2024 Volume 49 Issue 4 Pages 292-296
The biological properties of isofetamid, a new fungicide, were examined using pot tests in a greenhouse. In addition, we investigated the practical effects of isofetamid in field trials. In greenhouse pot tests, isofetamid exhibited high preventive efficacy against cucumber gray mold, powdery mildew, Corynespora leaf spot, and stem rot even at low concentrations. Isofetamid has excellent biological properties against cucumber gray mold and powdery mildew, such as rainfastness, residual activity, curative activity, translaminar activity, and transfer activity to undeveloped leaves, even at low concentrations. In field trials, isofetamid displayed high efficacy against tomato gray mold, cucumber powdery mildew, and lettuce stem rot. Therefore, the biological properties of isofetamid revealed in greenhouse tests contributed to its good performance in field trials.
Succinate dehydrogenase inhibitors (SDHIs) are important agricultural fungicides with a very broad spectrum of control. Since the introduction of SDHIs, such as boscalid and penthiopyrad, which are effective against ascomycetes, several new compounds have been developed and are widely used as foliage sprays and seed treatments for cereals, fruits, vegetables, turf, and ornamental plants.1–4) Therefore, this family of fungicides is an important component of disease control agents, along with demethylation inhibitors (DMIs) and quinone outside inhibitors (QoIs).5–8)
Isofetamid, N-[1,1-dimethyl-2-(4-isopropoxy-o-tolyl)-2-oxoethyl]-3-methylthiophene-2-carboxamide (code name: IKF-5411, trade names: KENJA®, ZENBY®, KRYOR®, HAREGI®, KABUTO®, ASTUN®) is a new fungicide developed by Ishihara Sangyo Kaisha, Ltd.9–11) We have previously reported that isofetamid is an SDHI (FRAC code 7). It specifically inhibits SDH activity in ascomycetes, whereas it exerts no impact on the activity of oomycetes, plants, and mammals.11) The structure of isofetamid is unique in that it contains a phenyl-oxo-ethyl thiophene amide, which is the first halogen-free compound among the SDHIs marketed since 2000.12,13) We have provided details regarding the fungicidal spectrum of isofetamid and its effect on the infection processes of Botrytis cinerea. Furthermore, we found that isofetamid did not exhibit cross-resistance to existing fungicides.11)
Although preliminary studies have reported that isofetamid has good biological activity in greenhouse tests against several important diseases such as gray mold, powdery mildew and leaf spot, its precise biological properties have not been investigated in detail. In this study, we focused on the biological properties of isofetamid in pot tests in a greenhouse. In addition, we investigated the practical effects of isofetamid in field trials.
Pot tests were used to evaluate the preventive efficacy of isofetamid in controlling different plant diseases of cucumber listed in Table 1, including gray mold, powdery mildew, leaf spot, and stem rot.
| Isofetamid concentration (µg/mL) | Control value (%) | |||
|---|---|---|---|---|
| Gray molda) | Powdery mildewb) | Leaf spotb) | Stem rota) | |
| Botrytis cinerea | Podosphaera xanthii | Corynespora cassiicola | Sclerotinia sclerotiorum | |
| 266.0 | 100 | 100 | 100 | 100 |
| 133.0 | 100 | 100 | 100 | 100 |
| 66.5 | 100 | 100 | 97.2 | 100 |
| 33.3 | 99.2 | 100 | 90.1 | 99.7 |
| 16.6 | 72.4 | 100 | 85.6 | 99.7 |
| 8.3 | 62.1 | 100 | 40.1 | 94.5 |
a) Lesion diameters of untreated plants were 22.9 mm (gray mold) and 36.1 mm (stem rot), respectively. b) Disease severities of untreated plants were 95% (powdery mildew) and 62% (leaf spot), respectively.
An isofetamid suspension concentrate (SC) of 400 g/L was used. Cucumber seedlings of cv. Sagami hanjiro was used for gray mold, powdery mildew, and stem rot tests, and cucumber seedlings of cv. Natsusuzumi were used to test Corynespora leaf spot. Botrytis cinerea, Sclerotinia sclerotiorum and Corynespora cassiicola isolates used in this study were maintained on PSA medium at 20°C. Podosphaera xanthii isolate used in this study was maintained on the cucumber plants at 20°C. Cucumber seedlings were sprayed on the adaxial surface of leaves with test solutions using a handgun sprayer (20–40 mL/0.2 m2; this water volume is equivalent to 1000–2000 L/ha). Inoculation was conducted after the solutions dried.
The treated seedlings were inoculated by spraying aqueous spore suspensions of P. xanthii (5.0×105 conidia/mL) and C. cassiicola (3.0–4.0×105 conidia/mL) with a handgun sprayer to test for powdery mildew and Corynespora leaf spots. The inoculated plants were incubated at 20°C for 7 days. The fungicidal activity was evaluated by visually observing the lesion area and was expressed in terms of disease severity (0–100%). The control values were calculated using the following equations:
 |
where T is the disease severity of the treated plot and C is the disease severity of the untreated plot.
Filter papers containing conidial suspensions of B. cinerea (1.0×105 conidia/mL, 100 µL) or ascospore suspensions of S. sclerotiorum (1.0×105 conidia/mL, 100 µL) were placed on the detached leaves of the treated seedlings to conduct gray mold and stem rot tests. The inoculated plants were incubated at 20°C for 3 to 7 days. The fungicidal activity was determined by measuring the diameter of each lesion. The control values were calculated using the following equations:
|
where T is the lesion diameter of the treated plot and C is the lesion diameter of the untreated plot.
2. Pot tests against cucumber gray mold2.1. Preventive activity and rainfastness2.1.1. Preventive activityAn isofetamid SC (400 g/L) was used. Cucumber seedlings (cv. Sagami hanjiro) were sprayed on the adaxial surface of the cotyledons with the test solutions using a handgun sprayer (20 mL/0.2 m2; this water volume is equivalent to 1000 L/ha). The treated seedlings were maintained until the solutions dried. Twenty-four hours after application, filter papers containing conidial suspensions of B. cinerea (1.0×106 conidia/mL, 100 µL) were placed on the detached leaves of treated seedlings. The inoculated leaves were incubated at 20°C for 3 days. The fungicidal activity was determined by measuring the diameter of each lesion. The control values were calculated using the following equations:
|
where T is the lesion diameter of the treated plot and C is the lesion diameter of the untreated plot.
2.1.2. RainfastnessCucumber seedlings were sprayed on the adaxial surface of the cotyledons with test solutions using the method described in Section 2.1.1. Twenty-four hours after application, the seedlings were treated with artificial rain (10 mm/hr) for 1 hr using an artificial rain generator (Daiki Rika Kogyo, DIK-6000). After drying, seedlings were inoculated with the pathogen. Inoculation and evaluation were conducted as described in Section 2.1.1.
2.2. Residual activityCucumber seedlings were sprayed on the adaxial surface of the cotyledons with test solutions using the method described in Section 2.1.1. After the application and drying of the test solution, the treated seedlings were kept in a greenhouse for 7 and 14 days before inoculation. Inoculation and evaluation were conducted as described in Section 2.1.1.
2.3. Curative activity2.3.1. Inhibition activity in lesion developmentFilter papers containing conidial suspensions of B. cinerea (1.0×106 conidia/mL, 100 µL) were placed on the detached cotyledons of cucumber seedlings. Twenty-four hours after inoculation, the inoculated leaves were dipped in test solutions for 10 sec. After application and drying of the solution, the treated leaves were incubated at 20°C for 24 hr. The evaluation was conducted using the method described in Section 2.1.1.
2.3.2. Inhibition activity in sporulationFilter papers containing conidial suspensions of B. cinerea (1.0×106 conidia/mL, 100 µL) were placed on the cotyledons of cucumber seedlings. Three days after inoculation, the seedlings were sprayed on the adaxial surface of the cotyledons with the test solutions using the method described in Section 2.1.1. After application and drying of the spray solution, the treated seedlings were incubated at 20°C for 7 days until the formation of conidia. After incubation, the leaves were washed with water (5 mL/leaf), and the conidia were counted using a hemocytometer. The fungicidal activity was determined based on the number of conidia present. The control values were calculated using the following equations:
|
where T is the number of conidia in the treated plot and C is the number of conidia in the untreated plot.
2.4. Translaminar activityCucumber seedlings were sprayed with test solutions on the adaxial or abaxial surface of the cotyledons using the method described in Section 2.1.1. Twenty-four hours after application, the cotyledons were cut off and the opposite side of the treated surface was inoculated with a conidial suspension of B. cinerea (1.0×106 conidia/mL, 100 µL). The inoculated leaves were incubated at 20°C for 3 days. The evaluation was conducted using the method described in Section 2.1.1.
3. Pot tests against cucumber powdery mildew3.1. Transfer activity to undeveloped leavesUndeveloped cucumber seedlings (0.3 leaf stage) (cv. Sagami hanjiro) were sprayed on the adaxial surface of the first leaves with the test solutions using the method described in Section 2.1.1. After application and drying of the test solution, the treated seedlings were kept in a greenhouse for 7 days before inoculation. The treated seedlings were inoculated by spraying aqueous spore suspensions of P. xanthii (5.0×105 conidia/mL) with a handgun sprayer. The inoculated leaves were incubated at 20°C for 7 days. The evaluation was conducted using the method described in Section 1.2
4. Field trials4.1. Tomato gray moldTomato gray mold test was conducted by JCPA (Japan Crop Protection Association) in a greenhouse in Mie Prefecture, Japan in 2014. The tomato seedlings (cv. House Momotaro) were transplanted into the field. Isofetamid (400 g/L SC, Kenja®, Ishihara Sangyo Kaisha Ltd.) and mepanipyrim (400 g/L SC, Furupica®, Kumiai Chemical Industry Co., Ltd.) were sprayed three times with an interval of 7 to 10 days (water volume: 3000 L/ha). Diseased eggplant fruits with conidia of B. cinerea were used as inoculation sources. The fungicidal activity was determined by counting the number of infected fruits.
4.2. Cucumber powdery mildewThe test was conducted by JCPA in a greenhouse in Kochi Prefecture, Japan in 2014. Cucumber seedlings (cv. ZQ7) were transplanted into the field. Isofetamid and TPN (40% wettable powder [WP], Daconil®, Sumitomo Chemical Co., Ltd.) were sprayed three times with an interval of 7 days (water volume: 2700 L/ha). This test was conducted without artificial inoculation. The fungicidal activity was determined by observing the lesion area on the leaves.
4.3. Lettuce stem rotThe lettuce stem rot test was conducted by JCPA in a greenhouse in Miyazaki Prefecture, Japan in 2012. The lettuce seedlings (cv. Cisco) were transplanted into the field. Isofetamid and thiophanate-methyl (70% wettable powder [WP], Topsin-M®, Nippon Soda Co., Ltd.) were sprayed four times with an interval of 7 to 8 days (water volume: 2500–3400 L/ha). Small pots containing apothecia formed from sclerotia were used as inoculation sources. Fungicidal activity was determined by observing the lesion area on plants.
In preventive activity tests, isofetamid displayed high efficacy (control value was more than 90) against cucumber gray mold, powdery mildew, Corynespora leaf spot, and stem rot even at low concentrations of 33.3 to 8.3 µg/mL (Table 1). Isofetamid completely suppressed the lesion formation at a low concentration of 8.3 µg/mL (Table 1) against cucumber powdery mildew. These results indicate that isofetamid showed high preventive efficacy against ascomycetes in pot tests and has the potential to control multiple diseases caused by ascomycetes in the field. However, its efficacy against diseases caused by basidiomycetes, such as soybean rust and rice sheath blight, was lower than that against ascomycetes in the pot test (data not shown).
2. Pot tests against cucumber gray mold2.1. Preventive activity and rainfastnessThe preventive activity and rainfastness of isofetamid against cucumber gray mold were compared. In the preventive activity tests, isofetamid displayed high efficacy (control value was more than 90) even at a low concentration of 31.3 µg/mL. To evaluate the rainfastness of isofetamid, the seedlings were treated with artificial rain (10 mm/hr ×1 hr) using an artificial rain generator 1 day after application. Below 62.5 µg/mL, the efficacy in the rainfastness test was slightly lower than that in the preventive activity test. However, almost no difference was observed in the efficacy between the rainfastness and the preventive activity at 125 µg/mL (Table 2). Therefore, isofetamid exhibits high preventive activity and good rain fastness against gray mold at 125 µg/mL.
| Isofetamid concentration (µg/mL) | Control value (%)a) | |||
|---|---|---|---|---|
| Preventive activity | Rainfastness (10 mm/hr×1 hr) | Residual activity | ||
| Inoculation a day post spray | Inoculation a day post spray | Inoculation 7 days post spray | Inoculation 14 days post spray | |
| 125.0 | 99 | 97 | 100 | 98 |
| 62.5 | 98 | 79 | 99 | 95 |
| 31.3 | 97 | 55 | 91 | 93 |
a) Lesion diameters of untreated plants were 27.8 mm (preventive activity and rain fastness), 24.8 mm (7 days residual activity) and 24.1 mm (14 days residual activity), respectively.
The preventive and residual activities of isofetamid against cucumber gray mold were compared. To evaluate the residual activity of the isofetamid, inoculation was carried out after maintenance for 7 or 14 days in a greenhouse. In the residual activity test, isofetamid displayed high efficacy (control value was more than 90) even at low concentrations of 31.3 µg/mL, and almost no difference in the efficacy between the residual activity and the preventive activity was confirmed (Table 2). These results indicate that isofetamid remained on the leaf surfaces after spraying.
2.3. Curative activityLesion development inhibition tests revealed that isofetamid exhibited high efficacy (control value was more than 70) against cucumber gray mold even at 62.5 µg/mL (Fig. 1). The lesion diameter of untreated leaves was 28.3 mm. This result indicates that isofetamid suppressed disease development during the early stages of infection. This activity is thought to be effective against gray mold fungi that have already developed on infected leaves.
Sporulation inhibition tests revealed that isofetamid exhibited very high efficacy (control value was more than 90) against cucumber gray mold even at 62.5 µg/mL. In the untreated control, abundant conidia were observed on leaf surfaces. The number of conidia on untreated leaves was 1.8×106 conidia. In contrast, isofetamid (500 and 62.5 µg/mL), treatment almost completely suppressed the formation of conidia (Fig. 1, Supplemental Fig. S1). These results suggest that isofetamid can control secondary infections and prevent the spread of the disease.
2.4. Translaminar activityWe next evaluated the translaminar activity of isofetamid, for which solutions were sprayed onto the adaxial or abaxial surfaces of cucumber seedling leaves. One day after the application, the opposite side of the solution-treated surface was inoculated with a conidial suspension. The lesion diameter of untreated leaves was 31.7 mm when inoculated on the abaxial surface and 30.1 mm when inoculated on the adaxial surface. Translaminar activity tests revealed that isofetamid displayed high efficacy (control value was more than 70) against cucumber gray mold even at a concentration of 125 µg/mL (Fig. 2) from the adaxial to the abaxial surface of the leaf and from the abaxial to the adaxial surface of the leaf. This result indicated that isofetamid rapidly penetrated the leaves after treatment. This activity could result in good efficacy under conditions in which a small volume of spray does not adequately cover the stems and leaves.
We evaluated the transfer activity of isofetamid to undeveloped leaves. The solutions were sprayed on young leaves (0.3 leaf stage). The seedlings were grown for 7 days and then inoculated. The disease severity of untreated leaves was 94. Isofetamid exhibited high efficacy (control value was more than 80) against cucumber powdery mildew even at a concentration of 133 µg/mL (Supplemental Fig. S2). This indicates that the isofetamid rapidly spreads to undeveloped leaves.
Based on these results of the pot tests, the practical concentration of isofetamid was found to be 267 µg/mL. Therefore, at this concentration, the efficacy of the isofetamid was confirmed in field trials, as described in the following section.
4. Field trialsIsofetamid demonstrated high efficacy against tomato gray mold, cucumber powdery mildew, and lettuce stem rot in field trials at a concentration of 267 µg/mL. In addition, its efficacy was higher than that of the reference fungicide (Supplemental Tables S1, S2, and S3). Therefore, the concentration of 267 µg/mL was found to be practical. These results confirmed that isofetamid is effective in controlling tomato gray mold, cucumber powdery mildew, and lettuce stem rot.
In this study, we demonstrated that the isofetamid has excellent biological properties such as rainfastness, residual activity, curative activity, translaminar activity, and transfer activity to undeveloped leaves in greenhouse tests. These biological properties seemed to contribute to its good practical performance in these field trials. Isofetamid was registered as a pesticide in Canada in 2014, introduced into the Japanese market in 2017, and has since been registered globally, contributing to the control of many important diseases.9–11)
The online version of this article contains supplementary materials (Supplemental Tables S1, S2, and S3, and Figs. S1 and S2) and are available at https:// www. jstage.jst.go.jp/browse/jpestics/.
