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

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).

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.

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 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 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.