Microbiological Research an Alternative Biocontrol Agent of Soil

Microbiological Research 221 (2019) 60–69 Contents lists available at ScienceDirect Microbiological Research journal homepage: www.elsevier.com/locate/micres An alternative biocontrol agent of soil-borne phytopathogens: A new antifungal compound produced by a plant growth promoting bacterium T isolated from North Algeria Bruna Agrilloa,b,1, Sara Mirinoa,1, Rosarita Tatèc, Lorena Gratinoa, Marta Gogliettinoa, ⁎ Ennio Coccaa, Nassira Tablid, Elhafid Nabtid, Gianna Palmieria, a Institute of Biosciences and BioResources (IBBR), National Research Council (CNR), Via Pietro Castellino 111, 80131, Naples, Italy b Materias S.r.l., Corso N. Protopisani n. 50, 80146, Naples, Italy c Institute of Genetics and Biophysics (IGB), National Research Council (CNR), Via Pietro Castellino 111, 80131, Naples, Italy d Laboratoire de Maitrise des Energies Renouvelables, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000, Bejaia, Algeria ARTICLE INFO ABSTRACT Keywords: Bacteria isolated from different environments can be exploited for biocontrol purposes by the identification of Pseudomonas protegens the molecules involved in the antifungal activity. The present study was aimed at investigating antifungal Antifungal protein protein compounds purified from a previously identified plant growth promoting bacterium, Pseudomonas pro- Biocontrol agent tegens N isolated from agricultural land in northern Algeria. Therefore, a novel protein was purified by chro- Alternaria matographic and ultrafiltration steps and its antifungal activity together with growth-inhibition mechanism was Conidia evaluated against different fungi by plate-based assays. In addition, stereomicroscopy and transmission electron Stereomicroscopy and TEM analyses microscopy (TEM) was performed to explore the inhibition activity of the compound on spore germination processes. The protein, showing a molecular mass of about 100 kDa under native conditions, was revealed to be in the surface-membrane fraction and displayed an efficient activity against a variety of phytopathogenic fungi, being Alternaria the best target towards which it exhibited a marked fungicidal action and inhibition of spore germination. Moreover, the compound was able to significantly decrease fungal infection on tomato fruits producing also morphological aberrations on conidia. The obtained results suggested that the isolated compound could represent a promising agent for eco-friendly management of plant pathogens in agriculture. 1. Introduction activities. However, the increasing use of chemical inputs causes several negative effects to the environment and human health besides adding The rhizosphere behavior is the result of the dynamic nature of the burdens to poor farmer resources (Adesemoye et al., 2009; De Weger soil and its dramatic changes, which affect directly the plant growth. On et al., 1995; Gerhardson, 2002; Liu et al., 2017). Recently, the use of a large scale, agriculture is subjected to various exogenous factors that plant growth promoting bacteria (PGPB) as agents stimulating plant can hinder its development, as in the case of abiotic and biotic stresses. growth and managing plant health, represented a promising alternative The latter represents all plant diseases caused by fungi, bacteria, (Bensidhoum et al., 2016; Berg, 2009; Saharan and Nehra, 2011). In- viruses, and nematodes, which are more aggressive and constitute tensive research on this class of microorganisms is currently underway serious problems, decreasing the final yields of crops (Borges et al., worldwide for developing new bio-fertilizers and BioControl Agents 2014). (BCAs) (Pal and Mc Spadden, 2006). In addition to their direct antag- Pathogenic microorganisms affecting plant health are a major and onistic activity, BCAs can indirectly exert their protective effect by chronic threat to food production and ecosystem stability worldwide. sensitizing the plant to microbial attack via the defense mechanisms As agriculture intensified over the past few decades, producers became activation. PGPB are associated with many plant species and are com- more and more dependent on agrochemicals as a relatively reliable monly present in several environments. From a microbiological point of strategy of crop protection helping with the economic stability of their view, PGPB living in irrigation well water may constitute a potential ⁎ Corresponding author. E-mail address: [email protected] (G. Palmieri). 1 These authors equally contributed to this manuscript. https://doi.org/10.1016/j.micres.2019.02.004 Received 23 November 2018; Received in revised form 29 January 2019; Accepted 8 February 2019 Available online 10 February 2019 0944-5013/ © 2019 Elsevier GmbH. All rights reserved. B. Agrillo, et al. Microbiological Research 221 (2019) 60–69 source of new biological compounds for plant growth enhancement and different time intervals. phytopathogens’ biocontrol (Bensidhoum et al., 2016; Handelsman and Stabb, 1996; Ramadan et al., 2015). This phenomenon is the result of 2.2. Antifungal activity and induction assays multiple interactions between pathogens, plants and antagonist agents. Indeed, sustainable agriculture and the reduction of pesticides are a The antifungal activity and the induction assay were performed as major concern for farmers to improve soil quality and plant and human described by Tabli et al. (2017). The antifungal activity was evaluated health. However, plant stimulated by inducing agents can develop an against different fungi strains (Aspergillus niger, Alternaria spp, Botrytis enhanced defensive state called Induced Systemic Resistance (ISR) cinerea and Penicillium spp) that were plated on PCA plates and in- (Bakker et al., 2007), which confers to plant more resistance to future cubated at 28 °C until sporulation (2–4 days) (Tabli et al., 2017). The attacks by pathogens through endogenous plant signaling molecules antifungal compound production was induced by pouring 300 μl (Ramamoorthy et al., 2001) such as salicylic acid (SA), jasmonic acid (1×108cells/ml) of the bacterial culture in wells (0.5cm in diameter) (JA) and ethylene (ET) (Jourdan et al., 2015). Additionally, PGPB may aseptically punched on the plates, previously scraped with fungi spores affect indirectly phytopathogens via competition for space and nutrient (2×104conidia/ml) and by incubating the plates for 48h at 28°C. occupation or through producing antibiotic metabolites (Beneduzi Therefore, the induced bacterial aliquots were withdrawn from the et al., 2012) such as lytic enzymes, antimicrobial peptides or proteins, holes and underwent to the extraction process. The antifungal activity polyketides, phenolic compounds and bio-surfactants (Fernando et al., was evaluated measuring the diameter of the inhibition zone, after 2 2005). Numerous mechanisms adopted by PGPB to enhance directly days of incubation at 28 °C. plant growth were described, such as biofilm formation (Ravishankar et al., 2013), nutrient uptake, nitrogen fixation (Carvalho et al., 2014), 2.3. Fungistatic and fungicidal tests mineral phosphate solubilization (Wang et al., 2017), phytohormones and sidérophores production (Gamalero and Glick, 2015), ACC dea- The experimental procedure was carried-out by cutting an agar disk minase synthesis (Saravanakumar and Samiyappan, 2007). (0.5 mm) from the inhibition zone in PCA plates previously scraped Specifically, many studies reporting on bacteria with potential ef- with the above mentioned fungi spores and placing it in the middle of a fects against phytopathogenic fungi and/or insects, i.e. Pseudomonas, fresh Malt Extract Agar (MEA) plate (pH 5.5) incubated at 28 °C for Cellulosimicrobium, Bacillus (Bensidhoum et al., 2016; Mokrani et al., 168 h until fungal growth. Agar disks cut from fungal growth zone were 2018; Nabti et al., 2013; Tabli et al., 2017), were recently conducted in transferred on a fresh MEA plate and used as control. Algeria. These microorganisms isolated from different environments can be exploited for biocontrol purposes by extraction and purification 2.4. Preparation of extracellular, intracellular and surface-membrane of the specific compounds involved in the antifungal activity. protein fractions The present work constitutes the continuation of a previous paper focused on the characterization of PGPB, isolated from irrigation well- Induced P. protegens N cultures were centrifuged at 8000 rpm, 4 °C, water of North-eastern Algeria, and on their inducible antifungal for 45 min. The pellet was used to isolate the membrane protein frac- compounds (Tabli et al., 2017). In this context, the purpose of this study tions, accordingly to Gogliettino et al. (2010) with some modifications. was to isolate and analyse the active antifungal protein produced by the The bacterial cell pellet was re-suspended in lysis buffer (50 mM Tris- previously identified PGPB Pseudomonas protegens N strain (Tabli et al., HCl, 2 mM EDTA, 1 mM PMSF, pH 7.5) and lysed by sonication (4 cy- 2017). The antifungal compound was purified through ultrafiltration cles with a 60 s pulse ON and 60 s OFF) using a Soniprep (B. Braun and chromatographic steps and it was in vitro testing for fungistatic Labsonic U). The unbroken cells were removed by centrifugation and/or fungicidal activity against different phytopathogenic fungi, i.e. (13,000 rpm at 4 °C for 30 min), whereas the supernatant represented Aspergillus niger, Alternaria spp, Botrytis cinerea, and Penicillium spp, the intracellular fraction. The surface-membrane protein fraction was Moreover, the effects of

Load more