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Article

Development of Procymidone and Difenoconazole Resistance in Alternaria alternata, the Causal Agent of Kiwifruit Brown Spot Disease

by
Yahui Liu
1,
Manfei Bao
1,
Yanxin Wang
2,* and
Chuanqing Zhang
1,*
1
College of Advanced Agricultural Sciences, Zhejiang Agriculture and Forest University, Hangzhou 311300, China
2
Extension Centre of Agriculture Technology of Hangzhou, Hangzhou 310020, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(14), 2245; https://doi.org/10.3390/plants14142245
Submission received: 1 July 2025 / Revised: 14 July 2025 / Accepted: 18 July 2025 / Published: 21 July 2025
(This article belongs to the Special Issue Fungicide Resistance in Plant Pathogens: Monitoring, Detection, Mechanisms and Management)
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Abstract

Brown spot, caused by Alternaria alternata, is the most important leaf fungal disease threatening kiwifruit production in China, and it is typically controlled through the application of fungicides, such as procymidone and difenoconazole. To date, fungicide resistance development has not yet been systematically reported for the pathogen of kiwifruit. A total of 135 single-conidium A. alternata isolates were collected from different cities in Zhejiang Province, China. Alternaria alternata developed prevailing resistance to procymidone and initial resistance to difenoconazole, with resistance frequencies of 60.7 and 13.3%, respectively. Positive cross-resistance was observed between procymidone and iprodione but not between procymidone and difenoconazole, tebuconazole, prochloraz, pydiflumetofen, pyraclostrobin, or thiophanate-methyl. Moreover, no cross-resistance was observed between difenoconazole and all other tested fungicides, including the two other demethylation inhibitors, tebuconazole and prochloraz. A fitness penalty was not detected in procymidone-resistant (ProR) or difenoconazole-resistant (DifR) isolates. However, double-resistant (ProR DifR) isolates had a fitness penalty, showing significantly decreased sporulation, germination, and pathogenicity. The P894L single point mutation, caused by the change from CCA to CTA at the 894th codon of Os1, was detected in ProR isolates. Molecular dynamic simulation showed that the P894L mutation significantly decreased the inhibitory activity of procymidone against AaOs1 in A. alternata. These results provide insight into the development and characteristics of fungicide resistance, offering guidance for the study and management of kiwifruit diseases.

1. Introduction

Kiwifruit (Actinidia chinensis Planch.), also known as Chinese gooseberry, is a small fruit that is rich in vitamin C, vitamin K, vitamin E, and potassium [1]. It is native to China and has become popular worldwide [2,3]. China, Italy, New Zealand, Chile, and Greece are the key producers, with a total annual production of 1.8 million tons [4,5]. In 2024, China became the largest global producer of kiwifruit, with a planting area exceeding 200,000 ha and an annual output of more than 3.8 million tons. As an important production and consumption region in China, Zhejiang Province faces significant challenges from foliar diseases [6]. Particularly, brown spot, also called black rot, is caused by Alternaria alternata and is the most devastating leaf disease threatening kiwifruit production. Alternaria alternata has also been reported to be the causal agent of fruit rot at both preharvest and postharvest stages in kiwifruit [7,8,9]. Alternaria alternata is a saprophytic fungal pathogen with a wide host range that includes tomato, apple, pear, orange, strawberry, melon, and tobacco [10,11,12].
Chemical control by fungicides is an important strategy for the management of kiwifruit diseases. Procymidone, a dicarboximide fungicide, classed into Fungicide Resistance Action Committee (FRAC) group 2, inhibits the two-component histidine kinase involved in the osmotic-regulatory signal transduction pathway in fungi [13,14]. It has been applied to control brown spot disease of kiwifruit for more than 10 years. Resistance to group 2 fungicides has been reported in plant pathogens such as Botrytis cinerea and Alternaria spp. [14,15,16]. Mutations in the osmotic-sensitive-1 (OS1) gene are commonly responsible for this resistance [14,17,18]. Difenoconazole (demethylation inhibitor (DMI), FRAC group 3) is also an important agent adopted to manage kiwifruit diseases, including brown spot. Due to the continuous applications of DMI fungicides, some pathogenic fungi have developed resistance, for which CYP51 mutations are the main molecular mechanisms [14,19,20]. Although significant decreases in control efficacy have been reported by growers and local technicians, the detection and characterization of fungicide resistance have not yet been systematically reported in pathogens of kiwifruit.
This study aimed to (I) assess procymidone and difenoconazole resistance of A. altenata on kiwifruit, and (II) evaluate cross-resistance, fitness, and related target mutations. These results provide critical insights for disease control and resistance management on kiwifruit production.

2. Results

2.1. Procymidone and Difenoconazole Resistance

Alternaria alternata, which causes brown spot disease on kiwifruit developed prevailing resistance to procymidone, a typical representative of dicarboximide fungicides, as indicated by a total resistance frequency (FR) of 60.7% (Figure 1). Among the four assessed cities, only isolates from Jinhua had an FR value of 33.3%, while isolates from Jiangshan, Shangyu, and Hangzhou had FR values of more than 50.0%. For the tested DMI, difenoconazole, the FR of 13.3% showed that resistance development was still at an initial stage. The FR values of isolates from Jiangshan, Shangyu, Hangzhou, and Jinhua were 8.8, 17.1, 16.7, and 8.3%, respectively (Figure 1).

2.2. Cross-Resistance Between Procymidone or Difenoconazole and Other Fungicides

A total of 20 isolates, including both sensitive and resistant isolates, were selected at random to further determine the 50% effective inhibitory concentration (EC50) to different fungicides. Procymidone-resistant (ProR) isolates were cultured on potato dextrose agar (PDA) plates amended with 16 μg/mL procymidone (Figure 2). Procymidone-sensitive (ProS) isolates had an EC50 of 0.23–0.75 μg/mL, with a mean of 0.41 μg/mL (Table S1, at Supplementary). The five ProR isolates had a mean EC50 of 25.86 μg/mL and a mean resistance factor (RF = EC50 of resistant isolate/mean EC50 of sensitive isolates) of 62.6. Positive cross-resistance was observed between procymidone and iprodione. The mean EC50 to iprodione for the same ProS and ProR isolates was 0.52 and 24.68 μg/mL, respectively, with a mean RF of 47.5. All tested isolates had an EC50 > 100 μg/mL for thiophanate-methyl, regardless of whether the isolate was sensitive or resistant to procymidone or difenoconazole. Spearman’s rank correlation showed that there was positive cross-resistance between procymidone and iprodione (ρ = 0.288, p < 0.001). However, no cross-resistance was observed between procymidone and difenoconazole (ρ = −0.069, p = 0.772), procymidone and tebuconazole (ρ = −0.099, p = 0.677), procymidone and prochloraz (ρ = 0.143, p = 0.547), procymidone and pydiflumetofen (ρ = −0.295, p = 0.206), procymidone and pyraclostrobin (ρ = 0.111, p = 0.640), and procymidone and thiophanate-methyl (ρ = −0.040, p = 0.867; Figure 3).
For difenoconazole, the difenoconazole-sensitive (DifS) isolates had EC50 values of 0.22–0.76 μg/mL, with a mean of 0.43 μg/mL (Table 1). The difenoconazole-resistant (DifR) isolates had a mean EC50 of 15.38 μg/mL and a mean RF of 35.8. No obvious cross-resistance was observed between difenoconazole and iprodione (ρ = −0.160, p = 0.500), pydiflumetofen (ρ =0.048, p = 0.841), pyraclostrobin (ρ = 0.171, p = 0.472), or thiophanate-methyl (ρ = 0.296, p = 0.205; Figure 4). No positive cross-resistance was observed between difenoconazole and the other two tested DMIs, tebuconazole and prochloraz. The DifS isolates had EC50 values of 0.68–21.1 μg/mL, with a mean of 3.42 μg/mL for tebuconazole. The DifR isolates had EC50 values of 0.95–2.12 μg/mL with a mean of 1.36 μg/mL. There was no significant difference in tebuconazole sensitivity between DifS and DifR. Similar results were found for prochloraz. Spearman’s rank correlation showed no cross-resistance between difenoconazole and tebuconazole (ρ = 0.052, p = 0.828) and prochloraz (ρ = 0.036, p = 0.880; Figure 4).

2.3. Fitness of Procymidone- or Difenoconazole-Resistant Isolates

There were significant differences in mycelial growth, sporulation, conidial germination, pathogenicity, and compound fitness index (CFI) among the 17 tested isolates (Table S2). However, when isolates with two phenotypes (ProS DifS and ProR DifS, ProS DifS, and ProS DifR) were compared, there were no significant differences between them (Figure 5 and Figure 6). However, the double-resistant (ProR DifR) isolates had significantly decreased sporulation, germination, and pathogenicity compared with isolates with one of the other three phenotypes, namely ProS DifS, ProR DifS, and ProS DifR. The CFI of ProR DifR also decreased significantly (Figure 7). These results indicate that there was no significant fitness penalty for the ProR DifS or ProS DifR isolates, but a significant fitness penalty was associated with double resistance (ProR DifR).

2.4. Target Mutations of Os1 and CYP51 in Procymidone- and Difenoconazole-Resistant Isolates

The target coding genes, Os1 and CYP51, for procymidone and difenoconazole, respectively, were amplified using PCR and compared. The P894L single point mutation, caused by the change from CCA to CTA at the 894th codon of Os1, was detected in ProR isolates, including two ProR DifR isolates. For CYP51 and difenoconazole resistance, single nucleotide polymorphisms at the 188th, 192nd, 237th, 307th, 412th, 434th, 448th, and 462nd codons were observed. However, no distinct relationships were observed between these amino acid mutations in CYP51 and difenoconazole resistance (Table 1).

2.5. Binding Affinity Decreased After P893L Mutation of Os1

Four complex systems, wild type (WT) + ADP, WT + procymidone, P894L_ADP, and P894L_procymidone were identified through molecular dynamic simulation. Eleven hydrogen bonds were formed between ADP and N865, N869, A924, D925, T929, T935, L937, and G938 of the AaOs1 WT. In addition, a salt bridge formed between ADP and K872 of WT (Figure 8A). For P894L, seven hydrogen bonds were formed between ADP and N869, A924, D925, T935, G936, or L939, and a π–π conjugate formed with F873 (Figure 8B). Thus, P894L had no effect on the binding affinity of ADP with the Os1 protein. In WT + procymidone and P894L_procymidone, the binding conformation was maintained through hydrophobic interactions and hydrogen bonds (Figure 8C,D). In WT, procymidone bound to the active center of AaOs1 through a hydrophobic interaction with F873, I909, I917, T203, L209, and F965 and hydrogen bonds with Q923 and A924. When P894L mutation occurred, the hydrogen bonds between procymidone and Q923 disappeared, and hydrophobic interactions were weakened. Thus, it was more difficult for procymidone to bind to the active center of AaOs1 in A. alternata. The P894L mutation significantly decreased the inhibitory activity of procymidone against AaOs1 in A. alternata.

3. Discussion

Procymidone, a FRAC group 2 fungicide, has been applied as the leading chemical to manage major plant diseases caused by Alternaria for approximately two decades (http://www.chinapesticide.org.cn/, accessed on 25 December 2024). The present study showed that pathogenic A. alternata, which is responsible for brown spot on kiwifruit, had developed widespread resistance to procymidone, with an FR value of 60.7%. Procymidone resistance in Alternaria spp. has been reported in China in crops such as garlic, tobacco, broccoli, Dendrobium officinale, and Fritillaria thunbergii [18,20,21,22,23]. Resistance to group 2 dicarboximides, including procymidone, iprodione, dimethachlon, and chlozolinate, has been reported in other major fungal plant pathogens, such as B. cinerea, Sclerotinia spp., Monilinia fructicola, and Stemphylium vesicarium [16,24,25,26,27]. However, resistance of pathogens on kiwifruit, including A. alternata, has not been reported or well understood.
Understanding the fitness penalty of the pathogen–fungicide combination will guide resistance management and disease control strategies [19,24,28]. Several studies have reported a fitness penalty for resistance to dicarboximide fungicides. For example, procymidone-resistant A. alternata had reduced mycelial growth, pathogenicity, and mycotoxin production [18,19]. In the present study, no significant decrease in growth, sporulation, germination, or pathogenicity was observed for ProR isolates compared with ProS isolates. No obvious fitness penalty for resistance to group 2 fungicides has been reported in previous studies [19,28,29,30]. However, a significant fitness penalty was associated with double-resistant (ProR DifR) isolates in this study.
Different mutations in the OS1 gene, which encodes a two-component histidine kinase in fungi, are generally to the molecular mechanism of resistance to this fungicide group [19,21,23,26,27]. Alternaria alternata on kiwifruit showed positive cross-resistance to iprodione, as previously reported [18,23,27]. Only the P894L mutation in OS1 was observed in ProR isolates. Molecular dynamic simulation showed that the P894L mutation decreased the inhibitory activity of procymidone against AaOs1. The P894L + S1277L double point mutation has been reported in A. alternata isolates from Fritillaria thunbergii, a widely cultivated medicinal plant [23].
DMIs (FRAC group 3), such as difenoconazole, are commonly adopted fungicides for the prevention and control of plant diseases caused by A. alternata. They target sterol 14α-demethylase (CYP51), the key enzyme in biosterol synthesis [14,19,31]. Resistance to DMIs has been described in many plant pathogens, such as Aspergillus spp., Blumeria graminis, Cercospora beticola, Colletotrichum spp., Fusarium spp., Magnaporthe oryzae, Monilinia fructicola, and Penicillium digitatum [14,19]. Amino acid mutation in CYP51 is the main resistance mechanism. In our previous study, the G462S substitution of CYP51 was shown to be the main factor for moderate resistance to tebuconazole in A. alternata from tobacco, and mechanisms other than CYP51-target mutation might involve isolates with low resistance to tebuconazole [20]. In this study of A. alternata from kiwifruit, the initial stage of resistance development to difenoconazole was detected, with an FR value of 13.3%. No cross-resistance was observed between difenoconazole and the other tested fungicides, including two DMIs, tebuconazole and prochloraz. No mutations in CYP51 were found in DifR isolates, although all tested DifR isolates were moderately resistant, with a mean RF of 35.8. Our present results indicated the difference in mechanisms of resistance to DMIs for A. alternata on kiwifruit with previous reports. No mutations in CYP51 have been previously reported, such as in difenoconazole-resistant mutants of Sclerotium rolfsii induced in the laboratory [32]. The I463V point mutation in CYP51A is associated with low difenoconazole resistance in Colletotrichum truncatum [33]. The molecular mechanism by which A. alternata on kiwifruit resists difenoconazole requires further investigation.
In summary, A. alternata on kiwifruit has developed resistance to dicarboximide fungicides associated with the P894L mutation in OS1. In the future, disease management practices of kiwifruit should reduce the use of group 2 dicarboximide fungicides, and strategies such as mixes or rotations with fungicides that have no cross-resistance should be adopted to delay the development of difenoconazole resistance.

4. Materials and Methods

4.1. Fungicides

The following technical-grade fungicides were used for testing in this study: difenoconazole (a.i. 95.4%, Zhejiang Tianyi Agricultural Chemical Co., Ltd., Hangzhou, China), tebuconazole (a.i. 97%, Zhejiang Yongnong Corporation, Shangyu, China), prochloraz (a.i. 99.5%, Tianfeng Biotech Corporation, Jinhua, China), procymidone (a.i. 98%, Heben Biotech Corporation, Wenzhou, China), iprodione (a.i. 96.1%, Tianfeng Biotech Corporation, Jinhua, China), pydiflumetofen (a.i. 98%, Syngenta, Basel, Switzerland), thiophanate-methyl (a.i. 97%, Zhejiang Welldone Chemistry Company, Hangzhou, China), and pyraclostrobin (a.i. 98%, BASF, Ludwigshafen, Germany). These chemicals were dissolved in methanol or acetone to obtain stock solutions of 104 μg a.i./mL, and then the prepared stock solutions were kept at 4 °C in the dark before further tests.

4.2. Origin of Single-Spore Alternaria alternata Isolates

From May to August 2020–2022, kiwifruit leaf samples with typical brown spot disease symptoms were collected from Jiangshan, Shangyu, Hangzhou, and Jinhua. Jiangshan is one of the top 10 main production cities in China. The sampled leaves were placed in plastic bags and stored at 4 °C before isolation. Each leaf was flushed with tap water, and small pieces of tissue (5 mm × 2 mm) were cut from the samples surface-sterilized with 75% ethanol for 30 s and 30% sodium hypochlorite solution for 3 min. Then the tissue was washed three times with sterile distilled water, placed onto PDA (200 g potato, 20 g glucose, 20 g agar, and 1 L distilled water) plates amended with 50 mg/L streptomycin, and incubated at 25 °C in the dark. A total of 135 single-spore A. alternata isolates were recovered from 33 orchards. Less than five isolates were collected from each orchard. Procymidone (dicarboximide fungicide, FRAC group 2) and difenoconazole (DMI, FRAC group 3) have been applied on kiwifruit for leaf disease management for approximately 20 and 10 years, respectively. All tested single-spore isolates were identified by pathogenicity on kiwifruit, morphological characteristics (Figure S1), and analysis of the internal transcribed spacer (ITS) sequence [9,20,23]. All isolates were kept on PDA slants at 4 °C in the dark.

4.3. Determination of Procymidone and Difenoconazole Resistance

The discriminatory dose method was adopted to identify ProS and ProR A. alternata isolates, as described in previous studies [23,34]. ProS isolates had a minimum inhibitory concentration (MIC) of <5 μg/mL for mycelial growth after incubation at 25 °C in the dark for 5 days, and ProR isolates had an MIC of >5 μg/mL. Similarly, as described in previous studies [20,35], DifS isolates had an MIC of <5 μg/mL, and DifR isolates had an MIC of >5 μg/mL. For each concentration, three PDA plates were used per isolate, and the assessments were repeated twice. The resistance frequency was calculated as FR/% = (No. of resistant isolates/total no. of tested isolates) × 100.

4.4. Determination of Fungicide Sensitivity and Cross-Resistance Analysis

Fungicide sensitivity was determined through the mycelium growth inhibition method. Each isolate was pre-cultured on PDA plates at 25 °C in the dark for 5 days, and 6-mm mycelial plugs were cut from the edge of the colony and placed in the center of PDA plates amended with different concentrations of the tested fungicides (Table 2). Three plates were adopted for each treatment, and the assay was repeated twice. After incubation at 25 °C for 5 days, the diameter of each colony was measured, and the EC50 value was calculated for each isolate–fungicide combination by linear regression of the percent inhibition of mycelial growth relative to the control versus the log10 transformation of the fungicide concentrations.
To identify cross-resistance among procymidone, difenoconazole, and other fungicides, 20 isolates, including sensitive and resistant isolates, were randomly selected and assessed. The logEC50 of procymidone or difenoconazole was adopted as the x-coordinate, and the logEC50 of the other tested fungicides was individually plotted as the y-coordinate to establish the linear regression equations. The Spearman rank correlation was then used to determine cross-resistance. p < 0.05 and ρ > 0.6 indicated strong positive cross-resistance between two fungicides [23,36].

4.5. Fitness Characterization

Isolates resistant or sensitive to procymidone and difenoconazole were chosen at random to characterize the fitness components, including mycelial growth, sporulation, conidial germination, and pathogenicity as previously described [16,20]. The mycelial growth ability was represented by the mean colony diameter 5 days after incubation on PDA plates at 25 °C in the dark. After incubation for 14 days on PDA plates at 25 °C with continuous light, conidia were recovered with 5 mL of ddH2O for each 9-cm PDA plate, and a hemocytometer was used to count the number of conidia, representing sporulation. Then, 100 μL suspensions at a concentration of 1 × 105 conidia per mL were spread onto water agar (WA) plates. After incubation for 18 h at 25 °C in the dark, each plate was evaluated, and the germination rate was calculated. To determine the pathogenicity, healthy kiwifruit leaves were inoculated with A. alternata mycelial plugs (6-mm diameter). PDA plugs without mycelia were adopted as the negative control. Five days after incubation at 25 °C with 12 h:12 h (light: dark), the mean lesion size was measured and calculated to represent the pathogenicity. Triplicate measurements were performed for each tested isolate. The compound fitness index (CFI) was calculated as follows: CFI = mycelial growth × conidia production × conidial germination × lesion diameter [20].

4.6. Analysis of Mutations in the Coding Gene of Fungicide-Targeted Proteins

Isolates resistant or sensitive to procymidone and difenoconazole were cultured in YEPD medium (3 g yeast extract, 20 g dextrose, and 20 g glucose in 1 L distilled water). After 2 days, genomic DNA was extracted from each isolate using the cetyl trimethyl ammonium bromide (CTAB) method. The gene sequences were PCR amplified using the primers (Table S3) and procedures previously described for Os1 and CYP51 [20,23]. The PCR products were sequenced by Sangon Biotech (Shanghai, China). The coding sequences were translated into amino acid sequences and aligned using the ClustalW multiple alignment through BioEdit (v7.2.6.1, San Diego, CA, USA)

4.7. Molecular Docking Analysis

Amino acid sequences of AaOs1(two-component osmosensing histidine kinase) (XP_018380446.1) were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/protein/, accessed on 1 July 2024) and submitted to SWISS-MODEL to construct protein models. To analyze the docking of difenoconazole to AaOs1 before and after P894L substitution, homologous models of the AaOs1 WT and AaOs1-P894L were constructed using the AlphaFold server, an automated protein structure homology modeling server. The structure of difenoconazole was downloaded from the PubChem database and used as the ligand. Autodock Tools 1.5.6 and Gromacs2023.2 were used to perform the molecular dynamic simulation as described by references [20,37,38,39].

4.8. Statistical Analysis

The dataset was analyzed using Data Processing System software, version 6.55 (developed by Hangzhou RuiFeng Information Technology Co., Ltd., Hangzhou, China). The least significant difference test was used to identify differences among multiple means, with the significance threshold set at p < 0.05.

5. Conclusions

Brown spot caused by A. alternata on kiwifruit developed prevailing resistance to procymidone while early stage of resistance to difenoconazole. Fitness penalty was detected only in double-resistant (ProR DifR) isolates. The P894L single point mutation was first reported through significantly decreasing the inhibitory activity of procymidone against AaOs1. No cross-resistance was observed between difenoconazole and tebuconazole or prochloraz. No mutations in CYP51 were found in DifR isolates, indicating the difference in A. alternata on kiwifruit with previous studies. These results provide understanding and guidance for the study and management of fungicide resistance in Alternaria and kiwifruit diseases.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14142245/s1, Table S1: Cross-resistance between procymidone and difenoconazole with six other fungicides; Table S2: Fitness characteristics of procymidone- and difenoconazole-sensitive and -resistant isolates; Table S3: Primers and sequences; Figure S1: Pathogenicity and morphological characteristics of Alternaria alternata causing brown spot disease in kiwifruit.

Author Contributions

Conceptualization, M.B., Y.L. and C.Z.; methodology, Y.L., M.B., Y.W. and C.Z.; software, Y.L. and C.Z.; validation, Y.L. and C.Z.; formal analysis, Y.L., Y.W. and C.Z.; investigation, M.B., Y.L. and C.Z.; resources, Y.W.; data curation, Y.L. and C.Z.; writing—original draft preparation, Y.L., Y.W. and C.Z.; writing—review and editing, Y.L., C.Z. and Y.W.; supervision, C.Z.; project administration, C.Z.; and funding acquisition, Y.W. and C.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data will be made available upon reasonable request to the corresponding authors.

Acknowledgments

We thank LetPub (Shanghai, China) for its linguistic assistance during the preparation of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Gorinstein, S.; Haruenkit, R.; Poovarodom, S.; Park, Y.S.; Vearasilp, S.; Vearasilp, S.; Suhaj, M.; Ham, K.-S.; Heo, B.-G.; Cho, J.-Y.; et al. The Comparative Characteristics of Snake and Kiwi Fruits. Food Chem. Toxicol. 2009, 47, 1884–1891. [Google Scholar] [CrossRef]
  2. Dwivedi, S.; Mishra, A.K.; Priya, S. Potential Health Beneffts of Kiwifruits: The King of Fruits. Sci. Technol. 2020, 5, 126–131. [Google Scholar]
  3. Wu, X.M. Genetic Breeding and the Industry Progress of Kiwifruit. N. Fruits 2010, 2, 1–4. [Google Scholar]
  4. Guroo, I.; Wani, S.A.; Wani, S.M.; Ahmad, M.; Mir, S.A.; Masoodi, F.A. A Review of Production and Processing of Kiwifruit. J. Food Process. Technol. 2017, 8, 699. [Google Scholar]
  5. Ward, C.; Courtney, D. Kiwifruit: Taking Its Place in the Global Fruit Bowl. Adv. Food Nutr. Res. 2013, 68, 1–14. [Google Scholar]
  6. Li, D.; Huang, W.; Zhong, C. Current Status of China’s Kiwifruit Industry and Development Recommendations for the “15th Five-Year Plan”. J. Fruit Sci. 2024, 41, 2149–2159. [Google Scholar]
  7. Kwon, J.H.; Cheon, M.G.; Kim, J.W.; Kwack, Y.B. Black Rot of Kiwifruit Caused by Alternaria alternata in Korea. Plant Pathol. 2011, 27, 298. [Google Scholar] [CrossRef]
  8. Pan, H.; Li, W.; Chen, M.; Lei, J.; Cao, H.; Xie, Y.; Zhong, C.; Li, L. Pathogen Identification of Kiwifruit Diseases in Jiangshan City, Zhejiang Province. China Fruits 2024, 11, 93–97. [Google Scholar]
  9. Liu, X.P. Investigation, Pathogen Identification and Control of Kiwifruit Leaf Spot Diseases in Jiangshan City; Zhejiang Agriculture and Forest University: Hangzhou, China, 2023; Volume 5. [Google Scholar]
  10. Dang, H.X.; Pryor, B.; Peever, T.; Lawrence, C.B. The Alternaria Genomes Database: A Comprehensive Resource for a Fungal Genus Comprised of Saprophytes, Plant Pathogens, and Allergenic Species. BMC Genom. 2015, 16, 239. [Google Scholar] [CrossRef] [PubMed]
  11. Huang, K.; Tang, J.; Zou, Y.; Sun, X.; Lan, J.; Wang, W.; Xu, P.; Wu, X.; Ma, R.; Wang, Q.; et al. Whole Genome Sequence of Alternaria alternata, the Causal Agent of Black Spot of Kiwifruit. Front. Microbiol. 2021, 12, 713462. [Google Scholar] [CrossRef]
  12. Meena, M.; Samal, S. Alternaria Host-speciffc (HSTs) Toxins: An Overview of Chemical Characterization, Target sites, Regulation and Their Toxic Effects. Toxicol. Rep. 2019, 6, 745–758. [Google Scholar] [CrossRef]
  13. Avenot, H.; Simoneau, P.; Lacomivasilescu, B.; Bataillesimoneau, N. Characterization of Mutations in the Two-Component Histidine Kinase Gene AbNIK1 from Alternaria brassicicola that Confer high Dicarboximide and Phenylpyrrole Resistance. Curr. Genet. 2005, 47, 234–243. [Google Scholar] [CrossRef]
  14. Francis, A.D.; Silvino, M.I.; Costa, S.D.S.G.; Valter, C.M.; Eduardo, A. Target and Non-target Site Mechanisms of Fungicide Resistance and Their Implications for the Management of Crop Pathogens. Pest Manag. Sci. 2023, 79, 4731–4753. [Google Scholar]
  15. Grabke, A.; Fernández-Ortuño, D.; Amiri, A.; Li, X.; Peres, N.A.; Smith, P.; Schnabel, G. Characterization of Iprodione Resistance in Botrytis cinerea from Strawberry and Blackberry. Phytopathology 2014, 104, 396–402. [Google Scholar] [CrossRef]
  16. Jahangir, M.F.; Xiao, X.; Zhu, F.X.; Fu, Y.P.; Jiang, D.H.; Guido, S.; Luo, C.X. Exploring Mechanisms of Resistance to Dimethachlone in Sclerotinia sclerotiorum. Pest Manag. Sci. 2016, 72, 770–779. [Google Scholar]
  17. Oshima, M.; Fujimura, M.; Banno, S.; Hashimoto, C.; Yamaguchi, I. A Point Mutation in the Two Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide-Resistance in Field Isolates of Botrytis cinerea. Phytopathology 2002, 92, 75–80. [Google Scholar] [CrossRef] [PubMed]
  18. Wei, L.L.; Chen, B.; Li, X.J.; Shi, H.P.; Xie, S.; Hu, H.; Chen, W.C.; Wei, L.H.; Wang, X.Y.; Chen, C.J. The HOG-Pathway Related AaOS1 Leads to Dicarboximide-Resistance in Field strains of Alternaria alternata and Contributes, Together with the Aafhk1, to Mycotoxin Production and Virulence. Pest Manag. Sci. 2024, 80, 2937–2949. [Google Scholar] [CrossRef]
  19. Yin, Y.N.; Miao, J.Q.; Shao, W.Y.; Liu, X.X.; Zhao, Y.F.; Ma, Z.H. Fungicide Resistance: Progress in Understanding Mechanism, Monitoring and Management. Phytopathology 2023, 113, 707–718. [Google Scholar] [CrossRef] [PubMed]
  20. Feng, J.; Cai, L.T.; Li, T.; Wang, H.C.; Zhang, C.Q. The G462S Substitution of AaCYP51 confers moderate resistance to tebuconazole in Alternaria alternata. Pest Manag. Sci. 2025, 81, 2891–2900. [Google Scholar] [CrossRef]
  21. Wang, B.R.; Lou, T.C.; Wei, L.L.; Chen, W.C.; Huang, L.B.; Ding, L.; Zhao, W.C.; Zhang, P.C.; Sun, P.; Chen, C.J.; et al. Biochemical and Molecular Characterization of Alternaria alternata Isolates Highly Resistant to Procymidone from Broccoli and Cabbage. Phytopath. Res. 2021, 3, 15. [Google Scholar] [CrossRef]
  22. Zhao, W.C.; Sun, C.X.; Wei, L.L.; Chen, W.C.; Wang, B.R.; Li, F.J.; Wei, M.D.; Lou, T.C.; Zhang, P.C.; Zheng, H.H.; et al. Detection and Fitness of Dicarboximide-Resistant Isolates of Alternaria alternata from Dendrobium officinale, a Chinese Indigenous Medicinal Herb. Plant Dis. 2021, 105, 2222–2230. [Google Scholar] [CrossRef]
  23. Wang, J.L.; Zhu, L.Y.; Zhang, C.Q. Resistance Development to Procymidone and Boscalid in Alternaria alternata Causing Black Spot Disease on Fritillaria thunbergii. Plant Dis. 2025, 109, 3614–3622. [Google Scholar] [CrossRef] [PubMed]
  24. Baibakova, E.V.; Nefedjeva, E.E.; Suska-Malawska, M.; Wilk, M.; Sevriukova, G.A.; Zheltobriukhov, V.F. Modern Fungicides: Mechanisms of Action, Fungal Resistance and Phytotoxic Effects. Annu. Res. Rev. Biol. 2019, 32, 1–16. [Google Scholar] [CrossRef]
  25. Alberoni, G.; Collina, M.; Lanen, C.; Leroux, P.; Brunelli, A. Field Strains of Stemphylium vesicarium with a Resistance to Dicarboximide Fungicides Correlated with Changes in a Two-Component Histidine Kinase. Eur. J. Plant Pathol. 2010, 128, 171–184. [Google Scholar] [CrossRef]
  26. Li, J.L.; Kang, T.H.; Talab, K.M.A.; Zhu, F.X.; Li, J.H. Molecular and biochemical characterization of dimethachlone. Pestic. Biochem. Physiol. 2017, 138, 15–21. [Google Scholar] [CrossRef]
  27. Ma, Z.; Luo, Y.; Michailides, T.J. Molecular characterization of the Two-Component Histidine Kinase Gene from Monilinia fructicola. Pest Manag. Sci. 2006, 62, 991–998. [Google Scholar] [CrossRef]
  28. Hawkins, N.; Fraaije, B. Fitness Penalties in the Evolution of Fungicide Resistance. Annu. Rev. Phytopathol. 2018, 56, 339–360. [Google Scholar] [CrossRef]
  29. Wang, H.C.; Zhang, C.Q. Multi-Resistance to Thiophanate-Methyl, Diethofencarb and Procymidone among Alternaria alternata Populations from Tobacco Plants, and the Management of Tobacco Brown Spot with Azoxystrobin. Phytoparasitica 2018, 46, 677–687. [Google Scholar] [CrossRef]
  30. Yang, L.N.; He, M.H.; Ouyang, H.B.; Zhu, W.; Pan, Z.C.; Sui, Q.J.; Shang, L.P.; Zhan, J.S. Cross-Resistance of the Pathogenic Fungus Alternaria alternata to Fungicides with Different Modes of Action. BMC Microbiol. 2019, 19, 205. [Google Scholar] [CrossRef]
  31. Becher, R.; Wirsel, S.G. Fungal Cytochrome P450 Sterol 14α-Demethylase (CYP51) and Azole Resistance in Plant and Human Pathogens. Appl. Microbiol. Biotechnol. 2012, 95, 825–840. [Google Scholar] [CrossRef]
  32. Jiang, C.F.; Zhou, L.; Wang, M.K.; Shen, S.R.; Cheng, W.F.; Zhao, Q.C.; Cui, K.D.; He, L. Sensitivity Determination and Resistance Mechanism of Sclerotium rolfsii to Difenoconazole. Pest Manag. Sci. 2025, 81, 2734–2741. [Google Scholar] [CrossRef] [PubMed]
  33. Shi, N.N.; Qiu, D.Z.; Chen, F.R.; Yang, Y.Q.; Du, Y.X. Analysis of the Difenoconazole-Resistance Risk and Its Molecular Basis in Colletotrichum truncatum from Soybean. Plant Dis. 2023, 107, 3123–3130. [Google Scholar] [CrossRef] [PubMed]
  34. Ma, Z.; Michailides, T.J. Characterization of Iprodione-Resistant Alternaria Isolates from Pistachio in California. Pestic. Biochem. Physiol. 2004, 80, 75–84. [Google Scholar] [CrossRef]
  35. Karaoglanidis, G.S.; Markoglou, A.N.; Bardas, G.A.; Doukas, E.G.; Konstantinou, S.; Kalampokis, J.F. Sensitivity of Penicillium expansum Field Isolates to Tebuconazole, Iprodione, Fudioxonil and Cyprodinil and Characterization of Fitness Parameters and Patulin Production. Int. J. Food Microbiol. 2011, 145, 195–204. [Google Scholar] [CrossRef] [PubMed]
  36. Sun, Y.N.; Shi, H.P.; Mao, C.X.; Wu, J.Y.; Zhang, C.Q. Activity of a SDHI Fungicide Penflufen and the Characterization of Natural-Resistance in Fusarium fujikuroi. Pestic. Biochem. Physiol. 2021, 179, 104960. [Google Scholar] [CrossRef]
  37. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef]
  38. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. Autodock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. Comput. Chem. 2009, 16, 2785–2791. [Google Scholar] [CrossRef]
  39. Adasme, M.F.; Linnemann, K.L.; Bolz, S.N.; Kaiser, F.; Salentin, S.; Haupt, V.J.; Schroeder, M. PLIP 2021: Expanding the Scope of the Protein–Ligand Interaction Profiler to DNA and RNA. Nucleic Acids Res. 2021, 49, 530–534. [Google Scholar] [CrossRef]
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Figure 1. Resistance frequency of Alternaria alternata causing brown spot disease on kiwifruit to procymidone and difenoconazole. The number following the sampled city represents the number of isolates obtained in that city. FR (%) = (No. of resistant isolates/total no. of tested isolates) × 100.
Figure 1. Resistance frequency of Alternaria alternata causing brown spot disease on kiwifruit to procymidone and difenoconazole. The number following the sampled city represents the number of isolates obtained in that city. FR (%) = (No. of resistant isolates/total no. of tested isolates) × 100.
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Figure 2. Growth of procymidone-resistant (ProR) (top) and procymidone-sensitive (ProS) (bottom) isolates with different procymidone concentrations. PDA without fungicide was used as controls (CK).
Figure 2. Growth of procymidone-resistant (ProR) (top) and procymidone-sensitive (ProS) (bottom) isolates with different procymidone concentrations. PDA without fungicide was used as controls (CK).
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Figure 3. Cross-resistance between procymidone and iprodione (A), difenoconazole (B), or tebuconazole (C) and between procymidone and prochloraz (D), pydiflumetofen (E), pyraclostrobin (F), or thiophanate-methyl (G). Spearman’s rank correlation calculations were performed to evaluate the cross-resistance between procymidone and other tested fungicides. p < 0.05 and ρ > 0.6 indicated strong positive cross-resistance between two fungicides.
Figure 3. Cross-resistance between procymidone and iprodione (A), difenoconazole (B), or tebuconazole (C) and between procymidone and prochloraz (D), pydiflumetofen (E), pyraclostrobin (F), or thiophanate-methyl (G). Spearman’s rank correlation calculations were performed to evaluate the cross-resistance between procymidone and other tested fungicides. p < 0.05 and ρ > 0.6 indicated strong positive cross-resistance between two fungicides.
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Figure 4. Cross-resistance between difenoconazole and iprodione (A) or tebuconazole (B) and between procymidone and prochloraz (C), pydiflumetofen (D), pyraclostrobin (E), or thiophanate-methyl (F). Spearman’s rank correlation calculations were performed to evaluate the cross-resistance between procymidone and other tested fungicides. p < 0.05 and ρ > 0.6 indicated strong positive cross-resistance between two fungicides.
Figure 4. Cross-resistance between difenoconazole and iprodione (A) or tebuconazole (B) and between procymidone and prochloraz (C), pydiflumetofen (D), pyraclostrobin (E), or thiophanate-methyl (F). Spearman’s rank correlation calculations were performed to evaluate the cross-resistance between procymidone and other tested fungicides. p < 0.05 and ρ > 0.6 indicated strong positive cross-resistance between two fungicides.
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Figure 5. Comparison of mycelial growth (A), sporulation (B), conidial germination (C), pathogenicity (D), and CFI (E) between procymidone-resistant (ProR) and procymidone-sensitive (ProS) isolates of Alternaria alternata from kiwifruit. DifS, difenoconazole-sensitive. Values are shown as the mean ± standard deviation (SD). ns indicates no statistical significance according to Student’s t-test.
Figure 5. Comparison of mycelial growth (A), sporulation (B), conidial germination (C), pathogenicity (D), and CFI (E) between procymidone-resistant (ProR) and procymidone-sensitive (ProS) isolates of Alternaria alternata from kiwifruit. DifS, difenoconazole-sensitive. Values are shown as the mean ± standard deviation (SD). ns indicates no statistical significance according to Student’s t-test.
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Figure 6. Comparison of mycelial growth (A), sporulation (B), conidial germination (C), pathogenicity (D), and CFI (E) between difenoconazole-resistant (DifR) and difenoconazole-sensitive (DifS) isolates of Alternaria alternata from kiwifruit. ProS, procymidone-sensitive. Values are shown as the mean ± standard deviation (SD). ns indicates no statistical significance according to Student’s t-test.
Figure 6. Comparison of mycelial growth (A), sporulation (B), conidial germination (C), pathogenicity (D), and CFI (E) between difenoconazole-resistant (DifR) and difenoconazole-sensitive (DifS) isolates of Alternaria alternata from kiwifruit. ProS, procymidone-sensitive. Values are shown as the mean ± standard deviation (SD). ns indicates no statistical significance according to Student’s t-test.
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Figure 7. Comparison of mycelial growth (A), sporulation (B), conidial germination (C), pathogenicity (D), and CFI (E) between the four phenotypes of Alternaria alternata isolates from kiwifruit. ProS, procymidone-sensitive; ProR, procymidone-resistant; DifR, difenoconazole-resistant; and DifS, difenoconazole-sensitive. Values are shown as the mean ± standard deviation (SD). Mean values with the same letters are not statistically different (p > 0.05) according to the least significant difference test.
Figure 7. Comparison of mycelial growth (A), sporulation (B), conidial germination (C), pathogenicity (D), and CFI (E) between the four phenotypes of Alternaria alternata isolates from kiwifruit. ProS, procymidone-sensitive; ProR, procymidone-resistant; DifR, difenoconazole-resistant; and DifS, difenoconazole-sensitive. Values are shown as the mean ± standard deviation (SD). Mean values with the same letters are not statistically different (p > 0.05) according to the least significant difference test.
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Figure 8. Interaction analysis of OS1. WT (A) and P894L (B) with ADP; WT (C) and P894L (D) with procymidone.
Figure 8. Interaction analysis of OS1. WT (A) and P894L (B) with ADP; WT (C) and P894L (D) with procymidone.
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Table 1. Amino acid mutations of Os1 and CYP51 between sensitive and resistant Alternaria alternata isolates from kiwifruit.
Table 1. Amino acid mutations of Os1 and CYP51 between sensitive and resistant Alternaria alternata isolates from kiwifruit.
IsolatePhenotypeOs1CYP51
894 **1277188192237307412434448462
AK-01ProS DifSP *SNVSAHEGG
AK-08PLNVSGHDGG
AK-13PSNVAAYESS
AK-22ProR DifSLSNISAYDGG
AK-123LSNVSGHEGS
AK-043LSKVAAYESS
AK-129ProS DifRPSKIAGYEGG
AK-205PLNISAHDSG
AK-024PSNVAGYDSS
AK-163ProR DifRLSNVAGYEGG
AK-037LLKIAGHDGS
* Proline (P), leucine (L), serine (S), lysine (K), asparagine (N), valine (V), isoleucine (I), alanine (A), glycine (G), tyrosine (Y), histidine (H), glutamic acid (E), and aspartic acid (D).** 894 indicates the 894th amino acid of Os1; the other numbers have a similar meaning.
Table 2. Fungicide concentrations used for sensitivity determination.
Table 2. Fungicide concentrations used for sensitivity determination.
FungicideConcentration (µg/mL)
Procymidone0, 0.25, 0.5, 1, 2, 4, 8, 16
Iprodione0, 0.25, 0.5, 1, 2, 4, 8, 16
Difenoconazole0, 0.3125, 0.625, 1.25, 5, 10, 40
Prochloraz0, 0.15625, 0.3125, 0.625, 1.25, 5, 20
Fenbuconazole0, 0.15625, 0.3125, 0.625, 1.25, 5, 20
Boscalid0, 0.3125, 0.625, 1.25, 2.5, 5, 20, 40
Fluxapyroxad0, 0.3125, 0.625, 1.25, 2.5, 5, 20, 40
Pyraclostrobin0, 0.3125, 0.625, 1.25, 2.5, 5, 20, 40
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Liu, Y.; Bao, M.; Wang, Y.; Zhang, C. Development of Procymidone and Difenoconazole Resistance in Alternaria alternata, the Causal Agent of Kiwifruit Brown Spot Disease. Plants 2025, 14, 2245. https://doi.org/10.3390/plants14142245

AMA Style

Liu Y, Bao M, Wang Y, Zhang C. Development of Procymidone and Difenoconazole Resistance in Alternaria alternata, the Causal Agent of Kiwifruit Brown Spot Disease. Plants. 2025; 14(14):2245. https://doi.org/10.3390/plants14142245

Chicago/Turabian Style

Liu, Yahui, Manfei Bao, Yanxin Wang, and Chuanqing Zhang. 2025. "Development of Procymidone and Difenoconazole Resistance in Alternaria alternata, the Causal Agent of Kiwifruit Brown Spot Disease" Plants 14, no. 14: 2245. https://doi.org/10.3390/plants14142245

APA Style

Liu, Y., Bao, M., Wang, Y., & Zhang, C. (2025). Development of Procymidone and Difenoconazole Resistance in Alternaria alternata, the Causal Agent of Kiwifruit Brown Spot Disease. Plants, 14(14), 2245. https://doi.org/10.3390/plants14142245

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