Article pubs.acs.org/JAFC
Enhanced Dissipation of Triazole and Multiclass Pesticide Residues on Grapes after Foliar Application of Grapevine-Associated Bacillus Species Varsha P. Salunkhe,†,§ Indu S. Sawant,*,† Kaushik Banerjee,† Pallavi N. Wadkar,† and Sanjay D. Sawant† †
ICAR−National Research Centre for Grapes, P.O. Manjri Farm, Pune 412 307, Maharashtra, India Department of Agrochemicals and Pest Management, Shivaji University, Kolhapur 416 004, India
§
ABSTRACT: Disease management in vineyards with fungicides sometimes results in undesirable residue accumulations in grapes at harvest. Bioaugmentation of the grape fructosphere can be a useful approach for enhancing the degradation rate and reducing the residues to safe levels. This paper reports the in vitro and in vivo biodegradation of three triazole fungicides commonly used in Indian vineyards, by Bacillus strains, namely, DR-39, CS-126, TL-171, and TS-204, which were earlier found to enhance the dissipation rate of profenophos and carbendazim. The strains utilized the triazoles as carbon source and enhanced their in vitro rate of degradation. Myclobutanil, tetraconazole, and flusilazole were applied in separate vineyard plots at field doses of 0.40 g L−1, 0.75 mL L−1, and 0.125 mL L−1, respectively. Residue analysis of field samples from the treated fields reflected 87.38 and >99% degradations of myclobutanil and tetraconazole, respectively, by the strain DR-39, and 90.82% degradation of flusilazole by the strain CS-126 after 15−20 days of treatment. In the respective controls, the corresponding percent degradations were 72.07, 58.88, and 54.28, respectively. These Bacillus strains could also simultaneously degrade the residues of profenofos, carbendazim, and tetraconazole on the grape berries and can be useful in multiclass pesticide residue biodegradation. KEYWORDS: Vitis vinifera, myclobutanil, tetraconazole, flusilazole, biodegradation, Bacillus, carbendazim, profenofos
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INTRODUCTION Powdery mildew, caused by the biotrophic fungus Erysiphe necator (Schw.) Burr (earlier Uncinula necator), is one of the most important fungal diseases of grapevines. In table grape vineyards located in Maharashtra state, India, powdery mildew infections can be seen almost throughout the year, except during the hot and dry months of summer season and for a brief period during the cold winter months.1 Thompson Seedless and other commercial table grape varieties grown in this region are highly susceptible to powdery mildew. In the tropical climate of Maharashtra, the shoots can grow unceasingly without fruiting. To curb vegetative growth and induce fruitfulness, the grapevines are pruned twice a year in the months of April and October, apart from other interventions. From April to September the vines are allowed to grow vegetatively, whereas the crop is taken on the shoots emerging after October pruning. The young clusters can be infected before the fruit set. Uncontrolled infections may subside during winter but suddenly erupt as the temperature rises after the short winter. Losses due to reduced yield and fruit quality can be very severe due to the favorable climatic conditions during the fruiting season. Furthermore, the inner canopy in the common extended “Y” trellis system often does not get proper fungicide coverage and the pathogen perpetuates in the inner foliage, providing a continuous source of inoculum for initiation of new infections on the growing shoot. Powdery mildew management, thus, is very challenging and constrains the grape growers to apply the recommended fungicides at frequent intervals to achieve the desired level of disease control. Myclobutanil [2-p-chlorophenyl-2-(1H-1,2,4,© 2015 American Chemical Society
triazole-1-ylmethyl)hexanenitrile], tetraconazole [(RS)-2-(2,4dichlorophenyl)-3- (1H-1,2,4-triazol-1-yl)propyl 1,1,2,2-tetrafluoroethyl ether], and flusilazole [bis(4-fluorophenyl) (methyl)(1H-1,2,4-triazol-1-ylmethyl)] are relatively broad spectrum systemic fungicides, belonging to the triazole family of chemicals, and are commonly used for the control of powdery mildew in vineyards. When attempting the chemical control measure, the biggest apprehension a grower faces is the possibility of accumulation of fungicide residues at harvest above the maximum residue limits (MRLs), set by the regulatory government agencies to guarantee consumer safety. To facilitate international trade, the European Union has set the MRLs of myclobutanil (1.0 mg/ kg), tetraconazole (0.5 mg/kg), and flusilazole (0.05 mg/kg) for table grapes. Triazoles are known to be fairly soluble in water, although they are not readily degradable, and have a limited sorption tendency. To ensure that the terminal residues are not higher than the specified MRLs, it is recommended that not more than two applications of myclobutanil and tetraconazole and only a single application of flusilazole are made in a growing season.2 Furthermore, the minimum waiting periods after application of the fungicide until harvest of grapes (preharvest interval, PHI) are specified as 30 days for myclobutanil and tetraconazole and 60 days for flusilazole. In real-life situations, it is sometimes seen that residues of triazole fungicides are detected above the specified MRLs in grapes at Received: Revised: Accepted: Published: 10736
May 22, October October October
2015 19, 2015 22, 2015 22, 2015 DOI: 10.1021/acs.jafc.5b03429 J. Agric. Food Chem. 2015, 63, 10736−10746
Article
Journal of Agricultural and Food Chemistry optimum physiological maturity stage.3 To comply with the specified MRLs, the harvesting is often delayed beyond the optimum physiological maturity until the produce is considered safe, which in turn incurs financial loss to the growers. Whereas the persistence of any pesticide in the environment is determined by its own overall chemical stability and physical characteristics, it is also affected by the physical, chemical, and biological characteristics of the environment4 and can be exploited for enhanced degradation. Among these, the biodegradation of pesticides by micro-organisms is being increasingly appreciated as an important,5 safer, and efficient technique for removal of the residues from any contaminated site. 6 Naturally occurring bacterial isolates capable of metabolizing organic compounds have received considerable attention as they provide the possibility of both environmentally friendly and in situ detoxification. Various studies have shown that a diverse group of micro-organisms can break down different classes of pesticides by enzymatic action and utilize the pesticide molecules as the source of carbon for their growth.6 It is reported that a wide range of micro-organisms under different environmental conditions are capable of degrading triazole group of fungicides,7−10 which are systemic in nature but are mainly accumulated in the peel.11 The residue accumulations on the surface of fruits make them amenable to biodegradation. Furthermore, the role of naturally occurring micro-organisms in reducing the incidences of plant diseases is well-known. Among many species of antagonistic bacteria, the members of Bacillus especially have gained favor as they enjoy a “generally regarded as safe” (GRAS) status granted by U.S. Department of Agriculture12 and are formulated, registered, and marketed as biopesticides for the control of a number of diseases on various crops.13 A patented formulation of Bacillus subtilis strain QST 713, marketed as Serenade, is also recommended for the control of powdery mildew and other diseases in grapes. These formulations are considered safe and have 0 day waiting periods (PHI).14 Many of these biocontrol Bacillus species, namely, B. subtilis, Bacillus amyloliquefaciens, and Bacillus pumilus, were shown to degrade different pesticides.15−17 The ability of Bacillus species in controlling plant diseases and degrading pesticide residues offers exciting possibilities of identifying a novel strain that can be applied in vineyards, near the end of the growing season, with dual benefit of protection of the grape bunches from powdery mildew disease and for the breakdown of any fungicide residues present on the berries. In an earlier study, we had seen that some biocontrol strains of Bacillus spp., isolated from grape ecosystem, are able to enhance the degradation of the organophosphate insecticide, profenofos, and the benzimidazole fungicide, carbendazim,18,19 offering exciting possibilities of in situ detoxification of multiclass residues. This study investigates the capabilities of these Bacillus strains for degradation of the residues of three triazole fungicides in vitro and in vivo on grape berries.
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myclobutanil 10% WP (Systhane, Dow Agroscience), tetraconazole 3.8% EV (Domark, Isagro Asia), carbendazim 50% WP (Bavistin, BASF India Ltd., Mumbai), and profenofos 50% EC (Curacron, Syngenta India Ltd., Mumbai, India) were used for the degradation studies. The certified reference standards of flusilazole, myclobutanil, tetraconazole, carbendazim, and profenofos, were purchased from Dr. Ehrenstorfer GmbH, Augsburg, Germany. Experiments were conducted in the liquid culture and on grape berries. For liquid culture studies, nutrient broth (M001) was obtained from Hi Media (Mumbai, India), and the medium was prepared as per the instructions provided by the manufacturer. The degradation study of triazole fungicides on grape berries was conducted on Vitis vinifera cv. Thompson Seedless in a 15-year-old vineyard of the Indian Council of Agricultural Research (ICAR)−National Research Centre for Grapes, Pune (latitude 18.31 N, longitude 73.55 E). The vineyard was planted at vine-to-vine spacing of 6 ft and row-to-row spacing of 10 ft with extended ‘Y’ canopy architecture. The vines were free from any physiological disorders, diseases, and infestations of insect pests. Bacillus Strains, Media, and Inoculum Preparation. The studies were done by using four Bacillus strains, DR-39, CS-126, TL171, and TS-204, obtained from the culture collection of this Centre. For studies with flusilazole and the pesticide mixture (multiresidue degradation), an additional strain, DR-38, was also included. The media used were nutrient agar (NA, M002) and nutrient broth (NB, M001) procured from Himedia, Mumbai, India, prepared as per the specifications. All of the cultures were preserved at 4 °C on NA plate until required. Inoculum was prepared by transferring a loop full of culture from a 24-h-old colony on NA plate to 100 mL of sterilized NB in an Erlenmeyer flask. The flasks were incubated on an orbital shaker at 28 °C and 150 rpm for 72 h. The cells were harvested by centrifugation at 1118g for 5 min, washed twice with sterile distilled water, and then resuspended in sterile distilled water to give a final count of about 1 × 108 CFU/mL. Preparation of Standard Solution. The stock solutions of the individual pesticide standards were prepared by accurately weighing 10 (±0.1) mg of each analyte in volumetric flasks (certified A class) and dissolving in 10 (±0.1) mL of methanol. These were stored in dark vials at 4 °C. A working standard mixture of 1 mg/L was prepared by appropriate dilution of the stock solution, from which the calibration standards 0.01, 0.05, 0.1, 0.25, and 0.50 mg/L were prepared by serial dilution with methanol/water (1:1, v/v). The calibration curves (solvent-based and matrix-matched) for flusilazole, myclobutanil, and tetraconazole were obtained by plotting the peak area against the concentration of the calibration standard. The recovery experiments were carried out on fresh untreated grape and NB medium by fortifying the samples (10 g/mL) in triplicate with each fungicide separately at three concentrations of 0.025, 0.05, and 0.5 mg/kg or mg/L. Method Validation. The performance of the analytical method was assessed in terms of the parameters of a single laboratory validation approach.20 Limits of detection and quantification (LOD and LOQ) were determined by considering a signal-to-noise ratio (S/ N) of 3 and 10, respectively, for the quantifier multiple reaction monitoring transition (MRM) ensuring that the qualifier MRM has a S/N of ≥3:1 at LOQ. The recovery experiments were carried out in all of the matrices at LOQ, 5 × LOQ, and 50 × LOQ levels (n = 6). The precision in terms of repeatability in recovery was estimated as %RSD. Utilization of Triazole Fungicides as Sole Carbon Source by Bacillus Strains. Culture tubes containing 10 mL of mineral salt medium (MSM) with 10 μg/mL carbon either from glucose or one of each fungicide were inoculated separately with 100 μL of inoculum of the five Bacillus strains and incubated on a shaking water bath (SW22, Julabo, Germany) at 28 °C and 150 rpm for 24 h under natural daylight. The absorbance was read at 630 nm on a spectrophotometer (Thermo Scientific, Evolution 201). Degradation Kinetics of Triazole Fungicides in Liquid Culture and Grape Berries. In Liquid Culture. Erlenmeyer flasks containing 150 mL of sterile NB were spiked with myclobutanil and tetraconazole at 10 μg/mL and flusilazole at 5 μg/mL of medium, separately. Flasks were inoculated with 100 μL of inoculum of Bacillus
MATERIALS AND METHODS
Chemicals, Reagents, Standards, and Matrices. Residue analysis grade ethyl acetate was obtained from Thomas Baker (Mumbai, India). Analytical reagent grade anhydrous sodium sulfate, acetonitrile, methanol, and water (HPLC grade) were procured from Merck (Mumbai, India). The cleanup reagents included primary− secondary amine (PSA, 40 μm, Agilent Technologies, Bangalore, India) and graphitized carbon black (GCB) (United Chemical Technology, Bristol, PA, USA). The commercially available agricultural formulations of flusilazole 40% EC (Nustar, DuPont) 10737
DOI: 10.1021/acs.jafc.5b03429 J. Agric. Food Chem. 2015, 63, 10736−10746
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Journal of Agricultural and Food Chemistry Table 1. Optimized LC-MS/MS Parameters with Retention Time for Pesticides
a
pesticide
RTa (min)
parent (m/z)
target (m/z)
DP
CEb (V)
CXPc (V)
qualifier (m/z)
CEb (V)
CXPc (V)
flusilazole myclobutanil tetraconazole carbendazim profenofos
9.43 9.92 9.14 2.20 11.22
316.04 289.07 372.00 192.13 372.90
247.0 125.2 159.0 160.2 302.9
13.0 30.0 30.8 13.0 46.0
23 29 39 27 25
4.00 4.00 6.50 4.00 18.00
165.10 69.70 70.00 132.18 97.00
29.30 33.00 35.30 30.80 43.00
4.00 4.00 4.00 4.00 16.00
RT, retention time. bCE, collision energy. cCXP, collision cell exit potential.
Table 2. Growth of Bacillus Strains on MSM Containing Glucose or Triazole Fungicides as Sole Carbon Source absorbance at 630 nm of Bacillus growth in MSM containing glucose or pesticide as carbon sourcea Bacillus strain DR-38 DR-39 CS-126 TL-171 TS-204 a
glucose 0.289 0.291 0.250 0.293 0.233
± ± ± ± ±
0.005 0.003 0.003 0.004 0.003
tetraconazole 0.254 0.271 0.225 0.253 0.226
± ± ± ± ±
flusilazole
myclobutanil
0.004 0.003 0.004 0.003 0.003
0.238 0.261 0.221 0.241 0.213
± ± ± ± ±
0.002 0.002 0.003 0.002 0.003
0.268 0.272 0.231 0.258 0.221
± ± ± ± ±
0.003 0.002 0.003 0.002 0.002
Each value is the mean of three replicates ± SD. Multiresidue Degradation Kinetics on Grape Berries. The study was conducted during February 2014 in the research vineyard of this Centre. Carbendazim, profenofos, and tetraconazole, which belong to three different chemical classes, were applied by foliar spray at the recommended application rates of 1.0 g/L, 2.5 mL/L, and 0.75 mL/L water. After 1 h, the vines were sprayed with a suspension of the five Bacillus strains separately to completely cover the fruits and foliage. Vines sprayed with pesticides but not with any Bacillus strain were maintained as control. Each treatment was replicated four times, and four vines per replicate were maintained in randomized blocks. Sampling and analysis techniques were the same as for the triazole fungicides. The 25th day sample corresponds to harvest. LC-MS/MS Analysis. The analysis was carried out on a highperformance liquid chromatograph (HPLC) (PerkinElmer 200) hyphenated to an API 2000 triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) probe. Separation was carried out by injecting 10 μL onto a C18 column (75 mm × 2 mm, 3 μm) with a flow rate 0.4 mL/min. The mobile phase was composed of (A) methanol/water (20:80, v/v) and (B) methanol/water (90:10, v/ v), each with 5 mM ammonium formate. Estimation was performed in positive mode by MRM. The MS/MS parameters with the corresponding retention times are presented in Table 1. Degradation Rate Kinetics and Data Analysis. The degradation kinetics of flusilazole, myclobutanil, and tetraconazole in the two matrices, liquid culture and grape berries, was determined by two models, a linear first-order model and a nonlinear first + first-order model.
strains DR-39, CS-126, TL-171, and TS-204, separately, and incubated on an orbital shaker (Orbitex, Scigenics Biotech, India) at 28 °C and 150 rpm in dark. Uninoculated NB spiked and unspiked with fungicides were maintained as controls. All treatments were replicated four times. For the studies with flusilazole, an additional strain, DR-38, was also used and the treatments were replicated three times. Aliquots (10 mL) were removed aseptically after 0, 1, 3, 5, 7, 10, and 15 days of incubation for residue analysis. A separate set of flasks was maintained for bacterial count enumeration. For residue analysis, an aliquot of 10 mL was extracted with 10 mL of ethyl acetate in the presence of 10 g of anhydrous sodium sulfate by vortexing (2 min) followed by centrifugation (5 min at 1006g). An aliquot of 2 mL was drawn from the supernatant and placed in a 10 mL test tube, and to it was added 10 μL of 10% diethylene glycol (in methanol) and mixed thoroughly by vortexing. This mixture was subsequently evaporated to near dryness under a gentle stream of nitrogen in a low-volume concentrator. The residues were dissolved in a mixture of 1 mL of methanol and 1 mL of 0.1% acetic acid in water by sonication (1 min) followed by vortexing (30 s). The solution was centrifuged at 2236g for 5 min after the addition of 25 mg of PSA. This supernatant was filtered through a 0.2 μm nylon membrane filter and then injected into LC-MS/MS. On Grape Berries. The studies were conducted during February 2013 in the research vineyard of this Centre. The agricultural formulations of myclobutanil, tetraconazole, and flusilazole were applied by foliar spray at the rates of 0.40 g/L, 0.75 mL/L, and 0.125 mL/L of water, respectively. After 1 h, the vines were sprayed with a suspension of the four Bacillus strains separately to completely cover the fruits and foliage. In studies with flusilazole, DR-38 was used as an additional strain. Vines sprayed with fungicides but not with any Bacillus strain were maintained as control. Each treatment was replicated four times, and four vines per replicate were maintained in randomized blocks. Berry samples were collected randomly from each replicate of the treated and control plots at regular time intervals on 0 (1 h after application of Bacillus inoculum) 1, 3, 5, 7, 10, 15, 20, and 25 days after spray. Bunches hidden inside the canopy were not included in the samples. The grape berries were separated from the pedicels, and a 1 kg sample was directly processed for analysis without any pretreatments. Berries were crushed thoroughly in a blender, and a 200 g sample was withdrawn and further blended. For extraction, a 10 g sample was taken to which 10 mL of ethyl acetate (+10 g anhydrous sodium sulfate) was added and homogenized for 2 min at 25000g followed by centrifugation at 447g for 5 min. The second step of extraction was the same as followed for extraction from the liquid culture.
first-order model
[A]t = [A]1 exp( −k1t ) first + first-order model
[A]t = [A]1 exp( −k1t ) + [A]2 exp( −k 2t ) were [A]t is the concentration (μg/mL for liquid culture and μg/g for grapes) of A at time t (days) , [A]1 and [A]2 are the initial concentrations of A at time 0 degraded through one first and another first-order process, and k1 and k2 are the degradation rate constants for 1 and 2 per day (day−1). The half-life denoted DT50 (degradation time 50%) in which the fungicide concentrations were reduced by 50% was obtained from the first and first + first-order models. The equation parameters were calculated by use of a commercially available program, Table Curve 2D (V5.01). In studies on the degradation of flusilazole in liquid culture, DT50 in spiked control was calculated by theoretically extrapolating the days of sampling using Table Curve 2D as the value could not be obtained until the last day of sampling. 10738
DOI: 10.1021/acs.jafc.5b03429 J. Agric. Food Chem. 2015, 63, 10736−10746
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Journal of Agricultural and Food Chemistry
Figure 1. Degradation of (a) myclobutanil, (b) tetraconazole, and (c) flusilazole by Bacillus strains in liquid culture with time. Each value is the mean of four replicates for myclobutanil and tetraconazole and for flusilazole, three replicates, with error bars indicating standard deviation from the mean.
Table 3. Kinetics of Myclobutanil Degradation in Liquid Culture Following Linear and Nonlinear Modelsa linear first-order model Bacillus strain DR-39 CS-126 TL-171 TS-204 control CD P = 0.05
R
2
0.87 0.96 0.87 0.96 0.95
−1
−1
nonlinear first + first-order model 2
k1 (day )
k2 (day )
DT50 (days)
R
9.128 9.092 8.172 9.250 9.213
NA NA NA NA NA
11.7 11.4 11.5 10.7 12.4
0.94 0.97 0.96 0.98 0.99
k1 (day−1)
k2 (day−1)
DT50b (days)
0.184 0.096 0.213 0.124 0.284
3.820 2.228 3.856 2.863 6.860
9.5 a 11.2 d 10.6 c 10.2 b 12.6 e
