Materials and methods

Sampling site. Seven drought-tolerant homozygous maize lines (CLQRQ108, CML384, CML445, CML544, DTMA90, DTMA224, DTMA256) and seven drought-susceptible homozygous maize lines (CML181, DTMA34, DTMA41, DTMA43, DTMA109, DTMA144, DTMA182) were sown, which were selected because they were inbred due to their uniform response to drought. Each line was sown with three replications at the experiment stations of the International Maize and Wheat Improvement Center (CIMMYT) in Tlaltizapán, Morelos (18.68 N; 99.11 O), El Batán, Texcoco, Mexico (19.53 N; 98.85 W), and Agua Fría, Puebla (20.5 N; 97.6 W) during the 2012 summer crop cycle (V-2012), 2012 winter cycle (I-2012) and 2013 autumn cycle (V-2013). Samples of the internal tissue formed in the apical root area of three plants per line were taken for each location and crop cycle 25, 52 and 75 days after sowing (dds) to be analyzed at CIMMYT’s Maize Pathology Laboratory at El Batán, State of Mexico. The size of the sample was determined by the method proposed by ^{Cochran (1982)}.

Isolation of endophytic root bacteria. Bacterial isolates from the internal tissue of maize roots that had been superficially sterilized were defined as endophytic strains. Symptomless maize roots were washed with sterile distilled water and cut in 2-cm pieces. 10 g of roots were sterilized superficially for 5 min in sterile glass jars and washed with 70% ethanol for 5 min, 0.53% sodium hypochlorite for 10 min and then rinsed three times with sterile distilled water. After the last washing with sterile distilled water, 100 µL from each jar were sown in Petri dishes containing NB culture medium (1L): 15 g bacteriological agar (BIOXON^® Mexico), 5 g peptone (BD DIFCO^TM USA), 3 g yeast extract (DIBICO^® Mexico, NaCl JT BAKER^® Mexico) and incubated at 28±1oC from 2 to 5 days. The absence of bacterial growth in the culture medium confirmed that the root surface had been efficiently sterilized. The roots were ground in cold mortars using 20 mL of a sterile buffer solution (50 mM KH₂PO₄, 150 mM NaCl, pH 7.6). The resulting suspensions were diluted in series (1:9) from 100 to 10-3; then 100 µL of each dilution and three replications were sown in NB medium and incubated at 28 ºC for 24 h under continuous light. The different bacterial morphotypes were counted and classified according to their color, shape, texture and type of growth. The bacterial growth was considered representative of cultivable endophytic bacteria in maize roots. Bacterial strains were preserved by freezing in NB medium and 20% glycerol for subsequent studies.

Population density of endophytic bacteria. The population density of bacteria per root tissue was estimated by directly counting the colonies on the plate. The number of bacterial colonies isolated from the 14 maize lines was counted using a colony counter (Quebec^®, Darkfield Colony Counter). The bacterial population of root tissue samples was expressed in terms of colony forming units (UFC g-1 of root tissue). The resulting data were converted to _Log10 UFC g^-1 of root and an analysis of variance (ANOVA) was performed using a factorial analysis design to separate the media by DMS (α=0,05) using the SAS statistical program (Statistical Analysis System version 9.1.3 SAS Institute, Cary, NC, USA) according to the crop cycle, location, days after sowing (dds) and the studied drought-tolerant or drought-susceptible maize lines.

Amplification of the 16S rDNA gene and identification of endophytic bacteria. Bacterial DNA was extracted using the protocol described by ^{Mahuku (2004)} with the following modifications: bacterial cells were obtained by growing them in NB medium at 28 °C for 48 h. After suspending the precipitate in 100 µL of 1X TE, 2 µL of RNAsa were added (1 mg mL-1) and then incubated in a water bath at 37 °C for 1 h. The DNA quality was verified by electrophoresis in agarose gel. Bacteria were identified by partially amplifying the 16S rADN gene using universal primers 27F (5´AGAGTTTGATCMTGGCTCAG-3´) and 1492R (5´TACGGHTACCTTGTTACGACTT-3´) under the PCR conditions described by ^{Galkiewicz and Kellogg (2008)}. DNA amplification and sequencing were carried out using Macrogen (DNA Sequency Service. Korean Biotechnology Company), and the sequences obtained were aligned with sequences kept at the GenBank of (NCBI) using the BLASTn program (http://www.ncbi.nlm.nih.gov/BLAST/).

Results and discussion

Taxonomic identification of endophytic bacteria. Through partial sequencing of the 16S rADN gene, 22 endophytic bacteria species were identified in roots of both groups of maize lines. The greatest abundance of bacteria was associated with the Proteobacteria phyla followed by Firmicutes and Bacteroidetes. Within Firmicutes, 100% of strains were identified as Bacillus sp., and within the class Gammaproteobacteria, 33% were identified as Pseudomonas sp.

The diversity and richness of bacteria naturally associated with maize roots is wide, and their estimation depends on the analytic method used. In this study, a dependent culture approach was used, and the results coincide with previous studies that found that the Proteobacteria phylum was the most predominant of the endophytic bacterial populations isolated from maize roots, stems and leaves (^{Rosenblueth and Martínez-Romero, 2006}). Other studies of the diversity of endophytic communities in maize roots using dependent and independent culture methods also found that the bacteria most frequently associated with this crop are Firmicutes (Bacillus), Gammaproteobacteria (Pseudomonas) (^{Pereira et al., 2011}) and Burkholderia spp. (^{Ikeda et al., 2013}). By analyzing these populations using gas chromatography and fatty acid profiles, Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens were identified as the most predominant species in maize stalks (^{Rai et al., 2007}), but when using pyrosequencing, the Proteobacteria, Bacteroidetes and Actinobacteria phyla were identified as being the most abundant in the maize rhizosphere (^{Li et al., 2014}).

Maize research has shown that these populations could be used as bacterial inoculants for controlling drought stress, and suggests that drought tolerance is induced by the production of phytohormones such as abscisic, gibberellic and indole-3-acetic acids, cytokinins, enzymes such as ACC deaminase, production of bacterial exopolysaccharides and systemic tolerance induction (^{Dimkpa et al., 2009}; ^{Kim et al., 2012}; ^{Timmusk et al., 2014}; ^{Yang et al., 2009}). In other crops, they have been identified as tolerance inductors in wheat (Triticum sativum) and legumes (Vigna radiata) when they were inoculated in seed, for they promoted an increase in the levels of regulation of genes related to drought tolerance and the activity of diverse enzymes (^{Kasim et al., 2013}; ^{Saravanakumar et al., 2011}).

Based on the frequency of the isolates, three groups of endophytic bacteria in the roots of drought tolerant and drought susceptible maize stand out in this study: 1) highly frequent (Bacillus subtilis, Bacillus megaterium and Pseudomonas geniculata), isolated during the three crop cycles at the three locations, 2) frequent (Bacillus firmus, Pseudomonas hibiscola and Sinorhizobium meliloti), isolated during the three crop cycles at two locations, and 3) low frequency (Acinetobacter soli, Stenotrophomonas maltophila and Burkholderia gladioli), isolated only at two locations and not in all crop cycles (Table 1).

The taxonomic and functional structure of bacterial communities in soil is influenced by biotic and abiotic factors such as soil physical and chemical characteristics, weather conditions, plant genotype and interaction with other soil prokaryotes and eukaryotes, a fact that suggests that those interactions are complex. In this study, the structure and large number of isolated bacteria may be associated with the interaction of several factors, including plant genotype, the genetic characteristics of bacterium, soil, temperature, crop cycle and maize plant phenology (^{Li et al., 2014}; ^{Oliveira et al., 2009}) that influence the colonization and dynamics of the endophytic bacterial community (^{Bodenhausen et al., 2013}). A specific plant-endophyte relationship was revealed by performing bacterial chemotaxis on host plant exudates as carbon sources that act as signaling molecules (^{Albareda et al., 2006}); it also showed that differences in exudate composition and patterns depend on the crop variety, development stage, exposure of the plant to stress and type of soil, which influence colonization by bacterial communities (^{Haichar et al., 2008}). Studies on maize indicated that root exudates are 65% sugar, 33% organic acids and 2% amino acids, and that changes in the quantity and quality of exudation patterns at different root growth and physiological stages influenced the biomass and the structure of bacterial communities by increasing nutrient activity and deposition by those microbial communities and favored plant growth (^{Baudoin et al., 2003}). Some maize studies that used a dependent culture approach have shown dynamic changes in the bacterial community of the rhizosphere at different crop growth stages (^{Cavaglieri et al., 2009}; ^{Nacamulli et al., 1997}).

In this study, specific bacteria were identified such as Burkholderia gladioli, which was isolated at a low frequency only in susceptible maize lines at two locations and crop cycles (Table 1).

Burkholderia gladioli was previously identified in roots of a wild maize ancestor (Zea mays ssp. parviglumis) and in modern maize genotypes as an endophyte showing antifungal properties (^{Shehata et al., 2016}). Also, Burkholderia sp. inoculation in maize had positive effects on drought tolerance (^{Fan et al., 2015}; ^{Naveed et al., 2014}).

Density of endophytic populations in maize lines. The density of endophytic bacteria in the 14 maize lines ranged from 1, 6 _Log10 UFC g^-1 of root. The ANOVA showed highly significant differences (**= p≤0,01) in the density of the endophytic population of B. subtilis, B. megaterium, P. hibiscola and S. meliloti in terms of location, crop cycle, days after transplanting (dds) and group (drought tolerance/ susceptibility) of maize lines (Table 2).

By location, the highest population densities recorded at El Batán were those of B. subtilis and P. hibiscola, while in Tlaltizapán they were B. megaterium and S. melilloti. By crop cycle and sampling date, B. megaterium and P. hibiscola were significantly different (p≤0,05) during the V-2012 cycle at 52 dds. In this study, a total of 22 endophytic bacteria were identified in roots of drought tolerant and drought susceptible maize lines. However, B. subtilis, B. megaterium, P hibiscola and S. meliloti population densities were significantly higher in drought tolerant maize lines than in susceptible lines (Table 2).

The density of bacterial populations is a key element, considered to be the greatest metabolism regulation mechanism in the interaction with the biotic and abiotic environment, through which it coordinates the expression of specialized genes, depending on cellular density. It has been demonstrated that this behavior, known as quorum sensing (QS) in bacteria associated with plants, regulates the expression of genes in the rhizosphere to synthesize secondary metabolites, antifungal compounds, antibiotics and extracellular enzymes involved in biocontrol (^{Somers et al., 2004}; ^{Whitehead et al., 2001}).

In this study, the total population density of B. subtilis in the maize lines sown at El Batán was 3, 4 _Log10 UFC g^-1 of root, and there was no significant difference by cycle or days after sowing, but it was significantly higher in drought-tolerant maize lines (Table 2, Figure 1).

Figure 1 B. subtilis population in 7 drought-tolerant maize lines and 7 drought-susceptible maize lines at 25, 52 y 75 dds during the 2012 summer crop cycle, 2012 winter crop cycle and 2013 summer crop cycle in three locations of Mexico. 

As an endophyte, B. subtilis is a microorganism of great interest in biotechnological applications as biocontrol agent for biotic and abiotic stresses because of its efficient colonization of plant roots which triggers different biocontrol and adaptation mechanisms in different environments (^{Marulanda et al., 2006}). Additionally, this demonstrates its high capacity to produce volatile organic compounds (VOCs) that act as signaling molecules to trigger a defense response through specific induced systemic resistance (ISR) (^{Farag et al., 2013}). These VOCs activate hormone production paths, including auxins, gibberellins, cytokinins and salicylic acid that foster host plant growth under stress conditions, mainly by increasing root biomass, which in turn improves water absorption (^{Zhang et al., 2007}). Another study showed that maize seed inoculated with selected B. subtilis strains provided important benefits as a growth promoter and increased nutrient absorption capacity (^{Canbolat et al., 2006})In Tlaltizapán, the population density of B. megaterium in maize lines was from 3, 5_Log10 UFC g^-1 of root during the three cycles and significantly higher (5 _Log10 UFC g^-1 of root) in the V-2012 cycle at 52 dds, and in drought tolerant maize lines (Table 2, Figure 2).

Figure 2 B. megaterium population in 7 drought-tolerant maize lines and 7 drought-susceptible maize lines at 25, 52 y 75 dds during the 2012 summer crop cycle, 2012 winter crop cycle and 2013 summer crop cycle in three locations of Mexico. 

Recent studies have shown that B. megaterium modifies the maize plant response to several abiotic stresses, and the importance of the population density of some Bacillus species in the drought tolerance and water transportation response in Retama sphaerocarpa, because it promotes root growth and water absorption capacity, as well as the levels of proline and indoleacetic acid (^{Marulanda et al., 2006}). In particular, those studies point out that the presence of B. megaterium in the host plant increases biomass and water content in the root (^{Marulanda et al., 2009}), a fact that suggests that these endophytic populations could be used in maize plants sown in arid and semi-arid areas. The biological function of other endophytic Bacillus species in maize are related to the plant’s efficient defense response against pathogens associated with the production of antifungal lipopeptides that induces the expression of defense genes (^{Gond et al., 2015}).

At El Batán, the population density of P. hibiscola in maize lines was from 3, 5 _Log10 UFC g^-1 of root during the three crop cycles and significantly higher (5 _Log10 UFC g^-1 of root) in the V-2012 cycle. It was not isolated in Agua Fría. There were no significant differences among the days after sowing (dds), but the density was significantly higher in drought-tolerant maize lines (Table 2, Figure 3).

Figure 3 P. hibiscola population in 7 drought-tolerant maize lines and 7 drought-susceptible maize lines at 25, 52 y 75 dds during the 2012 summer crop cycle, 2012 winter crop cycle and 2013 summer crop cycle in three locations of Mexico. 

P. hibiscola has not been cited as a maize endophyte. However, according to the phylogenetic affiliation of the P. hibiscola type ATCC 19867 strain based on a comparative analysis of 16S rADN gene sequences and chemical taxonomy profiles, it was reclassified as Stenotrophomonas sp. (^{Anzai et al., 2000}). S. maltophilia has been cited as a maize endophyte (^{McInroy and Kloepper, 1995}) and reported to act as a biological control agent against soil-borne pathogens such as Pythium spp., Fusarium spp. and Rhizoctonia solani. It has also been reported to produce indoleacetic acid, which regulates the development of sprouts and side roots in plants (^{Mehnaz et al., 2010}).

The population density of S. meliloti in drought-tolerant maize lines in Tlaltizapán was from 4, 6 _Log10 UFC g^-1 of root during the three cycles, and significantly higher (6 _Log10 UFC g^-1 of root) at 52 dds. It was not isolated in Agua Fría. There were no significant differences per crop cycle, but it was significantly higher in drought-tolerant maize lines (Table 2, Figure 4).

Figure 4 S. meliloti population in 7 drought-tolerant maize lines and 7 drought-susceptible at 25, 52 y 75 dds during the 2012 summer crop cycle, 2012 winter crop cycle and 2013 summer crop cycle in three locations of Mexico. 

S. meliloti is known to be a nitrogen-fixing nodular bacterium in plants of the Medicago genus, and involved in the drought tolerance and salinity response (^{Roumiansteva and Muntyan, 2015}). In Mexico, the presence of a great genetic diversity of native S. meliloti populations in alfalfa (Medicago spp.) was reported, and so far, new species have been identified in woody legumes of the Leucaena (^{Wang et al., 2002}) and Acacia genera (^{Toledo et al., 2003}). The Sinorhizobium genus was found in the maize rhizosphere (^{Rosenblueth and Martínez-Romero, 2006}), and Rhizobium etli bv. phaseoli as an endophyte in maize roots in Mexico (^{Gutiérrez-Zamora and Martínez-Romero, 2001}). However, there is no information about S. meliloti as an endophyte in maize roots, so the present study provides reference information about the natural endophytic colonization of S. meliloti in maize in Mexico. Alfalfa and maize intercropping is common in the studied locations, so this may explain the endophytic colonization of S. meliloti in maize roots. Studies conducted on rice (Oryza sativa) suggest that S. meliloti may produce a lumichrome signaling molecule in the rhizosphere of those plants and promote growth by inducing better root respiration, stomatal conductance, leaf transpiration and photosynthetic efficiency (^{Chi et al., 2010}). When it was applied at nanomolecular concentrations, it promoted legume and cereal growth and increased root biomass in the legume Lotus japonicus and in tomato (Solanum lycopersicum) (^{Gouws et al., 2012}).

The frequency of certain bacteria genera isolated from maize roots in this study may be related to the phylogeny of the maize lines. For example, among bacteria showing higher frequency and density in drought-tolerant maize lines, two Bacillus species stand out. Bacillus is considered an important endophytic bacterium that has been isolated from both Teozintle (a maize ancestor) and modern maize genotypes; it has been shown that the composition of the endophytic bacteria colony in maize seed varies and that it has been preserved through the evolution, ethnography and ecology of the maize plant as a host (^{Johnston-Monje and Raizada, 2011}).

In this research, we determined that some factors such as drought tolerance/susceptibility of the maize lines used, as well as the location, crop cycle and time (days after transplanting) significantly affected the density of cultivable endophytic bacteria in the roots of the maize genetic materials. Rhizobacteria as promoters of growth (PGPR), nutrition and plant disease management have been extensively studied, but their function in managing abiotic stresses such as drought has generated great interest in recent years (^{Dimpka et al., 2009}; ^{Grover et al., 2010}; ^{Kavamura et al., 2013}; ^{Yang et al., 2009}). The use of endophytic microorganisms may be a viable alternative against biotic and abiotic stresses. Strains isolated in this study are a microbial resource that is intimately associated with maize and has the potential to be used as a biotechnological tool in agriculture; these strains deserve to be evaluated in the future as microbial inoculants and inductors of drought stress tolerance, as well as to other biotic and abiotic factors in regions of Mexico where maize is produced under limiting conditions.

Conclusions

Cultivable root endophytic bacteria that were most frequently present in the 14 maize lines tested belong to the Proteobacteria and Firmicutes phyla. The highest population density of endophytic bacteria was found in drought-tolerant maize lines. Bacteria of the Bacillus, Pseudomonas and Sinorhizobium genera showed the highest frequency and population density in drought-tolerant maize lines at the three study locations and crop cycles. Sinorhizobium meliloti is able to endophytically colonize maize roots.