Maize growth promotion by inoculation with Azospirillum brasilense and metabolites of Rhizobium tropici enriched on lipo-chitooligosaccharides (LCOs)

Marks et al. AMB Expr (2015) 5:71 DOI 10.1186/s13568-015-0154-z ORIGINAL ARTICLE Maize growth promotion byinoculation withAzospirillum brasilense andmetabolites ofRhizobium tropici enriched onlipo-chitooligosaccharides (LCOs) Bettina Berquó Marks1, Manuel Megías2, Francisco Javier Ollero2, Marco Antonio Nogueira1, Ricardo Silva Araujo3 and Mariangela Hungria1* Abstract There is an increasing interest in the development and use of inoculants carrying plant growth-promoting bacteria (PGPB) in crops of agronomic interest. The great majority of the inoculants commercialized worldwide contain rhizobia for legume crops, but the use of PGPB as Azospirillum spp. for non-legume is expanding, as well as of inoculants combining microorganisms and microbial metabolites. In this study we evaluated the effects of inoculants containing Azospirillum brasilense with or without metabolites of Rhizobium tropici strain CIAT 899 highly enriched in lipo-chitooli- gosaccharides (LCOs) in six field experiments performed for three summer crop seasons in Brazil with maize (Zea mays L.). Inoculants and metabolites were applied either at sowing by seed inoculation, or by leaf spray at the V3 stage of plant growth. Improvement in shoot dry weight (SDW) and total N accumulated in shoots (TNS) by single, but especially by dual inoculation was observed in some of the experiments. Statistically significant increases in grain yield in relation to the non-inoculated control were observed in five out of six experiments when maize was inoculated with Azospirillum supplied with enriched metabolites of R. tropici applied by seed or leaf spray inoculation. The results give strength to the development of a new generation of inoculants carrying microorganisms and microbial molecules. Keywords: Nod factor, Zea mays, Inoculant, PGPB © 2015 Marks et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Introduction Inoculation of crops of agronomic interest with plant growth-promoting bacteria (PGPB)—especially those belonging to the group of rhizobia associated with legumes—represents a biotechnological practice consolidated worldwide (Bashan etal. 2014). In Brazil, for example, about 25 million doses of inoculants are commercialized every year, 95% of which for the soybean (Glycine max [L.] Merr.) crop (Hungria and Mendes 2015). The symbiotic interaction between rhizobia and the host legumes to establish the biological nitrogen fixation process involves an intense exchange of signals between the partners. The dialogue starts with the exudation of molecules by the plant—especially flavonoids—which act as signals to the rhizobia (Hungria etal. 1992; Hungria and Phillips 1993; de Rijke etal. 2006). The interaction occurs by means of a key protein in rhizobia—NodD— launching the expression of all other bacterial nodulation genes; in the following step, rhizobia reply with the synthesis and secretion of Nod Factors (Phillips 2000). Nod Factors are lipo-chitooligosaccharides (LCOs), which may comprise up to 60 different structural arrangements (D’Haeze and Holsters 2002), depending on the bacterial species and the environmental conditions (Folch-Mallol etal. 1996; Debellé etal. 2003; Estévez etal. 2009; del Cerro etal. 2015a, b). LCOs act directly in root colonization and cortex cell division (Spaink etal. 1998; Dardanelli etal. 2008). Open Access *Correspondence: [email protected]; biotecnologia.solo@ hotmail.com; [email protected] 1 Embrapa Soja, C.P. 231, Londrina, Paraná 86001-970, Brazil Full list of author information is available at the end of the article Page 2 of 11 Marks et al. AMB Expr (2015) 5:71 Intriguingly, it has been reported that LCOs can also promote growth of non-leguminous plants, one possible explanation being because they mimic the effects of plant hormones such as cytokinins and auxins (Rélic etal. 1993), resulting in increased seed germination and resistance to pathogens (Miransari and Smith 2009). With the current knowledge about the effects of LCOs, a new generation of inoculants based on, or enriched with LCOs has proven to be very effective with legumes, and is now advancing to the use with non-legumes (Marks etal. 2013; Smith etal. 2015). The technology of inoculation of non-legumes with non-symbiotic PGPB—whose main representative is Azospirillum spp.—is also being increasingly adopted in several countries, especially for crops such as maize (Zea mays L.) and wheat (Triticum aestivum L.) (Díaz- Zorita and Fernandez-Canigia 2009; Hartmann and Bashan 2009; Smith etal. 2015). In Brazil, inoculants containing Azospirillum brasilense strains Ab-V5 and Ab-V6 were exponentially employed by farmers in the past 5years for maize and wheat (Hungria etal. 2010; Hungria 2011). More recently, co-inoculation of soybean with rhizobia and azospirilla has also been adopted as an agronomic practice by several farmers, with reported positive effects on nodulation precocity (Chibeba etal. 2015) and increases in grain yield (Hungria etal. 2013, 2015b); positive effects on common bean (Phaseolus vulgaris L.) yield have also been reported (Hungria etal. 2013). Among the benefits of inoculation with non-symbiotic PGPB, the contributions of biological nitrogen fixation (Ashraf etal. 2011), production of phytohormones (Strzelczyk etal. 1994), phosphate solubilization (Rodriguez et al. 2004) and control of plant pathogens (Araujo etal. 2005; Wang etal. 2009) are commonly cited. Studies of the application of LCOs associated with Azospirillum to cereals crops are still incipient. Our research group has previously reported that the addition of concentrated metabolites (CM) from two strains of rhizobia containing LCOs resulted in significant increases in maize grain yield (Marks et al. 2013). Rhizobium tropici is a very interesting species that synthesizes a wide variety of LCOs, even in the absence of plant inducers (Estévez etal. 2009; del Cerro etal. 2015a, b), making it an interesting bacterium for metabolite production. In this study, metabolites of R. tropici strain CIAT 899 highly enriched in LCOs were obtained and applied along with A. brasilense in six field experiments performed in 3years, aiming at getting a better understanding of the combined effects Azospirillum and rhizobial molecules on the growth and yield of cereals. Materials andmethods Inoculant andlipo‑chitooligosaccharides (LCOs) preparation Liquid inoculants were prepared with A. brasilense strains CNPSo 2083 (=Ab-V5) and CNPSo 2084 (=Ab- V6). Strains are deposited in the Diazotrophic and Plant Growth Promoting Bacteria Culture Collection of Embrapa Soja (WFCC Collection #1213, WDCM Collection #1054). These two elite strains were identified in a previous selection program for the maize and wheat crops (Hungria etal. 2010; Hungria 2011) and are used in commercial inoculants in Brazil. Inoculant concentration was determined by spread-plating on NFb (Hungria and Araujo 1994; Döbereiner etal. 1995) and RC (Cassán etal. 2010) solid media and adjusted to the concentration of 2×108 CFU (colony forming units) mL−1 in all three crop seasons. Metabolites of R. tropici strain CIAT 899 enriched in LCOs were produced with a combination of procedures. Bacterium growth and extraction of the supernatant n-butanol were performed as described by Sanjuan et al. (1992). Purification was accomplished by solidphase chromatography, with SPE C18 Resprep, Teknokroma column, concentration and lyophilization were performed as described by Soria-Díaz etal. (2003) and Guasch-Vidal (2011). Prior to sowing, lyophilized metabolites of R. tropici CIAT 899 were re-suspended in a mixture of acetonitrile and water (20%). The concentration was adjusted to 0.1mLL−1, corresponding to approximately 10−9 M. The metabolites were added to the inoculant containing Azospirillum at the time of inoculation, either when applied to the seeds, or by spraying. Field experiments Sites description Six field experiments were conducted over a 3-year period, always in the summer crop season. Two experiments were conducted in 2012/2013, in Ponta Grossa, State of Paraná (southern region) and Cachoeira Dourada, State of Goiás (central-western region), two others in 2013/2014, in Rio Verde, State of Goiás (central-western region), and Maracaí, State of São Paulo (southeastern region), and two others in the crop season of 2014/2015, in Londrina, State of Paraná (southern region) and Ponta Grossa. Ponta Grossa (25°13′S, 50°1′W) is at 880m of altitude and has a Köppen-Geiger climate type Cfb (temperate with mild summer). Cachoeira Dourada (18°29′S; 49°28′W) is at 450m of altitude and has a climate type Aw (tropical with dry season in the winter). Rio Verde (17°47′S; 50°54′W) is at 730m altitude and has a climate type Aw, Maracaí (22°36′S; 50°40′O) is at 475m and has a climate type Cfa (tropical humid with warm summer) Page 3 of 11 Marks et al. AMB Expr (2015) 5:71 and Londrina (23°11′S, 51°11′W) is at 620 m altitude and has a climate type Cfa. The trials were performed on soils classified as Latossolo Vermelho Distrófico (Brazilian classification) (Typic Hapludox, USA Soil Taxonomy) (Ponta Grossa, Cachoeira Dourada, Rio Verde, Maracaí), and Latossolo Vermelho Eutroférrico (Brazilian classification) (Rhodic Eutrudox, USA Soil Taxonomy) in Londrina. At each site, 2 months before the experiments were established twenty soil samples (0–20 cm depth) were taken to evaluate chemical properties, granulometry and biological properties. For chemical analyses, the samples were previously dried (60°C for 48h), sieved (2mm), and analyzed as described before; soil granulometry was also analyzed as described before (Hungria etal. 2010; Hungria etal. 2015a). Population of free-living diazotrophic bacteria was estimated by the NMP method with dilutions in NFb semi-solid medium (Hungria and Araujo 1994; Döbereiner etal. 1995). Soil properties are shown in Table1. About 50days before starting the experiment, lime was applied to alleviate acidity when necessary, based on soil pH values. The amount of lime applied was estimated for a base saturation of 50%, to increase the pH to 5.5 or higher. Treatments, experimental design andfield management The maize hybrids used in the experiments were DOW 2B 707 HX (Dow AgroSciences) in 2012/2013, DKB-350-PRÓ (Dekalb) in 2013/2014, and DKB-350- PRÓ2 (Dekalb) in 2014/2015. Seeds were not surface disinfected. Two methods for the inoculation with A. brasilense strains CNPSo 2083 and CNPSo 2084 were tested. The first method consisted of seed inoculation at sowing and the second of leaf spray with the same inoculant at the V3 stage (third leave developed). The experiments consisted of five treatments: (1) noninoculated control (NI); (2) seed inoculation (SI) with Azospirillum at sowing; (3) SI + metabolites enriched with LCOs applied at sowing; (4) leaf spray inoculation (LSI) with Azospirillum strains at V3 stage; (5) LSI+enriched metabolites at the V3 stage. Seed inoculation was performed at a rate of 100mL 20kg−1 of seeds (2×108 CFUmL−1) while the spray inoculation was applied at a rate of 200mLha−1 (2×108 CFUmL−1) diluted in 100 L of water; 20kg of seeds give rise to a population of about 60,000 plants ha−1. Enriched metabolites were prepared at a concentration of 0.1mL L−1 and mixed with the inoculant before application at a rate of 50mL 20kg−1 of seeds when applied to the seeds and of 100mLha−1 when sprayed. As mentioned above, the main objective of our study was to verify the plant growth-promoting activity of the enriched bacterial metabolites. Therefore, all treatments received the same fertilization, consisting of 300kgha−1 of a formulation of 08-20-20 (corresponding to 24kg of N, 60kg of P and 60kg of Kha−1) at sowing and a topdressing fertilization at the V4 stage (four fully developed leaves), representing 75% of the usual dose of N-fertilizer recommended for the crop in Brazil, corresponding to 90kgNha−1 of urea (67.5kgNha−1). Plots measured 4.5m (width)×8m (length) (=27m2), with rows spaced by 0.9m and plots were separated by 2m terraces to prevent contamination by superficial runoff containing bacteria, metabolites or fertilizers. The experiments were set in a complete randomized block design with six replicates. Cultural and phytosanitary managements followed the technical recommendations for the maize crop (Embrapa 2011). The experiments were not irrigated. Plant sampling, harvesting andanalyses Between 29 and 57days after sowing (DAS), depending on the climatic conditions, five plants were randomly collected from each plot to evaluate the performance at the vegetative growth. Shoots were washed and dried to constant weight at 50°C for evaluation of shoot dry weight (SDW). Shoots were then ground (20mesh) and total N was determined by sulfuric digestion followed by semimicro Kjeldahl distillation method, as described before (Hungria etal. 2015a). At the time of physiological maturity, plant height (PH) was determined based on the average of six plants, and plant population was also estimated. Grain yields (kgha−1) were determined by harvesting a 6.3m2 area (0.9m wide×7m long) from the central portion of each plot. Grains were cleaned and weighed, the humidity evaluated in a grain moisture tester and the content corrected to 13% moisture. In 2013/2014 and 2014/2015 the N content of seeds was also determined, as described for shoots. It is worth mentioning that all field experiments were performed according to the Brazilian legislation required for the registration of commercial inoculants or technologies of inoculation for plant growth-promoting bacteria (MAPA 2011). Statistical analyses Data from each experiment were first submitted to tests of normality and homogeneity of variances for each variable and then to the analysis of variance (ANOVA). When significant differences were detected by the F test, Duncan’s multiple-range test at p≤0.05 and 0.10 (for inoculant products the Brazilian legislation accepts p≤0.10; MAPA 2011) was used as a multiple comparisons procedure. Page 4 of 11 Marks et al. AMB Expr (2015) 5:71 Table 1 Soil chemical properties, granulometry andpopulation offree-living diazotrophic bacteria atthe 0–20cm layer ofthe field sites beforesowing SB sum of bases, BS bases saturation=[(K+Ca+Mg)/Tcec]×100, where Tcec=K+Ca+Mg+total acidity at pH 7.0 (H+Al) a Estimated by the most probable number method by dilutions and counts in semi-solid NFb medium Site pH (CaCl2) Al (cmolc dm−3)H+Al (cmolc dm−3)K (cmolc dm−3)Ca (cmolc dm−3)Mg (cmolc dm−3)P (g dm−3) C (g dm−3) SB (cmolc dm−3)BS Granulometry (%) MPNa %Clay Silt Sand NMP soil g−1 2012/2013 Ponta Grossa 4.60 0.26 3.07 0.37 3.55 1.73 1.71 18.55 5.65 64.79 58.4 15.7 25.9 1.5 × 104 Cachoeira Dourada 5.40 0.00 7.89 0.15 2.02 1.30 0.80 30.50 3.47 30.54 57.8 18.2 24.0 4.5 × 106 2013/2014 Rio Verde 5.02 0.10 3.03 0.17 3.46 0.94 2.45 29.43 4.57 60.13 36.3 9.6 54.1 2.5 × 106 Maracaí 5.42 0.00 1.12 0.05 1.20 0.34 6.57 4.88 1.59 58.67 8.8 0.8 90.4 1.5 × 106 2014/2015 Londrina 5.20 0.00 3.67 0.50 3.91 1.75 11.10 11.10 6.16 62.66 71.0 8.2 20.8 9.0 × 106 Ponta Grossa 4.90 0.07 4.10 0.44 2.95 1.06 25.70 18.00 4.45 52.05 23.8 3.0 73.2 4.5 × 105 Page 5 of 11 Marks et al. AMB Expr (2015) 5:71 Results In the 2012/2013 crop season, in Ponta Grossa, grain yield of maize plants inoculated with A. brasilense and supplied with enriched metabolites either at sowing or at the V3 stage was significantly higher than the other treatments (Table2). In Cachoeira Dourada, shoot dry weight (SDW) and total N accumulated in shoots (TNS) were significantly increased when Azospirillum was inoculated on the seeds (Table2). The supplementation with enriched metabolites associated to seed inoculation with Azospirillum seemed to improve the same parameters at this site, even though this treatment did not show significantly higher SDW than the other treatments (Table2). In addition, in Cachoeira Dourada both treatments with Azospirillum inoculated on seeds along with enriched metabolites, and Azospirillum-inoculated by leaf spray added of enriched metabolites promoted higher yield than the other treatments (Table2). In 2013/2014, in Rio Verde, the supplementation of both treatments that received Azospirillum inoculation, either on seeds or by leaf spray, with enriched metabolites resulted in significant increases in the N content of the grains (TNG, Table3). In Maracaí, the best performance was achieved again in the treatment pulverized with A. brasilense supplied with enriched metabolites, resulting in greater yield, values of accumulation of N in grains (NG) and TNG, in general statistically higher than all other treatments (Table3). In Londrina, in 2014/2015, leaf spray with Azospirillum resulted in significant increases in SDW relative to the non-inoculated controls, either in the presence or in absence of enriched metabolites (Table4). Seed inoculation resulted in increased grain yield relative to the non-inoculated control, but no further increases were observed when metabolites were added; in contrast, leaf spray inoculation only resulted in yield increases when supplemented with enriched metabolites (Table 4). In Ponta Grossa, the addition of the enriched metabolites to both inoculation with Azospirillum by seeds or leaf spray resulted in increased grain yield when compared to the non-inoculated control and to the treatments inoculated only with Azospirillum (Table4). Discussion One of the main goals of new biotechnological products is to reduce the agricultural utilization of pesticides and/or chemical fertilizers, providing higher sustainability associated with enhanced environmental quality (Hameeda etal. 2006). In this study, we observed that when maize seeds were inoculated with A. brasilense strains CNPSo 2083 and CNPSo 2084, there were no increases in grain yield in the leaf spray treatment. When Azospirillum was applied on seeds, statistically significant increases were observed in two out of six experiments. Although this percentage is lower than usually reported (Okon and Labandera-Gonzalez 1994; Díaz-Zorita and Table 2 Effect ofAzospirillum brasilense strains CNPSo 2083 andCNPSo 2084 andof enriched metabolites ofR. tropici strain CIAT 899 applied tothe seeds atsowing or byleaf spray atthe V3 stage onplant growth (shoot dry weight, SDW; plant height, PH), shoot N (content [NS] andtotal N accumulated inshoots [TNS]) at57 and51days aftersowing (DAS), andgrain (yield) atthe physiological maturity ofmaize hybrid DOW 2B 707 HX Field experiments performed in Ponta Grossa and Cachoeira Dourada, Brazil, in the summer crop season of 2012/2013. Means (n=6) on the same column which are followed by different letters are significantly different (p≤0.10, Duncan test) n.s. statistically non-significant Treatment Ponta Grossa Cachoeira Dourada 57 DAS Maturity 51 DAS Maturity SDW (g pl−1) PH (cm) NS (gkg−1) TNS (mgN pl−1)Yield (kgha−1)SDW (g pl−1) PH (cm) N (gkg−1) TNS (mgN pl−1)Yield (kgha−1) Non-inoculated control 56.1ns 252ns 23.04ns 1292ns 8406 b 33.1 c 234ns 21.16ns 700 b 6310 b Seed inoculated (Azospirillum)58.3 260 23.86 1391 8850 b 44.1 a 242 21.00 926 a 6567 b Seed inoculated (Azospirillum + enriched metabolites) 52.7 253 22.64 1193 9225 a 40.8 ab 243 21.94 895 a 7373 a Leaf spray inoculation (Azospirillum)55.0 258 22.45 1235 8567 b 36.9 bc 241 21.10 779 b 6543 b Leaf spray inoculation (Azospirillum + enriched metabolites) 55.2 256 22.71 1254 9256 a 36.4 bc 238 21.06 766 b 7286 a Page 6 of 11 Marks et al. AMB Expr (2015) 5:71 Table 3 Effect ofAzospirillum brasilense strains CNPSo 2083 andCNPSo 2084 andof enriched metabolites ofR. tropici strain CIAT 899 enriched inLCOs applied tothe seeds atsowing or byleaf spray atthe V3 stage onplant growth (shoot dry weight, SDW; plant height, PH), shoot N (content [NS] andtotal N accumulated inshoots [TNS]) at32 and42days aftersowing (DAS), andgrain parameters (yield, N content ingrains [NG] andtotal N accumulated ingrains [TNG]) inmaize hybrid DKB- 350-PRÓ Field experiments performed in Rio Verde and Maracaí in the summer crop season of 2013/2014. Means (n=6) on the same column which are followed by different letters are significantly different (p≤0.10, Duncan test) n.s statistically non-significant Treatment Rio Verde Maracaí 32 DAS Maturity 42 DAS Maturity SDW (g pl−1)PH (cm) NS (gkg−1) TNS (mgN pl−1)Yield (kgha−1)NG (gkg−1) TNG (mgN pl−1)SDW (g pl−1)PH (cm) N (gkg−1) TNS (mgN pl−1)Yield (kgha−1)NG (gkg−1) TNG (mgN pl−1) Non-inoculated control 13.0ns 176ns 25.27ns 328ns 6465ns 11.77ns 76 b 40.3ns 153ns 22.49ns 806 b 3091 d 14.13 bc 44 c Seed inoculated (Azospirillum)12.1 174 26.74 324 6758 11.23 76 b 40.4 149 23.41 946 a 3486 bc 15.41 ab 54 b Seed inoculated (Azospirillum + enriched metabolites) 11.9 175 26.81 320 6769 13.93 94 a 40.8 151 23.35 953 a 3698 ab 14.41 abc 53 b Leaf spray inoculation (Azospirillum)11.8 176 28.08 331 6621 11.12 74 b 40.7 150 23.31 948 a 3278 cd 13.59 c 44 c Leaf spray inoculation (Azospirillum + enriched metabolites) 11.9 174 28.18 335 6792 13.92 94 a 43.7 149 23.39 1022 a 3958 a 15.79 a 62 a Page 7 of 11 Marks et al. AMB Expr (2015) 5:71 Table 4 Effect ofAzospirillum brasilense strains CNPSo 2083 andCNPSo 2084 andof metabolites ofR. tropici strain CIAT 899 enriched withLCOs applied tothe seeds atsowing or byleaf spray atthe V3 stage onplant growth (shoot dry weight, SDW; plant height, PH), shoot N (content [NS] andtotal N accumulated inshoots [TNS]) at32 and42days aftersowing (DAS), andgrain parameters (yield, N content ingrains [NG] andtotal N accumulated ingrains [TNG]) inmaize hybrid DKB- 350-PRÓ2 Field experiments performed in Londrina and Ponta Grossa in the summer crop season of 2014/2015. Means (n=6) on the same column which are followed by different letters are significantly different (p≤0.10, Duncan test) n.s statistically non-significant Treatment Londrina Ponta Grossa 29 DAS Maturity 53 DAS Maturity SDW (g pl−1) PH (cm) NS (gkg−1) TNS (mgN pl−1)Yield (kgha−1)NG (gkg−1) TNG (mgN pl−1)SDW (g pl−1)PH (cm) NS (gkg−1) TNS (mgN pl−1)Yield (kgha−1)NG (gkg−1) TNG (mgN pl−1) Non-inoculated control 7.5 b 233ns 31.78ns 238ns 8624 b 15.47ns 133 c 42.2ns 248ns 30.74ns 1297ns 7723 b 11.86ns 92 ns Seed inoculated (Azospirillum)7.9 ab 236 31.32 247 9472 a 15.76 149 abc 41.0 246 30.70 1259 7632 b 12.17 93 Seed inoculated (Azospirillum + enriched metabolites) 7.8 ab 239 32.17 251 9581 a 15.78 151 ab 42.2 249 30.33 1280 8453 a 12.18 103 Leaf spray inoculation (Azospirillum)8.3 a 228 32.32 268 8815 b 15.43 136 bc 40.8 248 30.91 1261 7426 b 12.31 91 Leaf spray inoculation (Azospirillum + enriched metabolites) 8.3 a 238 32.43 269 9426 a 16.50 156 a 45.9 256 30.47 1398 8250 a 12.11 100 Page 8 of 11 Marks et al. AMB Expr (2015) 5:71 Fernandez-Canigia 2009; Hungria etal. 2010), increments in yield were observed in all trials, and when a combined analysis was performed, there was a statistically significant gain of 358kgha−1 in relation to the non-inoculated treatment. However, when the Azospirillum inoculant was supplemented with LCO-enriched metabolites from R. tropici strain CIAT 899, either by seed inoculation or by leaf spray, statistically significant increases in grain yield in comparison to the non-inoculated control were observed in five out of six field experiments, and when compared to the single inoculation with Azospirillum, in three and five out of six experiments for seeds and leaf spray, respectively. The beneficial relationships between PGPB such as Azospirillum, and several plant species have been previously described (Okon, and Labandera-Gonzalez 1994; Bashan and de Bashan 2010; Cassán etal. 2013). Field experiments have shown increases in grain yield ranging from 5 to 75% (Okon and Labandera-Gonzalez 1994; Fuentes-Ramirez and Caballero-Mellado 2005; Castro- Sowinski etal. 2007; Rodrigues etal. 2008; Hungria etal. 2010). These increases are commonly attributed to root growth promotion, accomplished by phytohormones produced by the bacterium, with an emphasis on indole acetic acid, gibberellins and cytokinins (Tien etal. 1979). Moreover, it is inferred that the application of Azospirillum is also responsible for higher rates of absorption of water and minerals by the plant (Okon, and Kapulnik 1986; Dardanelli etal. 2008) and higher tolerance to abiotic stresses, such as drought and salinity (Cassán etal. 2009; Zawonski etal. 2011). The relationship between different soil microorganisms and the role of metabolites secreted by them on growth of other surrounding microbial species and plants has been the subject of numerous studies. For example, Massoud etal. (2009) studied the effects of the combined inoculation of mycorrhizal fungi, Bacillus circulans, Rhizobium sp., Azospirillum lipoferum, Azotobacter chroococcum and mineral rocks on common bean (Phaseolus vulgaris L.) plants. The inoculum consortium promoted higher nitrogenase activity and increased the availability of macronutrients, besides promoting plant growth, resulting in increased yield in comparison to the single inoculation (Massoud etal. 2009). The authors attributed these results at least partially to the exudation of beneficial molecules by the microorganisms (Massoud etal. 2009). The positive effects of molecules such as LCOs, exopolysaccharides (EPSs), and plant hormones on plant growth (hosts or non-hosts) may be associated with increased survival and capacity of plant infection by beneficial rhizospheric bacteria and fungi and/or with plant growth promotion (Marks etal. 2013). In a study with the legume model Medicago truncatula, application of LCOs of Sinorhizobium meliloti facilitated root infection by mycorrhizal fungi and stimulated lateral root hair development (Olah etal. 2005). It is possible that LCOs, although produced by rhizobia, have a direct influence on the rhizospheric microbial community by influencing interactions among microorganisms and promoting plant growth, including growth of non-host plants. The LCOs secreted by rhizobia are described as responsible for several physiological modifications in the root hairs of legumes. Such changes include alterations in ion flux, membrane depolarization of root cells, intra and extracellular alkalization, synthesis of phosphatidic acid and diacylglycerol, accumulation of reactive oxygen species, root hair deformations involving changes in actin cytoskeleton, cell division activation and induction of the expression of genes involved in nodulation (Mulder etal. 2006; Cooper 2007). All these changes allow the rhizobia to invade the host plant, leading to the formation of nodule primordia (Riely etal. 2004), and therefore, LCOs would behave as mitogenic and morphogenic agents (Rélic etal. 1993). However, intriguingly, LCOs have also been described as growth regulators of a wide variety of non-leguminous plants (Prithiviraj etal. 2003), inducing various physiological responses (Souleimanov etal. 2002), as increased seed germination, lateral root development and nutrient uptake (Smith etal. 2015). The study by Rélic etal. (1993) supports the hypothesis that the LCOs may act as plant hormones when applied to non-host plants. Previous studies with cells and plants of tobacco (Nicotiana sp.) (Baier etal. 1999), tomato (Solanum lycopersicum L.) (Staehelin etal. 1994) and carrot (Dacus carota L.) (De Jong etal. 1993) have shown that LCOs are activators of cell division and embryonic development of non-host plants. Marks etal. (2013) observed, in previous field experiments, an 11.4% increase in the grain yield of maize inoculated with the same strains of A. brasilense and supplied with concentrated metabolites of R. tropici that included LCOs. Although the mechanisms responsible for the benefits of LCOs in non-leguminous are not fully understood, the application of such molecules must somehow modify the hormone balance, affecting plant growth and development (Souleimanov etal. 2002). The most effective contribution of LCOs to non-leguminous plants might be the stimulation of root development, increasing the absorption of water and nutrients and resulting in improved plant growth and yields (Smith etal. 2015). In the field experiments performed in our study, the application of LCOs-rich rhizobial metabolites seems to have affected the N metabolism, increasing the N content of shoots and grains, and also influencing grain yield. Our results have also shown that the application of LCOs-rich rhizobial metabolites by leaf spray resulted Page 9 of 11 Marks et al. AMB Expr (2015) 5:71 in higher grain yields. Khan (2003) also observed that the leaf application of LCOs in maize stimulated photosynthesis, increased leaf area and shoot dry weight. In another study, Chen et al. (2006) applied LCOs of Bradyrhizobium japonicum to tomato leaves and observed the anticipation of flowering and fruiting and an increase in the number and weight of fruits under greenhouse conditions, as well as a 30% increase in the number and fruit weight in a field experiment. The benefits of LCOs leaf spray can be attributed to the fact that these molecules indirectly affect the photosynthesis and accelerate growth, probably by the stimulation of mitotic activity in meristematic tissue of leaves (Khan etal. 2008). It can also be inferred that the foliar application of LCOs promotes the suppression of innate immune responses, which possibly facilitates the microbial interactions, such as the invasion and colonization by endophytic bacteria (Liang etal. 2013). The rationale of the utilization of metabolites of R. tropici CIAT 899 enriched on LCOs was based on some interesting properties of this strain, which produces a broad variety of LCOs, even in the absence of inducing flavonoids, when subjected to abiotic stresses such as acidity (Morón etal. 2005) and salinity (Estévez etal. 2009; del Cerro etal. 2015a, b). R. tropici carries five copies of nod gene (Ormeño-Orrillo etal. 2012) and, recently, the synthesis of several LCOs structures related to nodD1, nodD2, nodD3, nodD4 and nodD5 genes has been elucidated (del Cerro etal. 2015a, b). The production of a large variety of LCOs by R. tropici CIAT 899 may represent a strategy for nodulation of several host plants under various environmental stressful conditions (Liang etal. 2013; del Cerro etal. 2015a, b). Consequently, it is possible that these LCOs also favor the systemic resistance, particularly in leaf spray, giving greater vigor to the plants, and resulting in increases in crops yields. Therefore, it is likely that LCOs have a broad spectrum of action in regulating plant growth, in addition to its primary function in nodulation of legumes. The results of our study reveal the biotechnological potential of adding microbial metabolites, in our case rhizobial metabolites enriched with LCOs to products for leaf spray and seed inoculation of non-leguminous plants, such as maize. This knowledge can be applied to the improvement of commercial products, taking into account the need for developing a new generation of inoculants carrying microorganisms and microbial metabolites. Authors’ contributions Idealized the experiments: MH, MM, FJO, MAN. Performed the experiments: MH, MAN. Analyzed the data: BBM, MM, FJO, MAN, RSA, MH. Wrote the manuscript: BBM, MH, RSA. All authors read and approved the final manuscript. Author details 1 Embrapa Soja, C.P. 231, Londrina, Paraná 86001-970, Brazil. 2 Depto de Microbiología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, Apdo Postal 41012 Seville, Spain. 3 Total Biotecnologia Indústria e Comércio S/A, Rua Emílio Romani 1190, CIC, Curitiba, Paraná 81460-020, Brazil. Acknowledgements This study was partially supported by Embrapa (02.13.08.001.00.00), Total Biotecnologia Indústria & Comércio S/A and CNPq (National Council for Scientific and Technological Development, Project Science without Borders # 400205/2012-5). B.B. Marks acknowledges a PhD fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil). M. Hungria and M.A. Nogueira are also research fellows from CNPq. Approved for publication by the Editorial Board of Embrapa Soja as manuscript 285/2015. Competing interests The authors declare that they have no competing interests. Received: 16 September 2015 Accepted: 17 October 2015 References Araujo FF, Henning AA, Hungria M (2005) Phytohormones and antibiotics produced by Bacillus subtilis and their effects on seed pathogenic fungi and on soybean root development. 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