2014 Volume 39 Issue 1 Pages 1-6
Fusarium head blight (FHB) is one of the important diseases of cereals caused by Microdochium nivale or several kinds of Fusarium species.1–3) It is important to control FHB not only to prevent yield loss, but also to avoid quality problems such as mycotoxin contamination.4,5) Fungicide application is the most effective strategy used to control FHB.
Metconazole [(1RS,5RS;1RS,5SR)-5-(4-chlorobenzyl)-2,2-dimethyl-1-(1H-1,2,4-triazol-1-ylmethyl)cyclopentanol], a demethylation inhibitor (DMI) in the ergosterol biosynthesis pathway, was discovered by the Kureha Corporation in 1986.6) It was initially registered as a cereal fungicide in France in 1993 and was globally used as a fungicide for various kinds of crops such as cereals, oilseed rape, turf, tree nuts, and fruits in more than 30 countries by 2012. Metconazole as a cereal fungicide exhibits excellent controlling activities against several diseases including leaf rust, Septoria blotch, and powdery mildew.4) It also controlled FHB with high efficacy, reduced mycotoxin concentrations in cereal grains,7–9) and increased yields.10,11) The sensitivity of the Fusarium graminearum species complex to metconazole and the efficacy of metconazole against isolates with different sensitivities have also been reported.12)
In field studies, optimal fungicide application timing for controlling FHB was shown to be different from that for controlling mycotoxin contamination.13) Some fungicides have also been reported to be ineffective against mycotoxin contamination in spite of their high efficacies against the symptoms of FHB in wheat.14)
On the other hand, the relationship between the application timing of fungicides and efficacy against FHB or control of deoxynivalenol (DON) in barley remains to be clarified. We previously examined the efficacy of metconazole against FHB and mycotoxin concentrations in grains using various application timings from the mid-flowering stage to the browning stage with single and double applications in a field trial of barley.
Several studies have also investigated fungicide application timing;15,16) however, few have assessed late application timing after the milking stage.13) In this study, the relationship between the application timing of metconazole and its efficacy against FHB, including a reduction in mycotoxin concentrations in the grains of wheat and barley, was investigated. To the best of our knowledge, this is the first report to examine the relationship between the application timing of metconazole and control against FHB and mycotoxin contamination.
Evaluating the application timing of metconazole on wheat was conducted in 2008. The experiment was performed in a field at the Kureha Corporation Agrochemical Research Center in Iwaki, Fukushima Prefecture, Japan. The wheat cultivar used in this study was Norin 61, one of the most popular Japanese soft winter cultivars. It is moderately resistant to FHB. Seeds were sown on December 2, 2007, in single rows that were 0.3 m apart and 40 m long. The plants were grown in accordance with standard agronomic practices in Fukushima, Japan, until harvest.
A similar evaluation of barley was conducted in 2011. The barley cultivar tested was Shunrai, which is a six-rowed hulled chasmogamous (open-flower type) cultivar popular in the Tohoku region of Japan. The seeds of this cultivar were sown on November 15, 2010. The experiment and cultivation were performed using methods similar to those described above.
2. Inoculation, monitoring of the spores of pathogens, and meteorological recordsInoculation in the field trial was performed using inoculated maize kernels, which generated ascospores over a long period during the testing season in the field. An isolate of F. graminearum s. str. H-3 (MAFF101551), a DON producer, was used as the inoculum. Pathogen-colonized maize kernels were prepared according to the method of Yoshida et al.17) and were distributed onto the testing field (approximately 30 g/m2) on a day estimated to be about 3 weeks before the heading stage of wheat and barley.
During the testing period, the mean, highest, and lowest temperature and amount of rainfall were recorded daily at an office of the Iwaki factory, Kureha Corporation, located approximately 700 m east of the testing field.
F. graminearum spore loads were monitored at the heading stage and anthesis timing using selective medium.17) Plates of the selection medium (9-cm-diameter plastic Petri dishes) were set at the height of the top level of spikes (80 cm high) at the center of the field and at another 4 points in the field. Plate media were then incubated, the colonized F. graminearum species complex was identified, and other monitoring methods were performed according to the methods established by Yoshida et al.13)
3. Application timing of metconazoleA flowable formulation of 18% metconazole (produced by Hokko Chemical Industry Co., Ltd.) was used in both the wheat and barley field trials. The formulation was diluted 2000-fold and sprayed at a rate of 1500 L/ha (135 g a.i./ha); i.e., a 300-mL solution was sprayed onto 2 m2 (one plot). All plots were assigned to a 4 m dabble-row 0.5 m wide (2 m2) and were arranged with 1 m interrow spacings.
Application timings in the wheat trial are shown in Table 1(1). The diluted suspension of metconazole was sprayed 4, 10, 17, 25, or 31 days after anthesis (DAA) for single application plots. Metconazole was commonly sprayed 4 DAA for double application plots and additionally at 10, 17, 25, or 31 DAA. The application timings; 4, 10, 17, 25, and 31 DAA corresponded to the mid-flowering stage, late-flowering stage, milking stage, yellowing stage, and browning stage, respectively. Each treatment had 4 replications.
| Growth stage | (1) Wheat trial in 2008 | (2) Barley trial in 2011 | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Mid-flowering | Late-flowering | Milking | Yellowing | Browning | Mid-flowering | Milking | Yellowing | Browning | |
| Date | 22-May | 28-May | 4-Jun | 12-Jun | 18-Jun | 9-May | 20-May | 1-Jun | 13-Jun |
| DAA* | 4 | 10 | 17 | 25 | 31 | 3 | 14 | 26 | 38 |
| Single application plots | ○ | ○ | |||||||
| ○ | ○ | ||||||||
| ○ | ○ | ||||||||
| ○ | ○ | ||||||||
| ○ | |||||||||
| Double application plots | ○ | ○ | ○ | ○ | |||||
| ○ | ○ | ○ | ○ | ||||||
| ○ | ○ | ○ | ○ | ||||||
| ○ | ○ | ||||||||
* DAA: Days after anthesis. ○: Application date.
The application timings in the barley trial are shown in Table 1(2). Metconazole was sprayed 3, 14, 26, or 38 DAA for single application plots. Metconazole was commonly sprayed 3 DAA and additionally at 14, 26, or 38 DAA for double application plots. The application timings; 3, 14, 26, and 38 DAA corresponded to the mid-flowering stage, milking stage, yellowing stage, and browning stage, respectively. Each treatment had 3 replications. Other methods of the barley trial were the same as those of the wheat trial.
4. Assessment of efficacy against FHB and analysis of mycotoxinsOne hundred spikes were randomly chosen from one plot and the disease severity of each spike was assessed using an index with a scale of 0 (no symptoms) to 100 (completely discolored), according to Ban and Suenaga’s method.18)
After harvesting the wheat and barley, the plants were dried for 1 week in a greenhouse and the grains were then threshed and collected. Grain samples from the replications were gathered and mixed together. DON and nivalenol (NIV) concentrations in the grains were analyzed at Kyowa Medex Co., Ltd. (Shizuoka, Japan) using approximately 100 g of each grain sample by the ELISA (enzyme-linked immunosorbent assay) method.19)
Meteorological data, the growth stage of wheat, application timings, the assessment date, and the harvesting date in 2008 were shown in Fig. 1. Relative high temperatures and some rainfall were observed during the flowering stage. Moderate rainfall was observed in the milking stage and late browning stage, whereas no rain fell in the yellowing stage. In the fungicide untreated plot, the mean disease index was 5.0 and the incidence was 43%. The trial was performed under the condition of a moderate disease level. DON and NIV concentrations in grains harvested from the untreated control were 1.9 and 0.3 ppm, respectively. The NIV concentration was lower than the DON concentration because the infected pathogen was a DON producer.
The relationship between the application timing of metconazole, the disease index of FHB, and the concentration of mycotoxins in wheat grains is shown in Fig. 2.
The efficacy of metconazole against FHB in single application plots was the highest when metconazole was applied at the mid-flowering stage. The later the application timings were shifted after the flowering stage, the lower the efficacies that were observed. The disease index when metconazole was applied after the late-flowering stage was higher than that in the untreated plot, whereas those of the yellowing stage and the browning stage were not. The incidences of spikes when metconazole was applied at the mid-flowering, late-flowering, milking, yellowing, and browning stages were 21, 29, 34, 37, and 44%, respectively.
On the other hand, the efficacy of metconazole for controlling DON concentrations in wheat grains was the highest when it was applied at the milking stage. DON concentrations were higher with later application timing than the milking stage; however, these were still lower than in the untreated control.
The efficacy of metconazole against FHB was almost the same for all application combinations in double application plots. The incidences of spikes after application at the mid-flowering stage followed by a second application at the late-flowering, milking, yellowing, or browning stage were 20, 24, 32, and 20%, respectively. The DON concentration in grain was relatively lower after the application of metconazole at the mid-flowering plus milking stages with than other application timings.
2. Efficacy of metconazole against FHB and the control of mycotoxins in barley grainMeteorological data, the growth stage of the barley, application timings, the assessment date, and the harvesting date are shown in Fig. 3.
Moderate rainfalls were observed between applications at the milking and yellowing stages and after the browning stage. In an untreated plot, the mean disease index was 15.2 and the incidence was 86%. This indicates that the trial was performed under conditions in which a high level of disease occurred. DON and NIV concentrations in grain harvested from the untreated control were 2.2 and 0.8 ppm, respectively.
The relationship between the application timing of metconazole, the disease index of FHB, and the concentration of mycotoxins in barley grain is shown in Fig. 4.
In single application plots, the efficacy of metconazole against FHB was the highest when it was applied at the mid-flowering stage. The disease index following application at the flowering stage was higher than that of the untreated plot, while those of the yellowing stage and the browning stage were not. Similar results were observed in the wheat trial. The incidences of spikes when metconazole was applied at the mid-flowering, milking, yellowing, and browning stages were 22, 82, 85, and 87%, respectively.
The efficacy of metconazole for controlling DON concentrations in barley grain was also the highest when it was applied at the mid-flowering stage. When metconazole was applied later than the mid-flowering stage, DON concentrations were relatively high, but were lower than those of the untreated plot. Different results were observed in the wheat trial.
The efficacy of metconazole against FHB in double application plots was similar for all application combinations. The incidences of spikes after the application of metconazole at the mid-flowering stage followed by a second application at the milking, yellowing, or browning stage were 20, 20, and 23%, respectively. The DON concentration in grain after application at the mid-flowering plus milking stages was almost the same as that after a single application at the mid-flowering stage. DON was below the detection limit (<0.1 ppm) in other double application combinations.
In addition to spore loads of pathogens at the heading stage and flowering stage, perithecia on corn kernels have always been observed until harvest. Inoculations were continuously performed in both the wheat and barley trials.
Rainfall and relatively high temperatures between the first (mid-flowering stage) and second application (late-flowering stage) may have stimulated the development of FHB in the wheat trial. Pathogen spore loads were observed at the heading stage and the flowering stage. It has been suggested that metconazole exerted a preventive effect when it was applied at the flowering stage and a curative effect when it was applied after the late-flowering stage. The results of disease severity and DON concentrations in grain indicate that the most appropriate application timing for FHB control is the mid-flowering stage (4 DAA) and that for DON control is the milking stage (17 DAA).
The application of metconazole at the flowering stage was clearly the most effective for controlling the symptoms of FHB, and its application at the yellowing or browning stage was effective for reducing DON contamination.
The most appropriate timing for infection by FHB pathogens in wheat was previously shown to be the flowering stage.16) Therefore, fungicide application against FHB is generally performed at the flowering stage. Concerning mycotoxin accumulation in wheat grain, Yoshida et al. reported that the concentration of mycotoxins largely increased after 20 DAA with infection occurring at earlier and later stages (20 DAA, the milking stage), causing high levels of contamination with mycotoxins without clear FHB symptoms.20) A similar phenomenon was observed in our wheat field trial in which the application of metconazole 17 DAA (the milking stage) prevented mycotoxin contamination in grain. Thus, it can be concluded that the optimal fungicide application timing for FHB is different from that of mycotoxin contamination.
Concerning fungicide application and the control of mycotoxin contamination, some fungicides such as thiophanate-methyl, tebuconazole, and metconazole significantly decreased DON levels, whereas other fungicides such as azoxystrobin were not effective against mycotoxins.9) Simpson et al. demonstrated that some fungicides stimulated DON accumulation in wheat grain infected with FHB fungi. The stimulation of or reduction in DON production in the presence of fungicides was shown to be influenced by complex interactions between water activities, concentrations, and the time of incubation.14) The high levels of mycotoxins in wheat grain can be produced after 20 DAA, even with early infection. Infection later than 20 DAA also caused mycotoxin contamination in grains. Control strategies at the later as well as early stages are necessary for reducing mycotoxin contamination.21) Pirgozliev et al. reported that when pathogens were inoculated at the mid-flowering stage (mixed spores of Fusarium culmorum, F. graminearum, Microdochium majus and Microdochium nivale), fungicide applications 2 days pre- and 2 days post-inoculation were the most effective at reducing FHB severity and DON concentrations. The efficacy of metconazole was the most consistent in their study.22) Yoshida et al. showed that the application timing of the fungicide, thiophanate-methyl, differentially affected FHB and mycotoxin concentrations, and also suggested that application timing around 20 DAA (the milking stage) was crucial for preventing mycotoxin contamination.13)
In the barley trial, rainfall and relatively high temperatures between the second (milking stage) and third (yellowing stage) applications may have stimulated DON contamination. Pathogen spore loads were observed at the heading stage and the flowering stage. From the results of disease severity and DON concentrations in the grain, the most appropriate application timing was shown to be the flowering stage (3 DAA).
Concerning the effect of inoculation timing and the susceptibility of various barley cultivars to FHB, chasmogamous (open-flower type) cultivars were susceptible to FHB and cleistogamous (closed-flower type) cultivars were resistant to FHB at the flowering stage, whereas cleistogamous cultivars were susceptible at 10 DAA. Inoculation timing and mycotoxin accumulation also differed between chasmogamous and cleistogamous cultivars. The accumulation of mycotoxins was larger with the inoculation 10 or 20 DAA than with the inoculation at anthesis, and chasmogamous cultivars accumulated more mycotoxins with inoculation at anthesis than did cleistogamous cultivars.23) This finding indicates that the strategy for controlling FHB, including mycotoxin contamination, differs in each type of cultivar. The chasmogamous cultivar was used in our study, and application timing at the flowering stage was shown to be the most effective for both FHB and mycotoxin control. Additional applications after the milking stage were also effective for mycotoxin control even when the symptoms of FHB were not prevented. To the best of our knowledge, this is the first study to investigate application timing for FHB and mycotoxin contamination in a field trial of chasmogamous barley.
Metconazole was recently shown to directly inhibit trichothecene production by F. graminearum s. str., Fusarium asiaticum, and Fusarium sporotricoides.24) This activity of metconazole likely contributes to the decreased mycotoxin concentration in grains especially after the milking stage.
It is clear that application timing at the flowering stage is important for FHB control, which is consistent with the findings of previous reports;13,15,16) however, optimal single application timing for mycotoxin control differed between our wheat and barley trials. Itakura et al. performed application timing trials of metconazole on wheat in Kanagawa and Hokkaido, Japan and observed different optimal application-timings for DON control.25) Mycotoxin contamination and optimal application timing for mycotoxin control presumably depend on the pathogens’ growth level in grains affected by meteorological or other factors.
Metconazole is effective against both FHB and mycotoxins in cereal grains. Application at the flowering stage is essential with an additional application at the milking stage being desirable when high disease pressure is forecasted. The application of metconazole at the flowering stage exhibited high efficacy against FHB, and an additional application after the milking stage contributed to a reduction in the mycotoxin concentration in grains.
