screening of plant growth promoting rhizobacteria as potential microbial inoculants

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    am, Butii S

    Accepted 5 May 2012

    Keywords:Phytopathogenic fungi

    cides for crop protection and development of pathogen resistance.The use of environmental friendlymicroorganisms has proved to beuseful in plant growth promotion due to their role in nutrientcycling (Bhattacharyya and Jha, 2012) and disease control. Plantgrowth promoting rhizobacteria (PGPR) inoculation has proven to

    and gaseous products including ammonia (Idris et al., 2007;Lugtenberg and Kamilova, 2009). The mechanism of antifungaleffects lies on the production of a variety of antimicrobialcompounds that act in different ways. The antagonistic effects arecaused by cytolysis, leakage of potassium ions, disruption of thestructural integrity of membranes, inhibition of mycelial growthand the protein biosynthesis (Quan et al., 2010).

    One of the most popular bacteria studied and exploited asbiocontrol agent is the Pseudomonas species (Ahmad et al., 2008).

    * Corresponding author.

    Contents lists available at

    Crop Pro

    .e l

    Crop Protection 40 (2012) 43e48E-mail address: (. Laslo).Plant-associated bacteria may indirectly benet the plants bypreventing the growth or activity of plant pathogens throughdifferent mechanisms (e.g. competition for space and nutrients,antibiosis, production of hydrolytic enzymes, inhibition ofpathogen-produced enzymes or toxins) and through inductionof plant defense mechanisms (Weyens et al., 2009). The diversity ofthe rhizospheric and nodule bacteria is very high (Gyrgy et al.,2010).

    Biological control of plant diseases is gaining attention due toincreased pollution concerns caused by the excessive use of pesti-

    in crop protection, growth promotion or biological disease control.The use of biocontrol bacteria isolated from the rhizosphere maypresent an alternative for plant disease prevention (Compant et al.,2005; Fernando et al., 2006; Fatima et al., 2009). In crop protection,integrated pest management involves the application of differentbacteria alone or in combination with other antagonistic agents(Spadaro and Gullino, 2005).

    One of the plant growth promoting mechanisms of rhizobac-teria is the antagonism against phytopathogenic microorganismsdue to the production of antimicrobial metabolites like side-rophores, antibiotics, cyanides, fungal cell wall degrading enzymesAntimycogenic effectSiderophoresRhizobacteriaGrowth rate inhibitionPhosphate solubilizationAmmonia production

    1. Introduction0261-2194/$ e see front matter 2012 Elsevier Ltd.doi:10.1016/j.cropro.2012.05.002bacteria, isolated from different monocotyledonic plants rhizosphere and soil, was tested against Fusa-rium oxysporum radicis-lycopersici, Sclerotium bataticola, Pythium ultimum, Fusarium graminearum, andAlternaria spp. The antifungal activity of these isolates was described based on the comparison of thegrowth rate inhibition. As the production of iron-chelating compounds is one of the mechanismsresponsible for the antimycotic effect, we tested the siderophore producing capacity of the isolatedstrains. Also, we assayed the ammonia production of these bacteria. This secondary metabolitecompound contributes to the biocontrolling property of these bacteria. Our examinations also includethe inorganic phosphate solubilization capacity of these isolates, which may improve the phosphorusuptake of plants. The results indicate that 17 bacterial isolates are able to produce siderophores and 30from them possess capacity of calcium-phosphate mobilization. The majority of the cultures were foundto have highly inhibitory effects against the mycelium growth of P. ultimum, F. oxysporum radicis-lyco-persici and F. graminearum, whereas others showed little activity. Only twelve bacteria showed no activityagainst the S. bataticola plant pathogen fungus.

    2012 Elsevier Ltd. All rights reserved.

    be a promising agricultural approach that plays an important roleReceived in revised form30 April 2012effects on phytopathogenic microorganisms. Suppression of phytopathogenic fungi by 47 differentArticle history:Received 1 November 2011

    Plant growth promoting bacteria can enhance and promote plant growth and development in differentways. These mechanisms include solubilization of phosphorus, nitrogen xation and biocontrollingScreening of plant growth promoting rhinoculants

    va Laslo a,*, va Gyrgy b, Gyngyvr Mara b, va Ta Politehnica University of Bucharest, Faculty of Applied Chemistry and Material Scienceb Sapientia University, Cluj-Napoca, Faculty of Sciences, Miercurea Ciuc 530104, Liberta

    a r t i c l e i n f o a b s t r a c t

    journal homepage: wwwAll rights reserved.obacteria as potential microbial

    s a, Beta brahmb, Szabolcs Lnyi b

    charest 060042, Splaiul Independentei, Nr. 313, Romaniaq, Nr. 1, Romania

    SciVerse ScienceDirect


    sevier .com/locate/cropro

  • otecMost of the identied Pseudomonas biocontrol strains produceantifungal metabolites such phenazines, pyrrolnitrin, pyoluteorinand cyclic lipopeptides like viscosinamide. It was demonstratedthat viscosinamide prevents the infection of sugarbeet by Pythiumultimum (Bloemberg and Lugtenberg, 2001). These bacterial strainsbeside the antagonistic effect also inuence the defense system ofplants (Maksimov et al., 2011).

    The siderophore-mediated competition for iron is one amongthe mechanisms responsible for the antagonistic activity of Pseu-domonas spp. The secreted iron-chelating compounds bind theferric ions (Fe3), and are taken up by microbial cells throughspecic recognition by membrane proteins (Srivastava and Shalini,2008). The presence of iron-chelating compounds makes thebacteria better competitors for iron, preventing this way thegrowth of the pathogen microorganisms. The Pseudomonas speciesproduce two different types of siderophore: pseudobactin andpyoverdin (Oldal et al., 2002).

    Siderophores produced by biocontrol bacteria have a higherafnity for iron than those produced by some fungal pathogens,allowing the former microbes to scavenge most of the availableiron, preventing the proliferation of fungal pathogens (Hillel, 2005).

    Some authors have reported that Pseudomonas uorescens ebelonging to the PGPR class e produces siderophores and havebiocontrol effect against P. ultimum, R. batatticola, Fusarium oxy-sporum. Other Pseudomonas species like Pythium stutzeri produceextracellular enzymes like chitinase and laminase capable of lysingthe mycelia of Fusarium solani (Kumar et al., 2002; Srivastava andShalini, 2008). Pseudomonas aeruginosa under iron-limitingconditions, produces three types of siderophores: pyoverdine,pyochelin and its precursor salicylic acid, and induces resistance toplant diseases caused by Botrytis cinerea on bean and tomato, Col-letotrichum lindemuthianum on bean (Hfte and Bakker, 2007).

    F. oxysporum causes vascular wilt and foot-, root- and bulbrotdiseases in a wide variety of economically important crops. Alter-naria spp., Sclerotium spp. cause leaf spots, root rot and stem rot,which also leads to serious yield losses (Chaiharn et al., 2009).

    The antifungal effect of PGPRs is inuenced by a lot of envi-ronmental and genetic factors. Biotic and abiotic environmentalsignals may have an important input on the regulation of biocontrolgenes in pseudomonads, e.g. on the repression of siderophorebiosynthesis. Together with low oxygen concentrations, the avail-able carbon and nitrogen sources that inuence the molecularmechanisms are involved in biocontrol activity (Haas and Dfago,2005).

    Another plant growth promoting activity of these bacteriaconsists in solubilization of inorganic insoluble phosphates, trans-forming them into bioavailable forms. This nutrient mobilizationmay enhance crop productivity because phosphorus is a macronu-trient for plants, required for growth and development. It is alsoinvolved in photosynthesis, energy transfer, signal transduction,macromolecular biosynthesis and respiration (Zaidi et al., 2009a,2010). The insoluble phosphate-content of the soils is high, due tothe excessive application of chemical fertilizers. A considerableamount of phosphorus is rapidly xed into less available formstrough complexation with aluminium or iron (in acidic soils) orwith calcium (in calcareous soils), before plant roots have hada chance to absorb it in orthophosphate form (Malboobi et al.,2009).

    Phosphate-solubilizing bacteria have been reported forpromoting plant growth and enhancing production yield (Rodrigezand Fraga, 1999; Khan et al., 2009). Secretion of organic acids andphosphatase enzymes are common mechanisms that facilitate theconversion of insoluble forms of phosphorous to plant accessibleforms (Kumari et al., 2009). The inorganic phosphate mobilization

    . Laslo et al. / Crop Pr44is realised due to organic acid production, proton release orproduction of chelating substances by the bacteria (Zaidi et al.,2009b). Some soil bacteria with phytase activity contribute to thephosphorus release from organic phosphates (Singh andSatyanarayana, 2011).

    The application of phosphorus biofertilizers in the form of plantgrowth promoting microorganisms can facilitate the availability ofaccumulated phosphates for plant growth and development bysolubilization. The bacteria involved in phosphorus solubilizationas well as better scavenging of soluble forms can enhance plantgrowth by increasing the efciency of biological nitrogen xation,enhancing the availability of other trace elements (Gyaneshwaret al., 2002).

    The broad aim of this study is the development of plant growthpromoting inoculants. To our best knowledge, only one recentresearch was made in the neighbouring geographical area (Djuricet al., 2011). In the present article, we screen bacteria for side-rophore production and antifungal activity against plant patho-genic soil borne fungi as follows: F.


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