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Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate
YPEST-03905; No of Pages 7 Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate Hancheng Wang a,1, Jin Wang b,1, Qingyuan Chen a,1, Maosheng Wang a, Tom Hsiang c, Shenghua Shang a, Zhihe Yu a,⁎ a b c
Key Laboratory of Molecular Genetics, Guizhou Academy of Tobacco Sciences, Guiyang 550081, PR China College of Life Science, Yangtze University, Jingzhou 434025, PR China School of Environmental Sciences, University of Guelph, Guelph, ON N1G2W1, Canada
a r t i c l e
i n f o
Article history: Received 30 September 2015 Received in revised form 16 November 2015 Accepted 28 November 2015 Available online xxxx Keywords: Azoxystrobin Biolog FF MicroPlate Kresoxim-methyl Mode of action Sensitivity
a b s t r a c t Azoxystrobin and kresoxim-methyl are strobilurin fungicides, and are effective in controlling many plant diseases, including Fusarium wilt. The mode of action of this kind of chemical is inhibition of respiration. This research investigated the sensitivities of Fusarium kyushuense to azoxystrobin and kresoxim-methyl, and to the alternative oxidase inhibitor salicylhydroxamic acid (SHAM). The Biolog FF MicroPlate is designed to examine substrate utilization and metabolic profiling of micro-organisms, and was used here to study the activity of azoxystrobin, kresoxim-methyl and SHAM against F. kyushuense. Results presented that azoxystrobin and kresoxim-methyl strongly inhibited conidial germination and mycelial growth of F. kyushuense, with EC50 values of 1.60 and 1.79 μg ml−1, and 6.25 and 11.43 μg ml−1, respectively; while not for SHAM. In the absence of fungicide, F. kyushuense was able to metabolize 91.6% of the tested carbon substrates, including 69 effectively and 18 moderately. SHAM did not inhibit carbon substrate utilization. Under the selective pressure of azoxystrobin and kresoximmethyl during mycelial growth (up to 100 μg ml−1) and conidial germination (up to 10 μg ml−1), F. kyushuense was unable to metabolize many substrates in the Biolog FF MicroPlate; while especially for carbon substrates in glycolysis and tricarboxylic acid cycle, with notable exceptions such as β-hydroxybutyric acid, y-hydroxybutyric acid, αketoglutaric acid, α-D-glucose-1-phosphate, D-saccharic acid and succinic acid in the mycelial growth stage, and βhydroxybutyric acid, y-hydroxybutyric acid, α-ketoglutaric acid, tween-80, arbutin, dextrin, glycerol and glycogen in the conidial germination stage. This is a new finding for some effect of azoxystrobin and kresoxim-methyl on carbon substrate utilization related to glycolysis and tricarboxylic acid cycle and other carbons, and may lead to future applications of Biolog FF MicroPlate for metabolic effects of other fungicides and other fungi, as well as providing a carbon metabolic fingerprint of F. kyushuense that could be useful for identification. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China is the biggest single tobacco market and accounts for more than 40% of the global tobacco consumption [1]. During tobacco seedling development period, various fungal pathogens attack seedlings in the greenhouse. In the last five years, Fusarium wilt of tobacco, caused by Fusarium kyushuense O'Donnell & T. Aoki, was frequently observed in some regions of Guizhou province in southwest China [2,3]. The pathogen attacks seedlings leading to symptoms of severe wilting, chlorosis and stunting with poorly developed root systems, and eventual death. Control of the disease is based on an integration of several cultural methods with the use of fungicides. For Fusarium diseases caused by Fusarium oxysporum f. sp. nicotianae in ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Yu). 1 Contribute equally to this article.
China, multisite inhibitors, such as chlorothalonil and mancozeb, and the target site-specific fungicides such as carbendazim have been used to control this disease [4–6]. Multisite inhibitors may be effective when applied in protective fashion [7]. Carbendazim was a major fungicide for Fusarium disease management twenty years ago, but carbendazim-resistant strains developed causing all benzimidazoles to lose their efficacy [8–10]. Recently, the strobilurin fungicide azoxystrobin and kresoxim-methyl have been registered in China and other countries to control Fusarium diseases, including wheat scab, potato Fusarium dry rot, and tomato Fusarium wilt [11–13]. They have also been used to manage many other disease management on tobacco in the USA and Germany [14–16]. Few studies have been published to describe the sensitivity to azoxystrobin and kresoxim-methyl by F. kyushuense, and because of the relatively high cost of these chemicals locally, they have not been used for tobacco disease management in China. Azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin and famoxadone [17–20] are Quinone outside inhibitor (QoI) fungicides (Fungicide Resistance Action Committee
http://dx.doi.org/10.1016/j.pestbp.2015.11.013 0048-3575/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
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H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
[FRAC] group 11) also known as strobilurins. Preliminary studies revealed that azoxystrobin and kresoxim-methyl inhibit mitochondrial respiration by blocking electron transfer at the cytochrome bc1 complex [21]. They have high activity against spore germination and less activity against mycelial growth because hyphae may make use of the terminal alternative oxidase (AOX) that bypasses the blockage site [22,23]. Salicylhydroxamic acid is the special inhibitor for the terminal alternative oxidase. The metabolic effects of azoxystrobin and kresoxim-methyl have not yet been fully elucidated, and more research is needed to understand the diverse secondary effects of strobilurins on fungal plant pathogens. Recently, the Biolog FF MicroPlate was introduced by Biolog Company for characterizing filamentous fungi (FF MicroPlate™ Instruction, Biolog, Hayward, CA, USA). The MicroPlate uses 95 biochemical tests to profile substrate utilization and phenotypic profiling of many microorganisms with each well containing substrates that change color with metabolic activity (Fig. 1) [24,25]. There have been few previous reports of its use for investigating mode of action of fungicides [26]. Therefore, the objectives of this study were: (i) to document the sensitivities of F. kyushuense to azoxystrobin, kresoxim-methyl and SHAM and (ii) to evaluate the phenotypic profiling of F. kyushuense under pressures of azoxystrobin, kresoxim-methyl and SHAM. The outcome of this study will provide useful information on the effects and mode of action of azoxystrobin and kresoxim-methyl. 2. Materials and methods 2.1. Pathogen, media and chemical preparation A strain of F. kyushuense with wild-type strobilurin sensitivity and pathogenicity to tobacco [2] was collected in 2013 from an infected
tobacco seedling in a commercial field in Guizhou province, China. A monoconidial isolate was obtained and used for tests. The isolate was grown and maintained on a lima bean agar medium (LBA, 60.0 g l−1 lima beans boiled for 1 h and strained, 16.0 g l−1 agar), in a controlled climate cabinet at 25 °C in the dark. For conidial production, agar plugs were removed from the edge of an actively growing culture and placed on LBA plates. After 7 days, conidia were washed off with distilled water, and filtered through a double-layer of sterile cheesecloth (Grade #40: 24 × 20 threads per inch) to remove mycelial fragments. The resulting conidial suspension was quantified with a hemacytometer and adjusted to 1 × 105 spores/ml for subsequent use. For long-term storage, 5-mm agar plugs from the leading edge of individual colonies were transferred into several sterile 1.5-ml microcentrifuge tubes containing 1 ml of 30% sterile glycerol, and tubes were stored at −20 °C in darkness. Stock fungicide solutions were prepared by dissolving technical grade azoxystrobin (a.i. 93%; Syngenta China Co., Ltd, Shanghai, China), kresoxim-methyl (a.i. 96%; BASF China Co., Ltd, Shanghai, China) and SHAM (a.i. 99.9%; Sigma-Aldrich China Co., Ltd, Shanghai, China) in methanol. Solutions were diluted as required and stored at 4 °C in the dark. The methanol concentration never exceeded 1% of the testing solution. This concentration of methanol was not observed to affect different life stages of F. kyushuense (data not shown). Controls always contained the same methanol concentration as the test samples in the experiments. Fungicides were added to LBA after autoclaving when the agar had cooled to approximately 50 °C. Filamentous Fungi inoculating fluid (FF-IF, catalog # 72106) (containing 2.5 g l−1 Phytagel and 0.3 g l−1 Tween 40) and FF MicroPlate test panels (catalog # 1006) containing 95 different carbon sources were purchased from Biolog Inc. (Hayward, CA, USA) and stored at 4 °C until needed.
Fig. 1. Layout of assays in the Biolog FF MicroPlate.
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
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Individual agar discs (5-mm-diameter) were removed from the edge of an actively growing culture and placed face up on the centre of a Petri dish (9-cm-diameter) containing LBA amended with azoxystrobin, kresoxim-methyl and SHAM at various concentrations, respectively. The final concentrations tested with three replications were for azoxystrobin and kresoxim-methyl 0, 0.01, 0.1, 1, 10 and 100 μg ml−1; and for SHAM 0, 50, 100 and 200 μg ml−1, respectively. The diameters of the colonies were measured after incubation for 6 days at 25 °C in the dark. The experiment was conducted twice.
were harvested with sterile cotton-tipped applicators as described above, suspended in distilled water to obtain a final concentration of 1 × 105 spores/ml. The test FF-IF broth (16 ml in one tube) was prepared as mentioned above to get final test concentrations of 0, 0.01, 0.1, 1 and 10 μg ml−1 for azoxystrobin and kresoxim-methyl, and of 0, 100 and 200 μg ml− 1 for SHAM, respectively. Each FF-IF broth was added with 0.2 ml of conidial suspension to make the final test suspensions. One hundred microliters of each test suspension was added to each well of the FF plates. Afterwards, plates were incubated in the OmniLog at 28 °C for 7 days with readings taken every 15 min as mentioned above.
2.3. Inhibition of conidial germination
2.7. Data analysis
Conidia were harvested as described above from 7-day-old cultures. Aqueous preparations of azoxystrobin, kresoxim-methyl and SHAM at different concentrations were added to an equal volume (500 μl) of the conidial suspension, respectively. A control conidial suspension was diluted with an equal volume (500 μl) of water only. The final concentrations tested for azoxystrobin and kresoxim-methyl were 0, 0.01, 0.1, 1, 5, 10, 50 and 100 μg ml−1, and for SHAM were 0, 50, 100 and 200 μg ml−1, respectively. Conidial suspensions were then incubated at 25 °C in the dark for 12 h. A conidium was scored as germinated if the germ tube had reached at least the length of the conidium. Germinated conidia were quantified on microscope slides at three spots by assessing 100 conidia per spot. The experiment was conducted twice.
All data were processed with the SIGMASTAT Statistical Software Package (SPSS Science, Chicago). The concentration of fungicide causing 50% (EC50) or 90% (EC90) reduction in mycelial growth or germination of conidia compared to the absence of the fungicide was estimated from the fitted regression line of the log-transformed percentage inhibition plotted against the log-transformed fungicide concentration [21]. Data analysis for metabolic profiling of F. kyushuense was conducted using Kinetic and Parametric software (Biolog). Phenotypes were determined based on the area under the kinetic curve of dye formation [27].
2.2. Inhibition of mycelial growth
3. Results 3.1. Inhibition of mycelial growth
2.4. Metabolic profiling of F. kyushuense during mycelial growth exposed to azoxystrobin, kresoxim-methyl and SHAM For metabolic profiling of F. kyushuense during mycelial growth with exposure to azoxystrobin, kresoxim-methyl and SHAM, conidia were produced on LBA plates as described above and collected with sterile cotton-tipped applicators from the agar plates, avoiding carryover of nutrients from the agar medium. Conidia together with mycelia were then suspended in distilled water and incubated at 25 °C in darkness for 12 h to prepare mycelial suspensions. The FF-IF broth (16 ml broth in 20 ml tubes) were amended with various concentrations of azoxystrobin and kresoxim-methyl to obtain final test concentrations of 0, 0.01, 0.1, 1, 10 and 100 μg ml−1, and with various concentration of SHAM to get final test concentrations of 0, 100 and 200 μg ml−1, respectively. Afterwards, to each FF-IF broth tube 0.2 ml of mycelial suspension and mixed was added. One hundred microliters of each test suspension was then added to each well of the FF plates under aseptic conditions. Plates were then incubated in the OmniLog at 28 °C for 7 days with readings taken every 15 min. Incubation and recording of phenotypic data were performed in the OmniLog station by capturing digital images of the microarrays and storing turbidity values in a computer file displayed as a kinetic graph. 2.5. Metabolic comparisons when adjusting for mycelial mass A mycelial size (about 3 to 3.5 cm in diameter on Petri dishes) on the non-fungicidal plates (about 3 day incubation on LBA plates) had a similar size to day 7 on the azoxystrobin-amended or kresoxim-methylamended media at 1 and 10 μg ml−1. To adjust for the metabolic activities of a smaller mycelial mass with growth inhibition by azoxystrobin and kresoxim-methyl, Biolog readings for a mycelial mass of a similar were compared to the mycelium exposed to 1 and 10 μg ml− 1 of azoxystrobin and kresoxim-methyl, respectively. 2.6. Metabolic profiling of F. kyushuense during conidial germination under the pressure of azoxystrobin, kresoxim-methyl and SHAM For metabolic profiling during conidial germination with exposure to azoxystrobin, kresoxim-methyl and SHAM, conidia of F. kyushuense
Azoxystrobin and kresoxim-methyl inhibited mycelial growth of F. kyushuense. With increasing fungicide concentration, mycelial growth was more greatly inhibited. The EC50 values were 6.25 μg ml−1 and 11.43 μg ml− 1, respectively; and EC90 values were N100 μg ml−1 for each chemical. In comparison, SHAM had no activity against mycelial growth of the fungus even at the highest concentration of 200 μg ml−1. Additionally, mycelial growth of F. kyushuense was not be completely inhibited by azoxystrobin and kresoxim-methyl even at the highest test concentration of 100 μg ml−1 (data not shown). 3.2. Inhibition of conidial germination Azoxystrobin and kresoxim-methyl were very active against germination of conidia, with EC50 values of 1.60 μg ml−1 and 1.79 μg ml−1, respectively. The germination ratios decreased greatly with increasing concentrations of the compound (data not shown). In comparison, SHAM did not inhibit conidial germination of the fungus even at the highest concentration of 200 μg ml−1 (data not shown). Germination of conidia was completely inhibited by azoxystrobin and kresoximmethyl at 50 μg ml−1. In the absence of the compound, the percentage germination of conidia after 12 h at 25 °C was around 95%. 3.3. Metabolic profiling of F. kyushuense during mycelial growth under pressures of azoxystrobin, kresoxim-methyl and SHAM The metabolic abilities of F. kyushuense were tested by using the Biolog system. Using the Biolog FF MicroPlate, 95 different carbon sources were tested. In the absence of a fungicide, the pathogen was able to metabolize 91.6% of tested carbon sources (87/95 tested, Wells A1-H12). Sixty-nine compounds were effectively utilized by F. kyushuense; eighteen substrates were metabolized at a mid-level; while the other eight compounds were not utilized by F. kyushuense. When incubated with azoxystrobin and kresoxim-methyl at 0.01 μg ml−1, and with SHAM at 100 and 200 μg ml−1, F. kyushuense exhibited the same metabolic profiling as that of control during mycelial growth stage; when incubated at 0.1 μg ml−1 (well below the EC50 of 6.25 μg ml−1) only the utilization of succinic acid mono-methyl ester (Well G5) was inhibited by azoxystrobin; when treated at 1, 10 and
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
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H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
Table 1 Metabolic profiling of Fusarium kyushuense during mycelial growth under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.1
1
10
100
Well
Substrate
0
0.1
1
10
100
A2 A4 A7 A9
Tween 80 N-Acetyl-β-D-Glucosamine Amygdalin
+/+ +/+ +/+ +/+
+/+ −/+ −/+ −/+
+/+ −/− −/+ −/−
−/+ −/− −/− −/−
C10 C11 C12 D1
Maltitol Maltose Maltotriose
L-Arabinose
+ + + +
D-Mannitol
+ + + +
+/+ +/+ +/+ +/+
−/− −/− −/− −/+
−/− −/− −/− −/−
−/− −/− −/− −/−
A10
D-Arabitol
+
+/+
−/−
−/−
−/−
D2
D-Mannose
+
+/+
+/+
−/−
−/−
A11
Arbutin
+
+/+
−/−
−/−
−/−
D3
D-Melezitose
+
+/+
+/+
−/−
−/+
A12
D-Cellobiose
+
+/+
+/+
−/+
−/+
D4
D-Melibiose
+
+/+
−/+
−/−
−/−
B3 B5
Dextrin D-Fructose
+ +
+/+ +/+
+/+ −/+
+/+ −/+
+/+ −/+
D5 D8
α-Methyl-D-Galactoside ß-Methyl-D-Glucoside
+ +
+/+ +/+
−/− −/−
−/− −/−
−/− −/−
B7
D-Galactose
+
+/+
−/−
−/−
−/−
D9
Palatinose
+
+/+
−/−
−/−
−/−
B9
Gentiobiose
+
+/+
−/+
−/+
−/+
D10
D-Psicose
+
+/+
−/−
−/−
−/−
B10
D-Gluconic
+
+/+
+/−
−/−
−/−
D11
D-Raffinose
+
+/+
−/−
−/−
−/−
B11
D-Glucosamine
+
+/+
−/+
−/+
−/−
D12
L-Rhamnose
+
+/+
−/−
−/−
−/−
B12
α-D-Glucose
+
+/+
−/+
−/+
−/+
E1
D-Ribose
+
+/−
−/+
−/−
−/−
C1
α-D-Glucose-1 -Phosphate
+
+/−
+/−
+/−
+/−
E2
Salicin
+
+/−
+/−
+/−
+/−
Acid
C3
D-Glucuronic
+
+/+
−/−
−/−
−/−
E3
Sedoheptulosan
+
+/+
−/−
−/−
−/−
C4
Glycerol
+
+/+
+/−
+/−
+/−
E4
D-Sorbitol
+
+/+
+/+
−/−
−/−
C5
Glycogen
+
+/+
+/−
+/−
+/−
E5
L-Sorbose
+
+/−
−/−
−/−
−/−
C6 C7 C8
m-Inositol 2-Keto-D-Gluconic Acid α-D-Lactose
+ + +
+/+ +/+ −/+
−/+ −/− −/−
−/− −/− −/−
−/− −/− −/−
E6 E7 E8
Stachyose Sucrose
+ + +
+/+ +/+ +/+
−/− −/+ −/−
−/− +/+ −/−
−/− −/+ −/−
Acid
D-Tagatose
‘+’ and ‘−’ mean that the substrate was metabolized and not metabolized by the pathogen when incubated under pressures of azoxystrobin and kresoxim-methyl examined using the Biolog FF MicroPlate, respectively.
100 μg ml− 1, the number of carbon substances metabolized by the pathogen significantly decreased, with 21, 17 and 17 substrates utilized (Table 1), respectively. Moreover, when incubated with concentrations of 1, 10 and 100 μg ml−1 azoxystrobin, F. kyushuense could still metabolize 9 substrates at high levels, including α-D-glucose-1-phosphate (Well C1) and β-hydroxybutyric acid (Well F4) (Table 1); there were five (tween-80, dextrin, glycerol, glycogen, and adenosine), two (tween-80 and dextrin) and three (glycerol, D-lactic acid methyl ester, and D-malic acid) substrates metabolized at mid-levels for each treatment, respectively; moreover, three substrates (fumaric acid, L-alanine and L-alanyl-glycine), four chemicals (sucrose, D-lactic acid methyl ester, L-alanine and L-alanyl-glycine) and one substrate (salicin) were
delayed metabolized by F. kyushuense, respectively. When incubated at 0.1, 1, 10 and 100 μg ml−1 of kresoxim-methyl, the number of carbon substances metabolized by the pathogen significantly decreased as well, with 63, 22, 9 and 8 substrates utilized (Table 1), respectively. Moreover, when incubated with concentrations of 1, 10 and 100 μg ml− 1 kresoxim-methyl, F. kyushuense could still metabolize 5 substrates at high levels, including tween 80 (Well A2), D-cellobiose (Well A12), dextrin (Well B3), D-fructose (Well B5) and α-D-glucose (Well B12); while five other common carbon substrates were metabolized at mid-levels or delayed metabolized by the pathogen, including gentiobiose (Well B9), D-mannose (Well D2), D-melezitose (Well D3), D-melibiose (Well D4) and sucrose (Well E7) (Table 1).
Table 2 Metabolic profiling of Fusarium kyushuense during conidial germination under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.01
0.1
1
10
Well
Substrate
0
0.01
0.1
1
10
A2 A4 A6 A7 A8
Tween 80 N-Acetyl-β-D-Glucosamine Adonitol Amygdalin
+/+ +/+ +/+ +/+ +/+
+/+ −/+ −/+ +/+ −/−
+/+ −/− +/− +/+ −/−
+/+ −/− −/− −/+ −/−
C7 C10 C11 C12 D1
2-Keto-D-Gluconic Acid Maltitol Maltose Maltotriose
D-Arabinose
+ + + + +
D-Mannitol
+ + + + +
+/+ +/+ −/+ −/+ +/+
−/+ −/+ +/+ −/+ +/+
−/− −/− −/− −/− −/−
−/− −/− −/− −/− −/−
A9
L-Arabinose
+
+/+
−/−
−/−
−/−
D2
D-Mannose
+
+/+
+/+
+/+
−/+
A10
D-Arabitol
+
+/+
+/+
−/−
+/−
D3
D-Melezitose
+
+/+
+/+
−/+
−/−
A11
Arbutin
+
+/+
+/+
+/−
+/−
D4
D-Melibiose
+
+/+
+/+
−/+
−/−
A12
D-Cellobiose
+
+/+
−/+
−/+
+/−
D5
α-Methyl-D-Galactoside
+
+/+
−/+
−/−
−/−
B3 B5
Dextrin D-Fructose
+ +
+/+ +/+
+/+ +/+
+/+ −/+
+/+ −/+
D8 D9
ß-Methyl-D-Glucoside Palatinose
+ +
+/+ +/+
−/+ −/+
−/− −/−
−/− −/−
B7
D-Galactose
+
+/+
−/+
−/−
−/−
D10
D-Psicose
+
+/+
−/+
−/−
−/−
B9
Gentiobiose
+
−/+
−/+
+/+
−/−
D11
D-Raffinose
+
+/+
+/+
−/−
−/−
B10
D-Gluconic
+
+/+
+/+
−/−
−/−
D12
L-Rhamnose
+
+/+
+/+
−/−
−/−
B11
D-Glucosamine
+
+/+
+/+
+/+
+/−
E1
D-Ribose
+
+/+
−/−
−/−
−/−
B12 C1
α-D-Glucose α-D-Glucose-1 -Phosphate
+ +
+/+ +/+
−/+ +/−
−/+ −/−
−/+ −/−
E2 E3
Salicin Sedoheptulosan
+ +
+/+ +/+
−/+ −/+
−/− −/−
−/− −/−
D-Glucuronic Acid Glycerol
+
+/+
+/+
+/−
−/−
E4
D-Sorbitol
+
+/+
+/+
−/−
−/−
C4
+
+/+
+/+
+/−
+/−
E5
L-Sorbose
+
+/+
−/−
−/−
−/−
C5 C6
Glycogen m-Inositol
+ +
+/+ +/+
+/+ −/+
+/− −/−
+/− −/−
E6 E7
Stachyose Sucrose
+ +
+/+ +/+
−/+ −/+
−/− −/+
−/− −/+
C3
Acid
‘+’ and ‘−’ mean that the substrate was metabolized and not metabolized by the pathogen when incubated under pressures of azoxystrobin and kresoxim-methyl examined using the Biolog FF MicroPlate, respectively.
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
5
Table 1 Metabolic profiling of Fusarium kyushuense during mycelial growth under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.1
1
10
100
Well
Substrate
0
0.1
1
10
100
E9
D
+
+/+
−/+
−/−
−/−
G7
L-Alaninamide
+
+/−
−/−
−/−
−/−
E10
Turanose
+
+/+
−/−
−/−
−/−
G8
L-Alanine
+
+/−
+/−
+/−
+/−
E11 E12
Xylitol
+/+ +/+
−/− −/−
−/− −/−
−/− −/−
G9 G10
L-Alanyl-Glycine
D-Xylose
+ +
L-Asparagine
+ +
+/− +/+
+/− −/−
+/− −/−
+/− −/−
F1
y-Aminobutyric Acid
+
+/+
−/−
−/−
−/−
G11
L-Aspartic
+
+/+
−/−
−/−
−/−
F2
Bromosuccinic Acid
+
+/+
−/−
−/−
−/−
G12
L-Glutamic
+
+/+
−/−
−/−
−/−
F3 F4
Fumaric Acid ß-Hydroxybutyric Acid
+ +
+/+ +/+
+/+ +/−
−/− +/−
−/− +/−
H1 H2
L-Ornithine
+ +
+/− +/−
−/− −/−
−/− −/−
−/− −/−
F5
y- Hydroxybutyric Acid
+
+/−
+/−
+/−
+/−
H3
L-Phenylalanine
+
+/−
+/−
+/−
+/−
F6
p-Hydroxy-phenylacetic Acid
+
+/−
−/−
−/−
−/−
H4
L-Proline
+
+/+
−/−
−/−
−/−
F7
α-Ketoglutaric Acid
+
+/−
+/−
+/−
+/−
H5
L-Pyroglutamic
+
+/+
−/−
−/−
−/−
F8
D-Lactic
Acid Methyl Ester
+
+/+
−/−
+/−
+/−
H6
L-Serine
+
+/−
−/−
−/−
−/−
F9
L-Lactic
Acid
+
+/+
−/−
−/−
−/−
H7
L-Threonine
+
+/−
−/−
−/−
−/−
F10
D-Malic
Acid
+
+/+
−/−
−/−
+/−
H8
2-Aminoethanol
+
+/−
−/−
−/−
−/−
F11
L-Malic
Acid
+
+/+
−/−
−/−
+/−
H9
Putrescine
+
+/+
−/−
−/−
−/−
F12 G1
D-Saccharic
+ +
+/+ +/−
−/+ +/−
−/− +/−
−/− +/−
H10 H11
Adenosine Uridine
+ +
+/+ +/+
+/− −/−
+/− −/−
+/− −/−
G3
Succinamic Acid
+
+/+
−/−
−/−
−/−
H12
Adenosine-5′-Monophosphate
+
+/+
−/−
−/−
−/−
G4 G5 G6
Succinic Acid Succinic Acid Mono-Methyl Ester N-Acetyl-L-Glutamic Acid
+ + +
+/+ −/− +/+
+/+ −/− −/−
+/− −/− −/−
+/− −/− −/−
-Trehalose
Quinic Acid Acid
Acid
Acid Gycyl-L-Glutamic Acid
Acid
(Well D1), D-mannose (Well D2), D-melezitose (Well D3), D-melibiose (Well D4) was also delayed.
3.4. Metabolic comparisons when adjusting for mycelial mass When 3-day-old cultures on non-fungicidal treatments were compared to 7-day-old cultures on fungicidal treatments (1 and 10 μg ml−1), the mycelial mass on Petri dishes was similar. Metabolic comparisons showed that, under apressure of 1 μg ml−1 azoxystrobin, the utilization of fumaric acid (Well F3), L-alanine (Well G8) and L-alanyl-glycine (Well G9) was slower to start than that of water treatment; under a pressure of 10 μg ml−1 azoxystrobin, the metabolization of sucrose (Well E7), D-lactic acid methyl Ester (Well F8), L-alanine (Well G8) and L-alanyl-glycine (Well G9) was delayed as well; under a pressure of 10 μg ml− 1 kresoxim-methyl, the utilization of D-mannitol
3.5. Metabolic profiling of F. kyushuense during the conidial germination stage with exposure to azoxystrobin, kresoxim-methyl and SHAM When exposed to azoxystrobin and kresoxim-methyl during conidial germination, F. kyushuense presented different metabolic profiles than that of the water check. With increasing azoxystrobin and kresoxim-methyl, less substrate was metabolized. When treated at 0.01 μg ml− 1 of azoxystrobin, the utilization of five substrates (maltotriose, D-lactic acid methyl ester, quinic acid, L-asparagine and
Table 2 Metabolic profiling of Fusarium kyushuense during conidial germination under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.01
0.1
1
10
E8
D-Tagatose
+
+/+
−/+
−/−
−/−
E9
D-Trehalose Turanose
+
+/+
−/+
−/−
E10
+
+/+
−/+
−/−
E11
Xylitol
+
−/+
+/+
−/−
−/−
E12
D-Xylose y-Aminobutyric Acid
+
+/+
−/−
−/−
−/−
F1
+
+/+
−/+
−/−
−/−
F2
Bromosuccinic Acid
+
+/+
−/+
−/−
−/−
G12
F3 F4
Fumaric Acid ß-Hydroxybutyric Acid
+ +
+/+ +/−
+/+ +/−
−/− +/−
−/− +/−
F5
y- Hydroxybutyric Acid
+
+/+
+/−
+/−
F6
p-Hydroxy-phenylacetic Acid
+
+/+
−/−
−/−
F7
α-Ketoglutaric Acid
+
+/+
+/−
F8
D-Lactic
Acid Methyl Ester
+
−/+
F9
L-Lactic
Acid
+
+/+
F10
D-Malic
Acid
+
F11
L-Malic
Acid Quinic Acid
D-Saccharic
F12 G1 G3 G4 G5
Acid Succinamic Acid Succinic Acid Succinic Acid Mono-Methyl Ester
Well
Substrate
0
0.01
0.1
1
10
G6
N-Acetyl-L-Glutamic Acid
+
+/+
−/+
−/−
−/−
−/−
G7
L-Alaninamide
+
+/+
−/−
−/−
−/−
−/−
G8
L-Alanine
+
+/+
−/+
−/−
−/−
G9
L-Alanyl-Glycine
+
+/+
−/−
−/−
−/−
G10
L-Asparagine
+
−/+
−/+
−/−
−/−
G11
L-Aspartic
+
+/+
−/+
−/−
−/−
L-Glutamic
+
−/+
−/+
−/−
−/−
H1 H2
L-Ornithine
+ +
+/+ +/+
−/− +/−
−/− −/−
−/− −/−
+/−
H3
L-Phenylalanine
+
+/+
+/−
−/−
−/−
−/−
H4
L-Proline
+
+/+
−/+
−/−
−/−
+/−
+/−
H5
L-Pyroglutamic
+
+/+
−/−
−/−
−/−
−/+
−/−
−/−
H6
L-Serine
+
+/+
−/−
−/−
−/−
−/+
−/−
−/−
H7
L-Threonine
+
+/+
−/−
−/−
−/−
+/+
+/+
−/−
−/−
H8
2-Aminoethanol
+
+/+
−/−
−/−
−/−
+
+/+
+/+
−/−
−/−
H9
Putrescine
+
+/+
−/−
−/−
−/−
+ +
−/+ +/+
−/+ +/−
−/− −/−
−/− −/−
H10 H11
Adenosine Uridine
+ +
+/+ +/+
+/+ −/+
−/− −/−
−/− −/−
+ + +
+/+ +/+ +/+
−/+ +/+ −/−
−/− +/− −/−
−/− +/− −/−
H12
Adenosine-5′-Monophosphate
+
−/+
−/+
−/−
−/−
Acid
Acid Gycyl-L-Glutamic Acid
Acid
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
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H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
L-glutamic
acid) was completely inhibited, and four other substrates (gentiobiose, maltose, xylitol and adenosine-5′-monophosphate) were also partly inhibited; while treated at 0.01 μg ml−1 of kresoximmethyl, only one substrate (β-hydroxybutyric acid) was partly inhibited (Table 2). When treated at 0.1, 1 and 10 μg ml−1 of azoxystrobin, F. kyushuense metabolized 32, 15 and 12 substrates (Table 2), respectively; the substrates effectively utilized by the pathogen included tween-80, arbutin, dextrin, glycogen, β-hydroxybutyric acid and α-ketoglutaric acid (Table 2), while those utilized at much lower levels included Nacetyl-β-D-glucosamine, D-arabitol, D-cellobiose, D-glucosamine, glycerol and succinic acid. In comparison, when treated at 0.1, 1 and 10 μg ml−1 of kresoxim-methyl, F. kyushuense metabolized 60, 12 and 7 substrates (Table 2), respectively; the substrates effectively utilized by the fungus included tween-80, β-cyclodextrin, α- D -glucose, D -mannose and D -ribose
(Table 2), while those used at much lower levels included amygdalin, D -fructose, D -melezitose and sucrose (Table 2). When exposed to 0.01 μg ml − 1 of kresoxim-methyl and to 100 and 200 μg ml−1 of SHAM, the pathogen presented similar fingerprints as that of the water check. 4. Discussion 4.1. Activity of azoxystrobin and kresoxim-methyl against conidial germination and mycelial growth of F. kyushuense It is important to understand the activity of a chemical against the pathogen at various life cycle stages, especially for mycelial growth and conidial germination, and how it might affect pathogenicity and infection cycles of the pathogen. Azoxystrobin and kresoxim-methyl reduced not only mycelial growth of F. kyushuense, but also conidial germination. Similar findings have been previously reported for other Fusarium pathogens, including Fusarium oxysporum, Fusarium graminearum and Fusarium culmorum [11,28,29]. Less sensitivity to mycelial growth might be due to less ATP that was needed for the pathogen at mycelial growth stage, while more ATP was needed at the stage of conidial germination. Additionally, the induction of the alternative oxidase respiratory pathway at the cytochrome bc1 target site has been proposed as the likely reason for the low mycelial sensitivity to strobilurins displayed by several pathogens [30]. Other studies on biological characteristics have shown that F. kyushuense is capable of growth over a wide range of temperatures, relative humidity values, pH values and carbon resources [3]. The greenhouse environment during tobacco seedling growth favors the infection and epidemiology of this Fusarium disease especially because of the temperature and humidity [3], and control measures are needed. Azoxystrobin and kresoxim-methyl should be used in a preventative manner for management of Fusarium wilt of tobacco Fusarium since mycelial growth is less well inhibited, but spore germination is fully inhibited. This study tested the interaction of the pathogen with azoxystrobin and kresoxim-methyl in vitro, whereas disease management in the field normally encompasses the interaction of the pathogen, environment and fungicide, so further studies in the field are needed. 4.2. Metabolic effects of azoxystrobin and kresoxim-methyl against F. kyushuense examined using the Biolog FF MicroPlate Azoxystrobin and kresoxim-methyl are structurally a strobilurin also known by its mode of action as a Qo inhibitor (QoI). These inhibit mitochondrial respiration by binding to the Qo site (the outer, quinone oxidizing pocket) of the cytochrome bc1 enzyme complex. The inhibition results in energy deficiency due to a lack of adenosine triphosphate (ATP) by blocking the electron transfer process in the respiration pathway [18]. For respiration in fungi, carbon substrates are first utilized and degraded to pyruvic acid through glycolysis. Afterwards, pyruvic acid goes through the tricarboxylic acid cycle. In the absence of azoxystrobin
and kresoxim-methyl, F. kyushuense was able to metabolize 91.6% of the tested carbon sources; while under exposure to azoxystrobin and kresoxim-methyl during mycelial growth and conidial germination stages of the pathogen, more than 80% of the carbon substrates could not be utilized. Some of carbon sources are directly related to glycolysis or the tricarboxylic acid cycle stages, and others may be not directly related. Though the pathways of electron transport in F. kyushuense are up until now still unclear, our findings in this study presented that both azoxystrobin and kresoxim-methyl significantly inhibit the carbon substrate utilization during glycolysis and/or tricarboxylic acid cycle stages. Under various concentrations of azoxystrobin and kresoxim-methyl tested during mycelial growth up to 100 μg ml−1, F. kyushuense continued to show high levels of metabolism for seven and five carbon sources, respectively; while for conidial germination up to 10 μg ml−1, the pathogen metabolized eight and four sources effectively, respectively. Among these substrates in azoxystrobin tests, three (α-ketoglutaric acid, y-hydroxybutyric acid and β-hydroxybutyric acid) were metabolized in all three mycelial growth analyses (1, 10 and 100 μg ml−1 of azoxystrobin) and all three conidial germination analyses (0.1, 1 and 10 μg ml− 1 of azoxystrobin); while in kresoxim-methyl tests, three (tween 80, dextrin, α-D-glucose) were metabolized in all three mycelial growth analyses (1, 10 and 100 μg ml− 1 of kresoxim-methyl) and all three conidial germination analyses (0.1, 1 and 10 μg ml− 1 of kresoxim-methyl). The metabolic fingerprints of F. kyushuense under various concentrations of these two strobilurins were quite different. Glucose was not metabolized in the presence of azoxystrobin, while metabolized in the presence of kresoxim-methyl at 100 μg ml−1 in mycelial growth stage and at 10 μg ml− 1 in conidial germination stage. There should be some different metabolic effects between azoxystrobin and kresoxim-methyl. Additionally, there could be some differences in metabolism between mycelial growth and conidial germination for F. kyushuense. The substrates α-ketoglutaric acid, y-hydroxybutyric acid and β-hydroxybutyric acid might be used extensively by F. kyushuense for the tricarboxylic acid cycle in both mycelial growth and conidial germination stages; while the use of other substrates might differ during different growth stages. 5. Conclusions The current study has shown some metabolic effects of azoxystrobin, kresoxim-methyl and salicylhydroxamic acid on the mycelial growth and conidial germination of F. kyushuense, and also provided a carbon metabolic fingerprint of F. kyushuense that could be useful for identification. The outcome of this analysis has also shown some side effects of the strobilurin fungicides azoxystrobin and kresoxim-methyl. Acknowledgments This work was supported by the National Natural Science Foundation of China (31360448, 31501679), Guizhou Tobacco Company (201305, 201336, 201436), and Guizhou Science Technology Foundation ([2011]2337). We also thank the anonymous reviewers for critical reviews of the manuscript. References [1] British American Tobacco Annual Report, 2012 8. [2] H.C. Wang, M.S. Wang, H.Q. Xia, S.J. Yang, Y.S. Guo, D.Q. Xu, Y. Xiang, W.H. Li, S.H. Shang, J.X. Shi, First report of Fusarium wilt of tobacco caused by Fusarium kyushuense in China, Plant Dis. 97 (2013) 424. [3] H.C. Wang, J. Wang, W.H. Li, N. Lu, M.S. Wang, H.Q. Xia, Biological characteristics of Fusarium kyushuense causing tobacco seedling stem rot, Acta Tabacaria Sin. 20 (2014) 65–70. [4] Y. Xu, F. Luo, L.Z. Yang, Y.D. He, C.L. Xiao, X.Y. Kong, Toxicity of several fungicides to watermelon Fusarium wilt, Chin. J. Trop. Agric. 30 (2010) 18–26. [5] Z.F. Wang, J.P. Li, J.J. Li, N.N. Hui, T.W. Zhou, L. Wang, Y.Q. Ma, X.R. Zhang, Identification of root rot pathogen of glycyrrhiza and indoor toxicity test, Acta Agric. Bor.-Occid. Sin. 22 (8) (2013) 98–102.
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Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate Hancheng Wang a,1, Jin Wang b,1, Qingyuan Chen a,1, Maosheng Wang a, Tom Hsiang c, Shenghua Shang a, Zhihe Yu a,⁎ a b c
Key Laboratory of Molecular Genetics, Guizhou Academy of Tobacco Sciences, Guiyang 550081, PR China College of Life Science, Yangtze University, Jingzhou 434025, PR China School of Environmental Sciences, University of Guelph, Guelph, ON N1G2W1, Canada
a r t i c l e
i n f o
Article history: Received 30 September 2015 Received in revised form 16 November 2015 Accepted 28 November 2015 Available online xxxx Keywords: Azoxystrobin Biolog FF MicroPlate Kresoxim-methyl Mode of action Sensitivity
a b s t r a c t Azoxystrobin and kresoxim-methyl are strobilurin fungicides, and are effective in controlling many plant diseases, including Fusarium wilt. The mode of action of this kind of chemical is inhibition of respiration. This research investigated the sensitivities of Fusarium kyushuense to azoxystrobin and kresoxim-methyl, and to the alternative oxidase inhibitor salicylhydroxamic acid (SHAM). The Biolog FF MicroPlate is designed to examine substrate utilization and metabolic profiling of micro-organisms, and was used here to study the activity of azoxystrobin, kresoxim-methyl and SHAM against F. kyushuense. Results presented that azoxystrobin and kresoxim-methyl strongly inhibited conidial germination and mycelial growth of F. kyushuense, with EC50 values of 1.60 and 1.79 μg ml−1, and 6.25 and 11.43 μg ml−1, respectively; while not for SHAM. In the absence of fungicide, F. kyushuense was able to metabolize 91.6% of the tested carbon substrates, including 69 effectively and 18 moderately. SHAM did not inhibit carbon substrate utilization. Under the selective pressure of azoxystrobin and kresoximmethyl during mycelial growth (up to 100 μg ml−1) and conidial germination (up to 10 μg ml−1), F. kyushuense was unable to metabolize many substrates in the Biolog FF MicroPlate; while especially for carbon substrates in glycolysis and tricarboxylic acid cycle, with notable exceptions such as β-hydroxybutyric acid, y-hydroxybutyric acid, αketoglutaric acid, α-D-glucose-1-phosphate, D-saccharic acid and succinic acid in the mycelial growth stage, and βhydroxybutyric acid, y-hydroxybutyric acid, α-ketoglutaric acid, tween-80, arbutin, dextrin, glycerol and glycogen in the conidial germination stage. This is a new finding for some effect of azoxystrobin and kresoxim-methyl on carbon substrate utilization related to glycolysis and tricarboxylic acid cycle and other carbons, and may lead to future applications of Biolog FF MicroPlate for metabolic effects of other fungicides and other fungi, as well as providing a carbon metabolic fingerprint of F. kyushuense that could be useful for identification. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China is the biggest single tobacco market and accounts for more than 40% of the global tobacco consumption [1]. During tobacco seedling development period, various fungal pathogens attack seedlings in the greenhouse. In the last five years, Fusarium wilt of tobacco, caused by Fusarium kyushuense O'Donnell & T. Aoki, was frequently observed in some regions of Guizhou province in southwest China [2,3]. The pathogen attacks seedlings leading to symptoms of severe wilting, chlorosis and stunting with poorly developed root systems, and eventual death. Control of the disease is based on an integration of several cultural methods with the use of fungicides. For Fusarium diseases caused by Fusarium oxysporum f. sp. nicotianae in ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Yu). 1 Contribute equally to this article.
China, multisite inhibitors, such as chlorothalonil and mancozeb, and the target site-specific fungicides such as carbendazim have been used to control this disease [4–6]. Multisite inhibitors may be effective when applied in protective fashion [7]. Carbendazim was a major fungicide for Fusarium disease management twenty years ago, but carbendazim-resistant strains developed causing all benzimidazoles to lose their efficacy [8–10]. Recently, the strobilurin fungicide azoxystrobin and kresoxim-methyl have been registered in China and other countries to control Fusarium diseases, including wheat scab, potato Fusarium dry rot, and tomato Fusarium wilt [11–13]. They have also been used to manage many other disease management on tobacco in the USA and Germany [14–16]. Few studies have been published to describe the sensitivity to azoxystrobin and kresoxim-methyl by F. kyushuense, and because of the relatively high cost of these chemicals locally, they have not been used for tobacco disease management in China. Azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin and famoxadone [17–20] are Quinone outside inhibitor (QoI) fungicides (Fungicide Resistance Action Committee
http://dx.doi.org/10.1016/j.pestbp.2015.11.013 0048-3575/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
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H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
[FRAC] group 11) also known as strobilurins. Preliminary studies revealed that azoxystrobin and kresoxim-methyl inhibit mitochondrial respiration by blocking electron transfer at the cytochrome bc1 complex [21]. They have high activity against spore germination and less activity against mycelial growth because hyphae may make use of the terminal alternative oxidase (AOX) that bypasses the blockage site [22,23]. Salicylhydroxamic acid is the special inhibitor for the terminal alternative oxidase. The metabolic effects of azoxystrobin and kresoxim-methyl have not yet been fully elucidated, and more research is needed to understand the diverse secondary effects of strobilurins on fungal plant pathogens. Recently, the Biolog FF MicroPlate was introduced by Biolog Company for characterizing filamentous fungi (FF MicroPlate™ Instruction, Biolog, Hayward, CA, USA). The MicroPlate uses 95 biochemical tests to profile substrate utilization and phenotypic profiling of many microorganisms with each well containing substrates that change color with metabolic activity (Fig. 1) [24,25]. There have been few previous reports of its use for investigating mode of action of fungicides [26]. Therefore, the objectives of this study were: (i) to document the sensitivities of F. kyushuense to azoxystrobin, kresoxim-methyl and SHAM and (ii) to evaluate the phenotypic profiling of F. kyushuense under pressures of azoxystrobin, kresoxim-methyl and SHAM. The outcome of this study will provide useful information on the effects and mode of action of azoxystrobin and kresoxim-methyl. 2. Materials and methods 2.1. Pathogen, media and chemical preparation A strain of F. kyushuense with wild-type strobilurin sensitivity and pathogenicity to tobacco [2] was collected in 2013 from an infected
tobacco seedling in a commercial field in Guizhou province, China. A monoconidial isolate was obtained and used for tests. The isolate was grown and maintained on a lima bean agar medium (LBA, 60.0 g l−1 lima beans boiled for 1 h and strained, 16.0 g l−1 agar), in a controlled climate cabinet at 25 °C in the dark. For conidial production, agar plugs were removed from the edge of an actively growing culture and placed on LBA plates. After 7 days, conidia were washed off with distilled water, and filtered through a double-layer of sterile cheesecloth (Grade #40: 24 × 20 threads per inch) to remove mycelial fragments. The resulting conidial suspension was quantified with a hemacytometer and adjusted to 1 × 105 spores/ml for subsequent use. For long-term storage, 5-mm agar plugs from the leading edge of individual colonies were transferred into several sterile 1.5-ml microcentrifuge tubes containing 1 ml of 30% sterile glycerol, and tubes were stored at −20 °C in darkness. Stock fungicide solutions were prepared by dissolving technical grade azoxystrobin (a.i. 93%; Syngenta China Co., Ltd, Shanghai, China), kresoxim-methyl (a.i. 96%; BASF China Co., Ltd, Shanghai, China) and SHAM (a.i. 99.9%; Sigma-Aldrich China Co., Ltd, Shanghai, China) in methanol. Solutions were diluted as required and stored at 4 °C in the dark. The methanol concentration never exceeded 1% of the testing solution. This concentration of methanol was not observed to affect different life stages of F. kyushuense (data not shown). Controls always contained the same methanol concentration as the test samples in the experiments. Fungicides were added to LBA after autoclaving when the agar had cooled to approximately 50 °C. Filamentous Fungi inoculating fluid (FF-IF, catalog # 72106) (containing 2.5 g l−1 Phytagel and 0.3 g l−1 Tween 40) and FF MicroPlate test panels (catalog # 1006) containing 95 different carbon sources were purchased from Biolog Inc. (Hayward, CA, USA) and stored at 4 °C until needed.
Fig. 1. Layout of assays in the Biolog FF MicroPlate.
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
3
Individual agar discs (5-mm-diameter) were removed from the edge of an actively growing culture and placed face up on the centre of a Petri dish (9-cm-diameter) containing LBA amended with azoxystrobin, kresoxim-methyl and SHAM at various concentrations, respectively. The final concentrations tested with three replications were for azoxystrobin and kresoxim-methyl 0, 0.01, 0.1, 1, 10 and 100 μg ml−1; and for SHAM 0, 50, 100 and 200 μg ml−1, respectively. The diameters of the colonies were measured after incubation for 6 days at 25 °C in the dark. The experiment was conducted twice.
were harvested with sterile cotton-tipped applicators as described above, suspended in distilled water to obtain a final concentration of 1 × 105 spores/ml. The test FF-IF broth (16 ml in one tube) was prepared as mentioned above to get final test concentrations of 0, 0.01, 0.1, 1 and 10 μg ml−1 for azoxystrobin and kresoxim-methyl, and of 0, 100 and 200 μg ml− 1 for SHAM, respectively. Each FF-IF broth was added with 0.2 ml of conidial suspension to make the final test suspensions. One hundred microliters of each test suspension was added to each well of the FF plates. Afterwards, plates were incubated in the OmniLog at 28 °C for 7 days with readings taken every 15 min as mentioned above.
2.3. Inhibition of conidial germination
2.7. Data analysis
Conidia were harvested as described above from 7-day-old cultures. Aqueous preparations of azoxystrobin, kresoxim-methyl and SHAM at different concentrations were added to an equal volume (500 μl) of the conidial suspension, respectively. A control conidial suspension was diluted with an equal volume (500 μl) of water only. The final concentrations tested for azoxystrobin and kresoxim-methyl were 0, 0.01, 0.1, 1, 5, 10, 50 and 100 μg ml−1, and for SHAM were 0, 50, 100 and 200 μg ml−1, respectively. Conidial suspensions were then incubated at 25 °C in the dark for 12 h. A conidium was scored as germinated if the germ tube had reached at least the length of the conidium. Germinated conidia were quantified on microscope slides at three spots by assessing 100 conidia per spot. The experiment was conducted twice.
All data were processed with the SIGMASTAT Statistical Software Package (SPSS Science, Chicago). The concentration of fungicide causing 50% (EC50) or 90% (EC90) reduction in mycelial growth or germination of conidia compared to the absence of the fungicide was estimated from the fitted regression line of the log-transformed percentage inhibition plotted against the log-transformed fungicide concentration [21]. Data analysis for metabolic profiling of F. kyushuense was conducted using Kinetic and Parametric software (Biolog). Phenotypes were determined based on the area under the kinetic curve of dye formation [27].
2.2. Inhibition of mycelial growth
3. Results 3.1. Inhibition of mycelial growth
2.4. Metabolic profiling of F. kyushuense during mycelial growth exposed to azoxystrobin, kresoxim-methyl and SHAM For metabolic profiling of F. kyushuense during mycelial growth with exposure to azoxystrobin, kresoxim-methyl and SHAM, conidia were produced on LBA plates as described above and collected with sterile cotton-tipped applicators from the agar plates, avoiding carryover of nutrients from the agar medium. Conidia together with mycelia were then suspended in distilled water and incubated at 25 °C in darkness for 12 h to prepare mycelial suspensions. The FF-IF broth (16 ml broth in 20 ml tubes) were amended with various concentrations of azoxystrobin and kresoxim-methyl to obtain final test concentrations of 0, 0.01, 0.1, 1, 10 and 100 μg ml−1, and with various concentration of SHAM to get final test concentrations of 0, 100 and 200 μg ml−1, respectively. Afterwards, to each FF-IF broth tube 0.2 ml of mycelial suspension and mixed was added. One hundred microliters of each test suspension was then added to each well of the FF plates under aseptic conditions. Plates were then incubated in the OmniLog at 28 °C for 7 days with readings taken every 15 min. Incubation and recording of phenotypic data were performed in the OmniLog station by capturing digital images of the microarrays and storing turbidity values in a computer file displayed as a kinetic graph. 2.5. Metabolic comparisons when adjusting for mycelial mass A mycelial size (about 3 to 3.5 cm in diameter on Petri dishes) on the non-fungicidal plates (about 3 day incubation on LBA plates) had a similar size to day 7 on the azoxystrobin-amended or kresoxim-methylamended media at 1 and 10 μg ml−1. To adjust for the metabolic activities of a smaller mycelial mass with growth inhibition by azoxystrobin and kresoxim-methyl, Biolog readings for a mycelial mass of a similar were compared to the mycelium exposed to 1 and 10 μg ml− 1 of azoxystrobin and kresoxim-methyl, respectively. 2.6. Metabolic profiling of F. kyushuense during conidial germination under the pressure of azoxystrobin, kresoxim-methyl and SHAM For metabolic profiling during conidial germination with exposure to azoxystrobin, kresoxim-methyl and SHAM, conidia of F. kyushuense
Azoxystrobin and kresoxim-methyl inhibited mycelial growth of F. kyushuense. With increasing fungicide concentration, mycelial growth was more greatly inhibited. The EC50 values were 6.25 μg ml−1 and 11.43 μg ml− 1, respectively; and EC90 values were N100 μg ml−1 for each chemical. In comparison, SHAM had no activity against mycelial growth of the fungus even at the highest concentration of 200 μg ml−1. Additionally, mycelial growth of F. kyushuense was not be completely inhibited by azoxystrobin and kresoxim-methyl even at the highest test concentration of 100 μg ml−1 (data not shown). 3.2. Inhibition of conidial germination Azoxystrobin and kresoxim-methyl were very active against germination of conidia, with EC50 values of 1.60 μg ml−1 and 1.79 μg ml−1, respectively. The germination ratios decreased greatly with increasing concentrations of the compound (data not shown). In comparison, SHAM did not inhibit conidial germination of the fungus even at the highest concentration of 200 μg ml−1 (data not shown). Germination of conidia was completely inhibited by azoxystrobin and kresoximmethyl at 50 μg ml−1. In the absence of the compound, the percentage germination of conidia after 12 h at 25 °C was around 95%. 3.3. Metabolic profiling of F. kyushuense during mycelial growth under pressures of azoxystrobin, kresoxim-methyl and SHAM The metabolic abilities of F. kyushuense were tested by using the Biolog system. Using the Biolog FF MicroPlate, 95 different carbon sources were tested. In the absence of a fungicide, the pathogen was able to metabolize 91.6% of tested carbon sources (87/95 tested, Wells A1-H12). Sixty-nine compounds were effectively utilized by F. kyushuense; eighteen substrates were metabolized at a mid-level; while the other eight compounds were not utilized by F. kyushuense. When incubated with azoxystrobin and kresoxim-methyl at 0.01 μg ml−1, and with SHAM at 100 and 200 μg ml−1, F. kyushuense exhibited the same metabolic profiling as that of control during mycelial growth stage; when incubated at 0.1 μg ml−1 (well below the EC50 of 6.25 μg ml−1) only the utilization of succinic acid mono-methyl ester (Well G5) was inhibited by azoxystrobin; when treated at 1, 10 and
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
4
H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
Table 1 Metabolic profiling of Fusarium kyushuense during mycelial growth under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.1
1
10
100
Well
Substrate
0
0.1
1
10
100
A2 A4 A7 A9
Tween 80 N-Acetyl-β-D-Glucosamine Amygdalin
+/+ +/+ +/+ +/+
+/+ −/+ −/+ −/+
+/+ −/− −/+ −/−
−/+ −/− −/− −/−
C10 C11 C12 D1
Maltitol Maltose Maltotriose
L-Arabinose
+ + + +
D-Mannitol
+ + + +
+/+ +/+ +/+ +/+
−/− −/− −/− −/+
−/− −/− −/− −/−
−/− −/− −/− −/−
A10
D-Arabitol
+
+/+
−/−
−/−
−/−
D2
D-Mannose
+
+/+
+/+
−/−
−/−
A11
Arbutin
+
+/+
−/−
−/−
−/−
D3
D-Melezitose
+
+/+
+/+
−/−
−/+
A12
D-Cellobiose
+
+/+
+/+
−/+
−/+
D4
D-Melibiose
+
+/+
−/+
−/−
−/−
B3 B5
Dextrin D-Fructose
+ +
+/+ +/+
+/+ −/+
+/+ −/+
+/+ −/+
D5 D8
α-Methyl-D-Galactoside ß-Methyl-D-Glucoside
+ +
+/+ +/+
−/− −/−
−/− −/−
−/− −/−
B7
D-Galactose
+
+/+
−/−
−/−
−/−
D9
Palatinose
+
+/+
−/−
−/−
−/−
B9
Gentiobiose
+
+/+
−/+
−/+
−/+
D10
D-Psicose
+
+/+
−/−
−/−
−/−
B10
D-Gluconic
+
+/+
+/−
−/−
−/−
D11
D-Raffinose
+
+/+
−/−
−/−
−/−
B11
D-Glucosamine
+
+/+
−/+
−/+
−/−
D12
L-Rhamnose
+
+/+
−/−
−/−
−/−
B12
α-D-Glucose
+
+/+
−/+
−/+
−/+
E1
D-Ribose
+
+/−
−/+
−/−
−/−
C1
α-D-Glucose-1 -Phosphate
+
+/−
+/−
+/−
+/−
E2
Salicin
+
+/−
+/−
+/−
+/−
Acid
C3
D-Glucuronic
+
+/+
−/−
−/−
−/−
E3
Sedoheptulosan
+
+/+
−/−
−/−
−/−
C4
Glycerol
+
+/+
+/−
+/−
+/−
E4
D-Sorbitol
+
+/+
+/+
−/−
−/−
C5
Glycogen
+
+/+
+/−
+/−
+/−
E5
L-Sorbose
+
+/−
−/−
−/−
−/−
C6 C7 C8
m-Inositol 2-Keto-D-Gluconic Acid α-D-Lactose
+ + +
+/+ +/+ −/+
−/+ −/− −/−
−/− −/− −/−
−/− −/− −/−
E6 E7 E8
Stachyose Sucrose
+ + +
+/+ +/+ +/+
−/− −/+ −/−
−/− +/+ −/−
−/− −/+ −/−
Acid
D-Tagatose
‘+’ and ‘−’ mean that the substrate was metabolized and not metabolized by the pathogen when incubated under pressures of azoxystrobin and kresoxim-methyl examined using the Biolog FF MicroPlate, respectively.
100 μg ml− 1, the number of carbon substances metabolized by the pathogen significantly decreased, with 21, 17 and 17 substrates utilized (Table 1), respectively. Moreover, when incubated with concentrations of 1, 10 and 100 μg ml−1 azoxystrobin, F. kyushuense could still metabolize 9 substrates at high levels, including α-D-glucose-1-phosphate (Well C1) and β-hydroxybutyric acid (Well F4) (Table 1); there were five (tween-80, dextrin, glycerol, glycogen, and adenosine), two (tween-80 and dextrin) and three (glycerol, D-lactic acid methyl ester, and D-malic acid) substrates metabolized at mid-levels for each treatment, respectively; moreover, three substrates (fumaric acid, L-alanine and L-alanyl-glycine), four chemicals (sucrose, D-lactic acid methyl ester, L-alanine and L-alanyl-glycine) and one substrate (salicin) were
delayed metabolized by F. kyushuense, respectively. When incubated at 0.1, 1, 10 and 100 μg ml−1 of kresoxim-methyl, the number of carbon substances metabolized by the pathogen significantly decreased as well, with 63, 22, 9 and 8 substrates utilized (Table 1), respectively. Moreover, when incubated with concentrations of 1, 10 and 100 μg ml− 1 kresoxim-methyl, F. kyushuense could still metabolize 5 substrates at high levels, including tween 80 (Well A2), D-cellobiose (Well A12), dextrin (Well B3), D-fructose (Well B5) and α-D-glucose (Well B12); while five other common carbon substrates were metabolized at mid-levels or delayed metabolized by the pathogen, including gentiobiose (Well B9), D-mannose (Well D2), D-melezitose (Well D3), D-melibiose (Well D4) and sucrose (Well E7) (Table 1).
Table 2 Metabolic profiling of Fusarium kyushuense during conidial germination under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.01
0.1
1
10
Well
Substrate
0
0.01
0.1
1
10
A2 A4 A6 A7 A8
Tween 80 N-Acetyl-β-D-Glucosamine Adonitol Amygdalin
+/+ +/+ +/+ +/+ +/+
+/+ −/+ −/+ +/+ −/−
+/+ −/− +/− +/+ −/−
+/+ −/− −/− −/+ −/−
C7 C10 C11 C12 D1
2-Keto-D-Gluconic Acid Maltitol Maltose Maltotriose
D-Arabinose
+ + + + +
D-Mannitol
+ + + + +
+/+ +/+ −/+ −/+ +/+
−/+ −/+ +/+ −/+ +/+
−/− −/− −/− −/− −/−
−/− −/− −/− −/− −/−
A9
L-Arabinose
+
+/+
−/−
−/−
−/−
D2
D-Mannose
+
+/+
+/+
+/+
−/+
A10
D-Arabitol
+
+/+
+/+
−/−
+/−
D3
D-Melezitose
+
+/+
+/+
−/+
−/−
A11
Arbutin
+
+/+
+/+
+/−
+/−
D4
D-Melibiose
+
+/+
+/+
−/+
−/−
A12
D-Cellobiose
+
+/+
−/+
−/+
+/−
D5
α-Methyl-D-Galactoside
+
+/+
−/+
−/−
−/−
B3 B5
Dextrin D-Fructose
+ +
+/+ +/+
+/+ +/+
+/+ −/+
+/+ −/+
D8 D9
ß-Methyl-D-Glucoside Palatinose
+ +
+/+ +/+
−/+ −/+
−/− −/−
−/− −/−
B7
D-Galactose
+
+/+
−/+
−/−
−/−
D10
D-Psicose
+
+/+
−/+
−/−
−/−
B9
Gentiobiose
+
−/+
−/+
+/+
−/−
D11
D-Raffinose
+
+/+
+/+
−/−
−/−
B10
D-Gluconic
+
+/+
+/+
−/−
−/−
D12
L-Rhamnose
+
+/+
+/+
−/−
−/−
B11
D-Glucosamine
+
+/+
+/+
+/+
+/−
E1
D-Ribose
+
+/+
−/−
−/−
−/−
B12 C1
α-D-Glucose α-D-Glucose-1 -Phosphate
+ +
+/+ +/+
−/+ +/−
−/+ −/−
−/+ −/−
E2 E3
Salicin Sedoheptulosan
+ +
+/+ +/+
−/+ −/+
−/− −/−
−/− −/−
D-Glucuronic Acid Glycerol
+
+/+
+/+
+/−
−/−
E4
D-Sorbitol
+
+/+
+/+
−/−
−/−
C4
+
+/+
+/+
+/−
+/−
E5
L-Sorbose
+
+/+
−/−
−/−
−/−
C5 C6
Glycogen m-Inositol
+ +
+/+ +/+
+/+ −/+
+/− −/−
+/− −/−
E6 E7
Stachyose Sucrose
+ +
+/+ +/+
−/+ −/+
−/− −/+
−/− −/+
C3
Acid
‘+’ and ‘−’ mean that the substrate was metabolized and not metabolized by the pathogen when incubated under pressures of azoxystrobin and kresoxim-methyl examined using the Biolog FF MicroPlate, respectively.
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
5
Table 1 Metabolic profiling of Fusarium kyushuense during mycelial growth under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.1
1
10
100
Well
Substrate
0
0.1
1
10
100
E9
D
+
+/+
−/+
−/−
−/−
G7
L-Alaninamide
+
+/−
−/−
−/−
−/−
E10
Turanose
+
+/+
−/−
−/−
−/−
G8
L-Alanine
+
+/−
+/−
+/−
+/−
E11 E12
Xylitol
+/+ +/+
−/− −/−
−/− −/−
−/− −/−
G9 G10
L-Alanyl-Glycine
D-Xylose
+ +
L-Asparagine
+ +
+/− +/+
+/− −/−
+/− −/−
+/− −/−
F1
y-Aminobutyric Acid
+
+/+
−/−
−/−
−/−
G11
L-Aspartic
+
+/+
−/−
−/−
−/−
F2
Bromosuccinic Acid
+
+/+
−/−
−/−
−/−
G12
L-Glutamic
+
+/+
−/−
−/−
−/−
F3 F4
Fumaric Acid ß-Hydroxybutyric Acid
+ +
+/+ +/+
+/+ +/−
−/− +/−
−/− +/−
H1 H2
L-Ornithine
+ +
+/− +/−
−/− −/−
−/− −/−
−/− −/−
F5
y- Hydroxybutyric Acid
+
+/−
+/−
+/−
+/−
H3
L-Phenylalanine
+
+/−
+/−
+/−
+/−
F6
p-Hydroxy-phenylacetic Acid
+
+/−
−/−
−/−
−/−
H4
L-Proline
+
+/+
−/−
−/−
−/−
F7
α-Ketoglutaric Acid
+
+/−
+/−
+/−
+/−
H5
L-Pyroglutamic
+
+/+
−/−
−/−
−/−
F8
D-Lactic
Acid Methyl Ester
+
+/+
−/−
+/−
+/−
H6
L-Serine
+
+/−
−/−
−/−
−/−
F9
L-Lactic
Acid
+
+/+
−/−
−/−
−/−
H7
L-Threonine
+
+/−
−/−
−/−
−/−
F10
D-Malic
Acid
+
+/+
−/−
−/−
+/−
H8
2-Aminoethanol
+
+/−
−/−
−/−
−/−
F11
L-Malic
Acid
+
+/+
−/−
−/−
+/−
H9
Putrescine
+
+/+
−/−
−/−
−/−
F12 G1
D-Saccharic
+ +
+/+ +/−
−/+ +/−
−/− +/−
−/− +/−
H10 H11
Adenosine Uridine
+ +
+/+ +/+
+/− −/−
+/− −/−
+/− −/−
G3
Succinamic Acid
+
+/+
−/−
−/−
−/−
H12
Adenosine-5′-Monophosphate
+
+/+
−/−
−/−
−/−
G4 G5 G6
Succinic Acid Succinic Acid Mono-Methyl Ester N-Acetyl-L-Glutamic Acid
+ + +
+/+ −/− +/+
+/+ −/− −/−
+/− −/− −/−
+/− −/− −/−
-Trehalose
Quinic Acid Acid
Acid
Acid Gycyl-L-Glutamic Acid
Acid
(Well D1), D-mannose (Well D2), D-melezitose (Well D3), D-melibiose (Well D4) was also delayed.
3.4. Metabolic comparisons when adjusting for mycelial mass When 3-day-old cultures on non-fungicidal treatments were compared to 7-day-old cultures on fungicidal treatments (1 and 10 μg ml−1), the mycelial mass on Petri dishes was similar. Metabolic comparisons showed that, under apressure of 1 μg ml−1 azoxystrobin, the utilization of fumaric acid (Well F3), L-alanine (Well G8) and L-alanyl-glycine (Well G9) was slower to start than that of water treatment; under a pressure of 10 μg ml−1 azoxystrobin, the metabolization of sucrose (Well E7), D-lactic acid methyl Ester (Well F8), L-alanine (Well G8) and L-alanyl-glycine (Well G9) was delayed as well; under a pressure of 10 μg ml− 1 kresoxim-methyl, the utilization of D-mannitol
3.5. Metabolic profiling of F. kyushuense during the conidial germination stage with exposure to azoxystrobin, kresoxim-methyl and SHAM When exposed to azoxystrobin and kresoxim-methyl during conidial germination, F. kyushuense presented different metabolic profiles than that of the water check. With increasing azoxystrobin and kresoxim-methyl, less substrate was metabolized. When treated at 0.01 μg ml− 1 of azoxystrobin, the utilization of five substrates (maltotriose, D-lactic acid methyl ester, quinic acid, L-asparagine and
Table 2 Metabolic profiling of Fusarium kyushuense during conidial germination under various pressures of azoxystrobin/kresoxim-methyl (μg ml−1). Well
Substrate
0
0.01
0.1
1
10
E8
D-Tagatose
+
+/+
−/+
−/−
−/−
E9
D-Trehalose Turanose
+
+/+
−/+
−/−
E10
+
+/+
−/+
−/−
E11
Xylitol
+
−/+
+/+
−/−
−/−
E12
D-Xylose y-Aminobutyric Acid
+
+/+
−/−
−/−
−/−
F1
+
+/+
−/+
−/−
−/−
F2
Bromosuccinic Acid
+
+/+
−/+
−/−
−/−
G12
F3 F4
Fumaric Acid ß-Hydroxybutyric Acid
+ +
+/+ +/−
+/+ +/−
−/− +/−
−/− +/−
F5
y- Hydroxybutyric Acid
+
+/+
+/−
+/−
F6
p-Hydroxy-phenylacetic Acid
+
+/+
−/−
−/−
F7
α-Ketoglutaric Acid
+
+/+
+/−
F8
D-Lactic
Acid Methyl Ester
+
−/+
F9
L-Lactic
Acid
+
+/+
F10
D-Malic
Acid
+
F11
L-Malic
Acid Quinic Acid
D-Saccharic
F12 G1 G3 G4 G5
Acid Succinamic Acid Succinic Acid Succinic Acid Mono-Methyl Ester
Well
Substrate
0
0.01
0.1
1
10
G6
N-Acetyl-L-Glutamic Acid
+
+/+
−/+
−/−
−/−
−/−
G7
L-Alaninamide
+
+/+
−/−
−/−
−/−
−/−
G8
L-Alanine
+
+/+
−/+
−/−
−/−
G9
L-Alanyl-Glycine
+
+/+
−/−
−/−
−/−
G10
L-Asparagine
+
−/+
−/+
−/−
−/−
G11
L-Aspartic
+
+/+
−/+
−/−
−/−
L-Glutamic
+
−/+
−/+
−/−
−/−
H1 H2
L-Ornithine
+ +
+/+ +/+
−/− +/−
−/− −/−
−/− −/−
+/−
H3
L-Phenylalanine
+
+/+
+/−
−/−
−/−
−/−
H4
L-Proline
+
+/+
−/+
−/−
−/−
+/−
+/−
H5
L-Pyroglutamic
+
+/+
−/−
−/−
−/−
−/+
−/−
−/−
H6
L-Serine
+
+/+
−/−
−/−
−/−
−/+
−/−
−/−
H7
L-Threonine
+
+/+
−/−
−/−
−/−
+/+
+/+
−/−
−/−
H8
2-Aminoethanol
+
+/+
−/−
−/−
−/−
+
+/+
+/+
−/−
−/−
H9
Putrescine
+
+/+
−/−
−/−
−/−
+ +
−/+ +/+
−/+ +/−
−/− −/−
−/− −/−
H10 H11
Adenosine Uridine
+ +
+/+ +/+
+/+ −/+
−/− −/−
−/− −/−
+ + +
+/+ +/+ +/+
−/+ +/+ −/−
−/− +/− −/−
−/− +/− −/−
H12
Adenosine-5′-Monophosphate
+
−/+
−/+
−/−
−/−
Acid
Acid Gycyl-L-Glutamic Acid
Acid
Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013
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H. Wang et al. / Pesticide Biochemistry and Physiology xxx (2015) xxx–xxx
L-glutamic
acid) was completely inhibited, and four other substrates (gentiobiose, maltose, xylitol and adenosine-5′-monophosphate) were also partly inhibited; while treated at 0.01 μg ml−1 of kresoximmethyl, only one substrate (β-hydroxybutyric acid) was partly inhibited (Table 2). When treated at 0.1, 1 and 10 μg ml−1 of azoxystrobin, F. kyushuense metabolized 32, 15 and 12 substrates (Table 2), respectively; the substrates effectively utilized by the pathogen included tween-80, arbutin, dextrin, glycogen, β-hydroxybutyric acid and α-ketoglutaric acid (Table 2), while those utilized at much lower levels included Nacetyl-β-D-glucosamine, D-arabitol, D-cellobiose, D-glucosamine, glycerol and succinic acid. In comparison, when treated at 0.1, 1 and 10 μg ml−1 of kresoxim-methyl, F. kyushuense metabolized 60, 12 and 7 substrates (Table 2), respectively; the substrates effectively utilized by the fungus included tween-80, β-cyclodextrin, α- D -glucose, D -mannose and D -ribose
(Table 2), while those used at much lower levels included amygdalin, D -fructose, D -melezitose and sucrose (Table 2). When exposed to 0.01 μg ml − 1 of kresoxim-methyl and to 100 and 200 μg ml−1 of SHAM, the pathogen presented similar fingerprints as that of the water check. 4. Discussion 4.1. Activity of azoxystrobin and kresoxim-methyl against conidial germination and mycelial growth of F. kyushuense It is important to understand the activity of a chemical against the pathogen at various life cycle stages, especially for mycelial growth and conidial germination, and how it might affect pathogenicity and infection cycles of the pathogen. Azoxystrobin and kresoxim-methyl reduced not only mycelial growth of F. kyushuense, but also conidial germination. Similar findings have been previously reported for other Fusarium pathogens, including Fusarium oxysporum, Fusarium graminearum and Fusarium culmorum [11,28,29]. Less sensitivity to mycelial growth might be due to less ATP that was needed for the pathogen at mycelial growth stage, while more ATP was needed at the stage of conidial germination. Additionally, the induction of the alternative oxidase respiratory pathway at the cytochrome bc1 target site has been proposed as the likely reason for the low mycelial sensitivity to strobilurins displayed by several pathogens [30]. Other studies on biological characteristics have shown that F. kyushuense is capable of growth over a wide range of temperatures, relative humidity values, pH values and carbon resources [3]. The greenhouse environment during tobacco seedling growth favors the infection and epidemiology of this Fusarium disease especially because of the temperature and humidity [3], and control measures are needed. Azoxystrobin and kresoxim-methyl should be used in a preventative manner for management of Fusarium wilt of tobacco Fusarium since mycelial growth is less well inhibited, but spore germination is fully inhibited. This study tested the interaction of the pathogen with azoxystrobin and kresoxim-methyl in vitro, whereas disease management in the field normally encompasses the interaction of the pathogen, environment and fungicide, so further studies in the field are needed. 4.2. Metabolic effects of azoxystrobin and kresoxim-methyl against F. kyushuense examined using the Biolog FF MicroPlate Azoxystrobin and kresoxim-methyl are structurally a strobilurin also known by its mode of action as a Qo inhibitor (QoI). These inhibit mitochondrial respiration by binding to the Qo site (the outer, quinone oxidizing pocket) of the cytochrome bc1 enzyme complex. The inhibition results in energy deficiency due to a lack of adenosine triphosphate (ATP) by blocking the electron transfer process in the respiration pathway [18]. For respiration in fungi, carbon substrates are first utilized and degraded to pyruvic acid through glycolysis. Afterwards, pyruvic acid goes through the tricarboxylic acid cycle. In the absence of azoxystrobin
and kresoxim-methyl, F. kyushuense was able to metabolize 91.6% of the tested carbon sources; while under exposure to azoxystrobin and kresoxim-methyl during mycelial growth and conidial germination stages of the pathogen, more than 80% of the carbon substrates could not be utilized. Some of carbon sources are directly related to glycolysis or the tricarboxylic acid cycle stages, and others may be not directly related. Though the pathways of electron transport in F. kyushuense are up until now still unclear, our findings in this study presented that both azoxystrobin and kresoxim-methyl significantly inhibit the carbon substrate utilization during glycolysis and/or tricarboxylic acid cycle stages. Under various concentrations of azoxystrobin and kresoxim-methyl tested during mycelial growth up to 100 μg ml−1, F. kyushuense continued to show high levels of metabolism for seven and five carbon sources, respectively; while for conidial germination up to 10 μg ml−1, the pathogen metabolized eight and four sources effectively, respectively. Among these substrates in azoxystrobin tests, three (α-ketoglutaric acid, y-hydroxybutyric acid and β-hydroxybutyric acid) were metabolized in all three mycelial growth analyses (1, 10 and 100 μg ml−1 of azoxystrobin) and all three conidial germination analyses (0.1, 1 and 10 μg ml− 1 of azoxystrobin); while in kresoxim-methyl tests, three (tween 80, dextrin, α-D-glucose) were metabolized in all three mycelial growth analyses (1, 10 and 100 μg ml− 1 of kresoxim-methyl) and all three conidial germination analyses (0.1, 1 and 10 μg ml− 1 of kresoxim-methyl). The metabolic fingerprints of F. kyushuense under various concentrations of these two strobilurins were quite different. Glucose was not metabolized in the presence of azoxystrobin, while metabolized in the presence of kresoxim-methyl at 100 μg ml−1 in mycelial growth stage and at 10 μg ml− 1 in conidial germination stage. There should be some different metabolic effects between azoxystrobin and kresoxim-methyl. Additionally, there could be some differences in metabolism between mycelial growth and conidial germination for F. kyushuense. The substrates α-ketoglutaric acid, y-hydroxybutyric acid and β-hydroxybutyric acid might be used extensively by F. kyushuense for the tricarboxylic acid cycle in both mycelial growth and conidial germination stages; while the use of other substrates might differ during different growth stages. 5. Conclusions The current study has shown some metabolic effects of azoxystrobin, kresoxim-methyl and salicylhydroxamic acid on the mycelial growth and conidial germination of F. kyushuense, and also provided a carbon metabolic fingerprint of F. kyushuense that could be useful for identification. The outcome of this analysis has also shown some side effects of the strobilurin fungicides azoxystrobin and kresoxim-methyl. Acknowledgments This work was supported by the National Natural Science Foundation of China (31360448, 31501679), Guizhou Tobacco Company (201305, 201336, 201436), and Guizhou Science Technology Foundation ([2011]2337). We also thank the anonymous reviewers for critical reviews of the manuscript. References [1] British American Tobacco Annual Report, 2012 8. [2] H.C. Wang, M.S. Wang, H.Q. Xia, S.J. Yang, Y.S. Guo, D.Q. Xu, Y. Xiang, W.H. Li, S.H. Shang, J.X. Shi, First report of Fusarium wilt of tobacco caused by Fusarium kyushuense in China, Plant Dis. 97 (2013) 424. [3] H.C. Wang, J. Wang, W.H. Li, N. Lu, M.S. Wang, H.Q. Xia, Biological characteristics of Fusarium kyushuense causing tobacco seedling stem rot, Acta Tabacaria Sin. 20 (2014) 65–70. [4] Y. Xu, F. Luo, L.Z. Yang, Y.D. He, C.L. Xiao, X.Y. Kong, Toxicity of several fungicides to watermelon Fusarium wilt, Chin. J. Trop. Agric. 30 (2010) 18–26. [5] Z.F. Wang, J.P. Li, J.J. Li, N.N. Hui, T.W. Zhou, L. Wang, Y.Q. Ma, X.R. Zhang, Identification of root rot pathogen of glycyrrhiza and indoor toxicity test, Acta Agric. Bor.-Occid. Sin. 22 (8) (2013) 98–102.
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Please cite this article as: H. Wang, et al., Metabolic effects of azoxystrobin and kresoxim-methyl against Fusarium kyushuense examined using the Biolog FF MicroPlate, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/10.1016/j.pestbp.2015.11.013