Accepted Manuscript Title: Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum Author: Waseem Raza Ling Ning Dongyang Liu Wei Zhong Qiwei Huang Shen Qirong PII: DOI: Reference:
S0944-5013(16)30048-9 http://dx.doi.org/doi:10.1016/j.micres.2016.05.014 MICRES 25911
To appear in: Received date: Revised date: Accepted date:
21-3-2016 25-5-2016 27-5-2016
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Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum
Waseem Raza, Ling Ning, Dongyang Liu, Wei Zhong, Qiwei Huang, Shen Qirong*
Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Wei Gang Road, No. 1, 210095, Nanjing, Jiangsu Province, P. R. China
Running title: Antibacterial effect of P. fluorescens volatiles
*Corresponding author
Qirong Shen
Email
[email protected]
Phone number
0086-13901586468
Fax number
0086-2584432420
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Graphical abstrct
Abstract The volatile organic compounds (VOCs) produced by soil microbes have a significant role in the control of plant diseases and plant growth promotion. In this study, we examined the effect of VOCs produced by Pseudomonas fluorescens strain WR-1 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum. The VOCs produced by P. fluorescens WR-1 exhibited concentration dependent bacteriostatic effect on the growth of R. solanacearum on agar medium and in infested soil. The VOCs of P. fluorescens WR-1 also significantly inhibited the virulence traits of R. solanacearum. The proteomics analysis showed that the VOCs of P. fluorescens WR-1 downregulated cellular proteins of R. solanacearum related to the antioxidant activity, virulence, inclusion body proteins, carbohydrate and amino acid synthesis and metabolism, protein folding and translation, methylation and energy transfer, while the proteins involved in the ABC transporter system, detoxification of aldehydes and ketones, protein folding and translation were upregulated. This study revealed the significance of VOCs of P. fluorescens WR-1 to control the tomato wilt
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pathogen R. solanacearum. Investigation of the modes of action of biocontrol agents is important to better comprehend the interactions mediated by VOCs in nature to design better control strategies for plant pathogens.
Keywords: Antibacterial; proteomics; Ralstonia solanacearum; virulence traits; volatile compounds.
1. Introduction The biological control of plant soil-borne diseases has emerged as an attractive alternative after decrease in the use of chemicals due to risks of environmental pollution and increase in the demand for organic food. Among different biocontrol agents, Pseudomonas strains especially P. fluorescens have their own importance. P. fluorescens strains have versatile metabolism, inhabit many environments, including plant, soil and water surfaces and have been successfully used to control different plant soil-borne pathogens (Ganeshani and Kumar, 2005). For example, P. fluorescens BE8 showed 55% control of Fusarium oxysporum f. sp. cucumerinum in a pot experiment (Szentes et al., 2013). Another P. fluorescens strain inhibited Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen in rice (Lingaiah and Umesha, 2013). The understanding of biocontrol mechanisms of action is very important to develop commercially efficient and successful biocontrol strategies against plant pathogens. The biocontrol mechanisms include production of antibiotics, hydrolytic enzymes and volatile organic compounds
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(VOC), competition for space and nutrients and induction of systematic resistance in plants (Raza et al., 2013; Faheem et al., 2015; Yunus et al., 2016). A lot of work has been done on the exploration and identification of antibiotics and hydrolytic enzymes produced by different P. fluorescens strains (Bangera and Thomashow, 1999; Rajmohan et al., 2002). The induction of systemic resistance in plants and competition for space and nutrients by P. fluorescens strains have also been studied (Murthy et al., 2014). The most ignored part is the production of VOCs by P. fluorescens strains and their role in the control of plant soil-borne disease. The VOCs, which are low molecular weight and high vapor pressure compounds, are generally produced by microbes through catabolic pathways, including glycolysis, proteolysis and lipolysis (Schulz and Dickschat, 2007). The VOCs produced by soil microbes have been reported to promote plant growth, exhibit antimicrobial and nematicidal activity and induce systemic resistance in crops (Raza et al., 2013; Audrain et al., 2015). For example, Yuan et al., (2012) showed 35% reduction in growth and complete inhibition of spore germination of Fusarium oxysporum f. sp. cubense by VOCs produced by Bacillus amyloliquefaciens NJN-6. In another report, Park et al., (2013) reported the strong induced resistance against Pseudomonas syringae in plants exposed to C13 VOC from Paenibacillus polymyxa E681. Some reports are available about the effect of VOCs of P. fluorescens strains on plant growth and pathogens, for example, Hernández-León et al., (2015) reported that P. fluorescens UM270 produced VOCs that showed antifungal and plant growth promoting activity. However, there is no information about the effect of VOCs produced by biocontrol P. fluorescens strains on the soil-borne bacterial pathogen Ralstonia solanacearum which is a major limiting factor in the production of many important crop plants around the world (Kelam, 1998). R. solanacearum can persist in soil for long periods along with infested plant debris and therefore, is difficult to eliminate
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completely (Swanson et al., 2005). P. fluorescens strains have been reported to control R. solanacearum causing bacterial wilt of tomato efficiently. Seleim et al., (2011) reported that P. fluorescens exhibited maximum control of bacterial wilt of tomato compared to other biocontrol strains. Similarly, Murthy et al., (2014) reported the induction of systemic resistance in tomato by P. fluorescens against R. solanacearum. In this study, the VOCs produced by a biocontrol strain P. fluorescens WR-1 were evaluated for their effects on the growth and pathogenicity traits of R. solanacearum using biochemical and proteomics techniques. The pathogenicity traits selected in this study included motility traits, antioxidant activity, production of exopolysaccharides and hydrolytic enzymes, biofilm formation and root colonization of tomato roots. This study extends our knowledge about the interactions of VOCs in nature, which would be helpful to develop safer fumigants to control plant diseases. 2. Materials and methods 2.1. Microbial strains A biocontrol strain P. fluorescens WR-1 (GeneBank accession No. JQ317786; Figure S1) and a bacterial pathogenic strain R. solanacearum QLRs-1115 [China general microbiology collection center (CGMCC] accession No. 9487], which exhibited high virulence in tomato were provided by our laboratory (Wei et al., 2011; Raza et al., 2012). The strain P. fluorescens WR-1 was routinely grown on nutrient agar (NA) medium while R. solanacearum strain was grown on tetrazolium chloride medium (TZC). Both strains were maintained on their respective medium supplemented with 30% glycerol at -80°C. 2.2. Antibacterial activity assay of VOCs produced by P. fluorescens WR-1 on agar media For the antibacterial activity assay, P. fluorescens WR-1 was grown in NA medium and R. solanacearum was grown in casamino acid-peptone-glucose (CPG) medium overnight at 30oC;
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next day, both strains were washed twice with sterilized water and adjusted to 1×107 colony forming units (CFU)/ml. The cell suspension of P. fluorescens WR-1 (5µl) was spot inoculated at two places onto the one compartment of divided Petri plates (85 mm diameter) containing modified minimal salt (MS) agar medium (1.5% agar, 1.5% sucrose, and 0.4% TSA (w/v)) and R. solanacearum was spot inoculated (5µl) at two places onto the other compartment of divided plates containing CPG agar medium. The plates were incubated at 30oC after sealing with parafilm. Three control treatments included: first control without the inoculation of P. fluorescens WR-1, second containing Escherichia coli DH5α in place of P. fluorescens WR-1 and in the third control, activated charcoal (2g) was placed between two compartments of divided plates spot inoculated with P. fluorescens WR-1 and R. solanacearum, respectively. After each 12 hours up to five days, R. solanacearum colonies were removed from the plates, suspended in sterilized water (1 ml), diluted by 500 times and spread on the CPG agar medium. The CFU/ml of R. solanacearum was calculated after incubation at 30oC for two days. To check the bacteriostatic or bactericidal role of VOCs of P. fluorescens WR-1 on R. solanacearum, three plates were again placed at 30oC after removing parafilm and P. fluorescens WR-1 along with agar medium from one side of the divided plates. To evaluate the antibacterial activity of VOCs produced by the different concentrations of P. fluorescens WR-1, water suspended cells of P. fluorescens WR-1 (1×107 CFU/ml) were prepared as described above in this section and spot inoculated (5µl) at 1, 2, 5 and 10 places and spread (50 µl) onto the one compartment of divided plates containing MS agar medium except control plates and R. solanacearum was spot inoculated (5µl) at one place in the middle of the other compartment of divided plates containing CPG agar medium. The CFU/ml of R. solanacearum was calculated after three days as described above in this section.
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To investigate the antibacterial potential of VOCs of P. fluorescens WR-1 grown on agar medium to inhibit R. solanacearum in infested soil, one compartment of divided Petri plates was inoculated with P. fluorescens WR-1 onto modified MS medium as described above in this section except control plates, while the other compartment was added with 7.5 g (dry weight) tomato diseased field soil (5.3×105 CFU/ml) from Yixing, Jiangsu province, China. After 14 days incubation at 30oC, the inhibition of R. solanacearum in soil was determined by calculating colony counts of R. solanacearum using the dilution plate technique on modified SMSA medium (Elphinstone et al., 1996). All the experiments described here had three replicates and were repeated twice. 2.3. Antibacterial activity assay of VOCs produced by P. fluorescens WR-1 in soil To evaluate the antibacterial activity of VOCs produced by P. fluorescens WR-1 in natural soil and sterilized soil, the healthy soil (pH 6.5, organic matter content 11.65 g/kg, and available N, P, K contents 41.3, 238.7, 177.5 mg/kg, respectively) was taken from a field in Yixing, China. The overnight culture of P. fluorescens WR-1 was diluted to 107 CFU/ml with sterilized water and 1 ml was mixed with 7.5 g (dry weight) natural soil or sterilized soil (121oC for 30 min) and added into the one compartment of divided plates, while the other compartment was either spot inoculated with R. solanacearum onto the CPG agar medium or added with R. solanacearum infested soil (7.5 g) as described in section 2.2. In control, natural soil or sterilized soil was used without the inoculation of P. fluorescens WR-1. The plates were sealed with parafilm and incubated at 30oC. After three days for CPG agar medium and after 14 days for R. solanacearum infested soil, the CFU/ml calculation of R. solanacearum was done as described in section 2.2. These experiments had three replicates and were repeated twice. 2.4. Ammonia and cyanide production assay
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Many P. fluorescens strains have been reported to produce ammonia and cyanide, which could be a reason of growth inhibition of R. solanacearum (Alstrom and Burns, 1989; Michel and Mew, 1998). Therefore, the ammonia and cyanide production by P. fluorescens WR-1 was evaluated using modified MS medium spot inoculated (2×5 µl) with both P. fluorescens WR-1 and R. solanacearum as described in section 2.2. For the ammonia production assay, the modified MS medium for R. solanacearum was added with 0.02g/L of bromthymol blue dye and for the cyanide production assay, a picrate/Na2CO3 paper strip was fixed to the underside of the divided Petri plate lid. In control, P. fluorescens WR-1 was not inoculated onto the one compartment of divided plates. The plates were sealed with parafilm and incubated at 30oC. After three days, the production of ammonia was observed by the color change of medium supplemented with bromthymol blue dye, while the production of cyanide was observed by the color change of paper strip from yellow to brown, or reddish brown compared to control (Alstrom and Burns, 1989; Michel and Mew, 1998). The growth inhibition of R. solanacearum (CFU/ml) was determined as described in section 2.2. These experiments had three replicates and were repeated twice. 2.5. Motility and chemotaxis traits assay For the motility traits assay, P. fluorescens WR-1 was spot inoculated onto one compartment of divided plates containing modified MS agar medium as described in section 2.2. While for the other compartment, overnight culture of R. solanacearum was washed twice, resuspended in sterilized water (1×107 CFU/ml) and 2 µl was spot inoculated onto the CPG medium containing 0.3%, 0.7% and 1.6% (w/v) agar for the swimming, swarming and twitching motilities, respectively. Later, the plates were sealed with parafilm and incubated at 30°C. After each 12 hours up to three days, swimming and swarming motility diameter (mm) was measured in four directions (Addy et al.,
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2012). While for the twitching motility, after two days incubated at 30°C, the morphology of the colony edges was observed under a light microscope (4× magnification) (Addy et al., 2012). For the chemotaxis assay, P. fluorescens WR-1 was spot inoculated onto one compartment of divided plates containing modified MS agar medium as described in section 2.2. While R. solanacearum was spot inoculated onto the other compartment containing chemotaxis buffer agar medium (10 mM phosphate buffer, 0.1 mM EDTA, 1 μM methionine, 10 mM lactic acid, 0.35% agar, pH 7.3) and at a 15 mm edge to edge distance, a 5 mm of the agar plug was removed from the medium and the hole was filled with 100 µl of Millipore membrane filtered (0.22 µm) tomato root exudates (Croze et al., 2011). Later, the plates were sealed with parafilm and incubated at 30°C. After each 12 hours up to 3 days, R. solanacearum cells, which had moved towards the root exudates, were removed and CFU/ml was determined as described in section 2.2. The root exudates of tomato were extracted after 30 days of plant growth as described by Raza et al., (2015a). These experiments had three replicates and were repeated twice. 2.6. Hydrolytic enzymes production assay To detect the hydrolytic enzymes produced by R. solanacearum, P. fluorescens WR-1 was spot inoculated onto the one compartment of divided Petri plate containing modified MS agar medium as described in section 2.2, while the other compartment of divided Petri plate was spot inoculated with 2 µl of water washed overnight culture of R. solanacearum (1010 cells/ml) on MS medium (0.8% agar) supplemented with sodium carboxymethyl cellulose (1%) for endoglucanase activity and with polygalacturonic acid (0.125%) for polygalacturonase activity. After two days, endoglucanase activity was detected by staining agar medium with a Congo red solution (1 mg/ml) for 15 min followed by NaCl (1M) solution for 15 min and polygalacturonase activity was determined by staining agar medium with 0.1% ruthenium red. The clearing zones around the colonies of R.
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solanacearum were measured in four directions. Later, the colonies of R. solanacearum were removed from the plates to calculate CFU/ml as described in section 2.2 and the results were determined as clearing zone (mm) per 107 CFU/ml. These experiments had three replicates and were repeated twice. 2.7. Antioxidant enzyme production assay For the estimation of the effect of VOCs of P. fluorescens WR-1 on the production of antioxidant enzymes, catalase and superoxide dismutase (SOD) by R. solanacearum, divided Petri plates were prepared with and without exposure to the VOCs as same as for growth inhibition assay. After three days, R. solanacearum colonies were washed twice and suspended in sterilized water. Total cellular proteins were extracted by sonication with the output power of 30% and a burst of 10s with the interval of 10s up to 5 min. After sonication, the mixture was centrifuged for 30 minutes (12000×g at 4oC) and the supernatant was used to determine the antioxidant enzymes. Total protein content was calculated using the Bradford method (Bradford, 1976). The SOD activity was determined using nitroblue tetrazolium (NBT) method and the amount of SOD enzyme that inhibited 50% of NBT was defined as one enzyme unit (Zhou et al., 2003). The catalase activity was determined using KMnO4 titration method and defined as the amount of H2O2 (µM) broke down per min by one mg of protein under the assay conditions (Chance and Maehly, 1955). These experiments had three replicates and were repeated twice. 2.8. Root colonization, exopolysaccharides (EPS) production and biofilm formation assays For the root colonization assay, tomato seeds with sterilized surface (Lycopersicon esculentum, cv. Jiangshu) were germinated on wet filter paper at 30°C and then transferred to plastic trays containing sterilizer vermiculite. The plants were irrigated after two days each up to ten days with half-strength Hoagland nutrient solution. The R. solanacearum cell suspensions with and without
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exposure to the VOCs of P. fluorescens WR-1 were prepared as described in section 2.2 and diluted to the concentration of 1×107 CFU/ml with sterilized water. Later, roots of tomato seedlings were collected, water washed, incubated in 5 ml cell suspension of R. solanacearum at room temperature and, after 30 min, rinsed with sterile water and blotted slightly. Later, the roots were cut, weighed and ground in sterile water and serial dilution plated on CPG agar medium to quantify the total R. solanacearum adhering to the roots. The cell numbers were normalized to seedling root fresh weight. For the EPS estimation, R. solanacearum colonies with and without exposure to the VOCs of P. fluorescens WR-1 were suspended in sterilized water and after measuring OD600 and cell counts on the CPG agar medium as described in section 2.2, were shaken at 40oC for 30 min and then centrifuged at 12,000×g for 10 min. Later, two volumes of ice cold ethanol were added in the cell free culture and incubated at 4oC for 24 hours. The EPS pellets were obtained by centrifugation (12,000×g for 10 min), dissolved in sterilized water and dialyzed at 4oC using a membrane (1000 Da molecular weight cutoff). After two days, the EPS was quantified by the phenol-sulfuric acid method (Dubois et al., 1956). For the biofilm formation assay, divided Petri plates were used. Biocontrol strain P. fluorescens WR-1 was spot inoculated onto the one compartment of the divided Petri plate containing MS agar medium as described in section 2.2, while the other compartment was used to inoculate R. solanacearum. For the inoculation of R. solanacearum, water agar (1.5%) was poured onto the other compartment of divided Petri plate and then the lower part of Eppendorf tube (2 ml) cut up to 250 µl capacity, was inserted upward in water agar when it was still hot. Later, 190 µl CPG broth was added into it and inoculated with 10 µl of 107 CFU/ml of water washed R. solanacearum overnight culture. In control, sterilized water (10 µl) was used. The plates were
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sealed with parafilm and placed at 30oC for 48 hours. The biofilm estimation was done using crystal violet staining method (O'Toole and Kolter, 1998). These experiments had three replicates and were repeated twice. 2.9. Collection and identification of VOCs produced by P. fluorescens WR-1 The VOCs produced by P. fluorescens WR-1 were identified using GC-MS (Trace DSQ, Finnigan) in triplicate. For that, P. fluorescens WR-1 was either spot inoculated (2×5 µl) onto MS agar medium or its 1 ml water suspended cells (107 CFU/ml) were mixed with sterilized soil or natural soil (7.5g, 25% moisture) in a 100 ml vial, respectively, as described in section 2.2 and incubated at 30oC. After three days, the solid-phase microextraction (SPME) fiber [Supelco (Bellefonte, PA) stable flex divnylbenzene/carboxen/polydimethylsiloxane (DCP, 50/30 µm)] was inserted in the vial and first incubated at 30oC for half an hour, and then incubated at 50oC. After 30 min, the SPME fiber was inserted in the injector of GC-MS and desorbed at 220°C (1 min) with an RTX-5MS column (30 m, 0.25 mm inside diameter, 0.25µm). The oven temperature protocol was 33°C (3 min), 180°C (10°C/min), 240°C (35°C/min) and then held for 5 min. The mass spectrometer was operated in the electron ionization mode at 70eV at 220°C with a scan from 50 to 500 m/z. The mass spectra of VOCs were compared with those in the NIST/EPA/NIH Mass Spectrometry Library with respect to the spectra in the Mainlib and/or Replib databases. The identified VOCs were further confirmed by comparing their retention times with the standard compounds. The standard compounds were also run as same as samples using SPME fibers. The pure standard compounds were purchased from Sigma, Tokyo Chemical Industry Co., Ltd. (TCI, Japan) and Aladdin Reagent Database, Inc. (Shanghai, China). The peak area of each compound was calculated in percentage relative to the total peak area of all VOCs in a particular treatment. 2.10. Antibacterial activity assay of VOCs produced by P. fluorescens WR-1
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The effect of each VOC produced by P. fluorescens WR-1 on the growth of R. solanacearum was evaluated in divided Petri plates. For that, the sterile filter paper discs containing the different concentrations (1-40 µg) of each pure VOC in 100 µl methanol were placed onto one compartment of divided plates, while the other compartment containing CPG agar medium was spot inoculated (2×5 µl) with the cell suspension of R. solanacearum as described in section 2.2. The VOCs in different concentrations were prepared from the stock solutions (1 mg/ml) and the stock solution of each VOC was prepared in methanol considering mass density (if VOC is liquid) and purity of commercially purchased VOCs. Two control treatments included, one containing methanol (100 µl) and second without any treatment. The plates were sealed with parafilm and incubated at 30oC. After three days, the colony counts of R. solanacearum (CFU/ml) were determined on CPG agar medium as described in section 2.2. The methanol did not show any growth inhibition of R. solanacearum compared to control (no treatment) so the results were expressed as percent inhibition compared to control (methanol). These experiments had three replicates and were repeated twice. 2.11. Protein extraction and 2D electrophoresis Total proteins of R. solanacearum with and without exposure to the VOCs produced by P. fluorescens WR-1 were extracted after three days incubation at 30oC in triplicate. FOCUS™ Bacterial Proteome (G-Biosciences) was used for the total protein extraction according to the instructions of the manufacturers. The quantification of proteins was done by Bradford method (Bradford, 1976). For 2D analysis, 20 µg protein was diluted to 250 µl of rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 50 mM dithiothreitol and 0.5% (v/v) carrier ampholytes, pH 3–10 nonlinear) and was applied to 13 cm immobilized pH gradient (IPG) strip, pH 3–10 NL in IPG box. After 12 hours, IPG strips were placed in Ettan IPGphor 3 manifold and isoelectric focusing (IEF) was done with consecutive steps at 100 V for 1h, 200 V for 1h, 500 V for 1 h, 1,000 V for 1 h and
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8,000 V for 7.5 h. Later, IPG strips were kept at -80oC or equilibrated immediately for 15 min in a buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS) containing 2% (w/v) dithiothreitol and later in buffer containing 2.5% (w/v) iodoacetamide. For second-dimension analysis, Dodeca Cell (Bio-Rad) with 12% acrylamide SDS-PAGE gels was used. Equilibrated IPG strips were put on top of 12% acrylamide SDS-PAGE gels, sealed in agarose containing a trace amount of bromophenol blue, and second-dimension separation was performed by electrophoresis at 100 mA until the front had reached the lower end of the gel. Proteins were detected by silver staining and analyzed by ImageMaster 2D Platinum 7.0 software (GE Healthcare). The protein extraction and 2D analysis was conducted in triplicate. 2.12. Mass spectrometry and Protein identification The proteins spots that showed more than two times changed expression level were excised from the gel and destained for ten minutes using 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. Later, the spots were repeatedly washed with water and acetonitrile and in-gel digested with modified trypsin (Roche Diagnostics) in 50 mM ammonium bicarbonate. After digestion, peptides were concentrated using C18-ZipTips (Millipore) and eluted directly on the MALDI target in 1 µl of a saturated solution of α-cyanohydroxycinnapinic acid in 50% acetonitrile (v/v). Peptides were analyzed using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) operated in reflectron mode at 20 kV accelerating voltage. MALDI-TOF mass spectra were calibrated internally with known trypsin peaks, and proteins were identified by searching masses of measured peptides against R. solanacearum proteins in nonredundant protein databases using the peptide mass fingerprint tool in Mascot (Matrix Science) allowing a mass tolerance of 40 ppm. 2.12. Statistical analysis
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One-way analysis of variance (ANOVA) was used to determine the significance among treatments and Duncan’s multiple-range test was used to find differences among treatments (P
