2024 Volume 49 Issue 2 Pages 130-134
The fungicidal properties of a new fungicide, isofetamid, were examined to assess its antifungal spectrum, mode of action, and effects on the infection process of Botrytis cinerea. Additionally, we investigated its fungicidal activity against isolates of B. cinerea resistant to existing fungicides. In mycelial growth inhibition tests, isofetamid exhibited excellent fungicidal activity against ascomycetes but showed no activity against basidiomycetes and oomycetes. Respiratory enzyme assay using mitochondria revealed that isofetamid inhibited succinate dehydrogenase activity prepared from B. cinerea and other ascomycetes fungi used in the study. On the other hand, the activity of mitochondria prepared from Pythium, potato and rat were not inhibited. Isofetamid inhibited also many stages of the infection processes in B. cinerea. Furthermore, it exhibited high fungicidal activity against B. cinerea isolates that were resistant to existing fungicides.
Succinate dehydrogenase inhibitors (SDHIs) are important agricultural fungicides. Since the introduction of SDHIs with a very broad control spectrum, such as boscalid and penthiopyrad, which are effective against ascomycetes, many new compounds have been developed and are widely used as foliage spray 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).5,6)
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®) (Fig. 1), is a new fungicide developed by Ishihara Sangyo Kaisha, Ltd., and is suggested to be an SDHI classified in FRAC code 7 from the similarities of the chemical structure to the existing SDHIs.7–10) Isofetamid was registered as a pesticide in Canada in 2014 and introduced into the Japanese market in 2017, and has since been registered worldwide, contributing to the control of various diseases. Although a preliminary study indicated that isofetamid inhibited succinate dehydrogenase activity of Botrytis cinerea, its effects against the enzyme activity of the other sensitive and insensitive fungi, along with plants and mammals were not investigated clearly.9,10) Furthermore, inhibitory activity against the infection cycle of the target fungi and existence of cross-resistance between the existing fungicides were not adequately evaluated.9,10)
In this study, we investigated antifungal spectrum and mode of action to elucidate the fungicidal properties of isofetamid. Furthermore, we investigated the effect of isofetamid on the infection processes of B. cinerea and the fungicidal activity of isofetamid against strains of B. cinerea resistant to existing fungicides, since isofetamid showed high antifungal activity against important plant pathogenic fungus B. cinerea.
Isofetamid suspension concentrate (SC) at 400 g/L was used in this study. Thirty plant pathogens (Table 1), which were taken from the collection in our laboratory, were maintained on potato sucrose agar (PSA) medium at 20°C in the dark. Mycelial discs (4 mm in diameter) of the test fungi grown on PSA medium were cut from the margins of the colony and placed on PSA medium containing different concentrations of isofetamid. After incubation at 20°C for 2–14 days, radial mycelial growth was measured. The recorded mycelial growth was compared with that of the controls. The activity was expressed as EC50 (the concentration inhibiting growth by 50%).
| Pathogen | EC50 (µg/mL) |
|---|---|
| Ascomycetes | |
| Alternaria alternata apple pathotype | 0.99 |
| Alternaria alternata Japanese pear pathotype | 0.99 |
| Alternaria brassicicola | 0.51 |
| Alternaria solani | 0.02 |
| Botrytis allii | 0.22 |
| Botrytis cinerea | 0.10 |
| Botrytis squamosa | 0.74 |
| Botrytis tulipae | 0.36 |
| Claviceps virens | 0.12 |
| Cochliobolus miyabeanus | 0.79 |
| Corynespora cassiicola | 0.35 |
| Monilinia mali | 0.19 |
| Mycosphaerella melonis | 0.65 |
| Mycovellosiella nattrassii | 0.66 |
| Phoma lingam | 0.07 |
| Pyrenophora graminea | 0.26 |
| Rhynchosporium secalis | 0.91 |
| Sclerotinia minor | 0.03 |
| Sclerotinia sclerotiorum | 0.01 |
| Sclerotinia trifoliorum | 0.01 |
| Septoria nodorum | 0.47 |
| Sphaerulina oryzina | 0.45 |
| Stromatinia cepivora | 0.01 |
| Trichoderma sp. | 0.41 |
| Venturia inaequalis | 0.79 |
| Basidiomycetes | |
| Atheria rolfsii | >50 |
| Helicobasidium mompa | >50 |
| Rhizoctonia solani | >50 |
| Oomycetes | |
| Pythium aristosporum | >50 |
| Pythium spinosum | >50 |
a) Potato sucrose agar medium was used.
Technical-grade active ingredients of isofetamid synthesized by Ishihara Sangyo Kaisha, Ltd., boscalid (Wako, Osaka, Japan) and antimycin A (Wako, Osaka, Japan) were used in this study. The compounds were dissolved in the solvent dimethyl sulfoxide. Mitochondria from B. cinerea, Alternaria solani, Corynespora cassiicola, Septoria nodurum, and Pythium aristosporum were prepared and used. Mitochondria from liver of Sprague-Dawley rats and tubers of potato (Solanum tuberosum cv. Norin No. 1) were also used in this study.
Fungal mitochondria were prepared as follows. After pre-incubation for 7 days in 500 mL potato sucrose (PS) liquid medium at 20°C, the mycelia of the fungi were centrifuged at 5,000 rpm for 10 min at 4°C, and the supernatant was decanted. Subsequently, the mycelia were homogenized in 100 mL of Buffer A (20 mM 3-(N-morpholino) propanesulfonic acid-potassium hydroxide (MOPS-KOH) buffer (pH 7.1) containing 0.3 M mannitol, 1 mM ethylene diamine tetraacetic acid (EDTA), and 0.1% (w/v) bovine serum albumin) with glass beads using bead beater on ice. Homogenization was performed for 15 sec, followed by incubation on ice for 2 min, which was repeated 10 times. Potato tubers were homogenized in Buffer A containing 0.1% 2-mercapto-ethanol using a blender.11) Next, homogenates of mycelia or tubers were centrifuged at 4,000 rpm for 20 min at 4°C. Subsequently, the supernatant was centrifuged at 8,000 rpm for 20 min at 4°C. The precipitate was re-suspended in approximately 20 mL of Buffer B (10 mM MOPS-KOH buffer (pH 7.1) containing 0.25 M sucrose and 1 mM EDTA) and homogenized on ice using a Teflon homogenizer. Mitochondria were obtained by centrifuging at 15,000 rpm for 30 min and were finally re-suspended in 1.5 mL of Buffer B. Rat liver mitochondria were prepared following the method of Myers and Slater.12)
Both NADH-cytochrome c oxidoreductase (complex I and III) and succinate-cytochrome c oxidoreductase (complex II and III) activities were spectrophotometrically assayed by measuring the increase in absorbance at 550 nm (i.e., the rate of cytochrome c reduction) at 25°C. The relative extent of cytochrome c reduction was determined using a SHIMADZU UV-2550 UV-visible spectrophotometer. The mitochondria of B. cinerea was used to determine NADH-cytochrome c oxidoreductase activity and succinate-cytochrome c oxidoreductase activity, and A. solani, C. cassiicola, S. nodorum, P. aristosporum, rat, and potato were used to determine succinate-cytochrome c oxidoreductase activity. As for succinate-cytochrome c oxidoreductase (complex II and III) activities, inhibition values of each sample were obtained assuming that percent inhibition of 1 µM antimycin A-treated samples was 100. From the relationships between inhibition value and inhibitor concentration in enzyme tests, IC50 (the concentration inhibiting enzyme activity by 50%) was determined.
Succinate dehydrogenase activity was spectrophotometrically assayed by measuring the rate of reduction of the 2,6-dichlorophenolindophenol dye at 600 nm. From the relationships between inhibition value and inhibitor concentration in enzyme tests, IC50 was determined. In this study, only the mitochondria of B. cinerea were used for the enzyme assay.
3. Inhibitory activity against infection processes of B. cinereaThe technical-grade active ingredient of isofetamid, synthesized by Ishihara Sangyo Kaisha, Ltd., was used in this study. The compound was dissolved in dimethyl sulfoxide. B. cinerea (B05.10) used in this study was maintained on PSA medium at 20°C. Conidia were obtained by gently scraping the cultures incubated for 14 days.
One milliliter of conidial suspension (1×104 conidia/mL) containing different concentrations of isofetamid was dispensed in a 24-well microplate. After incubating for 3 days at 20°C, the number of germinated conidia was counted using an inverted microscope, and inhibition rates were calculated.
One milliliter of conidial suspension (1×104 conidia/mL) was dispensed in a 24 well-microplate. After incubating for 3 days at 20°C, the length of germ tube was measured as an initial value using an inverted microscope. The water in each well was replaced with 500 µL of 1% yeast bacto acetate (YBA) liquid medium containing different concentrations of isofetamid. After incubating for a day at 20°C, the elongation of germ tube was measured as the final value. Elongation values were calculated by subtracting the initial value from the final value.
Subsequently, 1.5 mL of conidial suspensions (1×104 conidia /mL) in 50% PS liquid medium were dispensed into glass petri dishes. After incubating for 3 hr at 20°C, water in each petri dish was replaced with 1.5 mL of solution containing different concentrations of isofetamid. After incubating for 9 hr at 20°C, the number of appressoria was counted using an inverted microscope.
Mycelial discs (4 mm in diameter) of B. cinerea grown on PSA medium were cut from the margins of the colony and placed on YBA agar medium containing different concentrations of isofetamid. After incubating for 5 days at 20°C, radial mycelial growth was measured.
The activity of isofetamid was expressed in terms of its inhibition rate in all tests. The inhibition rate (IR) was calculated using the following equation:
 |
Isofetamid SC at 400 g/L, azoxystrobin 20% SC (Amister® 20; Syngenta), thiophanate-methyl 70% WP (TOPSIN-M® WP; NIPPON SODA), procymidone 50% WP (Sumilex® WP; SUMITOMO CHEMICAL), and fenhexamid 50% WP (Password® WG, BAYER) were used in this study. B. cinerea isolates used in this study were maintained on PSA medium at 20°C (Table 3). Conidia were obtained by gently scraping the cultures incubated for 14 days.
Cucumber seedlings (cv. Sagami hanjiro) were grown for approximately 20 days in a greenhouse. Seedlings were sprayed with chemical solutions adjusted to each concentration and dried. The seedlings were inoculated with a conidial suspension (1×105 spores/mL) using the following method. The treated leaves were cut and placed on wetting towels in petri dishes. Paper discs (8 mm in diameter) were placed on the leaves and 105 µL of spore suspension was dropped onto the paper discs. After incubating for 3 days at 20°C, the diameter of lesions was measured using venire calipers. The activities of fungicides were expressed in terms of IR, which was calculated as described in section 3.
The fungicidal activities of isofetamid against various fungi were investigated using a mycelial growth inhibition test. Isofetamid strongly inhibited mycelial growth of the all tested ascomycetes fungi including Alternaria spp., Botrytis spp., Claviceps virens, Cochliobolus miyabeanus, C. cassiicola, Monilinia mali, Mycosphaerella melonis, Mycovellosiella nattrassii, Phoma lingam, Pyrenophora graminea, Rhynchosporium secalis, Sclerotinia spp., S. nodorum, Sphaerulina oryzina, Stromatinia cepivora, Trichoderma sp., and Venturia inaequalis. The EC50 values of isofetamid against ascomycetes were <1 µg/mL. However, the EC50 values of isofetamid against basidiomycetes and oomycetes were >50 µg/mL (Table 1). These results indicate that isofetamid has a wide spectrum in ascomycetes and has the potential to control multiple diseases caused by ascomycetes at the same time in the field.
2. Effect on mitochondrial electron transportThe effect of isofetamid on mitochondrial electron transport was analysed. Isofetamid did not inhibit NADH-cytochrome c oxidoreductase (complex I and III) in the mitochondrial fraction of B. cinerea. In contrast, isofetamid and boscalid effectively inhibited succinate-cytochrome c oxidoreductase (complex II and III). Isofetamid indicated a 50% inhibition value of 0.0010 µg/mL for succinate-cytochrome c oxidoreductase activity (Supplemental Table S1).
To identify the target enzymes of isofetamid, the effects of isofetamid and boscalid on succinate dehydrogenase (complex II) activity were examined. Both chemicals inhibited succinate dehydrogenase activity efficiently (Supplemental Table S1). These results indicate that isofetamid is a SDHI similar to boscalid.
The effects of isofetamid on succinate-cytochrome c oxidoreductase (complexes II and III) activity of the mitochondrial fraction of other fungi shown in Tables 1, 2 were analysed. Isofetamid inhibited succinate-cytochrome c oxidoreductase activity of B. cinerea, A. solani, S. nodorum, and C. cassiicola at low concentrations but did not inhibit the activity of P. aristosporum. In addition, isofetamid did not inhibit the enzyme activity in potatoes and rats, which are non-target organisms (Table 2). These results indicate that although isofetamid has the potential to affect many species of ascomycetes, it has no effect on oomycetes, plants, and mammals at the enzyme assay level.
| Biological source | Isofetamid (µg/mL) |
|---|---|
| IC50 | |
| Botrytis cinerea | 0.0010 |
| Alternaria solani | 0.0028 |
| Septoria nodorum | 0.0071 |
| Corynespora cassiicola | 0.0007 |
| Pythium aristosporum | >1 |
| Potato | >1 |
| Rat | >1 |
Based on these results, we concluded that isofetamid is a succinate dehydrogenase inhibitor (FRAC code 7). Furthermore, isofetamid specifically inhibits succinate dehydrogenase activity in ascomycetes, whereas it has no impact on the activity of oomycetes, plants, and mammals. Therefore, the selectivity of fungicidal activity is determined clearly at the site of action level. Structure of isofetamid is unique in that it contains a phenyl-oxo-ethyl thiophene amide (Fig. 1) and isofetamid is the first halogen-free compound among the compounds classified in code 7 of FRAC marketed since 2000.7,8)
3. Inhibitory activity against infection processes of B. cinereaThe effect of isofetamid on infection process of the pathogen was investigated using B. cinerea, a pathogen of a commercially important diseases. The effects of isofetamid on conidial germination, germ tube elongation, appressorium formation, and mycelial growth were analysed. Isofetamid effectively inhibited these infection processes of B. cinerea and the EC50 values of conidial germination, germ tube elongation, appressorium formation, and mycelial growth were 0.4 µg/mL, 0.02 µg/mL, 0.04 µg/mL and 0.006 µg/mL, respectively (Supplemental Table S2). These results indicate that isofetamid strongly inhibits conidial germination, germ tube elongation, appressorium formation and mycelial growth of B. cinerea in in vitro conditions, suggesting that isofetamid inhibits efficiently many stages of infection processes of pathogenic fungi. These results are consistent with the high efficacy of isofetamid against various gray mold diseases observed in official field trials for pesticide registration.9,10)
4. Control efficacy against isolates of B. cinerea resistant to existing fungicidesThe efficacy of isofetamid against isolates of B. cinerea resistant to other existing fungicides was investigated using cucumber seedlings. Notably, BC39 was sensitive to all the tested fungicides (Table 3). Compared with BC39, BC42 was resistant to thiophanate-methyl, procymidone and azoxystrobin; BC56 was resistant to thiophanate-methyl, procymidone, azoxystrobin, and fenhexamid; B05.10 was resistant to only thiophanate-methyl. Isofetamid indicated consistently high efficacy against all these isolates, showing clearly that isofetamid was not cross-resistant to these fungicides (Table 3).
| Fungicide | EC95 (µg/mL) | |||
|---|---|---|---|---|
| BC39 | BC42 | BC56 | B05.10 | |
| Thiophanate-methyl | 14.1 | >400 | >400 | >400 |
| Procymidone | 18.3 | >400 | >400 | 18.4 |
| Azoxystrobin | 17.2 | >400 | >400 | 12.4 |
| Fenhexamid | 7.3 | 6.9 | >400 | 7.0 |
| Isofetamid | 32.2 | 14.5 | 17.6 | 11.4 |
Although multiple fungicide-resistant strains of B. cinerea exist in the field, isofetamid has no cross-resistance to methyl benzimidazole carbamates (MBC fungicides, FRAC code 1), QoI (FRAC code 11), KetoReductase inhibitors (KRI fungicides, SBI Class III, FRAC code 17), or dicarboximides (MAP/Histidine-Kinase in osmotic signal transduction fungicides, FRAC code 2).13) In addition, isofetamid did not exhibit cross-resistance to DMI (SBI Class I, FRAC code 3) (data not shown). We believe that isofetamid can be used for resistant management in chemical control of plant diseases.
We wish to thank Dr. Hahn, professor of University of Kaiserslautern, for providing Botrytis cinerea isolate BC56.
The online version of this article contains supplementary materials (Supplemental Table S1, Table S2), which are available at https://www.jstage.jst.go.jp/browse/jpestics/.
