Black blight caused by Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei is a serious problem in eggplant (Solanum melongena L.) production in Kochi Prefecture, Japan (Shimomoto et al. 2009). Dark brown spots approximately 1–2 mm in diameter appear on the leaves and gradually enlarge and coalesce, resulting in large, irregular lesions, followed by leaf detachment. Dark brown spots also appear on stems, and the stems eventually die above the lesions. The occurrence of this disease on leaves and stems causes large reduction of the amount of eggplant production. In diseased plants, small protrusions approximately 5 mm in diameter often appear on the fruit. These fruit are sorted as lower quality and are sold at lower prices than higher-quality fruit, which reduces farmers’ income. In 2021, the total area of eggplant production in Kochi Prefecture was 254 ha, of which 181 ha was affected by black blight (Ministry of Agriculture, Forestry and Fisheries of Japan 2022).
The application of protective fungicides is the primary method to control black blight. Pyribencarb is a quinone outside inhibitor (QoI) fungicide, which was developed by the Kumiai Chemical Industry and launched into the Japanese market in 2012. The target site of this fungicide is cytochrome b (cytb) of complex III in the electron transport system of the respiratory chain. Pyribencarb effectively controls the disease caused by C. cassiicola (Takagaki et al. 2014); therefore, this fungicide was registered for the control of black blight of eggplant in 2019. Since its registration, it has been widely used in eggplant production fields in Kochi Prefecture.
There are 20 QoI compounds currently (FRAC 2024), and there is a high risk of the occurrence of the pathogens resistant to this family of fungicides because of the highly site-specific mechanism of pathogen inhibition (Fernández-Ortuño et al. 2008; Gisi et al. 2002). To date, QoI resistance has been reported in more than 50 pathogen species (FRAC 2020). The development of QoI resistance in C. cassiicola has already been reported in several diseases, e.g., target spot on tomato (MacKenzie et al. 2020), Corynespora leaf spot on cucumber (Deng et al. 2023; Duan et al. 2019; Ishii et al. 2008;), and target spot on soybean (Wang et al. 2023). Previous reports have demonstrated that three mutations in the cytb gene leading to amino acid substitutions are responsible for QoI resistance in many plant pathogens (FRAC 2006). In QoI-resistant C. cassiicola, an amino acid change from G to A at position 143 (G143A), or F to L at position 129 (F129L), results from nucleotide mutations in the cytb gene (Duan et al. 2019; MacKenzie et al. 2020; Wang et al. 2023). Different substitutions in cytb confer different levels of resistance to QoI fungicides; compared with the F129L mutation, the G143A mutation is associated with stronger resistance to QoI fungicides (MacKenzie et al. 2020). In Kochi Prefecture, kresoxim-methyl-resistant isolates of C. cassiicola in eggplant have already been reported (Shimomoto et al. 2009). However, the occurrence of pyribencarb-resistant C. cassiicola in eggplant fields, the effectiveness of pyribencarb in controlling QoI-resistant C. cassiicola, and the molecular mechanisms of pyribencarb resistance have been unknown. The aim of this study was to investigate the occurrence of pyribencarb-resistant strains of C. cassiicola, explore the molecular mechanisms of resistance, and determine the effectiveness of pyribencarb for controlling resistant isolates.
A total of 99 mono-conidial isolates, which had been isolated from the leaves of eggplants with typical black blight symptoms from 34 eggplant fields in Kochi Prefecture, and were identified as C. cassiicola based on the morphological features of conidia, were used in this study (Table 1). These isolates were stored on PDA medium at 15 °C in the dark before the experiments.
The isolates were cultured on PDA medium at 25 °C for 5 days in the dark. Then, for analyses of the cytb gene sequence, DNA was extracted from a colony of each C. cassiicola isolate using a Maxwell RSC Instrument and the Maxwell RSC Plant DNA Kit (Promega, Madison, WI, USA). The complete cytb gene was amplified from DNA extracted from each isolate by PCR, with the primers CCbF (5′- TGAACTTCTCCTCATTCTCTATTATGAC-3′) and CCbR (5′-TATTTACTACGT AACTCTTAGTAAGAGC-3′). These primers were designed from the complete nucleotide sequence of C. cassiicola mitochondrial DNA deposited at the National Center for Biotechnology Information (NCBI) under the accession number OK054367 (Ma et al. 2021). The PCRs were conducted using Tks Gflex DNA Polymerase (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. The PCR products approximately 1.2 kb in size were sequenced by Eurofins Genomics (Tokyo, Japan) using the primers CCbF and CCbR, and the nucleotide sequences obtained were analyzed using Genetyx ver. 14 (Genetyx corporation, Tokyo, Japan). The results showed that all the isolates analyzed in this study had a cytb gene of 1,164 nt in length, exactly the same length as the cytb gene of C. cassiicola causing brown leaf spot disease on kiwi reported previously (the NCBI accession number NC_056323, Chen et al. 2019). The nucleotide sequence encoded a polypeptide of 387 amino acids (data not shown). Homology searches using the nucleotide and amino acid sequences of the cytb gene among all isolates showed that the guanine-to-cytosine substitution at position 428 from the 5′ end of the open reading frame, resulting in a glycine-to-alanine amino acid substitution at position 143 (G143A) of the amino acid sequence, was present in 68 of the 99 isolates (Table 1). No other substitutions were found (data not shown). The primary cause of QoI resistance is substitutions in the mitochondrial DNA cytb gene, with the G143A mutation presumed to hinder binding to QoIs, thereby conferring high resistance (FRAC 2006). The F129L substitution has also been detected in the cytb gene of C. cassiicola (MacKenzie et al. 2020), but it was not found in the isolates tested in this study. Although heteroplasmy (the mutual presence of distinct mitochondrial DNAs, G143 and A143, in different cytb gene alleles) has been reported in C. cassiicola (de Mello et al. 2022; Ishi et al. 2007), it was not detected in any of the isolates tested in this study (data not shown).
To evaluate the effectiveness of pyribencarb for controlling the resistant isolates, inoculation tests using potted eggplants were conducted with two wild-type isolates (CCE1 and CCE19) and two isolates harboring the G143A mutation (CCE59 and CCE71). After each fungal isolate was incubated on PDA plates at 25 °C for 7 days under black light irradiation (Toshiba, Tokyo, Japan), spores on the plates were suspended in distilled water (DW) and the concentration of conidia was adjusted to ca. 1.0 × 104 spores/ml. Potted eggplants (cv. Ryoma) were grown at 20 °C–28 °C in a greenhouse. When the plants reached the five-true-leaf stage, they were sprayed with pyribencarb or azoxystrobin at the registered concentration for eggplant production, i.e., a 2000-fold suspension in DW. In the control, plants were sprayed with DW only. After air-drying, the plants were inoculated with the spore suspension of each isolate (3 ml/plant), grown in the greenhouse for a further 10 days under high humidity, and then the disease severity of three leaves per plant was evaluated using the following scale, 0: no visible symptoms, 1: ˂10 lesions, 2: 11 to 50 lesions, 3: 51 to 200 lesions, 4: ˃200 lesions without leaf yellowing, 5: >200 lesions with leaf yellowing. These tests were conducted twice, with five plants per fungicide-isolate combination in the first test, and four in the second test. The average disease severities on the eggplants inoculated with isolate CCE1 after spraying with pyribencarb, azoxystrobin, and DW were 0.4, 0.9, and 2.9, respectively, in the first test, and 0.7, 0.6, and 3.3, respectively, in the second test. The average disease severities on the eggplants inoculated with isolate CCE19 after spraying with pyribencarb, azoxystrobin, and DW were 0.8, 0.9, and 3.2, respectively, in the first test, and 0.8, 0.6, and 3.2, respectively, in the second test. The average disease severities on the eggplants inoculated with isolate CCE59 after spraying with pyribencarb, azoxystrobin, and DW were 2.5, 3.5, and 3.5, respectively, in the first test, and 1.4, 3.2, and 3.4, respectively, in the second test. The average disease severities on the eggplants inoculated with isolate CCE71 after spraying with pyribencarb, azoxystrobin, and DW were 2.8, 4.1, and 4.3, respectively, in the first test, and 1.9, 3.5, and 3.9, respectively, in the second test (Table 2). The results of the inoculation tests demonstrated that pyribencarb and azoxystrobin were less effective in controlling isolates harboring G143A than in controlling wild-type isolates, indicating that the isolates with G143A are cross-resistant to pyribencarb and azoxystrobin. FRAC (2024) reported that all the QoI fungicides, FRAC cord 11, are in the same cross-resistance group. However, there were significant differences in average disease severities on eggplants inoculated with resistant isolates between the pyribencarb treatment and the control; but no differences between the azoxystrobin treatment and the control (Table 2). It has been reported that pyribencarb can control QoI-resistant strains of gray mold (Ozaki and Kida 2014). Further studies, e.g., field tests to evaluate the effectiveness of pyribencarb for controlling resistant isolates, and comparison of its efficacy with that of other fungicides registered for black blight, are needed to confirm that the application of pyribencarb is practically useful for controlling black blight in the eggplant production areas where resistant isolates are present.
We attempted to develop an in vitro fungicide sensitivity test method using mycelial homogenates or discs placed on pyribencarb-containing media. A 5 ml aliquot of a suspension of pyribencarb (Fantasista wettable powder, 40.0% a.i., Kumiai Chemical Industry, Tokyo, Japan) in sterilized DW and propyl gallate (Fujifilm Wako Chemicals, Osaka, Japan), an inhibitor of cyanide-insensitive alternative oxidase (Kaneko and Ishii 1999), in dimethyl sulfoxide (Fujifilm Wako Chemicals) were added into 1 L melted autoclaved PDA medium (Shimadzu Diagnostics, Tokyo, Japan) at 55 °C. The medium was poured into 230 mm × 80 mm square Petri dishes (Kenis, Osaka, Japan). In this way, PDA plates containing 0, 0.1 (for the test using homogenate only), 1.0, 10.0, and 100.0 µg/ml concentrations of pyribencarb based on a.i., and 4 mM propyl gallate were prepared. Similarly, PDA plates containing azoxystrobin (Amistar 20 Flowable, 20.0% a.i., Syngenta Japan, Tokyo, Japan) and propyl gallate, and PDA plates containing only propyl gallate, were also prepared. The 99 C. cassiicola isolates were precultured on PDA medium at 25 °C for 7 days. The test using mycelial homogenate was conducted following a previously described method (Watanabe et al. 2017); an agar block approximately 5 mm square was cut from the edge of a colony of each isolate and then smashed with zirconia beads (1 mm diameter) in a 2 ml plastic tube containing 600 µL sterilized distilled water using a Micro Smash device (Tomy Digital Biology, Tokyo, Japan) at 5,000 rpm for 5 s. Three 10 µL aliquots of the homogenate from each isolate were transferred onto each of the plates containing various additions at the concentrations described above. These tests were repeated three times. For the test using mycelial discs, three disks 5 mm in diameter were cut from the outer edge of each colony using an autoclaved plastic straw and placed upside down on each of the plates. The plates with both homogenates and disks were incubated at 25 °C in the dark for 4 days, and then the minimum inhibitory concentrations (MICs) of pyribencarb and azoxystrobin were determined. The results of the tests using mycelial homogenates showed that the MICs of pyribencarb against resistant and sensitive isolates ranged from 10 to ˃100 µg/ml and from ˂0.1 to 10 µg/ml, respectively; hence, it was impossible to differentiate between sensitive and resistant isolates on the pyribencarb-containing plates. The MICs of azoxystrobin against resistant and sensitive isolates were ˃100 and <0.1 to 10 µg/ml, respectively (Table 1); therefore, when mycelial homogenates of C. cassiicola were cultured on the plates containing 10 or 100 µg/ml azoxystrobin, it was possible to distinguish between pyribencarb and azoxystrobin-sensitive and -resistant C. cassiicola isolates. To our knowledge, this is the first report demonstrating the discrimination of resistant isolates of C. cassiicola from sensitive ones based on MICs. The results of the tests using mycelial disks showed that the MICs of both pyribencarb and azoxystrobin against sensitive isolates ranged from <1 to >100 µg/ml, and the MICs of pyribencarb and azoxystrobin against resistant isolates ranged from 100 to >100 µg/ml and from >100 µg/ml, respectively (Table 1); therefore, it was impossible to discriminate between isolates resistant to pyribencarb or azoxystrobin and sensitive isolates in these tests. Further research is required to understand why resistant isolates were not distinguishable from sensitive ones when mycelial homogenates were cultured on pyribencarb-containing media, and when mycelial discs were cultured on media containing pyribencarb or azoxystrobin.
Among the C. cassiicola isolates collected from diseased eggplants in 2022 and 2023, 90.8% were resistant to pyribencarb, revealing that resistant isolates were prevalent throughout eggplant production fields in Kochi Prefecture. This proportion was significantly higher than that detected in 2020 and 2021, i.e., 23.5% (Table 3). At present, pyribencarb is the only QoI agent registered for controlling eggplant black blight disease in Japan, being registered in 2019. It is very likely that the resistance rate has rapidly increased owing to its frequent use after registration.