Enhanced root uptake of acibenzolar-S-methyl (ASM) by tomato plants inoculated with selected Bacillus plant growth-promoting rhizobacteria (PGPR)

Applied Soil Ecology 77 (2014) 26–33

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Enhanced root uptake of acibenzolar-S-methyl (ASM) by tomato plants inoculated with selected Bacillus plant growth-promoting rhizobacteria (PGPR) Charalampos K. Myresiotis a , Zisis Vryzas b,∗ , Euphemia Papadopoulou-Mourkidou a a

Faculty of Agriculture, Pesticide Science Laboratory, Aristotle University of Thessaloniki, P.O.Box 1678, 54124 Thessaloniki, Greece Faculty of Agricultural Development, Laboratory of Agricultural Pharmacology and Ecotoxicology, Democritus University of Thrace, 193 Pantazidou, 682 00 Orestias, Greece b

a r t i c l e

i n f o

Article history: Received 9 July 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Keywords: Acibenzolar-S-methyl (ASM) CGA 210007 plant growth-promoting rhizobacteria (PGPR) tomato uptake

a b s t r a c t The combination of plant growth-promoting rhizobacteria (PGPR) and plant resistance inducers is an alternative crop protection approach in modern agricultural systems. Despite the numerous reports regarding the improved suppression of plant pathogens by their combined application, little is known about the interactions among these components. In the present study, the persistence behavior of the plant activator acibenzolar-S-methyl (ASM) in the rhizosphere of tomato plants and its root uptake as well as systemic translocation ability in aboveground parts after combined use with certain Bacillus PGPR strains (B. amyloliquefaciens IN937a, B. pumilus SE34, B. subtilis FZB24 and GB03) were investigated. Additionally, the population dynamics of the PGPR strain B. subtilis GB03 at the tomato root system and rhizosphere soil applied with or without the pesticide were studied. The results showed that the addition of PGPR inocula did not affect the dissipation rate of ASM in rhizosphere soil. Also, the formation of its major metabolite CGA 210007 in soil was rapid, since it was detected one hour after root drench and it was maintained at high levels during the sampling period without considerable variations among the bacterial treatments compared to the control. The uptake and systemic translocation of ASM and its metabolite CGA 210007 from root to shoot was rapid and maximum concentrations were observed at 48–96 h after its application. It was revealed that in plants treated with the PGPR strains B. subtilis GB03 and B. pumilus SE34 the uptake and systemic translocation of ASM and CGA 210007 in the aerial parts of the tomato plants was significantly higher compared to the control receiving no bacterial treatment. Also, the populations of the strain B. subtilis GB03 showed high colonizing ability in the root system and the rhizosphere soil. PGPR strains that lead to enhanced pesticide uptake by plants should be further evaluated as components in integrated management systems. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The use of synthetic pesticides is nowadays the most common method for controlling pests and plant pathogens in modern agriculture. However, the extensive use of pesticides can cause negative impacts on the environment and human health. Development of resistance in pest populations (Karaoglanidis et al., 2000; Myresiotis et al., 2007), the withdrawal of some of the most effective plant protection products (European Commission, Decision 2008/753/EC), the detection of pesticide residues in food and environment, (Vryzas et al., 2002) and also the high cost of pesticides and restrictions on availability, especially in developing countries, are the major limiting factors in pesticide use. Also, the preference

∗ Corresponding author: Tel.: +302552041120; fax: +302552041120. E-mail addresses: [email protected], [email protected] (Z. Vryzas). 0929-1393/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsoil.2014.01.005

of consumers for quality products free of pesticide residues has led the scientific community to explore new and more environmentally safe plant protection methods. A promising strategy for controlling plant diseases is the use of chemical compounds, with no direct anti-microbial effect, which stimulate the natural systemic acquired resistance (SAR) in plants. The biologically challenged SAR in plants is characterized by the accumulation of endogenous salicylic acid, which plays a key role in signal transfer during the infection by the pathogen and activates the expression of PR (Pathogenesis-Related) genes expression (Buonaurio et al., 2002; Hammerschmidt et al., 2001). Several substances including 2,6-dichloroisonicotinic acid (INA), beta-aminobutyric acid (BABA), probenazole, salicylic acid (SA), and synthetic analogs of salicylic acid such as acibenzolarS-methyl (S-methyl benzo[1,2,3]thiadiazole-7-carbothioate, CGA 245704, ASM), (Fig. 1) can activate defense mechanisms of plants and elicit SAR (Kunz et al., 1997; Walters et al., 2005). ASM is the first

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research data are available in the literature for the interactions among these components under actual agricultural practices. The objectives of the present study were: (i) to investigate the influence of specific PGPR inocula (B. amyloliquefaciens IN937a, B. pumilus SE34, B. subtilis FZB24 and GB03) on persistence behavior and fate of ASM in the rhizosphere of tomato plants under greenhouse conditions, (ii) to evaluate the root uptake and systemic translocation ability of ASM in aerial tomato parts, and (iii) to determine the population dynamics of the PGPR strain B. subtilis GB03 at the tomato root system and soil, spiked with or without ASM. Fig. 1. Structure of the synthetic SAR activator ASM and its major metabolite CGA 210007.

commercially available synthetic chemical developed as a SAR activator and is introduced by Syngenta in many countries, including Greece. It is a “plant activator” that belongs to the group of benzothiadiazoles (BTH) and is used to protect several plant species against a broad spectrum of diseases, including bacteria, fungi, and viruses (Anith et al., 2004; Buonaurio et al., 2002; Myresiotis et al., 2011). ASM has no anti-microbial activity, but induces host plant resistance by activating the SAR response found in most plants and it is applied as a foliar spray, seed treatment, root-dip and soil drench (Benhamou and Bélanger, 1998; Buonaurio et al., 2002; Buzi et al., 2004; Rohilla et al., 2001; Scarponi et al., 2001; Vallad and Goodman, 2004). The metabolic pathway of ASM in plant tissues proceeded via the hydrolysis of the parent molecule to the acidic metabolite benzo [1,2,3] thiadiazole-7-carboxylic acid (CGA 210007) (Fig. 1), substance that has been reported to provide systemic protection to plants (Buonaurio et al., 2002; Scarponi et al., 2001). Probably, the behavior of ASM in plants is analogous to various pesticides that are applied as esters, but exert their action in the acidic form (Scarponi et al., 2001). Another alternative approach for crop protection that has been recently adopted by researchers is the use of PGPR. PGPR are beneficial soil bacteria that exert their promoting effects on plant growth and development either directly or indirectly (Spaepen et al., 2009). Direct plant growth-promoting mechanisms include the enhancement of nutrient availability and use by nitrogen fixation, phosphate solubilization, and uptake of essential plant nutrients, and also may influence plant growth by synthesizing plant hormones like auxins, gibberellins, and cytokinins (Richardson et al., 2009; Spaepen et al., 2009; Vessey, 2003). On the other hand, PGPR can indirectly promote plant growth by protecting plants against phytopathogenic organisms with several indirect mechanisms such as induced systemic resistance (ISR), production of antimicrobial compounds, and competition for nutrients and colonization sites with pathogens (Kloepper et al., 2004). A large number of PGPR strains have been reported in the literature that can effectively control important soil-borne and foliar pathogens in many crops (Anith et al., 2004; Baysal et al., 2008; Myresiotis et al., 2011; Tahmatsidou et al., 2006). Additionally, the combination of biocontrol agents with SAR inducers is an alternative crop protection approach that has recently attracted the attention of researchers worldwide for integrated control of plant pathogens (Abo-Elyousr and El-Hendawy, 2008; Abo-Elyousr et al., 2009; Anith et al., 2004; Gent and Schwartz, 2005; Obradovic et al., 2005; Vallad and Goodman, 2004; Van Wees, 2000). Also, it has been reported in previous studies that the combined application of biocontrol agents with chemical SAR stimulators resulted in enhanced control efficacy compared with the individual applications (Abo-Elyousr et al., 2009; Anith et al., 2004; Gent and Schwartz, 2005; Myresiotis et al., 2012a; Van Wees, 2000). In spite of the numerous reports regarding the improved suppression of plant pathogens by their combined application, no

2. Materials and Methods 2.1 Chemicals Acetonitrile, methanol, and water (all HPLC grade) were obtained from Merck (Darmstadt, Germany). Acetic acid (purity 99.5%) was purchased from Carlo Erba (Milano, Italy), potassium dihydrogen phosphate (purity 99.5%) and sodium chloride (purity 99.8%) were obtained from Riedel-de Haën (Seelze, Germany). Solid-phase extraction was conducted through a polymeric reversed-phase material, Lichrolut EN cartridges (200 mg, 3 mL), Merck (Darmstadt, Germany). Analytical standard of ASM, (purity 99.5%) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and its acidic metabolite benzo[1,2,3]thiadiazole-7-carboxylic acid (CGA 210007), (purity 99.2%) was donated by Syngenta (Basel, Switzerland). Individual stock solutions (1 mg mL−1 ) of each compound were prepared by dissolving accurately weighed amounts in methanol and stored in darkness at -25 ◦ C. Working standard solutions were prepared by serial dilutions in methanol for the preparation of fortified samples and in mobile phase for the construction of calibration curves. All working standard solutions were stored under refrigerated conditions (4–5 ◦ C). 2.2 Bacterial strains and culture conditions The PGPR included in the present study were B. amyloliquefaciens strain IN937a, B. pumilus strain SE34, B. subtilis strain FZB24, and B. subtilis strain GB03. The former two strains were obtained from the culture collection of the Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA, while the latter were the commercial PGPR formulations of B. subtilis GB03 (Companion® ) and B. subtilis FZB24 (FZB24® ) purchased from the manufacturers Growth Products Ltd. (New York, USA) and ABiTEP GmbH (Berlin, Germany), respectively. Several mechanisms have been reported for plant growth promotion and biocontrol of phytopathogens by these PGPR strains. B. subtilis GB03 and B. subtilis FZB24 produce iturin-class lipopeptides that exhibit antibiosis against various plant pathogens (Kilian et al., 2000; Myresiotis et al., 2012a). In contrast, strains B. amyloliquefaciens IN937a and B. pumilus SE34 did not present any inhibitory effect against in vitro fungal growth indicating that antibiosis is not the main mechanism of their action (Myresiotis et al., 2012a). The promotion of plant growth and elicitation of induced systemic resistance (ISR) by these strains mainly through the production of volatile organic compounds (VOCs) such as 2,3-butanediol and acetoin has been demonstrated (Kloepper et al., 2004). Also, the production of phytohormones (Kilian et al., 2000) and the increased plant uptake of nitrogen using PGPR have been proposed for the promotion of plant growth (Adesemoye et al., 2009). The strains were maintained in tryptic soy broth (TSB, Difco Laboratories, Detroit, MI, USA) supplemented with 20% glycerol (Sigma-Aldrich, Steinheim, Germany) at -80 ◦ C for long-term

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storage. Inoculants were prepared by streaking each bacterial strain taken from ultracold storage onto tryptic soy agar plates (TSA, Difco Laboratories, Detroit, MI, USA), incubating the plates at 28 ◦ C for 24 h to check for purity, and subsequently transferring these single colonies to fresh TSB flasks and incubating them under shaking at 28 ◦ C for 24 h. The bacterial cultures were centrifuged at 8000 rpm for 10 min, and the pellet was resuspended in sterilized phosphate buffered saline (PBS) to obtain a final density of 1 × 108 cfu mL−1 . Bacterial concentration was determined using serial dilutions, plating, and counting the colony forming units (cfu) developed on the TSA plates (Myresiotis et al., 2012b). 2.3 Greenhouse experiments The studies were conducted in a glasshouse located in the experimental farm of Aristotle University (Thessaloniki, Greece). The tomato (Lycopersicon esculentum Mill.) cultivar ACE55 grown in pots was used as the test plant in this study. The pots (8 × 8 cm) were filled with a field soil (150 g/pot) and one seed was sown per pot at a depth of approximately 2.0 cm. The soil used in the present study (pH 7.7, organic matter 0.8%, sand 35.6%, silt 24.8%, and clay 39.6%) was collected from the surface layer (0–15 cm depth) of a wheat-cultivated field in the central Greece (04307473o N, 00354535o E), with no pesticide application history. The classification of this soil type is Entisol according to USDA soil taxonomy system (Soil Survey Staff, 1999). The seedlings were kept at 25 ± 2 ◦ C during the daytime and at 18 ± 2 ◦ C at night, with 14 h of photoperiod per day and irrigated at intervals of 3 d. The relative humidity ranged from 70% to 85%. The PGPR strains were applied individually by covering tomato seeds with bacteria by dipping the seeds in a 1:1 mixture consisting of 1% (w/v) methyl cellulose (Sigma-Aldrich, Steinheim, Germany) and 1 × 108 cfu mL−1 bacterial cell suspension in PBS buffer (ChinA-Woeng et al., 1998). Coated seeds were dried under a laminar flow hood. Control plants were grown from seeds coated with methyl cellulose and PBS without bacterial inoculum. Application of bacteria was repeated 20 d later, at the second-leaf stage, by drenching 20 mL of PGPR suspension (1 × 108 cfu mL−1 ) onto every pot around the base of each plant. The pesticide used in the study was a commercial formulation of ASM (Bion® 50 WG, Syngenta S.A.). The pesticide was dissolved in sterilized distilled water and stock solution at the concentration of 300 ␮g mL−1 was prepared. Tomato seedlings were drenched at their base with appropriate volume (30 mL) of ASM solution 2 d after the second PGPR treatment, i.e., 22 d after sowing. Control plants were drenched with sterile distilled water. Special attention was given to ensure the entire volume of the pesticide solution remained in the rhizosphere soil, by avoiding leaching. Treatments were arranged as a randomized complete block with 40 replicate plants per treatment. The treatments were as follows: 1) Control (plants with no bacterial treatment); 2) IN937a (plants inoculated with B. amyloliquefaciens IN937a); 3) SE34 (plants inoculated with B. pumilus SE34); 4) FZB24 (plants inoculated with B. subtilis FZB24), and 5) GB03 (plants inoculated with B. subtilis GB03). The experiment was repeated two times (once for the dissipation and root uptake of ASM and once for the enumeration of B. subtilis GB03 population dynamics). 2.4 Extraction procedure The extraction of ASM and CGA 210007 in soil samples was performed using a previously described method (Myresiotis et al., 2011). Both solutes were extracted from tomato plant samples with an acetonitrile based extraction procedure, according to a modified version of the method described by Scarponi et al. (2001). The aboveground part of each tomato plant was transferred into

50-mL centrifuge tubes, 25 mL of chilled acetonitrile was added and the sample was processed by homogenizer Polytron (Kinematica) for about 1 min. The tubes were closed hermetically and the mixture was mechanically shaken with a Vortex (Velp Scientifica, Milano, Italy) for 2 min. The extracts were centrifuged at 7000 rpm for 6 min, and the supernatants were transferred into 100 mL round-bottom flasks. The samples were concentrated to dryness using a rotary evaporator (Büchi, Switzerland) under reduced pressure at 40 о C and redissolved in 9 mL mixture of phosphate buffer (0.5 M KH2 PO4 , pH 3) : acetonitrile (70 : 30, v/v). Samples were cleaned up using solid phase extraction (SPE) carried on Lichrolut EN cartridges previously activated with 6 mL acetonitrile and 6 mL phosphate buffer solution (0.5 M KH2 PO4 , pH 3). Extracts were loaded on cartridges, followed by washing with 6 mL of phosphate buffer solution and analytes were eluted with 6 mL acetonitrile. The eluates were concentrated to dryness using a nitrogen stream at 30 ◦ C, redissolved in 0.5 mL acetonitrile, and after filtration through 0.45 ␮m PTFE filters were used for HPLC analysis.

2.5 Instrumental analysis Pesticide residue analysis was performed using a SpectraSYSTEM (Thermo Separation Products, Austin, TX, USA). HPLC system consisted of a P4000 tertiary solvent pump, an AS3000 autosampler equipped with a 100-␮L injection loop, and a UV6000LP diode array detector. Chromatographic separation was carried out on a Hypurity-C18 (Thermo Finnigan, USA) analytical column (4.6 mm × 150 mm, 5 ␮m) thermostated at 30 ◦ C. The injection volume was 20 ␮L and the elution was conducted under isocratic conditions at 1 mL min−1 . The mobile phase of the HPLC system consisted of a mixture containing acetonitrile: water (40 : 60, v/v) with 0.6 mL L−1 acetic acid. ASM and CGA 210007 were detected at 254 nm and 235 nm, respectively.

2.6 Method validation The developed analytical methods were evaluated for accuracy and precision with the analysis of fortified soil and tomato plant samples in triplicate. Soil samples were fortified at 0.02, 0.05, 0.1, 0.5, 1, 10, and 60 mg Kg−1 of ASM and 0.05, 0.1, 0.2, 0.5, 1, 2, and 4 mg Kg−1 of CGA 210007. Tomato samples were tested at 0.05, 0.1, 0.5, 1, and 30 mg Kg−1 spiking levels for both substances. The linearity of the detector response to the analyte was evaluated based on the correlation coefficient (r) values. The limits of detection (LOD) and quantification (LOQ) of the method were calculated according to Vryzas and Papadopoulou-Mourkidou (2002) criteria.

2.7 Experiment 1—Dynamics of ASM and its metabolite in plant and soil Totally nine samplings at intervals of 0, 1, 4, 8, 24, 48, 96, 168, and 240 h after ASM application were used for the determination of the parent compound and its major metabolite CGA 210007 in aboveground tomato plants and rhizosphere soil. The plants were taken out of the pots and the shoots were cut at the soil level and weighed to obtain the fresh weights of the aboveground biomass. Additionally, soil samples (5 g) were collected from the rhizosphere’s zone. The moisture content of the soil samples was determined by drying the soil in the oven at 105 ◦ C until constant weight. Tomato plants and soils were placed in Falcon tubes (50 mL) and analyzed directly or stored in zip-lock bags at -25 ◦ C until analysis.

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2.8 Experiment 2—Tomato rhizosphere colonization by B. subtilis GB03 This study was conducted to determine the colonization ability of tomato rhizosphere by the PGPR strain B. subtilis GB03 in natural field soil treated with ASM. Populations were enumerated at the root of tomato plants and also the rhizosphere soil. A chloramphenicol resistant mutant of B. subtilis GB03 (B. subtilis GB03-G7chlor + ) was used to differentiate between added PGPR and indigenous rhizobacteria. Specifically, chloramphenicol resistant mutants from the wild type strain B. subtilis GB03 were isolated by growing the strain on TSA amended with several concentrations of chloramphenicol (Hassen and Labuschagne, 2010; Kokalis-Burelle et al., 2006). The specific antibiotic mutants were evaluated for their colonization ability and plant growth-promoting characteristics which were similar to the parent wild-type strain (Myresiotis et al., 2012a). Aliquots of the bacterial culture suspension were transferred on agar plates containing 0, 0.1, 0.5, 1, 2, and 5 ␮g mL−1 chloramphenicol. Resistant mutants of B. subtilis GB03 to chloramphenicol were recovered from plates containing 5 ␮g mL−1 antibiotic by selecting the colonies with growth rates similar to the wild-type strain. The stability of resistance was confirmed by serial culturing on chloramphenicol TSA plates and also in TSB cultures containing 5 ␮g mL−1 antibiotic. For long-term storage, the strains were maintained in TSB supplemented with 20% glycerol at -80 ◦ C. Inoculum was prepared by streaking the bacterial strain taken from ultracold storage onto TSA plates amended with chloramphenicol, incubating at 28 ◦ C for 24 h, and then transferring these single colonies to TSB containing chloramphenicol and incubating them under shaking. Preparation of tomato seedlings and applications of B. subtilis GB03-G7chlor + resistant strain and pesticide were performed as described previously under “Greenhouse experiments”. Population dynamics in tomato rhizosphere by resistant strain were monitored at intervals of 0, 2, 4, 7, 12, and 21 d after ASM application, using serial dilutions, plating, and counting the cfu developed on TSA plates fortified with 5 ␮g mL−1 chloramphenicol. Rhizosphere samples were collected by removing the seedlings from pots and gently shaking them to remove excess soil. Soil samples (3 g) from the zone of rhizosphere were transferred to sterile Erlenmeyer flasks (250 mL) containing 100 mL of sterile distilled water and 0.05% (v/v) Tween 20 (Sigma-Aldrich, Steinheim, Germany) (Kinsella et al., 2009). After shaking at 140 rpm for 1 h, serial ten-fold dilutions were made (10−1 –10−5 ) and 100 ␮L of the dilutions were plated on TSA supplemented with chloramphenicol. The plates were incubated at 30 ◦ C for 72 h and then the number of colonies was measured and expressed in cfu g−1 soil. The colonization ability in the root system of tomato was determined at 0, 2, 4, 7, 12, and 21 d after pesticide application. The seedlings were delicately uprooted from the soil, washed thoroughly with tap water, and dried on tissue paper. Specifically, 0.2 g of the root tip of plants was homogenized in 1.8 mL of PBS buffer using a mortar and pestle (Zhang et al., 2011). The homogenate was serially diluted and plated onto TSA medium with 5 ␮g mL−1 chloramphenicol. After incubation at 30 ◦ C for 72 h the bacterial colonies were counted and expressed as cfu g−1 root.

2.9 Data analysis The half-life (t1/2 ) values were estimated based on first-order dissipation kinetics, Ct = C0 e−kt , where Ct is the concentration in the soil after time t, C0 is the apparent initial concentration, and k and t are the rate constant (d−1 ) and dissipation period in days, respectively (FOCUS, 2006). The experimental data were submitted to one-way analysis of variance (ANOVA). The differences among

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Table 1 Mean concentrations (mg/Kg) of ASM major metabolite (CGA 210007) in tomato rhizosphere soil. CGA 210007 (mg/Kg) Treatment *

Control IN937a SE34 FZB24 GB03

1h **

0.9a 0.9a 0.8a 0.8a 0.7a

4h

8h

24 h

48 h

96 h

168 h

240 h

1.7a 1.8a 1.7a 1.9a 1.7a

2.0a 2.2a 2.4a 2.4a 2.2a

3.1a 3.1a 3.1a 3.4a 3.3a

3.4a 3.1a 3.2a 3.2a 2.8a

3.0a 2.9a 3.0a 2.7a 2.8a

2.8a 2.3a 2.9a 2.5a 2.6a

2.8a 2.2a 2.8a 2.2a 2.2a

* Control: noninoculated plants; IN937a: plants inoculated with B. amyloliquefaciens IN937a; SE34: plants inoculated with B. pumilus SE34; FZB24: plants inoculated with B. subtilis FZB24; GB03: plants inoculated with B. subtilis GB03. ** Mean values followed by same letters in a column indicate nonsignificant differences among treatments according to Duncan’s multiple range test (P = 0.05).

the treatments were evaluated by Duncan test at a significance level of P = 0.05. The statistical analysis was performed using the statistical program SPSS 17.0 (SPSS, Chicago, USA).

3. Results 3.1 Validity of analytical method Retention times of ASM and CGA 210007 in both analytical methods (soil and plant tissue) were 11.98 and 2.78 min, respectively. The accuracy (as recoveries) and precision (as repeatability) of the methods were acceptable since recovery values for all solutes and at all fortification levels were between 74% and 120% in soil and between 76% and 96% in plant material, and respective RSDs were <11% and <8% (SANCO/10684/2009). The LOD of ASM and CGA 210007 in soil were 0.005 mg Kg−1 and 0.01 mg Kg−1 , respectively, while the respective LOQ values were 0.02 mg Kg−1 and 0.05 mg Kg−1 . The LOD and LOQ values in plant material were 0.02 and 0.05 mg Kg−1 for ASM and 0.03 and 0.05 mg Kg−1 for CGA 210007, respectively. The calibration curves were linear with correlation coefficients (r2 ) values >0.9997 for ASM and >0.9999 for CGA 210007 in the concentration range studied for each of the target analytes.

3.2 Persistence of ASM in rhizosphere of tomato plants The dissipation data of ASM in rhizosphere soil were fitted best to first-order kinetics (Ct = C0 e−kt ) with coefficients of determination (r2 ) higher than 0.906 in all treatments (Fig. S1). The t1/2 values of ASM were 7.7, 7.7, 8.9, and 8.3 h in the rhizosphere of tomato plants inoculated with the PGPR strains B. amyloliquefaciens IN937a, B. pumilus SE34, B. subtilis FZB24, and B. subtilis GB03, respectively. Insignificant differences (P > 0.05) in half-lives of control (7.8 h) and PGPR treated plants were observed suggesting that the inoculation of tomato plants with the PGPR strains did not affect the dissipation rate of ASM. The formation of ASM major metabolite in tomato rhizosphere soil is presented in Table 1. The acid derivative CGA 210007 was formed rapidly, since residues were found within 1 h after pesticide application, at concentrations between 0.7–0.9 mg Kg−1 . CGA 210007 residues were gradually enhanced in the rhizosphere up to 24 or 48 h from ASM treatment when the highest residue levels (3.1–3.4 mg Kg−1 ) were detected. Later, CGA 210007 residues were slightly decreased to 2.2–2.8 mg Kg−1 at 240 h after pesticide treatment. Nonsignificant differences (P > 0.05) among PGPR treated and control samples were observed in the formation of CGA 210007 in the rhizosphere of tomato in all sampling times (Table 1).

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Fig. 2. Total residues (mg Kg−1 ) of ASM and its major metabolite CGA 210007 in aboveground tomato parts after soil drench with Bion® (50% WG, ASM) in various PGPR treatments. Different small letters on the columns indicate significant differences of the means among treatments at the different assessment times, according to Duncan’s multiple range test (P = 0.05).

3.3 Root uptake and systemic translocation of ASM in tomato plants Tomato plants were cut at the soil level and weighed to obtain the fresh weights of the aboveground biomass at specific sampling times after ASM application (Table 2). The samples were extracted and for the calculation of pesticide residues and data analysis the fresh weight of each plant was included. The plant growth promoting effects of these Bacilli PGPR strains on tomato and the efficacy for phytopathogen suppression have been evaluated extensively in a previous study (Myresiotis et al., 2012a). In aerial tomato parts, the residues of ASM were determined as a total residue of the parent compound and its major degradation product CGA 210007 since both of them elicit SAR (Fig. 2). No pesticide residues were detected in samples of tomato plants cultivated in soil without ASM treatment. ASM and its metabolite were not detectable in tomato plants collected immediately after ASM application likely because its take time for chemicals applied into roots to reach the xylem and therefore an immediate uptake of active ingredient directly after chemigation is not expected. However, the translocation of residues from root system to aboveground parts was rapid since residue levels were detected between 0.66–3.00 mg Kg−1 and 0.17–0.37 mg Kg−1 for ASM and CGA 210007, respectively, within 1 h after treatment. Later, the residue levels of ASM

Table 2 Mean fresh weights (g) of the aboveground biomass of tomato plants (cv. ACE55) inoculated with the four Bacillus PGPR strains under greenhouse conditions. Shoot fresh weight (g) Treatment *

Control IN937a SE34 FZB24 GB03

1h **

1.6 1.9 1.8 2.6 2.1

4h

8h

24 h

48 h

96 h

168 h

240 h

1.5 2.4 1.8 2.6 2.0

1.5 2.7 1.8 2.6 2.1

1.5 2.8 1.8 2.7 2.3

1.7 2.8 1.8 2.7 2.3

1.7 2.8 1.8 2.8 2.3

1.7 3.0 1.9 2.8 2.4

1.9 3.1 2.0 3.1 2.5

* Control = plants with no bacterial treatment; IN937a = plants inoculated with B. amyloliquefaciens IN937a; SE34 = plants inoculated with B. pumilus SE34; FZB24 = plants inoculated with B. subtilis FZB24; and GB03 = plants inoculated with B. subtilis GB03. ** Values listed in the table are the mean values of three replicates.

in tomato parts were gradually reduced, while the concentration of CGA 210007 showed a significant increase. Significant differences among treatments were revealed from 8 to 240 h after ASM application. In particular, plants inoculated with the PGPR B. subtilis GB03 and B. pumilus SE34 presented residue levels significantly higher than the control plants (P < 0.05). Insignificant differences in total residue levels were observed between plants inoculated with the PGPR strain B. amyloliquefaciens IN937a and controls (P > 0.05). The maximum translocation of residues was detected between 48 h and 96 h. Specifically, plants inoculated with the PGPR strain B. subtilis GB03 showed the highest residues (37.9 mg Kg−1 ), followed by B. pumilus SE34 (35.5 mg Kg−1 ), and B. subtilis FZB24 (27.4 mg Kg−1 ) treated plants, while the maximum residue level in noninoculated controls was significantly lower (19.6 mg Kg−1 ), (P < 0.05). The highest concentration in plants treated with B. amyloliquefaciens IN937a was 22.3 mg Kg−1 . 3.4 Population dynamics of PGPR strain B. subtilis GB03 in tomato rhizosphere The population dynamics of the PGPR strain B. subtilis GB03 on tomato roots and rhizosphere soil treated with or without ASM 0, 2, 4, 7, 12, and 21 d after chemigation are presented in Figures 3 and 4, respectively. The selection of this strain was made due to the highest translocation of ASM residues previously described. Discrimination between added PGPR and indigenous rhizobacteria, in the rhizosphere soil and on tomato roots, was carried out using chloramphenicol resistant mutant (B. subtilis GB03-G7chlor + ) of the wild strain. As shown in Figure 3, the PGPR strain showed high colonizing ability of the tomato root tip applied individually or in combination with ASM. Specifically, immediately after chemigation the initial number of B. subtilis GB03-G7chlor + colonies was 3.1 × 106 cfu g−1 root. After 4 d of incubation, the population of the strain was maintained at the same level as the initial density (3.0 × 106 cfu g−1 root). Then the number of colonies was gradually increased reaching 1.4 × 107 cfu g−1 root at the last sampling time. Insignificant differences in the number of bacterial colonies were observed after ASM application at each sampling time, showing a parallel colonization pattern (Fig. 3).

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B. subtilis GB03-G7chlor+ B. subtilis GB03-G7chlor+ + acibenzolar-S-methyl

2.0E+07 1.8E+07 1.6E+07

CFU g-1

1.4E+07 1.2E+07 1.0E+07 8.0E+06 6.0E+06 4.0E+06 2.0E+06 1.0E+03

0

2

4

7

12

21

Time (d) Fig. 3. Colonization of tomato roots by the PGPR strain B. subtilis GB03-G7chlor+ in soil, applied individually or in combination with ASM, after 21 d of incubation under greenhouse conditions. Vertical bars indicate standard deviation of means.

Population of bacterial strain B. subtilis GB03-G7chlor + in the rhizosphere soil was 1.4 × 105 cfu g−1 soil on day zero (Fig. 4). After 4 d of incubation, the bacterial population decreased to 8.0 × 104 cfu g−1 and later the density of colonies was maintained at the same level (7.3 × 104 cfu g−1 ) until the end of the incubation time. Similar colonization pattern of the bacterial strain was observed in ASM treated soil nevertheless the number of colonies was consistently lower from the control with final concentration of bacteria at 3.7 × 104 cfu g−1 . Populations of B. subtilis GB03-G7chlor+ at the tomato root tip (1.4 × 107 cfu g−1 root) were significantly higher compared to the rhizosphere soil (7.3 × 104 cfu g−1 soil) after 21 d of incubation. 4. Discussion The use of synthetic SAR activators, such as ASM and PGPR can be key components in modern crop protection programs and implementation of such products appears as a promising prospect. In contrast to conventional pesticides, SAR activators do not cause direct antimicrobial activity and they can provide protection without exerting direct selection pressure on populations of the pathogen, preventing the risk of developing resistance

B. subtilis GB03-G7chlor+ B. subtilis GB03-G7chlor+ + acibenzolar-S-methyl

1.8E+05

1.6E+05 1.4E+05

CFU g-1

1.2E+05 1.0E+05 8.1E+04 6.1E+04 4.1E+04 2.1E+04 1.0E+03

0

2

4

7

12

21

Time (d) Fig. 4. Population survival of the PGPR strain B. subtilis GB03-G7chlor+ in tomato rhizosphere soil, applied individually or in combination with ASM, after 21 d of incubation under greenhouse conditions. Error bars indicate standard deviation of the mean.

31

(Vallad and Goodman, 2004). Furthermore, the combined application of synthetic inducers and rhizobacteria is considered to provide higher and broader disease control efficacy in many cases, suggesting an interaction between different mechanisms of action (Van Wees, 2000; Anith et al., 2004; Gent and Schwartz, 2005; AboElyousr et al., 2009; Myresiotis et al., 2012a). In the present study, combined application of four Bacillus PGPR strains with the plant disease resistance inducer ASM in the rhizosphere of tomato plants under greenhouse conditions was conducted in order to investigate the behavior and fate of ASM in the rhizosphere, as well its ability of uptake and systemic translocation of residues in aboveground tomato plants. The dissipation data of ASM in rhizosphere soil revealed that the inoculation of tomato plants with the PGPR strains B. amyloliquefaciens IN937a, B. pumilus SE34, B. subtilis FZB24, and B. subtilis GB03 did not affect the dissipation rate of ASM, presenting insignificant differences (P > 0.05) in half-life values of PGPR treated samples (7.7, 7.7, 8.9, and 8.3 h, respectively) and control (7.8 h). This is in accordance with a previous laboratory study conducted in natural field soil in the absence of plant which showed non-significant differences in half-lives between PGPR inoculated and uninoculated soil samples (Myresiotis, 2012). The formation of ASM’s major metabolite, CGA 210007 in tomato rhizosphere soil was rapid as detected from the first sampling, at concentrations ranging from 0.7 to 0.9 mg Kg−1 . In each treatment, the highest residue levels (3.1–3.4 mg Kg−1 ) were detected 24–48 h from ASM application and later the concentrations detected were slightly reduced. To our knowledge, this is the first report dealing with ASM dissipation in rhizosphere soil and the simultaneous formation of the acidic metabolite CGA 210007. According to the FOOTPRINT Pesticide Properties Data Base, ASM is characterized as non-persistent pesticide in soil with DT50 values ranged from 0.2 to 0.5 d under laboratory studies and from 1 to 14 d under field studies (FOOTPRINT: creating tools for pesticide risk assessment and management in Europe 2011). Despite the limited reported data on the persistence and translocation of ASM and its major metabolite CGA 210007 after foliar application (Buonaurio et al., 2002; Scarponi et al., 2001), this is the first report of ASM behavior after soil application. In a previous study, the acid metabolite CGA 210007 was formed in tomato plants within 2 h after ASM foliar application and no pesticide residues were detected 72 h after treatment (Scarponi et al., 2001). This is not in agreement with the present results since residues were analyzed until the last sampling (240 h after application). These data may explain the prolonged protection reported in ASM soil treatment compared to the foliage application. It has been reported that soil application of ASM causes increased expression of PR genes and therefore greater systemic protection and for longer period than foliar spraying (Francis et al., 2009; Graham and Leite, 2007). Previously, Buonaurio et al. (2002) reported a persistence of 5 d after the application of ASM in pepper leaves and rapid translocation to the untreated apical leaves, while the residue levels of metabolite CGA 210007 were lower than the detection limit of the analytical method. Furthermore, it was found that in plants treated with the PGPR strains B. subtilis GB03 and B. pumilus SE34 the uptake and systemic translocation of ASM and CGA 210007 in the aerial parts of the tomato plants was significantly higher compared to the control receiving no bacterial treatment. In previous studies, it has been well documented that the application of ASM leads to the stimulation of SAR through the production of PR proteins, without the accumulation of endogenous salicylic acid (Friedrich et al., 1996; Lawton et al., 1996). Consequently, the accumulation of high levels of ASM and CGA 210007 in the aboveground parts in plants treated with the PGPR may increase the expression of PR genes and thereby contribute to increased efficiency control observed during the combined applications (Abo-Elyousr et al., 2009; Anith et al.,

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2004; Gent and Schwartz, 2005; Myresiotis et al., 2012a). In order to confirm or reject this scenario, further investigation is required to determine the production of PR proteins. The enhanced pesticide residues in plant tissues may be due to several processes that take place between plants and rhizobacteria (Adesemoye et al., 2009; Lucy et al., 2004). It has been reported that the PGPR strains promote plant growth and enhance the total root surface area and therefore increase nutrient and water uptake by plant roots (Adesemoye et al., 2009; Adesemoye et al., 2010), a phenomenon which may explain the increased root uptake of ASM. The relation of plant growth promotion by PGPR and plant nutrient uptake is an ongoing investigation (Adesemoye et al., 2010). If this suggestion is correct, the PGPR should be principal components in integrated control of plant pathogens and could lead to lower rates of pesticides. Finally, in this investigation the population dynamics of the PGPR strain B. subtilis GB03 on tomato roots and rhizosphere soil with or without ASM, under greenhouse conditions were determined. The rhizobacterial strain showed high colonizing ability of the tomato root tip applied individually or in combination with ASM, showing similar colonization patterns. In the rhizosphere soil, the populations of the strain were lower compared to the root system, and a negative effect in the bacterial growth was observed in ASM treated soils. In a previous study, higher colonization at the tomato root tip compared to the soil by the strain B. subtilis GB03 has also been reported in experiments conducted in sterilized soil (Myresiotis et al., 2012a).

5. Conclusions We conclude that the addition of certain PGPR inocula did not affect the dissipation rate of ASM in rhizosphere soil. The major metabolite CGA 210007 was formed rapidly in the rhizosphere, since it was detected one hour after root drench and it was maintained at high levels during the sampling period without considerable variations among the bacterial treatments compared to the control. The uptake and translocation ability of ASM and its major metabolite CGA 210007 from root to shoot was rapid and maximum concentrations were observed at 48–96 h after ASM application. In plants treated with the PGPR strains B. subtilis GB03 and B. pumilus SE34 the uptake and systemic translocation of ASM and CGA 210007 in the aerial parts of the tomato plants was significantly higher compared to the control receiving no bacterial treatment. The population dynamics study of the strain B. subtilis GB03 revealed higher colonization in the root system compared to the rhizosphere soil. The results presented here suggest no effect of PGPR strains in the overall dissipation rate of ASM and in the formation of CGA 210007. Also, the PGPR-elicited enhanced uptake of ASM residues could lead to less use of pesticide. In further studies, the combination of biocontrol agents with resistance inducers or conventional pesticides in recommended and lower doses will be studied in order to evaluate the efficacy in controlling plant pathogens in the frame of intergraded control management by using the lower effective pesticide doses.

Acknowledgments This study is part of a Ph.D. thesis financially supported by the Alexander S. Onassis Public Benefit Foundation through a scholarship, which is gratefully acknowledged. We thank Professor J.W. Kloepper, Auburn University, AL, USA, for providing the PGPR strains B. amyloliquefaciens IN937a and B. pumilus SE34. We also wish to thank Syngenta Crop Protection, Inc., for providing Bion® (50 WG ASM) and its main metabolite CGA 210 007.

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