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No Antibiotic and Toxic Metabolites Produced by the Biocontrol Agent FEMS Microbiology Letters, 367, 2020, fnaa075 doi: 10.1093/femsle/fnaa075 Advance Access Publication Date: 29 April 2020 Research Letter R E S E A RCH L E T T E R – Environmental Microbiology No antibiotic and toxic metabolites produced by the Downloaded from https://academic.oup.com/femsle/article-abstract/367/9/fnaa075/5826813 by guest on 18 May 2020 biocontrol agent Pseudomonas putida strain B2017 Oriol Daura-Pich†,IkerHernandez´ †, Lola Pinyol-Escala, Jose M. Lara, Sonia Mart´ınez-Servat, Carolina Fernandez´ and Belen´ Lopez-Garc´ ´ıa*,‡ Futureco Bioscience S.A., Avinguda del Cad´ı 19–23, 08799 Olerdola´ (Barcelona), Spain ∗Corresponding author: Futureco Bioscience S.A., Av. Del Cad´ı, 19–23 – P.I. Sant Pere Molanta, 08799 Olerdola´ (Barcelona), Spain. Tel: +34 938182891 (# 039); E-mail: [email protected] One sentence summary: The non-production of antibiotics / toxic metabolites by Pseudomonas putida strain B2017 makes it a promising biocontrol microorganism for plant protection without side effects on environment, non-target organisms, and human health. Editor: Rustam Aminov †These two authors contributed equally to the present work. ‡Belen´ Lopez-Garc´ ´ıa, http://orcid.org/0000-0001-8131-3436 ABSTRACT Pseudomonas putida and closely-related species such as Pseudomonas fluorescens and Pseudomonas brassicacearum have been reported as potential biocontrol agents and plant growth-promoters. Recently, we have described the biocontrol activity of P.putida B2017 against several phytopathogens of agricultural relevance. In this study, its ability to produce potential antibiotic / toxic metabolites was assessed by functional, chromatography-mass spectrometry and genomic analysis. Our results show that B2017 is not able to synthesize surfactants and common antibiotics produced by Pseudomonas spp., i.e. pyrrolnitrin, 2,4-diacetylphloroglucinol, pyoluteorin and pyocyanin, but it produces pyoverdine, a siderophore which is involved in its biocontrol activity. The non-production of other metabolites, such as cyanide, safracin, promysalin and lipopeptides between others, is also discussed. Our data suggest that the mode of action of B2017 is not mainly due to the production of antimicrobial / toxic metabolites. Moreover, these features make P.putida B2017 a promising biocontrol microorganism for plant protection without side effects on environment, non-target organisms and human health. Keywords: secondary metabolites; siderophore; biosurfactant; agriculture; biological control agent; pseudomonads INTRODUCTION 2015; Mishra and Arora 2018). Pyrrolnitrin, synthesized by a four- gene cluster (prnABCD), is a phenylpyrrole with strong antifun- Fluorescent pseudomonads are biological control agents (BCAs) gal activity inhibiting fungal respiratory chains (Kirner et al. commonly isolated from soil and with the ability to produce a 1998). DAPG is toxic to a wide range of plant pathogenic fungi wide spectrum of bioactive metabolites (Haas and Defago 2005; and its core biosynthetic pathway is encoded by genes phlA, Couillerot et al. 2009; Deveau et al. 2016; Mishra and Arora 2018). phlB, phlC and phlD (Bangera and Thomashow 1999). Pyolute- The most common antibiotics produced by Pseudomonas spp. orin shows strong toxicity against oomycetes, such as Pythium are pyrrolnitrin, 2,4-diacetylphloroglucinol (DAPG), pyoluteorin ultimum. A total of eight genes were identified for pyoluteorin and phenazines that may play an important role in biocon- biosynthetic pathway in Pseudomonas fluorescens Pf-5 (pltA, pltB, trol (Howell and Stipanovic 1980; Shanahan et al. 1992; Pierson pltC, pltD, pltE, pltF, pltG and pltM) (Nowak-Thompson et al. 1999). and Pierson 1996; Haas, Blumer and Keel 2000; Chin-A-Woeng, Other low-molecular-weight metabolites with a broad-spectrum Bloemberg and Lugtenberg 2003; Neidig et al. 2011; Nandi et al. antimicrobial activity are phenazines. Five genes of the phz Received: 12 August 2019; Accepted: 28 April 2020 C FEMS 2020. All rights reserved. For permissions, please e-mail: [email protected] 1 2 FEMS Microbiology Letters, 2020, Vol. 367, No. 9 gene cluster—phzE, phzD, phzF, phzB and phzG—are strictly nec- Cultivation conditions for antibiotic production and essary for their biosynthesis (Blankenfeldt and Parsons 2014). extraction procedure In addition, all pseudomonads have a phzA gene encoding for a PhzB-paralogue that may be involved in the specific forma- To grow B2017, a fermentation process was done in an industrial tion of phenazine-1-carboxylic acid (PCA) instead of phenazine- bioreactor F3–100 (F3-industrial model, Bionet) with a working 1,6-dicarboxylic acid (PDC) (Mavrodi et al. 2010). Furthermore, volume of 100 L of LB medium (5 g/ L yeast extract, 10 g/ L tryp- ◦ the action of PhzM and PhzS are also strictly necessary for the tone and 10 g/ L NaCl) for 6 h at 28 C, pH 7 and agitation ramp biosynthesis of pyocyanin using PCA as precursor (Mavrodi et al. from 250 to 400 rpm. The inoculum was prepared in a lab biore- 2001;Parsonset al. 2007). actor F1–5 (F1-lab model, Bionet) for 24 h and the same parame- In addition, other antimicrobial / toxic metabolites have been ters. described in pseudomonads, including cyanide, promysalin, After fermentation, the cells were harvested by centrifuga- safracins and lipopeptides, between others (Kuiper et al. 2004; tion at 5000 g for 10 min (Hitachi CR22N), the culture supernatant ◦ Downloaded from https://academic.oup.com/femsle/article-abstract/367/9/fnaa075/5826813 by guest on 18 May 2020 Velasco et al. 2005; Raaijmakers, de Bruijn and de Kock 2006; was stored at 4 C and the cells were lyophilized (Heto PowerDry Roongsawang, Washio and Morikawa 2010;Liet al. 2011; Rokni- LL 3000 Freeze Dryer, Thermo Fisher Scientific, Waltham, MA Zadeh et al. 2013; Mishra and Arora 2018). Lipopeptides are bio- USA). surfactants with a wide range of biological activities, such as Secondary metabolites were extracted from both cells and antagonism against pathogenic microorganisms, biofilm forma- cell-free culture supernatant. Lyophilized bacterial cells were tion or triggering plant defense (Raaijmakers et al. 2010; Bon- quantified by measuring colony forming units per milliliter 10 nichsen et al. 2015). ( CFUs/mL). Bacterial cells (0.5–1.0 x 10 CFUs) were suspended Other compounds involved in the biocontrol activity of Pseu- in 8 mL of methanol and acidified to pH 2.5 with 1 N HCl. After domonas spp. are siderophores, small extracellular molecules 4 h-shaking at room temperature, the cell suspension was cen- that are able to sequester the limited iron pool in the rhizo- trifuged (5000 g, 20 min) and the supernatant was called EPM-C sphere competing with phytopathogens, which also require iron (from Extracted Potential Metabolites from Cells). to grow (Cornelis 2010, Ahmed and Holmstrom¨ 2014,Ringeland Culture supernatant was filtered by passing through a Bruser¨ 2018). Pyoverdines, the primary siderophores synthesized 0.22 μm pore size cellulose acetate filter to get 200 mL of cell- by fluorescent Pseudomonas species, are composed of three parts: free culture supernatant (CS). Then, CS was adjusted to pH 2.5 (i) a conserved dihydroxyquinoline chromophore, (ii) a variable with 1 N HCl and extracted three times with the same volume of peptide chain (6–12 amino acids) which differs substantially ethyl acetate (EtAc). The organic phases were combined, dried between species and even between strains of the same species by rotary evaporation and dissolved in 5 mL of methanol; this and (iii) an acyl side-chain attached to the 3-amino group of the extract was called EPM-S (from Extracted Potential Metabolites chromophore. from Supernatant). As a control, the same organic extraction Although there are many studies regarding the production was done on the culture medium LB without microbial inocu- ◦ and regulation of antimicrobial metabolites and siderophores lation. All fractions were kept at 4 C until their use. in fluorescent Pseudomonas spp., few works focus on Pseu- domonas putida. Hassan and coworkers demonstrated the pro- duction of pyoluteorin and siderophores by the P.putida NH-50, Surfactant activity although it was unable to produce pyrrolnitrin and DAPG (Has- The surfactant activity or the ability to collapse a droplet of san, Afghan and Hafeez 2011). By PCR detection using specific water was tested as described (Kuiper et al. 2004). In summary, primers for genes involved in the biosynthesis of pyrrolnitrin, 20 μL of sample CS were pipetted as a droplet onto parafilm. Sub- DAPG, pyoluteorin and phenazines, a diverse profile of antibi- sequently, 3 μL of the dye Congo Red (which had no influence otic production was shown among isolates of P.putida (Agrawal on the shape of the droplets) was added to stain the water and et al. 2015). supernatants for photographic purposes. The spreading of the Pseudomonas putida B2017 was previously described as effec- droplet on the parafilm surface was followed over hours until tive BCA against Fusarium oxysporum f.sp. radicis-lycopersici in the droplet became dry and the diameter was measured. tomato, Rhizoctonia solani and Pectobacterium atrosepticum in potato and Sclerotinia sclerotiorum in lettuce (Oliver et al. 2019). The risk assessment of microbial BCAs is always required before In vitro antimicrobial activity of metabolic fractions their market authorization (Mudgal et al. 2013; OECD 2018). The aim of this study was to assess the potential risk of using Metabolic fractions CS, EPM-C and EPM-S were assessed against B2017 as BCA in agriculture based on its ability to produce bacteria using a radial diffusion assay as previously described secondary metabolites including antibiotics or any other toxic (Murakami et al. 2004). Bacteria were overnight cultured in LB compound. medium at 28 ◦C and 200 rpm, and further refreshed for 4 h. From this exponential-phase culture, a bacterial work solution was adjusted to 0.01 of absorbance at 600 nm in agar media (1 % agar and 1 % triptone) and plated. After solidification, 30 μL aliquots MATERIALS AND METHODS of the tested fractions were loaded in 4 mm-wells done into agar Bacterial strains plates.
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