Purple corn‐associated rhizobacteria with potential for plant growth promotion

MR. ANTONIO CASTELLANO-HINOJOSA (Orcid ID : 0000-0002-5785-7625) Accepted Article Article type : Original Article Title Purple corn-associated rhizobacteria with potential for plant growth promotion Running headline Plant growth promoting rhizobacteria in corn crops Author names and affiliations A. Castellano-Hinojosa1,2, V. Pérez-Tapia1, E.J. Bedmar2, N. Santillana3 1 Department of Soil Microbiology and Symbiotic Systems. Estación Experimental del Zaidín, CSIC. E-419, 180080-Granada, Spain 2 Department of Microbiology, Faculty of Pharmacy, University of Granada. Campus Cartuja, 18071-Granada, Spain 3 Facultad de Ciencias Agrarias. Universidad Nacional de San Cristóbal de Huamanga, Ayacucho, Perú Corresponding author A. Castellano-Hinojosa, Department of Soil Microbiology and Symbiotic Systems Estación Experimental del Zaidín, CSIC, E-419, 18080-Granada, Spain E-mail: ach@ugr.es This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jam.13708 This article is protected by copyright. All rights reserved. Abstract Accepted Article Aims: Purple corn (Zea mays var. purple amylaceum) is a native variety of the Peruvian Andes, cultivated at 3000 m since pre-Inca times without N fertilization. We aimed to isolate and identify native plant growth promoting rhizobacteria (PGPR) for future microbial-based inoculants. Methods and Results: Eighteen strains were isolated from the rhizosphere of purple corn plants grown without N-fertilization in Ayacucho (Peru). The 16S rRNA gene clustered the 18 strains into 9 groups that contained species of Bacillus, Stenotrophomonas, Achromobacter, Paenibacillus, Pseudomonas and Lysinibacillus. A representative strain from each group was selected and assayed for N2-fixation, phosphate solubilization, indol acetic (IAA) and siderophores production, ACC deaminase activity and biocontrol abilities. Inoculation of purple corn plants with single and combined strains selected after a principal component analysis (PCA) caused significant increases in root and shoot dry weight, total C and N contents of the plants. Conclusions: PGPRs can support growth and crop production of purple corn in the Peruvian Andes and constitute the base for microbial-based inoculants. Significance and Impact of the Study: This study enlarges our knowledge on plantmicrobial interactions in high altitude mountains and provides new applications for PGPRs inoculation in purple amylaceum corn, which is part of the staple diet for the native Quechua communities. Keywords purple amylaceum corn, PGPRs, 16S rRNA gene, phylogenetic tree, PCA, bioinoculants. Introduction Corn (Zea mays L.) is a cereal crop widely used all over the world to feed people and animals due to its high nutritional value (Pérez-Montaño et al. 2014; Rosas-Castor et al. 2014). Purple corn (Zea mays var. purple amylaceum) is a corn variety native of the This article is protected by copyright. All rights reserved. Peruvian Andes, usually cultivated at 3000 m above sea level since pre-Inca times, which makes part of the staple diet of the Andean natives. In addition to its richness in Accepted Article phenolic compounds, purple amylaceum corn contains anthocyanin, of which the cyanidin derivatives constitute around 70% and lend the typical color to the cereal (Yang et al. 2009). Peru is the world´s leading producer and exporter of purple corn with a domestic production of 366,000 tonnes harvested from a cultivated land surface of about 250,000 ha (www.minagri.gob.pe/ data of Ministry of Agriculture and Irrigation of Peru). Peasants of the Andean region still grow purple amylaceum corn following traditional methods, mostly without chemical fertilization and no irrigation, and yet its production remains sustainable. The growing interest, however, of purple corn production is leading farmers to abandon their traditional methods and substitute them for intensive farming practices based on the use of chemical fertilizers. Achievement of higher yields, in contrast, has been shown to result in environmental negative impacts including eutrophization, soil and water contamination, loss of biodiversity, emission of greenhouse gases to the atmosphere, etc. (Galloway et al. 2008; Sutton et al. 2011; Erisman et al. 2015). This situation makes imperative to develop strategies to maintain, even to increase, purple corn productivity following Andean traditional agricultural practices. The importance of microbial-based inoculants in agriculture has increased in the last few years. There is evidence that bacteria in the rhizosphere of the plants have the capacity to stimulate their growth by a number of mechanisms leading to plant growth promotion, among them N2 fixation, inorganic phosphorus solubilization, production of phytohormones and siderophores, excretion of diverse compounds with antibiotic or lytic activity against pathogenic organisms, etc., and are now well known as plant growth promoter rhizobacteria (PGPR). Comprehensive reviews covering mechanisms related to plant promotion by PGPR traits have been published (Lugtenberg and Kamilova 2009; Compant et al. 2010; Hayat et al. 2010; Saharan and Nehra 2011; Gamalero and Glick 2011; Beneduzi et al. 2012, Glick 2014; Bashan et al. 2014; Calvo et al. 2014; Pérez-Montaño et al. 2014; Tkacz and Poole 2015, and references therein). The need to devise efficient, environment friendly, economical, and sustainable strategies for enhancing plant productivity is leading to develop biofertilizers for economically important crops (Bhattacharyya and Jha 2012). This article is protected by copyright. All rights reserved. Despite the interest of purple corn in Latin American countries, information about its plant-associated PGPR is scarce. Because the PGPR characteristics of a given strain can Accepted Article vary depending on the plant species and cultivar, soil type, environmental conditions, etc., (Ştefan et al. 2008; Ashrafuzzaman et al. 2009; Saharanand Nehra 2011) identification and selection of microbial strains to be used as potential plant growth promoters should be carried out under the corresponding specific ecological and environmental conditions where they will be used (Fischer et al. 2007; Verma et al. 2013). Therefore, the objectives of this study were (i) to isolate and identify native rhizobacterial strains from corn (Zea mays var. purple amylaceum), (ii) to evaluate their plant growth promoting traits, and (iii) to test the best PGPR-consortia under greenhouse conditions. Materials and methods Sample collection and isolation of rhizobacteria Purple corn plants (Zea mays L var. purple amylaceum) grown in an agricultural field near the locality of Cangari (12º 59´ 51” S; 74º 16´ 96” O) (Ayacucho, Peru), about 2500 m above sea level were used in this study. Plants were grown without any prior chemical fertilization. The soil had a clay texture and the following characteristics: pH (in water) 8.48, 24.9% sand, 25.5% silt, 49.6% clay, 1.83% organic matter, 0.09% total N, 9.9 ppm total P, and 152.3 ppm total K. Corn roots were taken from at least 25-30 plants grown at five different sites of the experimental field, kept on ice and brought to the laboratory for use. After cleaning of the bulk soil, the remaining adhering rhizospheric soil was carefully removed and pooled together. Samples (0.5 g) were placed in microtubes containing 1 ml sterile saline solution, shaken in a vortex for 30 s and centrifuged at 250 rpm for 1 minute in a microfuge. Then, 1 ml aliquots of the supernatant were taken, serially diluted and used for inoculation of Petri dishes containing tryptone soybean agar (TSA) medium. Cells were incubated at 30 °C until the appearance of colony forming units (CFUs). They were further selected by microscopic observation (Nikon CFI60) so that they represented all the different colony types appeared on the plates. This article is protected by copyright. All rights reserved. Sequencing and analysis of the 16S rRNA and nifH genes Accepted Article Genomic DNA was isolated from bacterial cells using the Real Pure Genomic DNA Extraction kit (Durviz, Spain), according to the manufacturer’s instructions. DNA concentration was determined using a Nanodrop spectrophotometer (NanoDrop ND1000, Thermo Fisher Scientific, USA). PCR amplifications of 16S rRNA gene were done using the two opposing fD1 and rD1 universal primers (Weisburg et al., 1991). The nifH gene was assessed by means of nested PCR using primers nifH (forA), nifH (forB), and nifH (rev) described by Widmer et al. (1999) with the modifications indicated by Villadas et al. (2007). Amplification products were purified using the Qiagen PCR product purification kit and subjected to sequencing using the same primers as for PCR amplification, with ABI Prism dye chemistry, and analysed with a 3130 xl automatic sequencer at the sequencing facilities of the Estación Experimental del Zaidín, CSIC, Granada, Spain. The 16S rRNA gene sequences were compared to those deposited in EzTaxon-e (Kim et al. 2012) and those of the nifH gene sequences with homologous sequences in GenBank using the Phydit program (Chun 2001). The phylogenetic trees were inferred using the neighbour-joining model (Saitou and Nei, 1987). MEGA 6.0 was used for all the phylogenetic analyses (Tamura et al. 2013). In vitro plant growth promoting activities N2-fixation Growth under microoxic conditions was performed as indicated earlier (CastellanoHinojosa et al. 2015) using 100 ml flasks containing N-free semisolid (0.3% agar) Burk medium (Wilson and Knight 1952). Flasks were inoculated, hermetically closed with screw caps and kept at 30ºC until appearance of a dense cellular film in the subsurface of the medium. Acetylene-dependent ethylene production was assayed as indicated earlier (Castellano-Hinojosa et al. 2015). In addition to Burk, the N-free Nfb (Döbereiner and Day, 1976), JMV (Reis et al., 2004) and LIGP (Reis et al., 1994) media were also used for detection of acetylene reduction activity. This article is protected by copyright. All rights reserved. Phosphate solubilization Accepted Article Quantitative estimation of P-solubilization was evaluated as described earlier (Mehta and Nautiyal 2001), using Pikovskaya’s (PVK) liquid medium containing Ca3(PO4)2. Briefly, bacteria were grown in 3 ml of PVK at 30ºC to reach a density of about 108 cells ml-1. Then, aliquots (1:1000, v/v) were used to inoculate flasks containing PVK medium and incubated for 14 d at 30 °C. Samples were taken every 2 d intervals and centrifuged at 12000 rpm for 1 minute in a microfuge. The phosphate content in the supernatant was estimated spectrophotometrically at 880 nm (Murphy and Riley 1962). The pH of the supernatant was determined using a pH-meter (model Crison Basic 20). Azospirillum brasilense C16 (Cárdenas et al. 2010) was used as a positive control. Siderophore production Chrome azurol S (CAS)-shuttle assay (Payne 1994) was used to quantify siderophores production. Bacteria were grown in Fe-free SM minimal medium (Meyer and Abdallah 1978) at 30ºC to reach about 108 cells ml-1. After centrifugation at 12000 rpm for 15 minutes in a microfuge, the supernatant was mixed (1:1, v:v) with CAS solution, allowed to stand for 20 minutes and used to assay the intensity of blue colour at 630 nm. SM minimal medium was used as blank. Siderophore production was estimated following the formula reported by Sayyed et al. (2005): siderophore units = [(ArAs)/Ar] × 100, where, Ar = absorbance of reference (minimal media + CAS assay solution) and As = absorbance of sample. IAA production Production of IAA and IAA-related compounds was determined spectrophotometrically as described by Gravel et al. (2007). Briefly, bacterial cultures were grown in Bergersen liquid minimal medium (Bergersen 1977) to reach about 108 cells ml-1. After centrifugation at 12000 rpm for 5 minutes, the supernatant was mixed with Salkowsky reagent (1:2, v/v) (Gordon and Weber 1951). Colour intensity was measured spectrophotometrically at 535 nm after 30 minutes. Auxin levels were estimated as IAA This article is protected by copyright. All rights reserved. equivalents, using standard curves prepared with pure IAA. Brevibacillus brevis BEA1 Accepted Article (Moreno et al. 2009) was used as a positive control. ACC deaminase activity Ability to hydrolyse 1-aminocyclopropane-1-carboxylic acid (ACC) was assayed according to Honma and Shimomura (1978) with the modifications described by Penrose and Glick (2003). Essentially, the bacterial cells were grown in Dworkin and Foster (DF) salts minimal medium (Dworkin and Foster 1958) containing ACC as the sole nitrogen source until they reached about 108 cells ml-1. The amount of αketobutyrate in the supernatant was determined after centrifugation at 3000 rpm for 10 minutes and determination of the absorbance of the samples at 540 nm. Standard curves were prepared with pure α-ketobutyrate. Antagonism Antifungal activity was determined following the assay described by Landa et al. (1997). Essentially, after growth of Fusarium oxysporum in potato dextrose agar (PDA) medium, a 0.9 cm-diameter agar plug was taken and placed in the middle of plates containing PDA medium that had been previously inoculated independently with each of the bacterial cultures grown in tryptone soybean broth (TSB) medium. After incubation at 25ºC, the diameter of the inhibition zones was recorded every 2 d for 15 d, and the percentage of inhibition relative to the control (without bacteria) was evaluated. Inhibition of mycelial growth was estimated following the formula % Inhibition = [(GcGs)/Gc] × 100, where, Gc = diameter of control mycelial growth and Gs = diameter of the bacterial growth. Antibiosis The ability to inhibit growth of F. oxysporum due to antibiotic or toxins production was evaluated as indicated by Shoebitz et al. (2009). A 0.9 cm-diameter agar plugs containing F. oxysporum mycelium was placed near the border of a plate containing This article is protected by copyright. All rights reserved. PDA medium and incubated at 25ºC for 48 h. Then, the strain to be tested was streaked in a straight line on to the opposite side of the plate. The inhibitory effect on fungal Accepted Article growth was evaluated every 2 d for 15 d at 25ºC, and the percentage of inhibition relative to the control (without bacteria) was evaluated as indicated above. Protein determination Protein concentration in bacterial samples was estimated according to Bradford (1976), with a standard curve of varying bovine serum albumin concentrations. Plant inoculation assays Seeds of Z. mays var. purple amylaceum were surface-sterilized, placed in Petri dishes containing 1% water-agar and germinated at 30°C in darkness. Then, seedlings were planted in sterile 5 kg pots (10 cm diameter x 12 cm height) filled with a mixture of sand/vermiculite (1:1, v/v) and inoculated with 1 ml (~ 108 cells ml−1). The plants were watered every 7 days with tap water and every 14 days with half strength the mineral nutrient solution described by Rigaud and Puppo (1975) supplemented with 10 mM KNO3. Uninoculated plants treated with undiluted (treatment C2) or half-strength (treatment C1) mineral solution were used as a control. Plants were grown for eight weeks under greenhouse conditions previously reported (Talbi et al. 2013). Plant dry weight was determined on samples that had been dried at 60ºC for 48 h and total carbon (TC) and total nitrogen (TN) were determined using a Leco TruSpec CN Elemental Analyser. Statistical analysis For each PGPR activity, the normality of data was assessed using the Shapiro-Wilk test. Since most data set failed to fit the normal distribution, the Kruskal-Wallis signed-rank test was chosen to search for significant differences among PGPR traits detected in bacterial strains. Bacteria for plant inoculation were selected using a covariance-based (N-1) principal component analysis (PCA) run to analyse relationships among mineral phosphate solubilization, siderophore production, nitrogen fixation, IAA production, This article is protected by copyright. All rights reserved. ACC deaminase activity and biocontrol activity. All the statistical analyses were carried Accepted Article out by the XLSTAT version 2013.1 software (Addinsoft). Results Sequencing and analysis of 16S rRNA A total of 18 CFUs differing in colony morphology were isolated from the rhizosphere of Z. mays var. purple amylaceum using TSA medium. The nearly complete sequence of the 16S rRNA gene clustered the 18 strains into 9 groups composed by 2 species of each genera Bacillus, Stenotrophomonas and Achromobacter and one species of each genera Paenibacillus, Pseudomonas and Lysinibacillus (Table 1). A phylogenetic tree based on 16S rRNA gene sequences from a representative strain of each group showed that they clustered with different type strains and pairwise alignments between globally aligned sequences revealed that strain A1 had 95.69% similarity to B. nealsonii DSM 15077T, strain A4 was 97.68% to S. maltophilia ATCC 434T, strain A6 showed 99.86% with B. simplex NBRC 15720T, strain A8 was 99.51% to L. fusiformis NBRC 15717T, strain A11 had 99.39% similarity with S. hibiscicola ATCC 19867T, strain A14 was 99.85% with A. spanius LMG 5911T, strain A15 was 99.71% to P. plecoglossicida FPC951T, strain A16 was 96.60% with P. validus JCM 9077T and strain A18 was 99.70% similar to A. marplatensis B2T (Table 1; Fig. 1). Analysis of PGPRs traits Two strains, A4 and A15, had subcellular growth when cultured under microoxic conditions, and they were the only representative strains which showed amplification of the nifH gene. Strains A4 and A15 yielded DNA fragments (data not shown) of about 370 base pair size that had 99.56% similarity with the sequence of the nifH gene from S. maltophilia strain ISSDS-782 and 99.65% with Pseudomonas sp. R1-73, respectively. Acetylene reduction activity by strains A4 and A15 could not be detected despite repeated attempts and utilization of several N-free media. All isolates were capable to solubilize tricalcium phosphate after 14 d incubation in PVK medium, though to a different extent (Table 2; Table S1). Strain A6 showed the highest value, 7.9 mg/ml PO4- content, with corresponding pH values of 4.2, This article is protected by copyright. All rights reserved. significantly higher than that of the reference strain A. brasilense C16 (5.4 mg/ml PO4content and a pH of 6.9). After growth in SM medium, siderophore production varied Accepted Article from 0.6 to 124.0 siderophores units/μg protein for strains A1 and A18, respectively, and production by A8 was not detected (Table 2). All the 9 strains produced IAA and IAA-related compounds after growth in minimal Bergersen medium (Table 2). Concentration of IAA varied among them, strain A15 being the largest producer (2.2 mg IAA/mg protein), even more than the reference strain B. brevis BEA1 used in this study (1.7 mg IAA/mg protein). Only 5 out of the 9 strains showed ACC deaminase activity. The values ranged between 363.6 and 463.6 μmol αketobutyrate mg protein x h corresponding to strains A18 and A14, respectively (Table 2). Mycelial growth of F. oxysporum was inhibited by strains A4 and A14 when the fungus and the bacteria were in contact during 15 d, with percentages of inhibition of 72.5%, and 65.9%, respectively after incubation for 15 d (Table 2; Table S2). Strain A14 also showed antibiosis activity against F. oxysporum, which suggests that the inhibitory effect is mediated by some diffusible metabolite segregated by the bacterium. Inhibition became evident from day 8 onwards, reaching an inhibition value of 73.1% (Table S2). Multivariate analysis PCA analysis including the variables of nitrogen fixation, mineral phosphate solubilization, siderophore production, IAA production, ACC deaminase activity, antibiosis and antagonism resulted in two new factors (Fig. 2). Factor 1 accounted for 49.19% of the total variation in the properties of the samples. Nitrogen fixation weighed the most heavily in forming factor 1 (Table 3). Factor 2 accounted for an additional 31.27% of the variation of the analysed variables and is described mainly by ACC deaminase activity and antibiosis, though a negative covariance was found between them (Table 3). PCA analyses showed that the strains A4, A14, A15 and A18 have the maximal contribution to the total variance. This article is protected by copyright. All rights reserved. Plant inoculation assays Accepted Article The results of seed inoculation assays showed that all treatment tested promoted growth of purple corn respect to control (Table 4). The dry weight of the inoculated seedlings (shoots and roots) was higher with respect to the uninoculated seedlings (Table 4). In both later parameters, the best consortia were A18, A4 + A14, A4 + A15 and A4 + A18. Total nitrogen content (%) significantly increased in purple corn plants inoculated with A4, A15 or A4 + A15 consortia, respect to the uninoculated plants. No-significant differences in the percentage of C were found when purple corn was inoculated (Table 4). Accession numbers Accession numbers of the nucleotide sequences used in this study are shown in the figure tree (Fig. 1). Discussion Continuous application of chemical fertilizers may result in irreparable damage to the overall ecosystem and environment. In addition, their prices and availability are limiting factors for crop production in developing countries around the world. Utilization of PGPR as biofertilizers is suggested as a beneficial, sustainable option for the improvement of nutrient availability, plant growth, and yields (Vessey 2003, Babalola 2010; Hayat et al. 2010; Bhattacharyya and Jha 2012; Bashan et al. 2014; PérezMontaño et al. 2014), and its use to reduce chemical fertilization without compromising yield is an important feature of research in the field of agriculture, microbiology and biotechnology (Minorsky 2008). Rhizobacterial strains vary widely in their PGPR characteristic and they may be specific to the plant species, plant cultivar, soil type and plant genotype (Lucy et al. 2004). Accordingly, knowledge of native bacterial population and their identification is important to obtain specific microbial strains which can be used as potential PGPRs to achieve higher yields under specific ecological and environmental conditions (Fischer et al. 2007); using indigenous PGPR is also an added advantage since adaptation to the natural conditions they are used to may enhance the plant-microbe interactions (Verma This article is protected by copyright. All rights reserved. et al. 2013). According to this rationale, this study was intended to the obtaining of bacterial fertilizers based on native rhizobacteria for purple corn inoculation. Out the 18 Accepted Article strains isolated from the rhizosphere of purple corn, 8 strains (44.44%) belonged to phylum Firmicutes including genera Bacillus, Lysinibacillus and Paenibacillus, and the remaining 10 strains (55.56%) were members of phylum Proteobacteria, of which genus Achromobacter (40%) and genera Pseudomonas and Stenotrophomonas (60%) grouped in classes Betaproteobacteria and Gammaproteobacteria, respectively (Table 1). These results agree with those previously published that indicate that Proteobacteria and Firmicutes were the most abundant members among the bacterial community found in roots of corn plants (Pereira et al. 2011; Grönemeyer et al. 2012; Arruda et al. 2013; Zahid et al. 2015; Rodríguez-Blanco et al. 2015). Fixed nitrogen is a limiting factor in most environments. Given the low N content in the soil from which rhizobacteria were isolated it could have been expected the presence of higher diversity in the number of N2-fixers. However, only strains A4 (S. maltophilia) and A15 (P. plecoglossicida) grew well in Burk medium. We were, however, not successful in demonstrating acetylene reduction activity despite repeated attempts and utilization of several N-free media. This may be due to the absence in the media of a specific factor required for acetylene reduction activity. Because growth in N-free media does not guarantee the ability of a strain to reduce atmospheric N2, the diazotrophic nature of the A4 and A15 strains was confirmed by identification of the nifH gene in their genome and more. It is possible that primers used in this study were not suitable for the remaining taxa present in the rhizosphere of purple corn. Ability to fix nitrogen has been demonstrated in S. maltophilia strains (Reinhardt et al. 2008) and until this study this ability failed in P. plecoglossicida strains (Kaur et al. 2014). In contrast to N2 fixation, because of the alkaline pH (8.48) of the soil, all of the strains solubilized phosphate though to a different extent (Table 2), with strains A4 (S. maltophilia) and A6 (B. simplex) expressing the highest values, even higher than those found in the control strain A. brasilense C16. Phosphate solubilizing activity has already been shown in species of genus Stenotrophomonas (e.g. Majeed et al. 2015) and Bacillus (e. g. Zahid et al. 2015). Except for strain A8, production of siderophores was observed in all of the strains tested (Table 2), a result which agrees with those previously published that indicate that member of the genera identified in this study are siderophore producers (Sayyed et al. 2005; Tian et al. 2009; Govindasamy et al. 2010; Schwartz et al. 2013; Santos et al. 2014). This article is protected by copyright. All rights reserved. In this work we have used a defined culture medium to analyse the production of IAA and IAA-related compounds to avoid possible effects due to the different C and N Accepted Article sources that can be used for bacterial growth. Bergersen minimal medium was chosen because all the nine strains were able to grow in that medium. In those conditions, similar amounts of IAA were produced by most strains, except A15 (P. plecoglossicida) which had significantly higher values (Table 2). Ability to produce IAA had been previously demonstrated in members of those genera (Park et al. 2005; Mohite 2013; Bharucha et al. 2013; Castanheira et al. 2014; Grady et al. 2016). ACC deaminase activity was shown in five out of the nine representative strains (Table 2) with values within the range for a bacterium to be considered as PGPRs (Penrose and Glick 2003). The species with the highest activities were A. spanius (strain A14) and A. marplatensis (strain A18), of which their ACC deaminase activity has been already reported (Nadeem et al. 2007; Shahzad et al. 2013). Fusarium species represent major group of fungal pathogens associated with maize disease (Pechanova and Pechan 2015), a reason why F. oxysporum was chosen to study the biocontrol activity of the rhizobacteria isolated in this study. Only strains A4 (S. maltophilia) and A14 (A. spanius) inhibited significantly the mycelial growth of F. oxyporum when the bacteria and the fungus were in contact (antagonism), with percentages of inhibition of 72.5% and 65.9%, respectively, after incubation for 15 days (Table 2). However, when the bacteria and the fungus were not in contact, only strain A4 inhibited significantly F. oxysporum growth. This result suggests that the biocontrol effect could be mediated by some diffusible metabolites produced by the bacterium. Antifungal activity of S. maltophilia and A. spanius against F. oxysporum has been previously demonstrated (Suma and Podile 2013; Fan et al. 2015). A4, A14, A15 and A18 PCA-selected (Fig. 2) strains were tested in a greenhouse assay using purple amylaceum corn plants inoculated individually with each one of the strains or with some bacterial consortia. The individual inoculation of purple corn seeds with A4, A14, A15 and A18 PCA- selected strains either individually or in double consortia caused a significant increase in root and shoot dry weight and a concomitant enhancement of total C and N contents of the plants (Table 4). Previous data also show that inoculation of corn with PGPR strains resulted in significant increases in shoot and roots dry matter (Kausar and Shahzad 2006; Nadeem et al. 2007; Bender et al. 2013; Kifle and Laing 2016a, 2016b). Taken together our results show that sustainable production of purple corn in the Peruvian This article is protected by copyright. All rights reserved. Andes can be supported by rhizosphere bacteria with PGPR characteristics, and could constitute the base for elaboration of microbial-based inoculants to improve growth of Accepted Article purple corn. Greenhouse observations in this work has to be tested under the conditions prevailing in the area purple corn is grown to ensure the selected bacterial consortium has a positive effect on plant growth and development. Conflict of interest The authors declare no conflict of interest. Acknowledgements This study was supported by the ERDF-cofinanced grant PEAGR2012-1968 from Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain). ACH is recipient of a grant of MECD (FPU 2014/01633). References Arruda, L., Beneduzi, A., Martins, A., Lisboa, B., Lopes, C., Bertolo, F. and Vargas, L.K. (2013) Screening of rhizobacteria isolated from maize (Zea mays L.) in Rio Grande do Sul State (South Brazil) and analysis of their potential to improve plant growth. Appl Soil Ecol 4, 15-22. Ashrafuzzaman, M., Hossen, F.A., Ismail, M.R., Hoque, M.A., Islam, M.Z., Shahidullah, S. M. and Meon, S. (2009) Efficiency of plant growth promoting rhizobacteria (PGPR) for the enhancement of rice growth. Afr J Biotechnol 8, 12471252. Babalola, O.O. (2010) Beneficial bacteria of agricultural importance. Biotechnol Lett 32, 1559-1570. Bashan, Y., de-Bashan, L.E., Prabhu, S.R. and Hernandez, J.P. (2014) Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998-2013). Plant Soil 378, 1-3. Bender, R.R., Haegele, J.W., Ruffo, M.L. and Below, F.E. (2013) Nutrient uptake, partitioning, and remobilization in modern, transgenic insect-protected maize hybrids. Agron J 105, 161-170. Beneduzi, A., Ambrosini, A. and Passaglia, L.M.P. (2012) Plant growth-promoting rhizobacteria (PGPR): their potential as antagonists and biocontrol agents. Genet Mol Biol 35, 1044-1051. This article is protected by copyright. All rights reserved. Bergersen, F.J. (1977) A treatise on dinitrogen fixation. In Hardy RWF, Silver WS, editors. Biology, section III, Silver, NewYork: Wiley, pp. 519-556. Accepted Article Biari, A., Gholami, A. and Rahmani, H.A. (2008) Growth promotion and enhanced nutrient uptake of maize (Zea mays L.) by application of plant growth promoting rhizobacteria in Arid region of Iran. J Bio Sci 8, 1015-1020. Bhattacharyya, P.N. and Jha, D.K. (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol 28, 1327-1350. Bharucha, U., Patel, K. and Trivedi, U.B. (2013) Optimization of indole acetic acid production by Pseudomonas putida UB1 and its effect as plant growth-promoting rhizobacteria on Mustard (Brassica nigra). Agric Res 2, 215-221. Bradford, M.M. (1956) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254. Calvo, P., Nelson, L. and Kloepper, J.W. (2014) Agricultural uses of plant biostimulants. Plant Soil 383, 3-41. Cárdenas, D.M., Garrido, M.F., Bonilla, R.R. and Baldani, V.L. (2010) Aislamiento e identificación de cepas de Azospirillum sp. en pasto guinea (Panicum maximum Jacq.) del Valle del Cesar. Pastos y Forrajes 33. Castanheira, N., Dourado, A.C., Alves, P.I., Cortés-Pallero, A.M., Delgado-Rodríguez, A.I., Prazeres, A., Borges, N., Sánchez, C., Barreto-Crespo, M.T. and Fareleira, P. (2014) Annual ryegrass-associated bacteria with potential for plant growth promotion. Microbiol Res 169, 768-779. Castellano-Hinojosa, A., Correa-Galeote, D., Palau J. and Bedmar E.J. (2015) Isolation of N2-fixing rhizobacteria from Lolium perenne and evaluating their plant growth promoting traits. J Basic Microbiol 56, 85-91. Chun, J. (2001) PHYDIT. (Version 3.1) (available at http://plaza.snu.ac.kr/~jchun/phydit/). Compant, S., Clément, C. and Sessitsch, A. (2010) Colonization of plant growthpromoting bacteria in the rhizo- and endosphere of plants: importance, mechanisms involved and future prospects. Soil Biol Biochem 42, 669-678. Dworkin, M. and Foster, J. (1958) Experiments with some microorganisms which utilize ethane and hydrogen. J Bacteriol 75, 592-603. This article is protected by copyright. All rights reserved. Erisman, J.W., Galloway, J.N., Dice, N.B., Sutton, M.A., Bleeker, A., Grizzetti, B. and Accepted Article De Vries, W. (2015) Nitrogen: Too much of a vital resource. Science Brief. WWF Netherlands: Zeist. Fan, Z.Y., Miao, C.P., Qiao, X.G. and Zheng, Y.K. (2016) Diversity, distribution, and antagonistic activities of rhizobacteria of Panax notoginseng. J Ginseng Res 40, 97104. Fischer, S.E., Fischer, S.I., Margis, S. and Mori, G.B. (2007) Isolation and characterization of bacteria from rhizosphere of wheat. World J Microbiol Biotechnol 23, 895-903. Galloway, J.N., Townsend, A.R., Erisman, J.W. Bekunda, M., Cai, Z., Freney, J.R., Martinelli, L.A., Seitzinger, S.P. and Sutton, M.A. (2008) Transformation of the nitrogen cycle: recent trends, questions and potential solutions. Science 320, 889892. Gamalero, E. and Glick, B.R. (2011) Mechanisms used by plant growth-promoting bacteria, in Bacteria in Agrobiology: Plant Nutrient Management. Maheshwari, D.K., (ed). (Berlin; Heidelberg: Springer-Verlag). pp. 17-46. Glick, B.R. (2014) Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res 169, 30-39. Gordon, S.A. and Weber, R.P. (1951) Colorimetric estimation of indole-acetic acid. Plant Physiol 26, 192-195. Govindasamy, V., Senthilkumar, M., Magheshwaran, V., Kumar, U., Bose, P., Sharma, V. and Annapurna, K. (2010) Bacillus and Paenibacillus spp. Potential PGPR for sustainable agriculture. In Plant Growth and Health Promoting Bacteria Microbiology Monographs. Maheshwari, D.K., (Ed). 18, 333-364. Grady, E.N., MacDonald, J., Liu, L., Richman, A. and Yuan, Z.C. (2016) Current knowledge and perspective of Paenibacillus: a review. Microb Cell Factor 15, 203. Gravel, V., Antoun, H. and Tweddell, R.J. (2007) Growth stimulation and fruit yield improvement of greenhouse tomato plants by inoculation with Pseudomonas putida or Trichoderma atroviride: possible role of indole acetic acid (IAA). Soil Biol Biochem 39, 1968-1977. Grönemeyer, J.L., Burbano, C.S., Hurek, T. and Reinhold-Hurek, B. (2012) Isolation and characterization of root-associated bacteria from agricultural crops in the Kavango region of Namibia. Plant Soil 356, 67-82. This article is protected by copyright. All rights reserved. Hayat, R., Ali, S., Amara, U., Khalid, R. and Ahmed, I. (2010) Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol 60, 579-598. Accepted Article Honma, M. and Shimomura, T. (1978) Metabolism of 1-aminocyclopropane-1carboxylic acid. Agric Biol Chem 42, 1825-1831. Kausar, R. and Shahzad, S.M. (2006) Effect of ACC-deaminase containing rhizobacteria on growth promotion of maize under salinity stress. J Agri Soci Sci 2, 216-218. Kaur, G. and Reddy, M.S. (2014) Influence of P-solubilizing bacteria on crop yield and soil fertility at multilocational sites. Eur J Soil Biol 61, 35-40. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S. and Chun, J. (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716-721. Kifle M.H. and Laing M.D. (2016a) Effects of selected diaoztrophs on maize growth. Front Plant Sci 7:1429. Kifle M.H. and Laing M.D. (2016b) Isolation and screening of bacteria for their diazotrophic potential and their influence on growth promotion of maize seedlings in greenhouses. Front Plant Sci 6:1225. Landa, B.B., Hervas, A., Bethiol, W. and Jimenez-Diaz, R.M. (1997) Antagonistic activity of bacteria from chickpea rhizosphere against Fusarium oxysporum f. sp. ciceris. Phytoparasitica 25, 305-318. Lucy, M., Reed, E. and Glick, B.R. (2004) Applications of free living plant growthpromoting rhizobacteria. Anton Leeuw 86, 1-25. Lugtenberg, F. and Kamilova, F. (2009) Plant-growth-promoting rhizobacteria. Annu Rev Microbiol 63, 541-556. Majeed, A., Abbasi, M.K., Hameed, S., Imran, A. and Rahim, N. (2015) Isolation and characterization of plant growth-promoting rhizobacteria from wheat rhizosphere and their effect on plant growth promotion. Front Microbiol 6, 198. Meyer, J.M. and Abdallah, M.A. (1978) The fluorescent pigment of Pseudomonas fluorescens: Biosynthesis, purification and physicochemical properties. J Gen Microbiol 107, 319-328. Mehta, S. and Nautiyal, C.S. (2001) An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr Microbiol 43, 51-56. Minorsky, P.V. (2008) On the inside. Plant Physiol 146, 323-324. This article is protected by copyright. All rights reserved. Mohite, B. (2013) Isolation and characterization of indole acetic acid (IAA) producing Accepted Article bacteria from rhizospheric soil and its effect on plant growth. J Soil Sci Plant Nutr 13, 638-649. Moreno, B., Vivas, A., Nogales, R. and Benitez, E. (2009) Solvent tolerance acquired by Brevibacillus brevis during an olive-waste vermicomposting process. Ecotoxicol Environ Saf 72, 2109-2114. Murphy, J. and Riley, I.P. (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27, 31-36. Nadeem, S.M., Zahir, Z.A., Naveed, M. and Arshad M. (2007) Preliminary investigations on inducing salt tolerance in maize through inoculation with rhizobacteria containing ACC deaminase activity. Can J Microbiol 53, 1141-1149. Park, M., Kim, C., Yang, J., Lee, H., Shin, W., Kim, S. and Sa, T. (2005) Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea. Microbiol Res 160, 127-133. Payne, S.M. (1994) Detection, isolation and characterization of siderophores. Methods Enzymol 235, 329-344. Pechanova, O. and Pechan, T. (2015) Maize-pathogen interactions: An ongoing combat from a proteomics perspective. Int J Mol Sci 16, 28429-28448. Penrose, D.M. and Glick, B.R. (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118, 1015. Pereira, P., Ibáñez, F., Rosenblueth, M., Etcheverry, M. and Martínez-Romero, E. (2011) Analysis of the bacterial diversity associated with the roots of maize (Zea mays L) through culture-dependent and culture-independent methods. ISRN Ecology 938546:10. Pérez-Montaño, F., Alias-Villegas, C., Bellogin, R.A., del Cerro, P., Espuny, M.R., Jiménez-Guerrero, I., López-Baena, F.J., Ollero, F.J. and Cubo, T. (2014) Plant growth promotion in cereal and leguminous agricultural important plants: From microorganism capacities to crop production. Microbiol Res 169, 325-336. Reinhardt, E.L., Ramos, P.L., Manfio, G.P., Barbosa, H.R., Pavan, C. and Moreira-Filho, C.A. (2008) Molecular characterization of nitrogen-fixing bacteria isolated from Brazilian agricultural plants at Sao Paulo state. Braz J Microbiol 39, 414-422. Rigaud, J. and Puppo, A. (1975) Indole-3-acetic acid catabolism by soybean bacteroids. J Gen Microbiol 88, 223-228. This article is protected by copyright. All rights reserved. Rodríguez-Blanco, A., Sicardi, M. and Frioni, L. (2015) Plant genotype and nitrogen Accepted Article fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize (Zea mays L.). Biol Fertil Soils 51, 391-402. Rosas-Castor, J., Guzman-Mar, J., Hernandez-Ramirez, A., Garza-Gonzalez, M. and Hinojosa-Reyes, L. (2014) Arsenic accumulation in maize crop (Zea mays): a review. Sci Total Environ 488-489, 176-187. Saharan, B.S. and Nehra, V. (2011) Plant growth promoting rhizobacteria: a critical review. Life Sci Med Res 21, 1-30. Saitou, N. and Nei, M. (1987) A neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 44, 406-425. Santos, S., Neto, I.F.F., Machado, M.D., Soares, H.M. V.M. and Soares, E.V. (2014) Siderophore production by Bacillus megaterium: effect of growth phase and cultural conditions. Appl Biochem Biotechnol 172, 549-560. Sayyed, R.Z., Badguja, M.D., Sonwane, H.M., Mhaske, M.M. and Chincholkar, S.B. (2005) Production of Microbial iron chelators (siderophores) by fluorescent pseudomonads. J Ind Biotechnol 4, 486-490. Schwartz, A.R., Ortiz, I., Maymon, M., Fujishige, N. A., Hanamoto, K, Diener A., Sanders, E.R., DeMason, D.A. and Hirsch, A.M. (2013) Bacillus simplex alters legume root architecture and nodule morphology when co-inoculated with Rhizobium. Agronomy 3, 595-620. Shahzad, S.M., Arif, M.S., Riaz, M., Ashraf, M. and Iqbal, Z. (2013) PGPR with varied ACC-deaminase activity induced different growth and yield response in maize (Zea mays L.) under fertilized conditions. Eur J Soil Biol 57, 27-34. Shoebitz, M., Ribaudo, C.M., Pardo, M.A., Cantore, M.L., Ciampi, L. and Curá, J.A. (2009) Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere. Soil Biol Biochem 41, 1768-1774. Ştefan, M., Mihasan, M. and Dunca, S. (2008) Plant growth promoting Rhizobacteria can inhibit the in vitro germination of Glycine Max L seeds. Scientific Annals of University "Alexandru Ioan Cuza" Iasi. Sect Genet Mol Biol 3, 105-110. Suma, K. and Podile, A.R. (2013) Chitinase A from Stenotrophomonas maltophilia shows transglycosylation and antifungal activities. Bioresource Technol 133, 213220. Sutton, M.A., Howard, C.M., Erisman, J.W., Bealey, W.J., Billen, G., Bleeker, A., Bouwman, A., Grennfelt, A.F., van Grinsven, P. and Grizzetti, B. (2011) The This article is protected by copyright. All rights reserved. challenge to integrate nitrogen science and policies: the European nitrogen Accepted Article assessment approach. In Sutton, M.A., Howard, C.M., Erisman, J.W., Billen, G., Bleeker, A., Grennfelt, P., van Grinsven, H. and Grizzetti, B. (Eds.) The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, Cambridge. pp. 82-96. Talbi, C., Argandoña, M., Salvador, M., Alchón, J.D., Vargas, C., Bedmar, E.J. and Delgado, M.J. (2013) Burkholderia phymatum improves salt tolerance of symbiotic nitrogen fixation in Phaseolus vulgaris. Plant Soil 367, 673-685. Tamura, K., Stecher, G., Peterson, D., Filipski A. and Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, 27252729. Tian, F., Ding, Y., Zhu, H., Yao, L. and Du, B. (2009) Genetic diversity of siderophoreproducing bacteria of tobacco rhizosphere. Brazilian J Microbiol 40, 276-284. Tkacz, A., and Poole, P. (2015) Role of root microbiota in plant productivity. J Exp Bot 66, 2167-2175. Verma, J.P., Yadav, J., Tiwari, K.N. and Kumar, A. (2013) Effect of indigenous Mesorhizobium spp. and plant growth promoting rhizobacteria on yields and nutrients uptake of chickpea (Cicer arietinum L.) under sustainable agriculture. Ecol Eng 51, 282-286. Vessey, J.K. (2003) Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255, 571-586. Villadas, P.J., Fernández-López, M., Ramírez-Saad, H.C. and Toro, N. (2007) Rhizosphere bacterial community in Eperua falcata (Caesalpiniaceae) a putative nitrogen-fixing tree from French Guiana rainforest. Microb Ecol 53, 317-327. Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697-703. Widmer, F., Shaffer, B.T., Porteous, L.A. and Seidler, R.J. (1999) Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range. Appl Environ Microbiol 65, 374-380. Wilson, P.W. and Knight, S.C. (1952) Experiments in Bacterial Physiology. In Burguess Publishing, Minneapolis. Yang, Z., Chen, Z., Yuan, S., Zhai, W., Piao, X. and Piao, X. (2009) Extraction and identification of anthocyanin from purple corn (Zea mays L.). Int J Food Sci Tech 44, 2485-2492. This article is protected by copyright. All rights reserved. Zahid, M., Abbasi, M.K., Hameed, S. and Rahim, N. (2015) Isolation and identification Accepted Article of indigenous plant growth promoting rhizobacteria from Himalayan region of Kashmir and their effect on improving growth and nutrient contents of maize (Zea mays L.). Front Microbiol 6, 207. Figure legends Fig 1 Neighbor-joining phylogenetic tree based on partial 16S rRNA sequences of strains isolated from the rhizosphere of Z. mays var. purple amylaceum and of related type strains. The analysis was based on 1370 nucleotides. Bootstraps values are indicated as percentages derived from 1000 replications. Values lower than 70 are not shown. Bar, 0.05 nucleotide substitution per 100 nucleotides. The tree is rooted on Natronomonas moolapensis 8.8.11T. Fig 2 Principal components analysis (PCA) of N2-fixation (▲), IAA production (▼), ACC deaminase activity (X), mineral phosphate solubilization (+), siderophore production (▉), antagonism (●) and antibiosis (♦), and ranking of the samples on PCA factors 1 and 2 This article is protected by copyright. All rights reserved. Table 1 Identification of rhizobacterial strains isolated from Z. mays var. purple Accepted Article amylaceum based on the amplification of 16S rRNA % similarity Strain* Closest relative species according to 16S rRNA gene sequence (according to EzTaxon-e) A1, A13 Bacillus nealsonii DSM 15077T 95.69 A4, A2 Stenotrophomonas maltophilia ATCC 434T 97.68 A6, A3 Bacillus simplex NBRC 15720T 99.86 A8, A5 Lysinibacillus fusiformis NBRC 15717T 99.51 A11, A12 Stenotrophomonas hibiscicola ATCC 19867T 99.39 A14, A17 Achromobacter spanius LMG 5911T 99.85 A15, A10 Pseudomonas plecoglossicida FPC951T 99.71 A16, A7 Paenibacillus validus JCM 9077T 96.60 A18, A9 Achromobacter marplatensis B2T 99.70 * Strains in bold were chosen as the representative of those with identical 16S rRNA gene sequence. This article is protected by copyright. All rights reserved. ccepted Articl Table 2 Soluble phosphate content, siderophore and IAA productions, ACC deaminase activity, antagonism and antibiosis of rhizobacterial strains isolated from Z. mays var. purple amylaceum plants. Numbers in a column followed by the same letter are not significantly different according to Kruskal-Wallis test (n = 4; P ≤ 0.05) Siderophore Strain ACC deaminase Content of solubilized production IAA production activity phosphate (mg ml-1) (siderophores units (mg protein-1) (μmol α ketobutyrate μg protein-1) mg protein-1 h-1) Antagonism Antibiosis (% inhibits fungal (% inhibits fungal growth) growth) A1 7.3c 0.6a 1.2c n. d. 17.3a 30.7c A4 7.8c 11.6b 1.5d 2.3a 72.5b 23.0b A6 7.9c 9.4b 1.5d n. d. 17.3a 23.0b A8 7.8c n. d.* 1.1b n. d. 13.9a 51.5d A11 3.2a 47.2d 0.8a 21.8b 10.4a 53.4d A14 3.8a 17.9c 0.7a 463.6e 65.9b 73.1e A15 7.3c 6.1b 2.2c 71.4c 17.3a 11.5a A16 6.9c 37.5d 1.6d n. d. 17.3a 26.9b This article is protected by copyright. All rights reserved. ccepted Articl A18 7.0c A. brasilense C16 5.4b B. brevis BEA1 5.4b 124.0f *n. d., not detected. This article is protected by copyright. All rights reserved. 1.2c 1.7d 363.6d 20.8a 54.1d Table 3 Loading factors of N2-fixation, IAA production, ACC deaminase activity, mineral phosphate solubilization, siderophore production, antagonism and antibiosis as Accepted Article derived from the principal components analysis (PCA). The covariance (N-1) test was used to calculate F1 and F2 F1 F2 Mineral phosphate solubilization 6.457 4.854 ACC deaminase activity -21.653 31.734 Nitrogen fixation 40.650 16.264 Siderophore production 6.593 3.512 IAA production 9.555 -7.531 Antibiosis -8.985 -13.640 Antagonism 11.552 -6.846 This article is protected by copyright. All rights reserved. Table 4 Shoot dry weight, root dry weight, total carbon and total nitrogen of Z. mays var. purple amylaceum plants inoculated with different rhizobacterial strains. Plants Accepted Article were watered with half-strength mineral solution supplemented with 10 mM KNO3. Uninoculated plants treated with undiluted (treatment C2) or half-strength (treatment C1) mineral solution were used as a control. Numbers in a column followed by the same letter are not significantly different according to Kruskal-Wallis test (n = 4; P ≤ 0.05) Shoot dry Root dry weight (mg) weight (mg) A4 3.04b A14 Treatment Total C (%) Total N (%) 3.02c 40.07b 1.98d 4.72d 4.11c 40.13b 1.56b A15 3.90c 5.56d 40.63b 1.95d A18 9.59g 4.05c 40.90b 1.51b A4 + A14 7.75f 5.28d 40.97b 1.92c A4 + A15 4.95d 6.43e 42.23b 1.99d A4 + A18 7.03e 3.46c 42.63b 1.82c A15 + A18 6.68e 3.79c 40.93b 1.91c C1 1.18a 1.23a 39.27a 1.11a C2 2.75b 2.49b 40.13b 1.80c Supporting information legends Table S1 Soluble phosphate content and pH values in supernatants of rhizobacterial strains isolated from Z. mays var. purple amylaceum. Cells were grown in PVK medium supplemented with tricalcium phosphate for 14 d. Numbers in a column followed by the same letter are not significantly different according to Kruskal-Wallis test (n = 4; P ≤ 0.05). A. brasilense C16 was used as a control This article is protected by copyright. All rights reserved. Table S2 Biocontrol activity of rhizobacterial strains isolated from Z. mays var. purple amylaceum. Numbers in a column followed by the same letter are not significantly Accepted Article different according to Kruskal-Wallis test (n = 4; P ≤ 0.05). F. oxysporum grown in the absence of bacteria was used as a control This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved.