Isolation of Bacillus strains

The strains were isolated from soil samples collected in the Yucatan Peninsula in Mexico. Soil samples were taken from natural vegetated areas by scrapping off surface material with a sterile spatula and about 200 g samples were obtained from 10 to 20 cm depth. All samples were placed in sterile plastic bags and stored at 4°C until processed. Isolation was carried out using the methodology described by ^{Basurto-Cadena et al. (2012)}. Briefly, 1 g of each sample was suspended in 10 mL sterile distilled water and pasteurized at 80°C for 20 min. Subsequently, 50 μL of the suspension was placed in microfuge tubes with 500 μL of distilled water.

Aliquots of 50 μL of the diluted suspensions were spread in duplicate on a thin layer of nutrient agar (NA) (Merck, Germany) in Petri dishes. The incubation was carried out under aerobic conditions at 30^oC for 48 h. After incubation the Bacillus-like colonies, roughly identified based on their morphology, were transferred and incubated in new Petri dishes for one week at the above conditions, before storing at 4°C. The colonies were counted and the results expressed as bacteria count per milliliter (CFU mL^-1). Prior to use, all isolates were reactivated in NA for five days.

Evaluation of antagonistic activity

Plant pathogenic fungi were kindly donated by Dr. Jairo Cristobal from the Laboratory of Plant Pathology at the Instituto Tecnológico de Conkal. Alternaria alternata and Curvularia lunata were originally isolated from Chamaedorea elegans, and in turn, Helminthosporium rostratum was isolated from Heliconia wagneriana. Antagonistic activity was evaluated by the inhibition of fungal colony growth in direct confrontation and by inhibition of conidial germination using the Bacillus culture filtrates.

Bioassays of direct confrontation were carried out in potato-dextrose-agar (PDA) medium. Briefly, discs of PDA (5 cm diameter) from axenic fungal culture were inoculated in the center of a 15 cm diameter Petri dish with PDA. Spore suspensions (1x10⁷ CFU mL^-1) of Bacillus strains were obtained in sterilized distilled water by scrapping with sterile scalpel blade 5-day-old bacterial cultures grown in NA. Bacterial spore suspensions were inoculated 2 cm from the margin of PDA discs that contained the fungal culture. All control fungal colonies were not confronted with the antagonists. Petri dishes with fungal cultures were incubated at 30°C. The inhibition of radial growth of fungal colony was daily evaluated and the percentage of mycelial growth inhibition was calculated with respect to the control colony according to ^{Korsten and Jager (1995)} as follows: GI(%)=((RC-RE)/RC)x100, where GI (%) is the percentage of mycelial growth inhibition, RC is the radius of the fungal colony in the control Petri dish, and RE is the radius of the fungal colony that was confronted with the antagonistic Bacillus.

For the bioassays of conidial germination, Bacillus strains were cultured in agar broth (BD, USA) for 72 h in an orbital shaker at 150 rpm and 30°C. Cultures were centrifuged at 7000 g for 10 min and the precipitate (vegetative cells and spores) was discarded. A previous study carried out in our laboratory showed that autoclaved culture filtrates from Bacillus spp. revealed antifungal activity on Colletotrichum gloeosporioides. Thus, in this study, filtered free-cell culture were autoclaved at 120 °C for 15 min and cooled down before using. Culture filtrates were mixed with conidial suspensions of fungi (1:1 v/v) obtained from colony grown in PDA. Mixture of culture filtrate and conidial suspensions were then inoculated in a PDA plate and observed for 12 h for conidial germination using a light microscope (LEICA DM500®, USA) at 100x magnification. Conidia were counted as germinated when the germ tube had at least the length of the germinated conidia, according to ^{Mahadtanapuk et al. (2007)}.

Extraction of DNA of Bacillus strains

All Bacillus strains were cultured in Brain Hearth Infusion Broth (BHI) (BIOXON, USA) in 10 mL tubes at 30 °C for 48 h. Cultures were centrifuged at 2500 g for 5 min and washed with saline solution (0.85 %). Pellet was collected and supernatant discarded. DNA extraction was carried out with the kit Master Pure® Gram Positive DNA purification (Epicentre Biotechnologies, USA). Briefly, pellet was suspended in 150 μL TE buffer (Tris-EDTA), then added with 1 μL of lisozime ready lyse and incubated at 37°C for 30 min. Subsequently, 159 μL of proteinase K was added and incubated at 65°C for 15 min; tubes were agitated every 5 min. Then, tubes were maintained for 2 min at 37 °C, followed by 5 min in ice, to cool down. Samples were precipitated in a 175 μL of MPC (protein precipitation reagent) solution, agitated vigorously and centrifuged at 10 000 g for 10 min. The supernatant was separated and mixed with 1 μL RNase in a new tube. Samples were incubated at 37 °C for 30 min and 500 μL isopropanol was added. Tubes were centrifuged at 10000 x g for 10 min, the supernatant eliminated and the pellet remixed to suspension in 500 μL of ethanol (ETOH 70 %). Tubes were again centrifuged at 13000 x g for 1 min, supernatant eliminated and the pellet remixed to suspension in 35 μL of TE buffer (10 mM Tris-HCl and 1 mM EDTA Na₂, pH 8.0; PROMEGA Corp., USA). The DNA was kept in a freezer at -20°C. DNA quality was confirmed by agarose gel electrophoresis 1.2 % (87 V for 60 min). Gel was stained with ethidium bromide (0.5 μg mL^-1) for 30 min. Image was taken using a photo documentation system (Canon-Biotop, USA).

Polymerase Chain Reaction (PCR) analysis for molecular identification

For molecular identification of the strains, gen amplification by polymerase chain reaction (PCR) was carried out using the partial genes 16S rRNA, species-specific rpoB and gyrA. The methodology for each case was as follows:

For PCR analysis of 16S rRNA gene, specific primers for the internal portion of the “B. subtilis group” 16S rRNA, Bsub5F (5’-AAG TCG AGC GGA CAG ATG G-3’) and Bsub3R (5’CCA GTT CCA ATG ACC CTC CCC-3’) were used (^{Wattiau et al., 2001}; ^{Geetha et al., 2007}). The reaction mixtures (25 μL) consisted of 12.5 μL master mix 1X (PROMEGA Corp., USA), 1 μL of the primer (0.4 mM) (IDT Technologies, USA), 9.5 μL DNAse-free water and 1 μL genomic DNA. The PCR program consisted of denaturation at 94°C for 12 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 2 min, extension at 72°C for 2 min and, a final extension at 72°C for 7 min.

For PCR analysis of rpoB gene specific primers corresponding to B. subtilis position 6-585; rpoB-f (5’-AGG TCA ACT AGT TCA GTA TGG AC-3’) and rpoB-r (5’-AAG AAC CGT AAC CGG CAA CTT-3’) were used (^{De Clerck et al., 2004}; ^{Geetha and Manonmani, 2010}). The reaction mix (25 μL) consisted of 12.5 μL master mix 1X (PROMEGA Corp., USA), 0.5 μL primer (0.2 mM) (IDT Technologies, USA), 10.5 μL DNAase-free water and 1 μL genomic DNA. The PCR program consisted of denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, alignments at 51°C for 45 sec, extension at 68°C for 50 min and, final extension at 68°C for 90 sec.

For PCR analysis gyrA gene, specific primers corresponding to B. subtilis, position 43-1070; gryA-f (5’-CAG TCA GGA AAT GCG TAC GTC CTT-3’) and gyrA-r (5’CAA GGT AAT GCT CCA GGC ATT GCT-3’) were used (^{De Clerck et al., 2004}; Geetha and Manonmani, 2008). The PCR reaction mixture (25 μL) consisted of 12.5 μL master mix 1X (PROMEGA, Corp., USA), 0.5 μL primer (10 mM) (IDT Technologies, USA), 10.5 μL DNAse-free water and 1 μL genomic DNA. The PCR program consisted of denaturation at 94°C for 2 min, 40 cycles denaturation at 94°C for 30 s, alignment at 51°C for 45 s, extension at 68°C for 50 min and, a final extension at 68°C for 10 min.

The resulted amplicons were visualized in agarose gel 1.2 %, purified with the kit Qiagen QIAquick (Qiagen Corp., USA) and sequenced in both directions using the corresponding primers (Bioscience, San Diego, CA. USA).

Phylogenetic analysis

Gene sequences were edited and aligned with the software BioEdit version 7.1.3.0 (^{Hall, 1999}). To obtain the identity, the sequences were compared with those of the database from GenBank (National Center for Biotechnology Information), using the program BLASTN (Nucleotid Basic Local Alignment Search Tool). All sequences were deposited in the GenBank.

To study the relationship of the isolates with other members of the genus Bacillus, sequences of the regions used for each gene were obtained from GenBank and multiple alignments were carried out in the program CLUSTALW (^{Larkin et al., 2007}). The evolutionary model for each gene was estimated and the analyses of Neighbor-Joining with Bootstrap of 1000 replicates were carried out in the program MEGA (Molecular Evolutionary Genetics Analysis) 5 (^{Tamura et al., 2011}).

A matrix of partitions, with the sequences of the three analyzed regions, was built. The homogeneity test suggested that the three regions show a similar phylogenetic signal; therefore, a combined of such partitions may be built. Bacillus sonorensis as external group was used. The analysis of Neighbor-Joining was carried out with analysis Bootstrap of 1000 replicates. A matrix of evolutionary distances was built using the program MEGA 5 to determine the highest evolutionary rate of the analyzed region.

Data analysis

Experimental data were statistically analyzed using ANOVA with a completely randomized design. The Tukey test was used to determine significant differences among means (p≤0.05), using SAS 9.1. Unless otherwise indicated, all measurements were carried out in triplicate. Prior to run, data in percent were transformed to arcsin [y=arcsin(sqrt x/100)].

Antagonistic activity of the Bacillus strains on three plant pathogenic fungi

The antagonistic activity of the strains by direct confrontation and by the use of the cell-free culture filtrate was evaluated in three plant pathogenic fungi: Alternaria alternata, Helminthosporium rostratum and Curvularia lunata. The strains used in this were molecularly identified as B. subtilis subsp. inaquosorum and B. subtilis subsp. subtilis. The results showed that the percentage of mycelial growth inhibition ranged from 32 to 78 % (Table 1). Moreover, the strain CBK36 presented the highest activity, causing more than 60 % inhibition of mycelial growth among the three evaluated fungi. In contrast, the strain CBMT51 showed the lowest effect, less than 40 % inhibition of colony growth in two of the tested fungi (Table 1).

Table 1 Effects of Bacillus strains on the percentage of mycelial growth inhibition of three plant pathogenic fungi. 

Mean values (± standard errors) in a column followed by different letters are significantly different (Tukey; p≤0.05). CV(%): Coefficient of variation, HSD: Tukey’s Honest Significant Differences

The antagonistic activity of B. subtilis on plant pathogenic fungi is reported in literature. In vitro effects, specifically on mycelial growth and conidial germination are reported. In vivo effects are also reported, particularly on incidence and severity of foliar spots and blights, postharvest fruit rots and root rots (^{Chaurasia et al., 2005}; ^{Guillén-Cruz et al., 2006}; ^{Zongzheng et al., 2009}; ^{Basurto-Cardena et al., 2012}).

The effect of culture filtrates on conidial germination of the fungi showed significant differences (p≤0.05). Culture filtrate of the strains CBCK36, CBMN22 and CBRF24 caused up to 37 % inhibition of conidial germination in at least two of the evaluated fungi. The culture filtrates of the strains CBCC2 and CBCK47 caused up to 46 % inhibition of conidial germination in A. alternata, but their effects on the other two fungi were lower (Table 2).

Table 2 Effect of the Bacillus culture filtrates on the inhibition of conidial germination in three plant pathogenic fungi. 

Mean values (± standard errors) in a column followed by different letters are significantly different (Tukey; p≤0.05). CV (%)=Coefficient of variation; HSD = Tukey’s Honest Significant Differences

Moreover, the culture filtrate of this strain showed also one of the highest effects on conidial germination of the evaluated fungi, which suggest that part of its activity is mediated by the production of antifungal compounds. Culture filtrates of B. subtilis have suppressed the mycelial growth as well as the conidial germination of plant pathogenic fungi (^{Zongzheng et al., 2009}; ^{Islam et al., 2012}). Notice that particularly the culture filtrates of the strains CBCK36, CBCC2 and CBCK47 showed more than 30 % inhibition of conidial germination at least in one of the tested fungi. As these culture filtrates were autoclaved prior to use, the antifungal compounds responsible for the effects were heat resistant. The identities of these compounds were not determined in this study, but we might hypothesize that heat resistant lipopeptides are likely involved. Heat resistant antifungal lipopeptides, like iturins, were extracted from the culture media of various strains of B. subtilis (^{Rezuanul et al., 2012}). Indeed, culture filtrates containing heat resistant antifungal compounds were used to suppress the incidence and severity of fungal disease in vivo (^{Cubeta et al., 1985}).

PCR amplification and phylogenetic analysis

To confirm the identity of the Bacillus isolates, sequence of partial regions of the 16S rRNA, gyrA and rpoB genes were obtained by PCR. Amplification of the hyper variable region of 16S rRNA, species-specific gyrA and rpoB genes of the seven Bacillus strains yielded amplicons of the 535 bp, 960-1005 bp and 546 bp, respectively (Figure 1).

Figure 1 Agarose gel (1.2 %) electrophoresis of the PCR amplification of rpoB DNA, 16S rRNA and gyrA DNA genes of seven native Bacillus subtilis. Lane M: 1 kb ladder Promega, 1: CBCC2, 2: CBMT2, 3: CBMN22, 4: CBRF24, 5: CBCK36, 6: CBCK47 and 7: CBMT51. 

On the basis of this analysis all the isolates were placed taxonomically as B. subtilis subsp. inaquosorum and B. subtilis subsp. subtilis on phylogenetic trees. ^{Chun and Bae (2000)} showed the use of the rpoB gene sequences in Bacillus. Later, ^{Geetha and Manonmani (2010)} also showed the utility of this gene sequence to identify the isolate B. subtilis subsp. subtilis VCRC B471. The protein rpoB was also used to infer relationships among archaea orders and Gram positive and Gram-negative bacteria (^{Matte-Tailliez et al., 2002}; ^{Morse et al., 2002}).

The highest BLASTN identity of each of the strain was confirmed and also the genus identity of Bacillus subtilis group (Table 3). The gene sequences 16S rRNA, rpoB and gyrA-based PCR had values in a range of 95 to 100 %, similar to those deposited in Genbank (Table 3).

Table 3 Maximum identity values (%) of identified Bacillus strains based on the analysis of 16S rRNA, gyrA and rpoB genes. 

The amplicons obtained for regions 16S rRNA, rpoB and gyrA were 535, 546 and 953 bp, respectively. The Neighbor-Joining analyses of each of the regions (Figures 2, 3 and 4) and the total evidence (Figure 5) showed that the isolates were separated into two clades. In all analyses, the isolates CBRF24 and CBMT2 were grouped in a different clade. Using the 16S rRNA region, the strains CBMT2, CBMN22, CBCK36, CBMT51 and CBCK47 formed a clade (Figure 2), with sequences of B. subtilis taken from GenBank. These isolates formed a clade with stand of 100 with B. subtilis subsp. subtilis; while with the analysis performed using the regions gyrA and rpoB, the mentioned strains formed a clade with B. subtilis subsp. inaquosorum.

Figure 2 Neighbor-joining tree with bootstrap values based on partial sequences of the 16S rRNA gene. Analysis made with a nucleotide substitution model of Jukes-Cantor. 

Figure 3 Neighbor-Joining tree with bootstrap values based on partial sequences of the gyrA gene. Analysis made with a nucleotide substitution model of Tamura-Nei with discrete gamma distribution. 

Figure 4 Neighbor-Joining tree with bootstrap values based on partial sequences rpoB gene. Analysis made with a nucleotide substitution model of Tamura-3 parameter using discrete gamma distribution. 

Figure 5 Neighbor-Joining tree with bootstrap values based on partial sequences of the 16S rRNA, gyrA and rpoB genes. Analysis made with a nucleotide substitution model of Tamura-3 parameter using discrete gamma distribution. 

The analysis of matrix partitions separated the isolates into two distinct clades, forming a clade with CBRF24 and CBMT2, completely independent of the other strains (Figure 5). Amplification, sequencing and sequence analysis of 16S rRNA, rpoB and gyrA genes for molecular identification of Bacillus species are reported only in separate studies (^{Geetha et al., 2007}; Geetha and Manonmani, 2008; ^{Geetha and Manonmani, 2010}). In our research, however, the first sequence analysis of 16S rRNA, gyrA and rpoB genes to locate taxonomically seven native strains of B. subtilis was successfully integrated. This molecular technique allowed us to infer the species of Bacillus and the existence of two new subspecies. Although 16S rRNA gene sequence was very useful for the determination of bacterial species, in the genus Bacillus some problems have arisen (^{Wattiau et al., 2001}; ^{Janda et al., 2007}).

Evolutionary distance matrices

The region that showed lower evolutionary distances was the 16S rRNA (from zero to 0.002) followed by the rpoB gene (up to 0.004). In contrast, the gyrA gene showed the highest evolutionary rate (up to 0.09), being even higher than that obtained from the matrix partitions, which includes information from the three regions analyzed (0.034 maximum).

The greatest distances were observed between the isolates CBRF24 and CBMT2. Thus, for the 16S rRNA, the proper determination of the B. subtilis subspecies could be difficult. In contrast, for the gyrA gene, its higher evolutionary rate allowed to allocate B. subtilis at the subspecies level. In particular, the comparative analysis of gyrA sequences proved to be robust for rapid classification and identification of B. subtilis and related taxa (^{Chun and Bae, 2000}), as shown in our study. The characterization of all strains at the subspecies level was not possible with the data obtained. The strains CBMT2 and CBRF24 might eventually form new subspecies, since the rest of the strains were grouped into clades with different subspecies.