Next Article in Journal
Data-Independent Acquisition (DIA) Is Superior for High Precision Phospho-Peptide Quantification in Magnaporthe oryzae
Previous Article in Journal
Morphological Characteristics and Molecular Evidence Reveal four New Species of Russula subg. Brevipedum from China
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JoF
Volume 9
Issue 1
10.3390/jof9010062
jof-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Mechanism of Pydiflumetofen Resistance in Fusarium graminearum in China

by
Feng Zhou
1,2,3,4,
Huan-Huan Zhou
1,4,
Ao-Hui Han
1,4,
Kou-Yun Guo
1,4,
Tian-Cheng Liu
1,4,
Yan-Bing Wu
4,*,
Hai-Yan Hu
1,* and
Cheng-Wei Li
1,3,*
1
Henan Engineering Research Center of Crop Genome Editing/Henan International Joint Laboratory of Plant Genetic Improvement and Soil Remediation, Henan Institute of Science and Technology, Xinxiang 453003, China
2
Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, China
3
School of Food Science and Engineering, Henan University of Technology, Zhengzhou 450001, China
4
Henan Engineering Research Center of Green Pesticide Creation and Pesticide Residue Monitoring by Intelligent Sensor, Henan Institute of Science and Technology, Xinxiang 453003, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2023, 9(1), 62; https://doi.org/10.3390/jof9010062
Submission received: 31 October 2022 / Revised: 5 December 2022 / Accepted: 27 December 2022 / Published: 30 December 2022
(This article belongs to the Section Fungal Pathogenesis and Disease Control)
Browse Figures
Versions Notes

Abstract

:
Fusarium head blight (FHB), which is primarily caused by Fusarium graminearum, is a widespread and devastating disease of wheat. In the absence of resistant varieties, the control of FHB relies heavily on the application of fungicides, and the new generation SDHI fungicide, pydiflumetofen, has recently been registered in China for the control of FHB in wheat. The current study explored three genetically stable, highly resistant laboratory mutants (S2-4-2R, S27-3R, and S28-2R, with EC50 values of 25.10, 28.57, and 19.22 μg/mL, respectively) to investigate the potential risks associated with pydiflumetofen resistance. Although the mycelial growth of the mutants differed little compared to their parental isolates, the study found that the resistant mutants exhibited significantly reduced (p < 0.05) levels of sporulation and pathogenicity, which suggests a significant fitness cost associated with pydiflumetofen resistance in F. graminearum. Sequence analysis of the Sdh target protein identified numerous amino acid substitutions in the predicted sequences of the four subunits: FgSdhA, FgSdhB, FgSdhC, and FgSdhD. Indeed, the mutants were found to have a series of substitution in multiple subunits such that all three exhibited five identical changes, including Y182F in the FgSdhA subunit; H53Q, C90S, and A94V in FgSdhB; and S31F in FgSdhC. In addition, gene expression analysis revealed that all of the FgSdh genes had significantly altered expression (p < 0.05), particularly FgSdhA and FgdhC, which exhibited remarkably low levels of expression. However, the study found no evidence of cross-resistance between pydiflumetofen and tebuconazole, fludioxonil, prochloraz, fluazinam, carbendazim, pyraclostrobin, or difenoconazole, which indicates that these fungicides, either in rotation or combination with pydiflumetofen, could mitigate the risk of resistance emerging and provide ongoing control of FHB to ensure high and stable wheat yields.

1. Introduction

Wheat (Triticum aestivum L.) is a critical grain crop that provides the staple food for approximately 40% of the global population [1], and ensuring sustainable wheat production is therefore crucial to world food security. In 2020, China was the leading producer of wheat, with an annual yield in excess of 130 million tons, and yet was still the second largest importer, which illustrates the importance of maintaining good harvests to both sustain domestic consumption as well as to reduce pressure on global wheat markets (https://worldpopulationreview.com/country-rankings/wheat-production-by-country, accessed on 30 October 2022). However, the production of wheat in China is under constant threat from Fusarium head blight (FHB), which is a widespread and devastating disease [2]. The FHB complex, which is primarily caused by Fusarium graminearum, not only results in yield loss and smaller grains but can also dramatically affect grain quality as a consequence of contamination with harmful mycotoxins such as deoxynivalenol (DON), which pose a threat to both human and animal health [2,3]. In the absence of effective and stable wheat varieties with high resistance to F. graminearum, the application of fungicides remains the most effective strategy to control FHB [3]. However, unfortunately inappropriate and over use of such compounds for an extended period of time has already led to the emergence of resistance to some of the most effective fungicides, including carbendazim and tebuconazole, within field populations of F. graminearum in China [3,4,5]. It is therefore extremely important to continuously monitor for signs of resistance in order to adjust chemical control strategies accordingly, and the need for alternative novel compounds for the management of FHB is increasing in urgency.
Pydiflumetofen is a new-generation succinate dehydrogenase inhibitor (SDHI), which was recently developed by Syngenta Crop Protection and registered in China for the control of FHB in 2020 (http://www.chinapesticide.org.cn/hysj/index.jhtml, accessed on 30 October 2022). The mode of action of pydiflumetofen and other SDHI fungicides has been studied in detail, and it is generally accepted that they block the ubiquinone binding site of the succinate dehydrogenase (SDH) enzyme, also known as succinate ubiquinone oxidoreductase, which plays an essential role in the tricarboxylic cycle in the fungal mitochondria [6,7]. The SDH of F. graminearum consists of at least four nuclear-encoded subunits, including FgSdhA (FGSG_13136), FgSdhB (FGSG_05610), FgSdhC (FGSG_09012), and FgSdhD (FGSG_00743), which are all located in the mitochondria [8]. Although pydiflumetofen exhibits high activity against F. graminearum in artificial culture (Table 1), the observation from a recent study that the risk of developing resistance to SDHI fungicides should be categorized as medium to high is of great concern [9]. Previous research has shown that several amino acid changes in the sequences of the various Sdh subunits have been associated with resistance to SDHI fungicides in plant pathogenic fungi, including Botrytis cinerea and Fusarium asiaticum [10,11,12]. The main objectives of the current study were therefore to investigate the potential for pydiflumetofen resistance in F. graminearum and more specifically to (i) explore the fitness parameters of pydiflumetofen-resistant mutants of F. graminearum; (ii) identify the molecular basis of the observed resistance; and (iii) assess the potential for cross-resistance between pydiflumetofen and other commonly used fungicides.

2. Materials and Methods

2.1. Fungicides, Isolates, and Medium

Stock solutions of the technical-grade fungicides (Table S1) used in the current study were prepared in appropriate solvents and stored at 4 °C for no longer than 2 weeks before use. Pydiflumetofen, tebuconazole, fludioxonil, prochloraz, fluazinam, pyraclostrobin, and difenoconazole were dissolved in acetone, whilst the carbendazim was dissolved in 0.1 mol/L hydrochloric acid (HCl). Serial dilutions were freshly prepared for each experiment, and mycelial growth assays performed to confirm that the solvents had no effect on the growth of F. graminearum at the range of concentrations tested (data not shown).
The three wild-type F. graminearum isolates used in the current study, S2-4-2, S27-3, and S28-2, which were highly sensitive to pydiflumetofen, with EC50 values of 0.0235, 0.0486, and 0.2354 μg/mL, respectively, were originally collected from wheat ears exhibiting typical symptoms of FHB growing in the fields of China (Table 1). Repeated exposure to pydiflumetofen under laboratory conditions following the protocol of a previous study [13] resulted in the selection of three genetically stable mutants, S2-4-2R, S27-3R, and S28-2R, which were highly resistant to pydiflumetofen, with EC50 values of 25.10, 28.57, and 19.22 μg/mL, respectively (Table 1). Both the wild-type isolates and the resistant mutants were routinely maintained on potato dextrose agar (PDA: 20 g/L glucose, 200 g/L potato, and 20 g/L agar) at 24 °C.

2.2. Mycelial Growth, Sporulation, and Pathogenicity of Pydiflumetofen-Resistant Mutants of F. graminearum

The biological characteristics of the three resistant F. graminearum mutants (S2-4-2R, S27-3R, and S28-2R) were evaluated in comparison to their wild-type parental isolates (S2-4-2, S27-3, and S28-2), with their mycelial growth on PDA; sporulation in mung bean broth (MBB), which was prepared by boiling 40 g of mung bean seeds in one liter of distilled water for 30 min; and pathogenicity on wheat spikelets assessed according to the methods detailed in a previous study [13,14]. For mycelial growth assay, mycelial plugs (5 mm in diameter) of the test isolates were taken from the edge of 2-day-old colonies and transferred to fresh PDA plates that were then incubated at 24 °C with a 12 h photoperiod, and the resulting colonies were observed daily and the diameter of each measured at 24 h, 48 h, and 72 h post inoculation (hpi), and each isolate was represented by at least eight individual plates. For sporulation assay, the test isolates were initially established by transferring 5 mm mycelial plugs from 2-day-old PDA cultures to flasks containing 30 mL MBB. After 3 days of incubation at 24 °C with shaking (120 rpm), the resulting spores were harvested and counted using a hemocytometer (Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd., Shanghai, China), and each isolate was represented by at least 3 flasks. Meanwhile, for the pathogenicity assay, colonies were initially established by transferring 5 mm mycelial plugs from 2-day-old PDA cultures to flasks containing 30 mL MBB. After 3 days incubation at 24 °C with 120 rpm shaking, the resulting spores were harvested and resuspended in serial dilutions of sterile water and counted using a hemocytometer. The pathogenicity of the six F. graminearum isolates was assessed using wheat ears grown under greenhouse conditions, and the test was conducted using intact wheat ears (Cultivar Bainong 307), which were inoculated with 10 μL of spore suspension (1 × 105 spores/mL) at the beginning of the heading and flowering growth stage. Water-inoculated wheat ears served as negative controls. The severity of disease symptoms were evaluated for 14 days post inoculation (dpi) from the previous studies [14]. Each isolate was represented by at least 10 wheat spikelets.

2.3. Cloning and Sequencing of Four FgSdh Genes

Mycelium samples were prepared in accordance with the method of a previous study [13], and total genomic DNA extracted using the Omega bio-tek Fungal DNA Kit (Omega bio-tek Inc., Guangzhou, China) following the instructions of the manufacturer. The resulting DNA was used as a template for polymerase chain reaction (PCR) amplification of the four full-length Sdh genes, FgSdhA, FgSdhB, FgSdhC, and FgSdhD, using the following primer sets, which were designed with primer premier software (ver.6.0., PREMIER Biosoft, Palo Alto, CA, Canada): FgSdhA-F/FgSdhA-R, FgSdhB-F/FgSdhB-R, FgSdhC-F/FgSdhC-R, and FgSdhD-F/FgSdhD-R, respectively (Table S2). The PCR was performed using 50.0 μL reaction mixtures containing 25.0 μL 2×ES Taq Master Mix, 1.5 μL of template DNA, 2.0 μL each primer, and 21.5 μL ddH2O (CoWin Biosciences, Cambridge, MA, USA) and processed using a 96-well thermal cycler (Applied biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with the following program: an initial denaturation at 95 °C for 2 min, following by 35 cycles of melting at 95 °C for 30 s, annealing at 58 °C for 120 s in the case of FgSdhA (57.5 °C for FgSdhB, 58 °C for FgSdhC, and 57 °C for FgSdhD), and extension at 72 °C for 120 s in the case of FgSdhA (50 s for FgSdhB, 40 s for FgSdhC, and 45 s for FgSdhD), with a final extension at 72 °C for 10 min. The resulting PCR products were then purified and cloned into the pMD19-T vector and sequenced commercially (Wuhan Genecreate Biotechnology Co. Ltd., China). The sequence data obtained were analyzed using the DNAMAN software package (ver.8.0. Lynnon Biosolf, California, USA), with the predicted amino acid sequences being used to create the multiple sequence alignments that were utilized to identify amino acid differences between the sequences of the various Sdh subunits from the three resistant mutants and their wild-type parental isolates, as detailed in a previous study [13].

2.4. Relative Expression of Four FgSdh Genes in Pydiflumetofen-Resistant Mutants of F. graminearum

Total RNA was extracted from fresh mycelial samples cultured in both the absence and presence of pydiflumetofen (0.1 μg/mL) using a fungal RNA kit (Omega bio-tek, Switzerland) following the protocol of the manufacturer. The first-strand cDNA was then synthesized using the PrimeScript RT reagent kit (TaKaRa, Kusatsu, Japan), with the resulting cDNA being used as a template for the qPCR amplification of partial sequences (approximately 200 bp in length) of the FgSdhA, FgSdhB, FgSdhC, and FgSdhD genes with the RT-FgSdhA-F/RT-FgSdhA-R, RT-FgSdhB-F/RT-FgSdhB-R, RT-FgSdhC-F/RT-FgSdhC-R, and RT-FgSdhD-F/RT-FgSdhD-R primer sets, respectively (Table S2). The real-time PCR itself was performed using reaction mixtures containing SYBR Green I fluorescent dye and processed using the QuantStudio 6 Flex PCR detection system (ThermoFisher, Waltham, MA, USA) with the following program: an initial denaturation at 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 34 s, and dissociation at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The relative expression of each gene was then determined following the protocol of a previous study [13,14] using actin as the reference gene with the RT-actin-F/RT-actin-R primer set (Table S2). Each isolate combination was represented by three biological samples, with the resulting values being used to calculate the mean expression and standard error (SE).

2.5. Cross-Resistance between Pydiflumetofen and Other Commonly Used Fungicides

The mycelial growth assay used to assess the growth of the pydiflumetofen-resistant mutants was also used to investigate any potential cross-resistance between pydiflumetofen and other commonly used fungicides. The assay was conducted using PDA medium amended with a range of active ingredient concentrations: 0, 0.0005, 0.0015, 0.0045, 0.0135, 0.0405, 0.1215, 0.3645, 1.0935, and 3.2805 μg/mL for tebuconazole, fludioxonil, prochloraz, fluazinam, pyraclostrobin, and difenoconazole as well as pydiflumetofen when assessing the wild-type parental isolates and 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μg/mL for carbendazim as well as pydiflumetofen with the pydiflumetofen-resistant mutants. Each treatment/isolate combination was represented by three separate plates, and the entire experiment was performed twice.

2.6. Statistical Analysis

The data collected in the mycelial growth, sporulation, and pathogenicity experiments as well as the results from gene expression analysis were subjected to analysis of variance (ANOVA) using SPSS software (ver. 17.0; SPSS Inc., Chicago, IL, USA), with significant differences between treatments being determined using Fisher’s least significant difference test (p ≤ 0.05).

3. Results

3.1. Mycelial Growth and Sporulation of Three Pydiflumetofen-Resistant Mutants of F. graminearum

Although the growth rate of the three pydiflumetofen-resistant mutants (S2-4-2R, S27-3R, and S28-2R) was slightly depressed in the early stages of growth (at 24 h and 48 h), no significant differences (p < 0.05) were detected in the diameter of the colonies formed on PDA at 72 h (Figure 1 and Figure S1), which infers that pydiflumetofen resistance had little effect on mycelial growth. However, all of the pydiflumetofen-resistant mutants were found to have significantly reduced levels of sporulation (p < 0.05) in comparison to their parental isolates (Figure 2), with the sporulation of one (S28-2R) being reduced by 50% and another (S2-4-2R) to almost zero, which indicates that there is some fitness cost associated with pydiflumetofen resistance in F. graminearum.

3.2. Pathogenicity of Pydiflumetofen-Resistant Mutants of F. graminearum on Wheat Spikelets

All of the pydiflumetofen-resistant mutants in the current study were found to have reduced pathogenicity compared to their wild-type parental isolates (Figure 3). However, there was a high degree of variation between the three mutants, with the difference in one mutant, S2-4-2R, not being significant, while the other two had significantly reduced pathogenicity (p < 0.05), resulting in 40% less disease in S27-3R and 80% less in S2-4-2R (Figure 3).

3.3. Sequence Analysis of Four FgSdh Genes in Pydiflumetofen-Resistant Mutants of F. graminearum

Numerous point mutations (Figure S2) that resulted in amino acid changes to the predicted sequences of the FgSdh subunits of the pydiflumetofen-resistant mutants were identified by comparing the FgSdh sequences from the mutants with those of their parental isolates, which, incidentally, were the same as the corresponding sequences of the F. graminearum wild-type species (PH-1) in the GenBank database. Interestingly many identical amino acid changes were found to occur in multiple subunits of all three mutants (S2-4-2R, S27-3R, and GY-1R). For example, all of the mutants shared the Y182F and S31F substitutions in their predicted FgSdhA and FgSdhD sequences, respectively (Table 2). Similarly, all of the mutants shared three substitutions (H53Q, C90S, and A94V) in their FgSdhC sequences, while two mutants (S27-3R and S2-4-2R) were found to have an identical change (E39G) in their FgSdhB sequence (Table 2). In addition to these shared amino acid changes, some of the mutants exhibited further substitutions. For example, GY-1R had three single-amino-acid substitutions (H101R, A185V, and W180K) in its FgSdhA, FgSdhC, and FgSdhD subunits respectively, while S2-4-2R had just one further change (A185L) in its FgSdhC sequence, which, interestingly, occurred at the same location as the A185V substitution in the FgSdhC sequence of GY-1R (Table 2). Taken together, these results paint a rather complicated picture of amino acid changes associated with pydiflumetofen resistance in the F. graminearum mutants investigated in the current study.

3.4. Relative Expression of Four FgSdh Genes in Pydiflumetofen-Resistant Mutants of F. graminearum

The results of the qPCR experiments (Figure 4) were a little difficult to interpret, as the genetic background of the parental isolates (S2-4-2, S27-3, and GY-1) seems to influence gene expression even before considering any altered expression that might occur in the resistant mutants. For example, the expression of FgSdhA followed the same general pattern in all of the parental isolates, with pydiflumetofen treatment resulting in a significant increase in the expression in all three isolates, while quite a different pattern of expression was observed for FgSdhB in response to the fungicide, with expression being dramatically down-regulated in S2-4-2, dramatically up-regulated in S27-3, and little changed in GY-1. However, some general trends could be observed in the pydiflumetofen-resistant mutants. For example, all of the mutants exhibited remarkably low expression of both their FgSdhA and FgSdhC genes, which appeared to be down-regulated even further by the presence of pydiflumetofen in the case of FgSdhC. Similarly, all of the mutants exhibited a shared pattern of expression with regard to their FgSdhB and FgSdhD genes, which resulted in high levels of expression in the absence of pydiflumetofen that were significantly higher (p < 0.05) than the parental isolates in the case of FgSdhD but also significant down-regulation (p < 0.05) in the presence of the fungicide. Indeed, the mutants seemed to exhibit more consistent patterns of gene expression than the wild-type parental isolates, indicating that the shared amino acid changes detailed in the preceding section might have had an effect on gene expression that superseded the divergence in expression resulting from the genetic background the mutants inherited from their parental isolates.

3.5. Cross-Resistance between Pydiflumetofen and Other Commonly Used Fungicides

Although the current study found that pydiflumetofen was a highly effective fungicide against wild-type isolates of F. graminearum (S2-4-2, S27-3, and S28-2), with EC50 values of 0.0235, 0.0486, and 0.2354 μg/mL, respectively, its efficacy was greatly reduced in the resistant mutants (S2-4-2R, S27-3R, and S28-2R), which had values of 25.10, 28.57, and 19.22 μg/mL (Table 1). However, despite this dramatic change to pydiflumetofen sensitivity, no evidence of cross-resistance was found between pydiflumetofen and the other fungicides tested, which included tebuconazole, fludioxonil, prochloraz, fluazinam, carbendazim, pyraclostrobin, and difenoconazole (Table 1).

4. Discussion

The succinate dehydrogenase inhibitor (SDHI) group of fungicides is highly effective against a broad range of fungi and widely used to control many plant diseases [10,15,16,17]. However, since SDHI fungicides have a single target site, many plant pathogens have already developed resistance. Indeed, there is growing evidence that SDHI resistance is primarily caused by amino acid mutations in the vicinity of the ubiquinone-binding pocket, especially in the SdhB, SdhC, and SdhD subunits [12,18,19]. Such mutations have been documented in many plant pathogens including Mycosphaerella graminicola [20], Alternaria alternata and Alternaria solani [19,21], Zymoseptoria tritici [21], Blumeriella jaapii [22], and Clarireedia jacksonii [23] although different pathogens may exhibit different resistance mechanisms to the same fungicide.
The new-generation SDHI fungicide, pydiflumetofen, has been registered in China since 2020, primarily for the control of FHB in wheat (http://www.chinapesticide.org.cn/hysj/index.jhtml, accessed on 30 October 2022). However, to date, there has been little investigation into potential resistance mechanisms in F. graminearum. The current study found that although three laboratory mutants of F. graminearum were highly resistant to pydiflumetofen, this resistance was accompanied by a certain cost to fitness, including significantly reduced sporulation and reduced pathogenicity in wheat spikelets even though mycelial growth was little affected (Figure 1, Figure 2 and Figure 3). These results are in contrast to a previous study of F. asiaticum, which found that pydiflumetofen-resistant mutants were completely unimpaired with regard to mycelial growth, sporulation, and virulence [12], and could suggest a lower risk associated with SDHI resistance in F. graminearum compared to F. asiaticum. Meanwhile, current studies provide further evidence of a fitness cost associated with pydiflumetofen resistance in F. graminearum but also indicate that there can be some degree of variation in different mutants, which could possibly be related to the molecular biology underlying the specific resistance mechanism of each mutant.
Many previous studies have documented amino acid substitutions associated with SDHI resistance in plant pathogenic fungi, particularly the conserved histidine residues in the ubiquinone binding site of the Sdh complex [24], for example, the H272L/R/Y mutations in the SdhB subunit of B. cinerea [10,25,26,27], the H132R mutation in the SdhD subunit of both B. cinerea and Sclerotinia sclerotiorum [28,29], and the H277Y/R/L, H134R, and H133R/P/T mutations in the SdhB, SdhC, and SdhD subunits, respectively, of A. alternata [30,31]. Single-amino-acid mutations have also been noted in previous studies of pydiflumetofen resistance in Fusarium species, including the A83V or R86H/C substitutions in the SdhC subunit of F. graminearum [8] as well as the H248Y and A64V or R67K substitutions in the SdhB and SdhC subunits, respectively, of F. asiaticum [12]. In contrast, the current study identified a collection of amino acid substitution in multiple subunits such that all three F. graminearum mutants exhibited five identical changes, including Y182F in the FgSdhA subunit; H53Q, C90S, and A94V in FgSdhB; and S31F in FgSdhC (Table 2). In addition, several mutants also exhibited further mutations, including H101R in FgSdhA, E39G in FgSDhB, W180K in FgSdhD, and A185V/L in FgSdhC, the latter of which resulted in two mutants having amino acid changes in all four of their Sdh subunits. To the best of our knowledge, this is the first report of these amino acid changes being associated with pydiflumetofen resistance in Fusarium as well as the first report of changes in multiple subunits or in the SdhA subunit, with the majority of changes linked with SDHI resistance generally occurring in subunits B, C, and D [24]. In addition, the current study found that the expression of all four FgSdh subunits was significantly altered (p < 0.05) compared to that of the parental wild-type isolates, with FgSdhA and FgSdhC in particular showing remarkably low levels of expression (Figure 4). However, it is unclear whether this altered expression contributed to the observed pydiflumetofen resistance or was merely a consequence of the primary resistance mechanism. Further studies, including site-directed mutagenesis for each amino acid change, both in isolation and in combination, are therefore required to evaluate the contribution of each to the resistance mechanism occurring in the F. graminearum mutants of the current study.
Although the multiple mutations and fitness cost reported in the current study suggest that the risk of pydiflumetofen resistance in F. graminearum is not particularly high, previous studies have found that the risk of resistance to SDHI fungicides in plant pathogenic fungi is medium to high [11,32,33]. Indeed, recent reports have shown that the in vitro selection of pydiflumetofen-resistant mutants can occur spontaneously in both F. graminearum and F. asiaticum, without the need for complex mutagenic procedures [8,12]. Consequently, great care should be taken in developing management strategies that both provide effective control of FHB in the field but also limit the potential for fungicide resistance to emerge. It was therefore extremely encouraging that the current study found no evidence of cross-resistance between pydiflumetofen and any of the other fungicides assessed, including tebuconazole, fludioxonil, prochloraz, fluazinam, carbendazim, pyraclostrobin, and difenoconazole, which indicates that the use of pydiflumetofen either in rotation or combination with these fungicides could provide ongoing control of FHB and ensure high and stable wheat yields in China for many years to come.

Supplementary Materials

The following are available online at: https://www.mdpi.com/article/10.3390/jof9010062/s1, Figure S1: Mycelial growth of three pydiflumetofen-resistant mutants of F. graminearum and their sensitive parental isolates; Figure S2: Multiple sequence of the FgSdhA, FgSdhB, and FgSdhC genes in pydiflumetofen-resistant mutants of F. graminearum and their sensitive parental isolates; Table S1: Fungicides used in the current study; Table S2: Primers used in the current study.

Author Contributions

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

Funding

This study was sponsored by the national natural science foundation of China (No. 32001860), the key scientific research projects of colleges and universities in Henan province (No. 22A210017), the key scientific and technological research project of Henan province (No. 222102110027), and the special project for local science and technology development guided by the central government of Henan province (No. 20221343034).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Li, H.J.; Murray, D.; McIntosh, A.; Zhou, Y. Breeding new cultivars for sustainable wheat production. Crop J. 2019, 7, 715–717. [Google Scholar] [CrossRef]
  2. Xia, X.B.; Zhang, X.; Zhang, Y.C.; Wang, L.R.; An, Q.; Tu, Q.; Wu, L.; Jiang, P.; Zhang, P.; Yu, L.X.; et al. Characterization of the WAK gene family reveals genes for FHB resistance in bread wheat (Triticum aestivum L.). Int. J. Mol. Sci. 2022, 23, 7157. [Google Scholar] [CrossRef]
  3. Wen, Z.Y.; Wang, J.R.; Jiao, C.; Shao, W.Y.; Ma, Z.H. Biological and molecular characterizations of field fludioxonil-resistant isolates of Fusarium graminearum. Pestic. Biochem. Phys. 2022, 184, 105101. [Google Scholar] [CrossRef]
  4. Zhang, Y.J.; Zhang, X.; Chen, C.J.; Zhou, M.G.; Wang, H.C. Effects of fungicides JS399-19, azoxystrobin, tebuconazole, and carbendazim on the physiological and biochemical indices and grain yield of winter wheat. Pestic. Biochem. Phys. 2010, 98, 151–157. [Google Scholar] [CrossRef]
  5. Qian, H.W.; Du, J.; Chi, M.Y.; Sun, X.M.; Liang, W.X.; Huang, J.G.; Li, B.D. The Y137H mutation in the FgCYP51B protein confers reduced sensitivity to tebuconazole in Fusarium graminearum. Pest Manag. Sci. 2018, 74, 1472–1477. [Google Scholar] [CrossRef]
  6. Piqueras, C.M.; Latorre, B.A.; Torres, R. Effectiveness of isofetamid, a new succinate dehydrogenase inhibitor fungicide, in the control of grapevine gray mold. Cien. Investig. Agric. 2014, 41, 365–374. [Google Scholar] [CrossRef] [Green Version]
  7. Rehfus, A.; Miessner, S.; Achenbach, J.; Strobel, D.; Bryson, R.; Stammler, G. Emergence of succinate dehydrogenase inhibitor resistance of Pyrenophora teres in Europe. Pest Manag. Sci. 2016, 72, 1977–1988. [Google Scholar] [CrossRef]
  8. Sun, H.Y.; Cui, J.H.; Tian, B.H.; Gao, S.L.; Zhang, X.X.; Chen, H.G. Resistance risk assessment for Fusarium graminearum to pydiflumetofen, a new succinate dehydrogenase inhibitor. Pest Manag. Sci. 2020, 76, 1549–1559. [Google Scholar] [CrossRef]
  9. Gao, Y.Y.; He, L.F.; Zhu, J.M.; Cheng, J.G.; Li, B.X.; Liu, F.; Mu, W. The relationship between features enabling SDHI fungicide binding to the Sc-sdh complex and its inhibitory activity against Sclerotinia sclerotiorum. Pest Manag. Sci. 2020, 76, 2799–2808. [Google Scholar] [CrossRef]
  10. Yin, Y.N.; Kim, Y.K.; Xiao, C.L. Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple. Phytopathology 2011, 101, 986–995. [Google Scholar] [CrossRef]
  11. Sierotzki, H.; Scalliet, G. A review of current knowledge of resistance aspects for the next-generation succinate dehydrogenase inhibitor fungicides. Phytopathology 2013, 103, 880–887. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Chen, W.C.; Wei, L.L.; Zhao, W.C.; Wang, B.R.; Zheng, H.H.; Zhang, P.C.; Lou, T.C.; Duan, Y.B.; Hou, Y.P.; Zhou, M.G.; et al. Resistance risk assessment for a novel succinate dehydrogenase inhibitor pydiflumetofen in Fusarium asiaticum. Pest Manag. Sci. 2021, 77, 538–547. [Google Scholar] [CrossRef]
  13. Zhou, F.; Cui, Y.X.; Wang, B.L.; Zhou, Y.D.; Li, S.W.; Zhang, Y.T.; Zhang, K.; Chen, Z.Y.; Hu, H.Y.; Li, C.W. Baseline sensitivity and potential resistance mechanisms for Fusarium pseudograminearum to fludioxonil. Plant Dis. 2022, 106, 2138–2144. [Google Scholar] [CrossRef] [PubMed]
  14. Zhou, F.; Zhou, H.H.; Cui, Y.X.; Hu, H.Y.; Liu, Q.L.; Liu, R.Q.; Wu, Y.B.; Li, C.W. Mechanism of Fusarium graminearum resistant to the phenylpyrrole fungicide fludioxonil (In Chinese). Chin. J. Pestic. Sci. 2022, 24, 1393–1401. [Google Scholar]
  15. Hou, Y.P.; Mao, X.W.; Wang, J.X.; Zhan, S.W.; Zhou, M.G. Sensitivity of Fusarium asiaticum to a novel succinate dehydrogenase inhibitor fungicide pydiflumetofen. Crop Prot. 2017, 96, 237–244. [Google Scholar] [CrossRef] [Green Version]
  16. Guo, X.F.; Zhao, B.; Fan, Z.J.; Yang, D.Y.; Zhang, N.L.; Wu, Q.F.; Yu, B.; Zhou, S.; Kalinina, T.; Belskaya, N.P. Discovery of novel thiazole carboxamides as antifungal succinate dehydrogenase inhibitors. J. Agric. Food Chem. 2019, 67, 1647–1655. [Google Scholar] [CrossRef]
  17. Duan, Y.B.; Xiu, Q.; Li, H.R.; Li, T.; Wang, J.X.; Zhou, M.G. Pharmacological characteristics and control efficacy of a novel SDHI fungicide pydiflumetofen against Sclerotinia sclerotiorum. Plant Dis. 2019, 103, 77–82. [Google Scholar] [CrossRef] [Green Version]
  18. Avenot, H.F.; Michailides, T.J. Progress in understanding molecular mechanisms and evolution of resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in phytopathogenic fungi. Crop Prot. 2010, 29, 643–651. [Google Scholar] [CrossRef]
  19. Landschoot, S.; Carrette, J.; Vandecasteele, M.; Baets, B.D.; HÖfte, M.; Audenaert, K.; Haesaert, G. Boscalid-resistance in Alternaria alternata and Alternaria solani populations: An emerging problem in Europe. Crop Prot. 2017, 92, 49–59. [Google Scholar] [CrossRef]
  20. Scalliet, G.; Bowler, J.; Luksch, T.; Kirchhofer-Allan, L.; Steinhauer, D.; Ward, K.; Niklaus, M.; Verras, A.; Csukai, M.; Daina, A.; et al. Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola. PLoS ONE 2012, 7, 4. [Google Scholar] [CrossRef] [Green Version]
  21. Mallik, I.; Arabiat, S.; Pasche, J.S.; Bolton, M.D.; Patel, J.S.; Gudmestad, N.C. Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase-inhibiting fungicides in Alternaria solani. Phytopathology 2014, 104, 40–49. [Google Scholar] [CrossRef] [PubMed]
  22. Steinhauer, D.; Salat, M.; Frey, R.; Mosbach, A.; Luksch, T.; Balmer, D.; Hansen, R.; Widdison, S.; Logan, G.; Dietrich, R.A.; et al. A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici. PLoS Pathog. 2019, 15, e1007780. [Google Scholar] [CrossRef] [PubMed]
  23. Outwater, C.; Proffer, T.; Rothwell, N.; Peng, J.Y.; Sundin, G.W. Boscalid resistance in Blumeriella jaapii: Distribution, effect on field efficacy, and molecular characterization. Plant Dis. 2019, 103, 1112–1118. [Google Scholar] [CrossRef] [PubMed]
  24. Lee, J.; Elliott, M.; Kim, M.; Yamada, T.; Jung, G. A rapid molecular detection system for SdhB and SdhC point mutations conferring differential SDHI resistance in Clarireedia populations. Plant Dis. 2021, 105, 660–666. [Google Scholar] [CrossRef]
  25. Ackrell, B.A. Progress in understanding structure-function relationships in respiratory chain complex II. FEBS Lett. 2000, 466, 1–5. [Google Scholar] [CrossRef] [Green Version]
  26. Leroux, P.; Gredt, M.; Leroch, M.; Walker, A.S. Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea, the causal agent of gray mold. Appl. Environ. Microb. 2010, 76, 6615–6630. [Google Scholar] [CrossRef] [Green Version]
  27. Veloukas, T.; Markoglou, A.N.; Karaoglanidis, G.S. Differential effect of Sdh B gene mutations on the sensitivity to SDHI fungicides in Botrytis cinerea. Plant Dis. 2013, 97, 118–122. [Google Scholar] [CrossRef] [Green Version]
  28. Fernández-Ortuño, D.; Chen, F.; Schnabel, G. Resistance to pyraclostrobin and boscalid in Botrytis cinerea isolates from strawberry fields in the Carolinas. Plant Dis. 2012, 96, 1198–1203. [Google Scholar] [CrossRef] [Green Version]
  29. Wang, Y.; Duan, Y.; Wang, J.; Zhou, M. A new point mutation in theiron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum. Mol. Plant Pathol. 2015, 16, 653–661. [Google Scholar] [CrossRef]
  30. Wang, Y.; Duan, Y.B.; Zhou, M. Molecular and biochemical characterization of boscalid resistance in laboratory mutants of Sclerotinia sclerotiorum. Plant Pathol. 2015, 64, 101–108. [Google Scholar] [CrossRef]
  31. Avenot, H.; Sellam, A.; Karaoglanidis, G.; Michailides, T. Characterization of mutations in the iron-Sulphur subunit of succinate dehydrogenase correlating with boscalid resistance in Alternaria alternata from California pistachio. Phytopathology 2008, 98, 736–742. [Google Scholar] [CrossRef]
  32. Avenot, H.; Sellam, A.; Michailides, T. Characterization of mutations in the membrane-anchored subunits AaSDHC and AaSDHD of succinate dehydrogenase from Alternaria alternata isolates conferring field resistance to the fungicide boscalid. Plant Pathol. 2009, 58, 1134–1143. [Google Scholar] [CrossRef] [Green Version]
  33. Miyamoto, T.; Ishii, H.; Stammler, G.; Koch, A.; Ogawara, T.; Tomita, Y.; Fountaine, J.M.; Ushio, S.; Seko, T.; Kobori, S. Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid. Plant Pathol. 2010, 59, 873–881. [Google Scholar] [CrossRef]
Jof 09 00062 g001 550
Figure 1. Mycelial growth of three pydiflumetofen-resistant mutants of F. graminearum. The average colony diameter of three resistant mutants (R) and their wild-type parental isolates was measured after 24-, 48-, and 72-h incubation on PDA at 24 °C. Data are the means of eight replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) after 24, 48, and 72 h incubation according to Fisher’s least significant difference test (p ≤ 0.05).
Figure 1. Mycelial growth of three pydiflumetofen-resistant mutants of F. graminearum. The average colony diameter of three resistant mutants (R) and their wild-type parental isolates was measured after 24-, 48-, and 72-h incubation on PDA at 24 °C. Data are the means of eight replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) after 24, 48, and 72 h incubation according to Fisher’s least significant difference test (p ≤ 0.05).
Jof 09 00062 g001
Jof 09 00062 g002 550
Figure 2. Sporulation of three pydiflumetofen mutants of F. graminearum. The sporulation of the resistant mutants (R) and their wild-type parental isolates was assessed after 3 day’s incubation in MBB at 24 °C with shaking (130 rpm). Data are the means of three replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) according to Fisher’s least significant difference test (p ≤ 0.05).
Figure 2. Sporulation of three pydiflumetofen mutants of F. graminearum. The sporulation of the resistant mutants (R) and their wild-type parental isolates was assessed after 3 day’s incubation in MBB at 24 °C with shaking (130 rpm). Data are the means of three replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) according to Fisher’s least significant difference test (p ≤ 0.05).
Jof 09 00062 g002
Jof 09 00062 g003 550
Figure 3. Pathogenicity of three pydiflumetofen-resistant mutants of F. graminearum. The top panels (A) show the lesions produced by both the resistant mutants (R) and their parental wild-type isolates on wheat spikelets at 14 dpi, while the graph below (B) shows the percentage of diseased grains in the spikelets. Data are the means of three replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) according to Fisher’s least significant difference test (p ≤ 0.05).
Figure 3. Pathogenicity of three pydiflumetofen-resistant mutants of F. graminearum. The top panels (A) show the lesions produced by both the resistant mutants (R) and their parental wild-type isolates on wheat spikelets at 14 dpi, while the graph below (B) shows the percentage of diseased grains in the spikelets. Data are the means of three replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) according to Fisher’s least significant difference test (p ≤ 0.05).
Jof 09 00062 g003
Jof 09 00062 g004 550
Figure 4. (A–D) Relative expression of four FgSdh genes in pydiflumetofen-resistant mutants of F. graminearum. The relative expression of the FgSdh genes in the resistant mutants (R) and wild-type parental isolates was compared in both the absence and presence of pydiflumetofen (0.1 μg/mL), using actin as the reference gene. Data are the means of three replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) in the absence and presence of pydiflumetofen (0.1 μg/mL) according to Fisher’s least significant difference test (p ≤ 0.05).
Figure 4. (A–D) Relative expression of four FgSdh genes in pydiflumetofen-resistant mutants of F. graminearum. The relative expression of the FgSdh genes in the resistant mutants (R) and wild-type parental isolates was compared in both the absence and presence of pydiflumetofen (0.1 μg/mL), using actin as the reference gene. Data are the means of three replicates ± standard error (SE). Different lowercase letters above the columns indicate significant differences (p ≤ 0.05) in the absence and presence of pydiflumetofen (0.1 μg/mL) according to Fisher’s least significant difference test (p ≤ 0.05).
Jof 09 00062 g004
Table
Table 1. Cross-resistance between pydiflumetofen and other commonly used fungicides.
Table 1. Cross-resistance between pydiflumetofen and other commonly used fungicides.
FungicidesPydiflumetofen-Sensitive Isolates (EC50, μg/mL)Pydiflumetofen-Resistant Mutants (EC50, μg/mL)
S2-4-2S27-3S28-1S2-4-2RS27-3RS28-1R
Pydiflumetofen0.0240.0490.23525.10028.57019.220
Tebuconazole0.1190.0830.0740.4260.0520.632
Fludioxonil0.0030.0030.0050.0100.0120.014
Prochloraz0.0030.0020.0040.0100.0110.013
Fluazinam0.0110.0090.0120.0110.0090.015
Carbendazim0.4290.5520.6360.4860.2650.612
Pyraclostrobine0.0410.0450.1260.0220.0210.046
Difenoconazole0.1030.2860.6450.1900.0080.641
Table
Table 2. Point mutations in Sdh genes associated with pydiflumetofen resistance in F. graminearum and F. asiaticum.
Table 2. Point mutations in Sdh genes associated with pydiflumetofen resistance in F. graminearum and F. asiaticum.
GenesMutantsNucleotide MutationsAmino Acid ChangesSpecies/Reference
SdhAS27-3RA833T, C834TY182FF. graminearum/Current study
SdhAS2-4-2RA833T, C834TY182FF. graminearum/Current study
SdhAGY-1RA539G, A833T, C834TH101R, Y182FF. graminearum/Current study
SdhBS27-3RA116GE39GF. graminearum/Current study
SdhBS2-4-2RA116GE39GF. graminearum/Current study
SdhBGY-1R//F. graminearum/Current study
SdhB//H248YF. asiaticum/Chen et al., 2021
SdhCS27-3RC270G, G380C, C392TH53Q, C90S, A94VF. graminearum/Current study
SdhCS2-4-2RC270G, G380C, C392T, G664C, C665TH53Q, C90S, A94V, A185LF. graminearum/Current study
SdhCGY-1RC270G, G380C, C392T, C665TH53Q, C90S, A94V, A185VF. graminearum/Current study
SdhC//A83VF. graminearum/Sun et al., 2020
SdhC//R86H/CF. graminearum/Sun et al., 2020
SdhC//A64VF. asiaticum/Chen et al., 2020
SdhC//R67KF. asiaticum/Chen et al., 2020
SdhDS27-3RC92TS31FF. graminearum/Current study
SdhDS2-4-2RC92TS31FF. graminearum/Current study
SdhDGY-1RC92T, G678AS31F, W180KF. graminearum/Current study
/: Indicates no data available.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhou, F.; Zhou, H.-H.; Han, A.-H.; Guo, K.-Y.; Liu, T.-C.; Wu, Y.-B.; Hu, H.-Y.; Li, C.-W. Mechanism of Pydiflumetofen Resistance in Fusarium graminearum in China. J. Fungi 2023, 9, 62. https://doi.org/10.3390/jof9010062

AMA Style

Zhou F, Zhou H-H, Han A-H, Guo K-Y, Liu T-C, Wu Y-B, Hu H-Y, Li C-W. Mechanism of Pydiflumetofen Resistance in Fusarium graminearum in China. Journal of Fungi. 2023; 9(1):62. https://doi.org/10.3390/jof9010062

Chicago/Turabian Style

Zhou, Feng, Huan-Huan Zhou, Ao-Hui Han, Kou-Yun Guo, Tian-Cheng Liu, Yan-Bing Wu, Hai-Yan Hu, and Cheng-Wei Li. 2023. "Mechanism of Pydiflumetofen Resistance in Fusarium graminearum in China" Journal of Fungi 9, no. 1: 62. https://doi.org/10.3390/jof9010062

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop