Fusarium head blight (FHB) is a devastating disease in wheat and other small grain cereals and is caused by Fusarium graminearum
species complex (FGSC), including at least 16 distinct and cryptic species, and some species have specific geographical distributions [1
]. In China, FGSC causing FHB is mainly composed of F. asiaticum
and F. graminearum
]. The disease can not only cause serious yield and quality losses in many wheat-growing regions, but also FHB pathogens can produce a series of trichothecene mycotoxins in FGSC-infected wheat grains, including deoxynivalenol (DON) and its acetylated derivatives (3AcDON, 15AcDON) and nivalenol (NIV), thus posing a grave threat to the safety and health of humans and animals [5
]. DON has been demonstrated to be the most common contamination associated with FGSC-infected wheat grains and can cause hematic and anorexic syndromes as well as neurotoxic and immunotoxic effects in mammals. Additionally, DON has also been reported as an important virulence factor of FGSC [6
The control of FHB always depends on chemical fungicides. Previous studies have shown that the resistance of benzimidazole is already widespread in China, especially in eastern China, and that there is a high resistance risk of FGSC to phenamacril [8
]. Accordingly, it is of great importance to discover and develop novel fungicides that exhibit inhibitory effects on the fungal growth and DON biosynthesis of FHB pathogens. Succinate dehydrogenase inhibitors (SDHIs) are a new class of chemical fungicides. Previous studies have demonstrated that SDHIs target enzyme complex II of the mitochondrial respiratory electron transport chain, namely succinate dehydrogenase (SDH) or succinate quinone reductase (SQR) in phytopathogenic fungi [11
]. The enzyme complex II is also an important functional part of the tricarboxylic acid (TCA) cycle and is linked to mitochondrial respiratory electron transport chain for catalysis of the coupling reaction from succinic acid oxidation to fumaric acid and reduction from ubiquinone to ubiquinol. It includes four subunits: Flavoprotein (SdhA), iron-sulfur protein (SdhB), and two other integral membrane proteins (SdhC and SdhD) [13
]. In terms of chemical structure, SDHIs contain an amide group (-CONH-). Most of the newly developed fungicides are based on the original reactive group as a backbone. At present, SDHIs have been widely applied for controlling many plant diseases [15
]. However, SDHIs are rarely used to control FHB, especially in the control of DON production in wheat grains. In this study, the effects of five SDHIs, fluopyram, flutolanil, boscalid, benzovindiflupyr, and fluxapyroxad, in inhibiting mycelial growth, spore germination of FGSC, and DON biosynthesis of F. asiaticum
were determined. This study also evaluated the expression of TRI5
gene, which is the DON biosynthesis-associated gene. In addition, the impacts of five SDHIs on DON biosynthesis-associated biological characteristics such as pyruvic acid, acetyl-CoA, ATP, citric acid and activities of several key enzymes were evaluated in vitro. Finally, the effect of these five SDHIs on toxisomes was investigated using a confocal laser scanning microscope.
FHB caused by FGSC is an economically important fungal disease on various cereals [27
]. In addition to the loss of yield, the mycotoxins produced by FGSC in infected cereals pose a grave threat to the safety and health of humans and animals [4
]. Since most wheat cultivars are susceptible to FGSC, the application of chemical fungicides has been a principal tool for controlling FHB in the last 40 decades. Previous studies have reported that the resistance of carbendazim is already widespread in China [8
]. In addition, carbendazim can stimulate DON biosynthesis of FGSC, and carbendazim resistance can cause increase in DON production of FGSC [28
]. A novel cyanoacrylate fungicide phenamacril exhibits a specific activity against Fusarium
spp. and an inhibitory effect on DON production [10
]. However, phenamacril has a high resistance risk in FGSC [10
]. Previous studies have reported a strong correlation between FHB control efficacy and DON contamination [32
]. Therefore, it is necessary to find novel fungicides for controlling FHB and DON contamination caused by FGSC.
Succinic dehydrogenase inhibitors (SDHIs) studied in this paper are respiratory inhibitors. They inhibit the transmission of electrons from succinic acid to ubiquinone by completely or partially occupying the ubiquinone site of the substrate, thus hindering the energy metabolism of bacteria, inhibiting the growth of pathogens, and achieving the purpose of controlling diseases [12
]. Currently, only one SDHI fungicide carboxin has been registered for the control of FHB in China. Previous studies reported that SDHIs have good effects in the prevention and control of other diseases. For example, pydiflumetofen can effectively inhibit Sclerotinia sclerotiorum
, with an average EC50
value of 0.0250 μg/mL [18
]. In addition, boscalid and isopyrazam have a better inhibitory effect on mycelial growth of Aspergillus flavus
species. Meanwhile, boscalid can reduce the toxin contamination of A. flavus
In China, FGSC mainly includes F. asiaticum
and F. graminearum
. In this study, the sensitivity of seven F. asiaticum
and six F. graminearum
strains to five SDHIs (fluopyram, flutolanil, boscalid, benzovindiflupyr, and fluxapyroxad) was determined. We found that the five SDHIs did not differ in inhibiting the mycelial growth of FGSC, except for fluopyram. However, these five SDHIs exhibited a higher activity in inhibiting spore germination than mycelial growth. The results showed that the five SDHIs have potential in controlling FHB caused by FGSC in the field. Additionally, fluopyram exhibited a better inhibitory effect on either mycelial growth or spore germination in comparison to the other four SDHIs. In addition to fungicidal activity, we also found that these SDHIs can decrease DON production in F. asiaticum
in vitro. At present, the biosynthetic pathway of DON has been extensively studied, and nearly all DON biosynthesis-involved genes (TRI
genes) have been identified [21
]. The trichothecene precursor synthase gene TRI5
is a key enzyme in step one of DON biosynthesis [19
]. In this study, five SDHIs caused a decrease in the TRI5
gene expression. Moreover, we also observed that the five SDHIs could disrupt the formation of the complete spherical structure of toxisomes in F. asiaticum
. The results revealed that the five SDHIs not only exhibited an inhibitory effect on spore germination in FGSC, but can also decrease DON biosynthesis in F. asiaticum
. Thus, the five SDHIs have a potential in either controlling FHB or reducing DON contamination in F. asiaticum
The pyruvic acid produced in glycolysis is first transported into the mitochondria and oxidatively decarboxylated under aerobic conditions to form acetyl-CoA [43
]. Meanwhile, acetyl-CoA is the major substrate for the biosynthesis of a variety of secondary metabolites, including trichothecenes [19
]. Therefore, the content of pyruvic acid and acetyl-CoA in the strain should be positively correlated with the content of DON. Previous studies have shown that the production of pyruvic acid controlled by hexokinase supplies the main substrate for the biosynthesis of many secondary metabolites, such as trichothecene, DON, fumonisins, penicillin, and aflatoxin [20
]. As expected, the content of pyruvic acid and acetyl-CoA were significantly reduced as affected by the five SDHIs. Pyruvic acid is the final product of glycolysis, and the decrease in its content indicated that the five SDHIs inhibited the glycolysis pathway. To verify this hypothesis, we performed qRT-PCR analysis of three key genes in the glycolysis pathway. We found that the relative expression of hexokinase and 6-phosphate fructokinase significantly decreased, and the relative expression of pyruvate kinase was significantly increased after treatment with the five SDHIs, resulting in inhibition of the glycolysis pathway. However, the relative expression of 6-phosphate fructokinase increased after treatment with benzovindiflupyr, possibly due to other action sites of benzovindiflupyr. The up-regulation of pyruvate kinase may be due to the product activation caused by the decrease of pyruvic acid content.
SDHIs affect the activity of succinate dehydrogenase, catalyzing the oxidation of succinic acid to fumaric acid in the TCA cycle. Isocitrate dehydrogenase is the rate-limiting enzyme of the TCA cycle [24
]. Therefore, we determined the citric acid content, isocitrate dehydrogenase, and succinate dehydrogenase activities to investigate the regulatory effect of the five SDHIs in the TCA cycle. We found that the five SDHIs inhibited succinate dehydrogenase and isocitrate dehydrogenase activities, causing the decrease of the TCA cycle. In addition, the five SDHIs caused increase in citric acid content. Citric acid is the product of the first step of the TCA cycle [22
], and its accumulation is due to the inhibition of downstream reactions. In addition, the accumulation of citric acid inhibits the glycolysis pathway [23
]. SDHIs are respiratory inhibitors, which inhibit energy metabolism in bacteria [13
]. Therefore, out of curiosity on whether SDHIs have the same effect on F. asiaticum
, we determined the ATP content of F. asiaticum
treated with five SDHIs. As expected, the ATP content was significantly reduced.
In summary, these five SDHIs exhibited inhibitory effects on the spore germination of FGSC. Importantly, SDHIs can decrease DON biosynthesis in F. asiaticum in vitro. This may be attributed to the inhibitory effects on glycolysis, TCA cycle, and energy metabolism caused by SDHIs. Thus, the results of the study will provide valuable information for wheat protection programs against the toxigenic fungi responsible for FHB and the consequent DON contamination in wheat grains.
4. Materials and Methods
4.1. Fungicides, Fungal Strains and Culture Conditions
Technical-grade fluopyram, flutolanil, boscalid, benzovindiflupyr, and fluxapyroxad were kindly provided by Bayer (Shanghai, China), Nihon Nohyaku Co. (Shanghai, China), BASF (Shanghai, China), Syngenta (Beijing, China) and BASF (Shanghai, China), respectively. These fungicides were dissolved in methanol at 10 g/L and stored at 4 °C prior to further use.
Thirteen FGSC strains were isolated from the infected wheat ears in the field and stored in the Fungicide Biology Laboratory, Nanjing Agricultural University (Nanjing, China). These strains were identified by PCR assay as previously described [44
]. The strain 2021, BM-1, BM-4, BM-13, BM-14, BM-17 and BM-20 are identified as F. asiaticum
, and the strains BM-2, BM-3, BM-5, BM-7, BM-9 and BM-10 are identified as F. graminearum
. F. asiaticum
is dominant in eastern China, the most serious region affected by FHB. In addition, the strain 2021 was isolated from the infected wheat ear in 2000, and its genome was sequenced and analyzed in our laboratory. Thus, the strain 2021, as an F. asiaticum
model strain, was selected for further research in this study.
Potato dextrose agar (PDA, 200 g/L potato, 20 g/L glucose and 20 g/L agar) was used for colony morphology examination and sensitivity test for five succinate dehydrogenase inhibitors in vitro. Water agar (WA, 16 g/L agar) was used for the determination of spore germination [45
]. Mung bean broth (MBB, 30 g/L mung bean) was used for sporulation assays [46
]. Yeast extract peptone dextrose medium (YEPD, 10 g/L peptone, 20 g/L glucose and 3 g/L yeast extract) was used for conidial germination. Glucose yeast extract peptone medium (GYEP, 1 g/L peptone, 50 g/L glucose and 1 g/L yeast extract) was employed for DON production.
4.2. Fungicide Sensitivity Tests Based on Mycelial Growth and Spore Germination
Prior to fungicide sensitivity tests, the preliminary experiments were performed to optimize fungicide concentration gradients. For fungicide sensitivity tests, at least five concentrations for each fungicide were determined, and inhibition rate for all fungicide concentrations ranged 10% to 90%, and inhibition rate for medial concentration close to 50%. For mycelial growth, PDA plates were amended with fluopyram to obtain final concentrations of 2.5, 5, 10, 20, and 40 μg/mL and amended with the other four SDHIs (flutolanil, boscalid, benzovindiflupyr, and fluxapyroxad) to obtain final concentrations of 31.25, 62.5, 125, 250, and 500μg/mL, respectively. Inverted mycelial plugs (5 mm in diameter) cutting from the edge of an actively growing colony were transferred to 9 cm Petri dishes containing PDA media amended with the above described fungicide concentrations. Plates without fungicides were used as control. After incubation for 3 days in a growth chamber (25 °C), the colony diameters in two perpendicular directions for each PDA plate were measured and averaged. The EC50 values (effective concentration for 50% inhibition of mycelial growth) were calculated with the probit regression of the percentage of inhibition against the logarithmic value of fungicide concentrations.
For spore germination, WA plates were amended with fluopyram to obtain final concentrations of 0.125, 0.25, 0.5, 1, and 2 μg/mL and amended with the other four SDHIs (flutolanil, boscalid, benzovindiflupyr, and fluxapyroxad) to obtain final concentrations of 0.5, 1, 2, 4, and 8 μg/mL, respectively. Five mycelial plugs of each strain from the edge of 3-day-old colonies on PDA plates were transferred to a 50 mL flask containing 20 mL of mung bean broth. Conidia were filtered with two layers of lens wiping paper and collected by centrifuging at 5000 rpm for 5 min after culturing at 25 °C for 3 days in a shaker (175 rpm, 12 h of illumination every day). The conidia were suspended with sterile water, and the concentration was adjusted to 1 × 106
/mL. Then, 100 μL of conidia suspension was spread on WA plate containing the above described fungicide concentrations. After incubation for 5–6 h at 25 °C in the dark, the number of germinated conidia was measured. A conidium was considered germinated if the germ tube was at least half the length of the conidium. A total of 100 conidia were scored for each dish. The EC50
values (effective concentration for 50% inhibition of conidia germination) were estimated from the probit regression of the percentage of inhibition against the logarithmic value of fungicide concentrations [48
]. Each concentration had three replicates, and the experiment was repeated twice.
4.3. RNA Extraction and Reverse Transcription PCR
The RNA simple Total RNA Kit (Tiangen, Beijing, China) was used to extract the total RNA from mycelia, and reverse transcription PCR was performed with the HiScript II qRT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) as previously described [49
]. The RNA integrity was validated by agarose gel electrophoresis and absorbance determination.
4.4. DON Production and TRI5 Gene Expression
For DON production, spore suspensions of the strain 2021 were prepared and diluted to 5 × 104
/mL. 1 mL of spore suspensions were added into 100mL GYEP [50
]. After culturing in the dark at 28 °C for 24 h, the five SDHIs were added into the cultures and the final concentrations were 0.54 μg/mL for fluopyram, 2.96 μg/mL for flutolanil, 1.52 μg/mL for boscalid, 1.79 μg/mL for benzovindiflupyr, and 3.16 μg/mL for fluxapyroxad, respectively (the EC50
values are from the spore germination inhibition method and are listed in Table 2
). After the incubation of an additive for 6 days, the culture liquid was collected and the mycelia were dried and weighed. DON production in the culture liquid was measured using the DON ELISA Kit (Wise, Zhenjiang, China) according to a previous study [7
]. The DON ELISA Kit uses an indirect competitive ELISA method to detect the DON content in strains and cereals. Compared with the previous HPLC method, it is faster and easier to operate, while it also has higher detection accuracy. Its detection range is from 10 μg/L to 135 μg/L. DON production ability in shake culture was expressed as the amount of DON produced per dry weight of mycelia (μg/g). The experiment was repeated three times independently, with each treatment having three replicates.
gene expression, the conidia of 2021 were added to GYEP (5 × 104
conidia per 100 mL GYEP). After culturing for 24 h at 28 °C in the dark, the concentrations of five SDHIs were then added to the cultures as described above. After the incubation of an additive for 2 days, mycelia were collected for extraction of total RNA as previously described. TRI5
gene expression was determined by qRT-PCR using the primers listed in Table 3
, as described in Section 4.3
. The experiment was repeated twice, with each treatment having three replicates.
4.5. Expression of Key Genes in Glycolysis Pathway
To determine the expression levels of key genes in glycolysis, the conidia of 2021 were added to YEPD (1 × 105
conidia per 100 mL YEPD). After culturing for 24 h at 25 °C, the concentrations of five SDHIs were added to the cultures as described above. After the incubation of an additive for 2 days, mycelia were collected for extraction of total RNA as previously described. The expression levels of key genes in glycolysis were determined by qRT-PCR with the primers listed in Table 3
. All data were normalized to actin gene expression, and relative changes in gene expression levels were analyzed with the CFX Manager Software (3.1, Bio-Rad, Hercules, CA, USA), which automatically sets the baseline. The experiments were repeated three times, with each treatment having three replicates.
4.6. Determination of Pyruvic acid, Acetyl-CoA, ATP and Citric Acid
The conidial suspensions were added to YEPD (1 × 105 conidia per 100mL YEPD) and incubated for 24 h, then the concentrations of five SDHIs were added to the cultures as described above. After incubation with an additive for 2 days, the mycelia were collected and used for the determination of pyruvic acid, acetyl-CoA, ATP, and citric acid. Pyruvic acid and acetyl-CoA were assayed using a pyruvic acid content test kit (Solarbio, BC2205, Beijing, China) and an acetyl-CoA content test kit (Solarbio, BC0980, Beijing, China), respectively. ATP and citric acid production were assayed using an ATP assay kit (Beyotime, S0026, Nanjing, China) and a citric acid content test kit (Solarbio, BC2150, Beijing, China), respectively. In short, 0.05g of mycelia were added to the corresponding lysis buffer of different detection kits. After the lysis of mycelia, pyruvic acid, acetyl-CoA, ATP, or citric acid production were determined according to the manufacturer’s instructions. The experiments were performed three times independently.
4.7. SDH, ICDHm and NADH Dehydrogenase Activities
The mycelia were collected as described in Section 4.6
and used for the activities of SDH and ICDHm. SDH and ICDHm activities were determined using a succinate dehydrogenase activity assay kit (Solarbio, BC0950, Beijing, China) and an isocitrate dehydrogenase mitochondrial activity assay kit (Solarbio, BC2160, Beijing, China), respectively. The experiments were performed three times independently, with each treatment having three replicates.
NADH dehydrogenase activity was defined as the rate of decomposition of NADH. This experiment reflects the change of NADH by measuring the change of absorbance at 340 nm [52
]. The mycelia were collected as described in Section 4.6
. The mycelia (0.05 g) were ground with 1 mL of PBS phosphate buffer. The extracts were centrifuged for 10 min at 10,000 rpm at 4 °C, and the supernatant was ultrasonically broken. Finally, 100 μL of NADH (40 μmol) was added to 100 μL supernatant, and the changes in absorbance at 340 nm for 15 min were recorded using a spectrophotometer. The experiments were performed three times independently, with each treatment having three replicates.
4.8. Microscopic Examinations
In order to observe the morphological changes of the toxisomes, the strain 2021-TRI1-GFP labeled with TRI1-GFP was cultured in GYEP at 28 °C for 24 h, then the different concentrations of five SDHIs were added to the cultures as described above and cultured at 28 °C for 48 h. All samples were mounted on glass slides and sealed with cover glasses. Images of toxisomes were obtained at room temperature using a LEICA TCS SP8 confocal laser-scanning microscope (LEICA, laser: At 488 nm). The experiment was performed three times independently.
4.9. Statistical Analysis
All the data in this study were analyzed with the SPSS 14.0 software (SPSS Inc. Chicago, IL, USA) to obtain statistical variances between repeated experiments. Fisher’s LSD test (p = 0.05) was used to obtain the standard errors and determine whether there were significant differences among the biological characteristics.