Monitoring of Benzimidazole Resistance in Botrytis cinerea Isolates from Strawberry in Korea and Development of Detection Method for Benzimidazole Resistance

Fungal strains and plant

In 2022, 78 strains of B. cinerea were isolated from 78 different strawberry farms, which were affected by gray mold disease in Korea. Single isolate was collected from each strawberry farm and identified based on ITS and NEP2 gene sequences (unpublished data). The isolated strains, along with their respective geographic origins, are provided (Supplementary Table 1). They were cultured on potato dextrose agar (PDA; MBcell, Seoul, Korea) at 25°C under dark conditions conducive for mycelial growth. The strains were then stored at −80°C in 25% glycerol solution. To evaluate the fungicide sensitivity of the strains on strawberries, the ‘Sulhyang’ variety, which has the highest cultivation rate in Korea was used (Jeong et al., 2018). To assess the effectiveness of benzimidazole fungicides against the isolated strains, benomyl (DuPont, Newark, DE, USA) was chosen as a representative agent.

In vitro sensitivity assay of B. cinerea strains to benzimidazole

The B. cinerea strains were inoculated onto PDA media and incubated at 25°C for 4 days. Agar plugs (5-mm-diameter) from colonies of the isolates were inoculated onto PDA media containing benomyl at concentrations of 0, 0.1, 1, 10, 100, and 1,000 mg/l. After incubation for 3 days at 25°C, the diameter of the colonies was measured using a digital caliper (Mitutoyo, Kawasaki, Japan). Two separate experiments and two replicates (Petri plates) per each experiment were conducted for each strain or concentration. The data were obtained by subtracting the lengths of 5mm agar plugs from mycelial diameters. The EC₅₀ (effective concentration for 50% inhibition) values for each strain were calculated using the LL.3 model from the drc package (Ritz et al., 2015) in R software (version 4.2.3, R Core Team 2023, R Foundation for Statistical Computing, Vienna, Austria).

PCR amplification and sequencing of β-tubulin (TUB2)

DNA was extracted from the mycelia grown on a PDA medium using the CTAB method (Cubero et al., 1999). The TUB2 gene was amplified using nPfu-Forte DNA polymerase (Enzynomics Inc., Daejeon, Korea). A total of 20 μl PCR reaction mixture was prepared, consisting of 2 μl of 10× nPfu-Forte buffer, 0.5 μl of 10 pmol/μl forward primer (5′-ATG CGT GAG ATT GTA TGT ATT TC-3′), 0.5 μl of 10 pmol/μl reverse primer (5′-CTA TTC CTC GCC CTC AAT TG-3′), 2 μl of 2 mM dNTP mixture, 1 μl of 100 ng genomic DNA, 0.2 μl of 2.5 units/μl nPfu DNA polymerase, and 13.8 μl of UltraPure DNase/RNase-free distilled water (ThermoFisher Scientific, Waltham, MA, USA). The PCR reaction was carried out using the MiniAmp PCR system by Applied Biosystems (ThermoFisher Scientific) with an initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 48°C for 30 s, and extension at 72°C for 1 min. A final extension step was performed at 72°C for 5 min. Subsequently, enzymatic purification was performed using the ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems, Waltham, MA, USA). The amplicons were sequenced by a commercial sequencing service provider (Macrogen Inc., Seoul, Korea).

Sensitivity assays of B. cinerea strains to benzimidazole on strawberry leaves and fruits

Detached strawberry leaves were sterilized with 1% NaOCl for 3 min. The sterilized leaves were then rinsed thrice with sterile ddH₂O for 1 min. After naturally drying to remove the remaining moisture, the leaves were soaked for 3 min in benomyl solutions at concentrations of 250 mg/l (recommended concentration), 125 mg/l (half the recommended concentration), and 500 mg/l (double the recommended concentration). The control group was treated with sterile ddH₂O. After drying, the treated leaves were inoculated with agar plugs (5mm-diameter) taken from the edge of fungal colonies. The inoculated leaves were cultured at 25°C under dark conditions for 5 days, and the resulting lesions were measured using a digital caliper. The relative lesion length was calculated as follow:

‘Lt’ represents the average length of lesions when treated with benomyl, while ‘L0’ represents the average length of lesions when treated with ddH₂O. For the strawberry fruits, the same surface disinfection with NaOCl was performed, followed by the same method as described above, including benomyl treatment. Fungal spore suspensions obtained after 7 days of cultivation were adjusted to a concentration of 1 × 10⁶ spores/ml using a hemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany) and a microscope (Olympus BX51, Tokyo, Japan). Sterile pipette tips were used to create consistent wounds on the benomyl-treated strawberry fruits, and 20 μl of the spore suspension was inoculated into the wounds. Subsequently, the inoculated fruits were cultured at 25°C under dark conditions for 4 days, and the length of the lesions were measured using a digital caliper. The relative lesion length was calculated using the aforementioned formula. This experimental procedure was performed in triplicate.

Control efficacy assay of benzimidazole against B. cinerea strains in strawberry farm

Field efficacy of benzimidazole against benzimidazole sensitive and resistant B. cinerea strains were assayed. For the study, strawberry seedlings were cultivated in a greenhouse, located in Gwangju, South Korea, to promote fruit formation. The strawberry plants, which had never been exposed to any fungicides, were sprayed with 12.5 ml of benomyl at the recommended concentration of 250 mg/l using a sprayer A5–700 (APOLLO INDUSTRIAL Co., Ltd., Siheung, Korea). The control group, on the other hand, was sprayed only with 12.5 ml of sterile ddH₂O. After allowing the sprayed strawberries to dry for a day, 2.5 ml of spore suspensions of each strain, at a concentration of 1 × 10⁵ spores/ml, were sprayed using the sprayer. Two weeks after inoculation, the severity of gray mold disease on each strawberry plant, especially the fruit portion, was visually assessed and scored on a scale of 0 (no disease) to 5 (severe disease), expressed as a percentage. The experiment was conducted twice with five replicates. Using these results, control efficacy was calculated according to the following formula (Paul et al., 2021):

Where A1 represents the disease severity in strawberries treated only with sterile ddH₂O, without benomyl treatment, and A2 represents the disease severity in strawberries treated with benomyl.

Sampling for diagnosis of benzimidazole resistance using TaqMan qPCR analysis

Diseased samples of strawberry leaves and tomato fruits were collected from two greenhouses at two different locations, respectively, for diagnosis of benzimidazole resistance. One of the greenhouses was situated in Gwangju, where strawberries had been previously grown and tomato cultivation was in progress (35°10′05.7″N, 126°.49′22.1″E), while the other was located in Damyang, where strawberry cultivation was still in progress (35°15′32.2″N, 126°55′46.1″E). The greenhouse in Gwangju was visited to gather tomato fruits for sampling in May 2023, and in Damyang, strawberry leaves were collected for sampling in July 2023.

TaqMan qPCR analysis

TaqMan qPCR analysis was performed using a CFX96 Real-Time System (Bio-Rad, Carlsbad, CA, USA). Each reaction consisted of a mixture of 10 μl of SsoAdvanced Universal Probe Supermix (Bio-Rad), 400 nM of each primer, 200 nM of the probe, 1 μl of genomic DNA, and 6 μl of DNase-free water. The primer and probe information are described in Table 1. The probes were designed with different single base modifications to detect point mutations conferring resistance to benzimidazole. The probe for detecting the wild type without mutations was labeled with Cy5 fluorescence; the probe for detecting E198V mutation, with Texas Red; the probe for detecting E198A mutation, with FAM; and the probe for detecting E198G mutation, with HEX. The initial denaturation was carried out at 95°C for 3 min, followed by 45 cycles of denaturation at 95°C for 15 s, and annealing at 63°C for 30 s. For the qPCR sensitivity assay, DNA of CMML22-BC67 was tested with concentrations ranging from 100,000,000 fg/μl (100 ng) to 10 fg/μl (10-fold serial dilutions) (Supplementary Fig. 1).

Table 1

TaqMan probes and primers’ designs for benzimidazole resistance assessment in Botrytis cinerea strains

Statistical analysis

Statistical analysis of this study was conducted using R software (v4.2.3, R Development Core Team 2023). After calculating parameters of relative growth and disease severity, Tukey’s honestly significant difference test was performed to evaluate the significance. The significance level was set at *P < 0.05, **P < 0.01, ***P < 0.001.

In vitro benzimidazole sensitivity assay

In vitro benzimidazole sensitivity assays of 78 strains collected from 78 farms resulted in the differentiation of 14 sensitive strains and 64 resistant strains. The strains with an EC₅₀ value of 300 mg/l or higher were classified as the resistant group, while those with an EC₅₀ value of less than 1 mg/l were classified as sensitive. The EC₅₀ values of the resistant group ranged from 377.62 mg/l (CMML22-BC04) to 5,955.72 mg/l (CMML22-BC29). For the sensitive group, EC₅₀ values ranged from 0.04 mg/l (CMML22-BC25) to 0.15 mg/l (CMML22-BC94) (Fig. 1). The investigation covering a wide geographic area with five different regions revealed that Gyeongsang Province had 93.75% resistant isolates; Jeolla Province, 87.50%, Chungcheong Province, 85.71%; Gangwon Province, 68.75%; and Gyeonggi Province, 75.00%.

Fig. 1

Benzimidazole sensitivity of Botrytis cinerea strains isolated from strawberries grown in 78 different farms in Korea. The EC₅₀ values of B. cinerea strains to a benzimidazole fungicide, benomyl, were divided into 14 benzimidazole sensitive strains and 64 resistant strains. Strains with EC₅₀ values equal to or below 0.147 mg/l, including CMML22-BC94, were classified as sensitive and marked in blue. On the other hand, strains with EC₅₀ values equal to or above 377 mg/l, including CMML22-BC04, were classified as resistant strains. Among the resistant strains, those with the E198A mutation in the TUB2 gene were marked in red, while those with the E198V mutation were marked in yellow.

Benzimidazole sensitivity tests on strawberry leaves and fruits

The effectiveness of benzimidazole fungicides in controlling B. cinerea infection in strawberry leaves and fruits and the phenotypic differences between sensitive and resistant strains were examined. It was observed that even when treated with half of the recommended concentration of benomyl (125 mg/l) typically used in farms, disease control was effectively achieved in the sensitive strain when inoculated on strawberry leaves. However, no control of the disease was observed in the resistant strains even when exposed to double the recommended concentration of benomyl (500 mg/l) (Fig. 2). Furthermore, in strawberry fruits, it was evident that the sensitive strains responded positively to the treatment with the recommended concentration of benomyl (250 mg/l), whereas no disease control was observed in the resistant strains (Fig. 3).

Fig. 2

Sensitivity of benzimidazole sensitive and resistant Botrytis cinerea strains on strawberry leaves against benomyl. (A) Pictures of lesions on strawberry leaves caused by B. cinerea strains in response to benomyl at different concentrations. (B) Relative growth of B. cinerea strains on strawberry leaves treated with benomyl. In all three concentrations of benomyl treatment, the sensitive strain CMML22-BC67 exhibited significantly lower disease severity compared to the resistant strains. Error bars represent the standard error of the mean (***P < 0.001).

Fig. 3

Sensitivity of benzimidazole sensitive and resistant Botrytis cinerea strains on strawberry fruits against benomyl (250 mg/l). (A) Pictures of strawberry fruits caused by B. cinerea strains in response to benomyl. (B) Relative growth of B. cinerea strains on strawberry fruits treated with benomyl. Significant differences were observed only between the sensitive strains and the resistant strains. Statistical differences of means ± standard errors were tested by Tukey’s honestly different test (P < 0.001).

Control efficacy of benzimidazole against B. cinerea strains in strawberry farm

The disease severity of strawberry plants caused by sensitive and resistant strains in response to benzimidazole were observed in strawberries grown in a greenhouse setting. Disease severity caused by the sensitive strain CMML22-BC67 was significantly reduced from 66.66% to 13.33% by application of 250 mg/l of benomyl (**P < 0.01). However, the severity caused by resistant strains (CMML22-BC03, -BC27, -BC30, and -BC42) was not significantly reduced even after benomyl treatment. For the disease severity of resistant strains, CMML22-BC03 slightly decreased after treatment of benomyl from 86.66% to 80.00%; CMML22-BC27 remained 100.00%; CMML22-BC30 increased from 45.00% to 71.42%; and CMML22-BC42 increased from 80.00% to 88.88% (Fig. 4). In the case of the sensitive strain, CMML22-BC67, the control efficacy was 80.00%, indicating a high effectiveness of benomyl. However, for the resistant strain, CMML22-BC03, the control efficacy was only 7.69%, indicating a very low effectiveness of benomyl. Additionally, other resistant strains, CMML22-BC27, -BC30, and -BC42, showed a control efficacy of 0%, indicating that they were not controlled at all by benomyl.

Fig. 4

Assessment of gray mold disease severity in strawberry farm. (A) Pictures of strawberry gray mold caused by benzimidazole sensitive and resistant Botrytis cinerea strains after treatment of sterile ddH₂O or benomyl (250 mg/l). The sensitive strain CMML22-BC67 demonstrated effective disease control, while CMML22-BC42 showed no response to the treatment. (B) Disease severity of the strawberry plants caused by the sensitive strain CMML22-BC67 and resistant strains, CMML22-BC03, CMML22-BC27, CMML22-BC30, CMML22-BC42, before and after the treatment of 250 mg/l of benomyl. This graph is represented as a crossbar plot, where the line inside the box indicates the mean value, and the range of the box represents the standard error. The severity caused by the sensitive strain was significantly reduced after the treatment of benomyl (**P < 0.01).

Mutation in TUB2 associated with benzimidazole resistance

Mutations were investigated in the amino acid sequence of β-tubulin from the 1st to the 238th position. The results revealed no mutations in the sensitive strains, whereas a mutation, E198A or E198V, was identified in the resistant strains. Among the resistant strains, 37 strains contained the E198A mutation, while 27 strains contained the E198V mutation (Table 2).

Table 2

Mutation within β-tubulin for each Botrytis cinerea strain and the average EC₅₀ value for each genotype

Detection of benzimidazole resistant B. cinerea using TaqMan qPCR analysis

To confirm the presence of mutations in β-tubulin associated with resistance to benzimidazole, TaqMan qPCR analysis was performed on the sensitive strain CMML22-BC67 and the resistant strains CMML22-BC03 and CMML22-BC42, which harbor E198V and E198A mutations in TUB2, respectively. The results showed that only the Cy5 fluorescence of the probe targeting the TUB2-198E was detected in CMML22-BC67 (Fig. 5A), while the Texas Red fluorescence of the probe targeting the TUB2-198V was detected in CMML22-BC03 (Fig. 5B), which acquired resistance due to the E198V mutation. Additionally, the FAM fluorescence of the probe targeting the TUB2-198A was detected in CMML22-BC42, where the E198A mutation had occurred (Fig. 5C).

Fig. 5

Detection of benzimidazole resistant Botrytis cinerea strains using TaqMan qPCR analysis. Genomic DNA extracted from each strain was mixed individually with primer pairs and supermix. Subsequently, each of these mixtures was mixed with four distinct fluorescent detection probes, including Cy5, FAM, Texas Red, and HEX. Each of these four mixtures was subjected to three technical replicates as part of the experimental procedure. (A) In the benzimidazole sensitive strain CMML22-BC67, only Cy5 fluorescence was detected. (B) In the resistant strain CMML22-BC03 harboring the E198V mutation, Texas Red was detected. (C) In the resistant strain CMML22-BC42 carrying the E198A mutation, FAM was detected.

Diagnosis of benzimidazole resistance in strawberry and tomato growing farms using TaqMan qPCR analysis

DNA was extracted from strawberry leaves sampled in Damyang and subjected to TaqMan qPCR analysis using the same method, resulting in the appearance of Texas Red fluorescence after 27 cycles in 4 individual samples (Fig. 6A). Similarly, DNA extraction was performed from four sampled tomatoes in a Gwangju farm, followed by TaqMan qPCR analysis. The results revealed the detection of Texas Red fluorescence corresponding to E198V in all samples after 25 cycles (Fig. 6B). Diagnosis of B. cinerea populations in strawberry and tomato farms by TaqMan qPCR analysis revealed both harbored resistance to benzimidazole.

Fig. 6

Benzimidazole resistance diagnosis of Botrytis cinerea within greenhouses located in Gwangju and Damyang, Korea. (A) Results of TaqMan qPCR analysis using genomic DNA extracted from strawberry leaves collected in Damyang showed that among four fluorescent probes, TexasRed fluorescence, which detects E198V, was detected in all samples. (B) Results of diagnosis using genomic DNA extracted from tomato fruits collected in Gwangju showed that TexasRed was detected in all samples.