L⁻¹ pyraclostrobin (Table 4, Fig. 3). Marzani (2011) found that among the QoI fungicides they tested on a collection of Ptt isolates, pyraclostrobin and picoxystrobin were highly inhibitory, whilst the efficacy of other compounds was less pronounced. In the current study, only one isolate of Ptm (SK64) had a growth inhibition of <30% in response to 0.15 mg L⁻¹ pyraclostrobin (Table 4, Fig. 3). This is the same isolate that also exhibited increased insensitivity to propiconazole, suggesting that this isolate is tolerant to both products. However, when evaluated against a higher discriminatory dose of 0.45 mg L⁻¹ pyraclostrobin, growth of all isolates was inhibited by >50%. Since this dose was only 23× the average EC₅₀, it appears that none of the isolates in the current study had qualitative insensitivity to pyraclostrobin. In contrast, such extreme insensitivity to pyraclostrobin has been reported previously in western Canada for both Ascochyta rabiei on chickpea and Mycosphaerella pinodes on field pea (Gossen & Anderson 2004; Chang et al. 2007; Wise et al. 2008, 2009; Thaher 2011; Bowness et al. 2016). Two isolates of A. rabiei had an EC₅₀ value almost 704× greater than that of baseline isolates (Wise et al. 2009). Similarly, the mean EC₅₀ of M. pinodes pyraclostrobin-insensitive isolates was nearly 1500× that of sensitive controls (Bowness et al. 2016). Mutations (G143A and F129L) in the mitochondrial target gene cytochrome b have been found to be associated with the insensitivity of many plant pathogens to QoIs (Grasso et al. 2006; Sierotzki et al. 2007). The G143A mutation, which is stronger and more frequent, results in qualitative insensitivity to the fungicide, while the F129L mutation results in moderate insensitivity (Gisi et al. 2000; Sierotzki et al. 2007). In P. teres, only the F129L mutation has been reported to confer pyraclostrobin insensitivity (Semar et al. 2007; Sierotzki et al. 2007; Marzani et al. 2013). The current findings are consistent with those of other studies (Sierotzki et al. 2007; Marzani et al. 2013) which reported there was no qualitative or extreme insensitivity to pyraclostrobin in the net blotch fungus.

No significant differences were observed in the propiconazole or pyraclostrobin sensitivity of the Ptt and Ptm isolates from Alberta, Saskatchewan and Manitoba. Recently, Akhavan et al. (2016b) reported that Ptt and Ptm isolates from these provinces are genetically similar, with a large fraction of the genetic differentiation occurring within as opposed to between provinces.

Campbell & Crous (2002) found that Ptm isolates were less sensitive to propiconazole, tebuconazole, triadimenol, bromuconazole and flusilazole than were Ptt isolates, and concluded that Ptm may have developed more insensitivity as a result of a different evolutionary path. Scott et al. (1992) reported that two applications of propiconazole were required to reliably control SFNB caused by Ptm, while only one application was sufficient for control of NFNB caused by Ptt. Similar, but weaker trends (P < 0.10) were found in the current study for propiconazole or pyraclostrobin sensitivity, where slightly higher EC₅₀ values were observed for Ptm versus Ptt.

Propiconazole has been used for the control of diseases of cereals and other crops in western Canada since the early 1990s (Xue et al. 1994; Thomas 1997), while pyraclostrobin has been applied commercially to cereals only since the early 2000s (BASF 2003; Government of Canada: Health Canada Pest Management Regulatory Agency 2011). In western Canada, barley stubble probably contains both the sexual and asexual stages of the net blotch pathogens, and with fungicide use by barley farmers, fungal populations are frequently exposed to these products. Therefore, the routine application of these site-specific compounds on the genetically diverse populations of Ptt and Ptm poses a high risk for the development of insensitivity to both fungicides, particularly since the sensitivity to fungicides such as propiconazole has a high heritability rate (Talas & McDonald 2015). Moreover, no fitness penalty was observed for mutant Ptt isolates with increased insensitivity to strobilurins (Marzani 2011). Campbell & Crous (2002) also suggested that sexual recombination could increase the rate of insensitivity of Ptm isolates toward DMI fungicides in South Africa. If the pathogen develops insensitivity, then with many asexual cycles during the following growing season, the insensitive individual may multiply and constitute a significant portion of a whole new emerging P. teres population. The high gene flow reported among the provincial populations of both forms of the net blotch pathogen in western Canada (Akhavan et al. 2016b) suggests there is a risk of the transfer of fungicide insensitivity from one population to another. In Europe, it was shown that the frequency of M. graminicola haplotypes with insensitivity to QoI fungicides (due to the G143A mutation) increased rapidly via strong fungicide selection pressure, and that the pathogen then spread in a west-to-east direction via wind dispersal of ascospores (Torriani et al. 2009).

The identification of Ptt and Ptm isolates insensitive to propiconazole, and of a Ptm isolate with increased insensitivity to both propiconazole and pyraclostrobin, suggest the need for increased fungicide stewardship in western Canada. Farmers should avoid relying exclusively on a single or the same few fungicide active ingredients, and employ other strategies to reduce the risk of fungicide insensitivity developing in Ptt and Ptm populations. Fungicide mixtures, comprising QoIs and DMIs or the novel SDHI formulations, were found to have great efficacy for net blotch disease management (Marzani 2011). Ideally, a broad combination of control methods, in addition to the application of fungicides, should be used to manage net blotch of barley.

In the current study, we assessed the sensitivity of Ptt and Ptm for the DMI fungicide propiconazole and the QoI fungicide pyraclostrobin. Our research mainly focused on detecting insensitivity and comparing differences between the two forms of the pathogen. Given the occurrence of insensitive isolates, our results underscore the need for continued monitoring of the fungicide sensitivity of P. teres populations in western Canada. In addition, future studies are needed to investigate the genetic mechanisms underlying these insensitivities, and to also evaluate the relative fitness of insensitive isolates; this research will help with the early detection of fungicide insensitivity, and allow for more extensive evaluation of insensitivity development in western Canadian populations of Ptt and Ptm.

Supplemental meterial

Supplemental data for this article can be accessed online here: http://dx.doi.org/10.1080/07060661.2017.1282541.

Acknowledgements

The authors would like to thank Dr Andy Tekauz, Mr James Tucker, Ms Colleen Kirkham, Mr Meconnen Beyene, Ms Noryne Rauhala, Ms Jackie Busaan (Agriculture and Agri-Food Canada), Dr Kequan Xi, and Dr Krishan Kumar (Alberta Agriculture and Forestry) for assistance with the collection of isolates, and Dr Tiesen Cao, and Dr Urmila Basu (University of Alberta) for technical advice and insight. Finally, the authors thank the Western Grains Research Foundation, Alberta Barley Commission, Rahr Malting Inc., Canadian Wheat Board, Agriculture and Agri-Food Canada, and the University of Alberta for financial support of this research.

Additional information

Funding

This work was supported by the Western Grains Research Foundation.