Screening of plant growth promoting rhizobacteria as potential microbial inoculants

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<ul><li><p>iz</p><p>am, Butii S</p><p>Accepted 5 May 2012</p><p>Keywords:Phytopathogenic fungi</p><p>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</p><p>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).</p><p>One of the most popular bacteria studied and exploited asbiocontrol agent is the Pseudomonas species (Ahmad et al., 2008).</p><p>* Corresponding author.</p><p>Contents lists available at</p><p>Crop Pro</p><p>.e l</p><p>Crop Protection 40 (2012) 43e48E-mail address: lasloeva@yahoo.com (. 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).</p><p>Biological control of plant diseases is gaining attention due toincreased pollution concerns caused by the excessive use of pesti-</p><p>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).</p><p>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</p><p>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.</p><p> 2012 Elsevier Ltd. All rights reserved.</p><p>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</p><p>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</p><p>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</p><p>a r t i c l e i n f o a b s t r a c t</p><p>journal homepage: wwwAll rights reserved.obacteria as potential microbial</p><p>s a, Beta brahmb, Szabolcs Lnyi b</p><p>charest 060042, Splaiul Independentei, Nr. 313, Romaniaq, Nr. 1, Romania</p><p>SciVerse ScienceDirect</p><p>tection</p><p>sevier .com/locate/cropro</p></li><li><p>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).</p><p>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).</p><p>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).</p><p>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).</p><p>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).</p><p>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).</p><p>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).</p><p>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</p><p>. 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).</p><p>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).</p><p>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. oxysporum radicis-lycopersici,Sclerotium bataticola, P. ultimum, Fusarium graminearum and Alter-naria spp. We assayed the ammonia production capacity andinorganic phosphate solubilization trait of these isolates.</p><p>2. Materials and methods</p><p>2.1. Isolation of the bacterial strains</p><p>The bacterial strains were isolated from different mono-cotyledonous plants (Carex spp., Zea mays L.) rhizosphere and soilon Kings B selective media for Pseudomonas species. From the soiland rhizosphere samples serial dilutions were prepared in gnoto-biotic conditions, and a volume of 0.1 ml was spread on Kings Bselective media. The inoculated Petri-dishes were incubated 48 h atthe temperature of 28 C. On the basis of morphological andphysiological characteristics we worked with 47 selected cultures,12 bacterial pure cultures originated from rhizosphere and 35 fromsoil.</p><p>2.2. Evaluation method for siderophore production</p><p>Siderophore production was detected by the universal methodof Schwyn and Neilands (1987). This assay is based on a competi-tion for iron between the ferric (Fe3) complex of an indicator dye,chrome azurol S (CAS), and a chelator or siderophore produced bythe microorganism. The iron is removed from CAS by the side-rophore, which apparently has a higher afnity for ferric ions(Fe3). The positive reaction results in a colour change of CASreagent (usually from blue to orange).</p><p>The bacterial cultures were grown in Kings B broth (containingproteose peptone 20 g, glycerol 10 ml, K2HPO4 1.5 g, MgSO4 1.5 g, in1000 ml distilled water) for 24 h at 28 C, followed by an inocula-tion with 5 ml bacterial suspension of the modied, CAS containingKings B agar plates and incubation for 72 h at 28 C. For the side-rophore producing colonies the original blue colour of mediumwaschanged in orange.</p><p>2.3. Antifungal effect assay</p><p>Isolated bacterial cultures were tested for growth inhibitoryeffect on the mycelium growth of F. oxysporum radicis-lycopersici,S. bataticola, P. ultimum, F. graminearum, and Alternaria spp. oncomplex agar medium (containing peptone 10 g, D-dextrose 40 g,yeast extract 10 g, agar 18 g). The test fungi were grown andmaintained on Czapek Dox agar (containing sucrose 30 g, NaNO3,3 g, K HPO 1 g, KCL 0.5 g, MgSO $7H O 0.5 g, FeSO $7H O 0.01 g,</p><p>tion 40 (2012) 43e482 4 4 2 4 2agareagar 15 g in 1000 ml distilled water) (Atlas, 2010).</p></li><li><p>phate solubilization (Malboobi et al., 2009).</p><p>resistance.</p><p>0</p><p>20</p><p>40</p><p>60</p><p>80</p><p>100</p><p>BC 1BC</p><p> 2BC</p><p> 3BC</p><p> 4BC</p><p> 5BC</p><p> 6BC</p><p> 7BC</p><p> 8BC</p><p> 9BC</p><p> 10BC</p><p> 11BC</p><p> 12BC</p><p> 13BC</p><p> 14BC</p><p> 15BC</p><p> 16BC</p><p> 17BC</p><p> 18BC</p><p> 19BC</p><p> 20BC</p><p> 21BC</p><p> 22BC</p><p> 23BC</p><p> 24BC</p><p> 25BC</p><p> 26BC</p><p> 27BC</p><p> 28BC</p><p> 29BC</p><p> 30BC</p><p> 31BC</p><p> 32BC</p><p> 33BC</p><p> 34BC</p><p> 35BC</p><p> 36BC</p><p> 37BC</p><p> 38BC</p><p> 39BC</p><p> 40BC</p><p> 41BC</p><p> 42BC</p><p> 43BC</p><p> 44BC</p><p> 45BC</p><p> 46BC</p><p> 47</p><p>Isolated bacteria</p><p>Inhi</p><p>bitio</p><p>n ra</p><p>te %</p><p>bac</p><p>. Laslo et al. / Crop Protection 40 (2012) 43e48 45The bacterial cultureswere grown 24 h at 28 C in Kings B broth,0.1 ml of each liquid cultures were spread with surface streaking oncomplex agars plates. In the next step agar disc of each fungus (withdiameter of 8 mm) was placed in the middle of agar discs con-taining the spread cultures and incubated at 28 C for seven days,measuring the diameter change of the fungal mycelium. At thesame time, for each fungal species control experiments were made(they were grown without bacteria). The diameter of the fungalcolony was measured after a seven-day incubation period at 28 C.</p><p>For the determination of the inhibitory effect of the bacterialisolates we calculated the inhibition rate (IR%):</p><p>IR% 100,C BC</p><p>where C is the diameter of the control fungal mycelium and B thediameter of the fungal mycelium grown in the presence of thebacteria (Oldal et al., 2002).</p><p>2.4. Detection of ammonia production</p><p>Bacterial cultures were tested for the production of ammonia inpeptone water. Freshly grown cultures (incubated 24 h at 28 C)were inoculated in 10 ml peptone water and incubated for 48e72 hat 30 2 C. After incubation, Nesslers reagent (0.5 ml) was addedin each tube. Development of brown to yellow colour will bea positive test for ammonia production (Dunca et al., 2007).</p><p>2.5. Determination of phosphate-solubilizing capacity</p><p>Evaluation of tricalcium phosphate solubilization of isolated</p><p>Fig. 1. Inhibition rate of the testedstrains was assessed using Pikovskayas agar (containing agar 15 g,</p><p>0</p><p>20</p><p>40</p><p>60</p><p>80</p><p>100</p><p>BC </p><p>1BC</p><p>2BC</p><p>3BC</p><p>4BC</p><p>5BC</p><p>6BC</p><p>7BC</p><p>8BC</p><p>9BC</p><p>10BC</p><p>11BC</p><p>12BC</p><p>13BC</p><p>14BC</p><p>15BC</p><p>16BC</p><p>17BC</p><p>18BC</p><p>19BC</p><p>20BC</p><p>21BC</p><p>22BC</p><p>23BC</p><p>2B</p><p>Isolated b</p><p>Inhi</p><p>bitio</p><p>n r</p><p>ate</p><p>%</p><p>Fig. 2. Inhibition rate of the tested bacteria aga3.2. Antifungal effect assays</p><p>The results of inhibition of mycelium growth of the assayedphytopathogenic fungi are shown in the diagrams below (Figs.1e5).Among the 47 isolates, almost all showed antagonistic effect against3. Results and discussion</p><p>3.1. Siderophore production tests</p><p>The production of siderophores, low molecular weight metalchelators, was detected in 36.2% of the isolates conferring thema competitive advantage to biocontrol agents and contributes todisease suppression due to the limited supply of essential traceminerals in natural habitats. The 17 isolates from the assayedbacteria able to produce siderophores may secrete directly anti-microbial compounds caused by a stimulated biosynthesis. In thedevelopment of the antag...</p></li></ul>

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