Microbial strains
The antifungal activity tests against F. verticillioides were performed with four bacterial strains from the maize silks from the Coleção de Microrganismos Multifuncionais e Fitopatogênicos of the Embrapa Milho e Sorgo, City of Sete Lagoas, Minas Gerais state, Brazil. The strains were collected in the localities of Sete Lagoas-MG, Sidrolândia-MS and Sertaneja-PR, in the year 2016. The Fusarium verticillioides isolate CML2743 used in the antagonism tests was from the Laboratório de Fitopatologia of the Embrapa Milho e Sorgo.
Molecular identification of the bacterial strains
The partial 16S rRNA gene sequence was used for identifying the four bacterial strains. Genomic DNA extraction was performed by the Wizard® Genomic DNA Purification kit (Promega, USA). PCR amplification of the 16S rRNA was carried out with the bacterial universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5′-GGTTACCTTGTTACGACTT-3) designed by Turner et al. (1999). PCR reactions consisted of 20 ng of bacterial genomic DNA plus 2.0 µL 10X PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 0.8 µL of each primer (10 μM), 1.5 µL dNTP (2,5 mM each), 0,6 μL of MgCl2 (50mM), 0,2 µL of Taq DNA polymerase (5 U/µL) (Invitrogen, USA) and the total reaction volume adjusted to 20 µL with ultrapure water. The PCR amplification was performed in a model Veriti® 96-Well Thermal Cyclers (Applied Biosystems, EUA) with the following conditions: one step of 2 min at 95 ºC for DNA denaturation, followed by 30 cycles of 30 s at 94 ºC, 30 s at 59 ºC and 90 s at 72 ºC, and a final extension step at 72 ºC for 10 min. The PCR products were analyzed by 1.0% (wt/vol) agarose gel electrophoresis and documented using the Gel Logic 200 system (KODAK Company, USA). The nucleotide sequences were determined on both strains using the PCR primers in the ABI PRISM 3500xL Genetic Analyzer Sequencer (Applied Biosystem, USA). The sequences alignments were made with the Sequencher 4.1.4 program (Genes Codes Corporation), and the alignment research tool (BLAST) was used to find similarities with sequences in the NCBI database (http://www.ncbi.nlm.nih.gov/). The edited sequence of each species was deposited in the Gene Bank and received the following accession number: Bacillus velezensis strain CT02 (MK461847), Bacillus velezensis strain IM14 (MK461831) Pseudomonas aeruginosa (MK461572) and Achromobacter xylosoxidans (MK461853).
In vitro antifungal activity against Fusarium verticillioides
The antagonistic test was carried out by previously growing the four bacterial strains ISD04, IPR45, CT02, and IM14 and F. verticillioides CML2743 on potato dextrose agar (PDA) medium. After, a 5 mm diameter from actively growing fungus was transferred to the center of a new plate containing PDA medium, and 10µL of bacterial suspension (108 UFC/mL) was applied in four equidistant points, near the periphery of the culture. The plates were incubated at 28 °C under the 12h photoperiod for seven days. The antifungal activity was estimated by measuring the radial growth rate (mm) of the fungus in the confrontation test after the fungus of the control plate reached the entire medium surface. The inhibition ratios were calculated using the following formula: Inhibition ratio (%) = (radial mycelial growth of control - radial mycelial growth with antagonist) / radial mycelial growth of the control × 100.
Antifungal activity of the cell-free supernatant
Pure cultures of the bacterial strains were inoculated in liquid Tryptic Soy Broth (TSB), incubated at 28 °C, and constant agitation rate of 90 rpm for 72 h. Afterward, the culture was centrifuged at 6.000 rpm, and the supernatant filtered through a 0.22 µm pore membrane. Then, streptomycin (20 mg/L) was added to the supernatant and used as a growth medium for F. verticillioides. The TSB medium-plus antibiotic inoculated with culture discs of the fungus, and non-inoculated TSB medium with antibiotic was used as controls. The incubation was performed at 28 °C without shaking for ten days to allow the mycelial growth ofF. verticillioides. The fungal mycelium was recovered by filtration on Whatman paper filters (n.4) and dried at 60 °C until constant weight. The mycelial growth inhibition rate (%) was determined by the dry weight percentage of the mycelial biomass concerning the control (100%).
Effect of cell-free supernatant on conidia germination and hyphae development of F. verticillioides
The bacterial culture filtrates were used to test the inhibitory activity against F. verticillioides conidia germination and hyphae growth. Conidia of the fungal pathogen were obtained from a seven-day culture at 25 °C and 12 h photoperiod in the BDA medium. The conidia suspension was filtered through gauze to remove any large fragments of mycelia, and the concentration was adjusted to 1 x 104 conidia/mL by counting in a Neubauer chamber. Then, the conidial suspension and each bacterial culture filtrate were mixed in equal proportion and incubated for 24 h at 26 °C under a 12 h photoperiod. The control consisted of TSB medium inoculated with F. verticillioides. The effect of each bacterial supernatant on conidia germination and hyphae development was evaluated by observation under an optical microscope. For the conidia germination test, 100 conidia of each treatment were counted, in triplicate, and those germ tubes with twice the size of the conidia were considered germinated (Abou-Jawdah et al. 2002). The percentages of inhibition were calculated by comparison with the fungal culture without bacterial inoculation (control). The experiment was repeated three times.
Hydrolytic enzymes production by the bacterial strains and calculation of the enzyme index
The concentration of bacterial culture grown in TSB for 72 h was adjusted to approximately 108 CFU mL1 and inoculated in the specific medium for each enzyme. After microbial growth for about 48 h, the enzymatic activity was measured by a clear zone surrounding colonies. The enzymatic index (EI) was calculated by EI = diameter (mm) of the discolored halo/diameter (mm) of the colonies.
Cellulase
To determine the cellulase production, the four bacterial strains were grown in minimal M9 culture medium (10 g/L carboxymethylcellulose, 5 g/L yeast extract, 12.8 g/L Na2HPO4 .7H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 5 g/L MgSO4.7H2O, 0.01 g/L CaCl2 .2H20, 15 g/L agar). After microbial growth, 10 mL of Congo Red (1g/L) was distributed on each plate. After 15 min, the plates were washed with 5M NaCl and observed for a yellowish area around the colonies (Teather and Wood 1982).
Pectinase
For pectinase activity, the bacterial strains were grown in the M9 medium plus citrus pectin with pH adjusted to 8.0 and incubated as described above. After the culture growth, ten mL of Lugol were added to the plates and washed with deionized water. A colorless halo around the colonies indicated the pectinase production (Beg et al. 2000).
Protease
The protease production was evaluated in culture medium containing 5 g/L tryptone, 2.5 g/L yeast extract, 2.5 g/L NaCl, 1 g/L of glucose, and 16 g/L agar. After sterilization, 100 mL of boiled skimmed milk were distributed on each plate. Protease activity was expressed by the formation of a colorless halo around the colonies (Beg et al. 2000).
Lipase
For lipase, the four strains were grown in a medium containing 5 g/L peptone, 1 g/L yeast extract, 4 g/L NaCl, 15 g/L agar, 31.25 mL/L olive oil, 0.01 g/L rhodamine B with the pH adjusted to 7.0. After the bacterial growth, the presence of a blue halo around the colonies was visualized by ultraviolet radiation (Savitha et al. 2007).
Chitinase
Chitinase activity was performed, as indicated by Trudel and Asselin (1989), with modifications. A 10µL of the supernatant of bacterial culture was added in a 2mm wells in plates containing agarose gel (1.6%) in sodium acetate buffer (0.1M, pH 5.0) and 0.01% (P/V) of glycol chitin solution. After the incubation at 30 °C for 48 h, the plates were stained with 0.1% calcofluor in 0.5M Tris-HCL buffer, pH 8.9 for 10 min, and washed with distilled water. The formation of a clear halo detected by ultraviolet light (300nm) was indicative of chitinase production.
Growth of Fusarium verticillioides in maize seeds microbiolized with the bacterial strains
Maize seeds with no antifungal treatment were disinfested with a 3% sodium hypochlorite solution for 5 min, followed by a wash with sterile distilled water. Then, the seeds were disinfected according to Daniels (1983) by placing the seeds in a 70% (v/v) ethanol solution for 10 min and then transferred to flasks containing sterile deionized water for 4 h. Subsequently, the seeds were transferred to another flask containing sterile deionized water and kept in a water bath at 60 °C for 5 min. Then, the seeds were placed under direct light for 24 h, followed by frozen at -20 °C for 24 h to inhibit seed germination. Afterward, the seeds were infected with a suspension of 1 x 106 conidia/mL of F. verticillioides. The antagonist bacterial strains were grown in liquid TSB medium for 72h, and the bacterial cell concentrations were adjusted to approximately 108 CFU mL-1. The maize seeds were microbiolized by immersion in bacterial suspensions for 24 h at 28 °C with shaking at 130 rpm. The seeds immersed only in TSB served as the negative control. An additional control with seeds disinfected without inoculations was included to assess the effectiveness of the disinfection method. The seeds were germinated in boxes (11 x 11 x 3cm) containing filter paper, moistened with sterile distilled water. Sixty seeds were distributed in three replicates of 20 seeds per treatment and incubated with a photoperiod of 12 h of light at 26 °C for eight days. The evaluation of F. verticillioides incidence was carried out by examining the seeds in the Zeiss Stemi 2000 binocular stereomicroscope with a 50X magnification objective.
Effects of seeds microbiolization with bacterial strains in reducing stalk rot disease
The bacterial strains were grown in Lysogeny Broth (LB) medium at 35 °C for 72h and shaking at 300 rpm. Each culture was centrifuged at 2.000 rpm, and the pellet resuspended in a 20% sucrose solution. For microbiolization, seeds of maize hybrids BRS 1010, susceptible to F. verticillioides were treated with bacterial culture plus 50% sucrose (w/vol) and incubated at 80 rpm and grown for 30 min at room temperature. Then, the seeds were mixed in starch and dried for 24 h at 30 °C (Figueiredo et al. 2010). The treated seeds were planted in pots containing 5 kg of soil, and 30 days after planting, the fungus was inoculated by stem punctures using sterile toothpick immersed in the conidial suspension (1 x 106 conidia / mL). Five seeds from each treatment were planted in each of three pots. They consisted of 1) seeds treated with each bacterial antagonist inoculated with F. verticillioides, 2) seeds without bacterial treatments inoculated with F. verticillioides, and 3) seeds without treatment and toothpicks puncture without F. verticillioides, As a control, seeds treated with each bacterial strain without F. verticillioides inoculation were included in the experiment. After 45 days of infection, the stalk rot severity was determined according to the Symptom Score Scale described by Nicoli et al. (2015).
Statistical analysis
The analysis of variance (ANOVA) was used to check the collected data, followed by the Scott-Knott means comparison test at p <0.05. All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD).