Isolation and screening of biocontrol bacteria
Bacterial strains were isolated from corn rhizosphere soil in Harbin, China (45° 41′ N, 126° 37′ E). The pathogenic fungus F. graminearum strain YJH2 (GenBank accession: MG548651), which causes corn stalk rot, was maintained in the Plant Pathology Laboratory of Northeast Agricultural University in Harbin, China. The culture media used were as follows: potato dextrose agar medium (PDA), nutrient agar medium (NA), and Luria–Bertani medium (LB). Using the standard dilution plate method for isolation of biocontrol bacteria strains, bacteria were isolated and cultured on beef extract-peptone medium plates (Li et al. 2019). The obtained bacterial strains were screened against F. graminearum, using the plate confrontation method (Hu et al. 2013). The tested bacterial strains were activated on the NA medium at 28 °C for 48 h and inoculated 3 cm from the center of a PDA plate (diameter 9 cm), using the parallel streak method. F. graminearum mycelial disks (diameter 0.7 cm) were then inoculated in the center of the PDA plates with three replicates. The PDA plates were incubated at 26 °C for 5 days. The maximum and minimum radii of F. graminearum colonies were measured to determine the antagonistic activity of the tested bacterial strains (Huang et al. 2017).
Antifungal spectrum
Using the described confrontation method, the antifungal spectrum of selected biocontrol bacterial strain (B. velezensis BM21) against 10 species of pathogenic fungi was assessed and the specific operation method is as above.
Identification of biocontrol bacteria
Tested bacterial strains were activated on the NA medium at 28 °C for 24 h. The morphological characteristics of the bacterium were observed on the NA medium. Physiological and biochemical characteristics were determined following the procedures of Schaad et al. (2001) and Dong and Cai (2001).
16S ribosomal DNA sequencing
Genomic DNA of tested biocontrol bacteria was extracted, using a DNA Extraction Kit (Kangweishiji CW Bio, Beijing, China). The 16S ribosomal RNA (rRNA) gene was amplified using the universal primers 27F (5′-AGAGTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The 50-μL PCR mixtures contained 25 μL PCR Taq mixture (Tiangen Biotech, Beijing, China), 2 μL of 10 mM primer 1492R, 2 μL of 10 mM primer 27F, 19 μL sterile deionized water, and 2 μL DNA template. The PCR protocol consisted of initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 60 s, 58 °C for 60 s, and 72 °C for 90 s, and a final extension step at 72 °C for 10 min (Frank et al. 2008). The PCR product was detected by agarose gel electrophoresis and directly sequenced by Shanghai Biological Engineering Co., Ltd. (Shanghai, China). Based on 16S rRNA sequence data, phylogenetic trees for the identified bacterial strain were constructed using the neighbor-joining method with 1000 bootstrap replicates with MEGA 6 software (Tamura et al. 2013).
Detection of production site of antifungal active substances
A small amount of activated bacterium strain was selected with a bacteriostatic hook and inoculated into the LB medium (liquid loading 300 mL L−1) at 28 °C and shaken at 170 rpm for 7 days. The suspension was then divided into two portions. One portion of the suspension was filtered through a bacterial filter (YY3014236, Millipore, USA). The second portion was ruptured using an ultrasonic cell crusher (Shanghai Hannuo Instruments Co., Ltd., Shanghai, China), then filtered through a bacterial filter. The activity of mixture extracts and extracellular antifungal substances in the filtrate was determined using a mycelial growth rate method (Li et al. 2016b). The aseptic filtrate containing the mixed and extracellular antifungal substances was added to the PDA medium to attain filtrate concentrations of 1%, 5%, and 10%. F. graminearum mycelial disks (diameter 0.7 cm) were inoculated in the center of PDA plates with three replicates. The quantitative LB medium was added to PDA as the control. The PDA plates were incubated at 26 °C for 5 days. The experiment was repeated twice. The colony diameter was measured to determine the inhibitory effect on F. graminearum.
Effect of sterile extracellular culture supernatant on conidial germination of F. graminearum
Activated F. graminearum was inoculated on rice straw medium (dry straw 30 g, glucose 5 g, and distilled water 1 L) to induce conidial production at 26 °C for 10 days without light. Conidia were diluted to generate a conidial suspension (108 cfu mL−1). Aseptic extracellular culture supernatant was added to conidial suspension to attain concentrations of 1, 5, and 10%. The LB medium was added to the conidial suspension as the control with three replicates. The conidial suspensions were incubated at 26 °C in an incubator. Once the percentage spore germination of the control exceeded 60%, the number of germinated conidia was scored for each treatment (100 spores per treatment). The experiment was repeated twice.
Effect of sterile extracellular culture supernatant on conidial production of F. graminearum
Five-day-old F. graminearum disks (diameter 7 mm) were transferred to rice straw medium plates and were incubated at 26 °C until the colonies had grown to 4 cm diameter. Aseptic extracellular culture supernatant was diluted to concentrations of 1, 5, and 10%, then added to the rice straw medium plates (20 mL per dish) for 20 min. The surface hyphae on the plates were scraped off, and the surrounding medium without hyphae was cut off. The excess solution on the plates was poured into a waste liquid cylinder and incubated at 26 °C for 72 h with three replicates. The LB liquid medium was added to plates as the control. The experiment was repeated twice. The concentration of the conidial suspension was measured using cell counting methods.
Effect of sterile extracellular culture supernatant on mycelial growth of F. graminearum
Aseptic extracellular culture supernatant was added to the PDA medium to attain concentrations of 1%, 5%, and 10%. F. graminearum disks (diameter 7 mm) were inoculated in the center of PDA plates with 3 replicates. An equal volume of the LB medium was added to PDA as the control. The PDA plates were incubated at 26 °C for 5 days. The experiment was repeated twice. The colony diameter was measured to determine the inhibitory effect on mycelial growth of F. graminearum.
Morphological and ultra-structural changes in mycelial cells induced by sterile extracellular culture supernatant
Fresh mycelia with some medium, cultured on PDA medium for 24 h, were picked up and added into sterile extracellular culture supernatant to attain concentrations of 1, 5, and 10%, then incubated at 26 °C for 24 h. Control treatments were prepared using an equal volume of the LB medium. Sample preparation for microscopic examination followed the methods of Ooi et al. (2011). The samples were observed under an optical microscope (90i, Nikon, Tokyo, Japan) and a transmission electron microscope (H-7650, Hitachi, Tokyo, Japan).
Thermal stability
Aseptic extracellular culture supernatant was treated by heating in a water bath at 20, 40, 60, 80, 100, and autoclaved at 121 °C for 20 min with untreated blank samples as the control. Using a mycelium growth rate method, the treated aseptic extracellular culture supernatant was added to the quantitative PDA medium to attain a concentration of 5%. The experiment was repeated twice. The antifungal activity of the treated broth was assessed using the same procedure described for the non-treated sterile extracellular culture supernatant.
pH stability
Using a mycelium growth rate method, aseptic extracellular culture supernatant was added to PDA media differing in pH (pH 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) to attain a concentration of 5%. The experiment was repeated twice. The antifungal activity of the treated broth was assessed using the same procedure described for the non-treated sterile extracellular culture supernatant.
Ultraviolet stability
Aseptic extracellular culture supernatant was irradiated for 10, 20, 30, or 60 min within a distance of 1 m from an ultraviolet lamp (100 μW cm−2), respectively. Using a mycelium growth rate method, treated aseptic culture supernatant was added to the quantitative PDA medium to attain a concentration of 5%. The experiment was repeated twice. The antifungal activity of the treated broth was assessed, using the same procedure described for the non-treated sterile extracellular culture supernatant.
Application of Bacillus velezensis strain BM21
Lipopeptide-producing fermentation medium contains sucrose 200 g, NH4NO3 2 g, KH2PO4 3 g, Na2HPO4 10 g, MgSO4·7H2O 0.2 g, yeast extract 0.2 g, CaCl2 0.7 μg, MnSO4·4H2O 1 μg, and distilled water 1 L.
Seed liquid of B. velezensis strain BM21 activated for 24 h was added to the lipopeptide-producing fermentation medium (liquid loading 300 mL L−1) with 5% inoculum and incubated at 28 °C for 48 h at 180 rpm. The pH value of the BM21 fermentation medium was then adjusted to 8.0 using 0.1 M NaOH, and the bacteria were removed by centrifugation for 10 min at 12000 r/min. The pH value of the sterile supernatant was adjusted to 2.0 with 0.1 M HCl and refrigerated overnight at 4 °C for completion of precipitation. The precipitate was collected and washed twice with HCl at pH 2.0 and extracted five times with methanol.
The lipopeptide extract was obtained by decompression drying of the methanol extract solution in a rotary evaporator (RE-52AA, Shanghai Ya Rong Biochemical Instrument Co. Ltd., Shanghai, China) at 35 °C. The lipopeptide extract was diluted to saturation with methanol for storage. After soaking 200 g sorghum grains in sterile water for 12 h, the sorghum grains were boiled for 30 min, transferred to flasks (50 g 250 mL−1), and treated twice with damp-heat sterilization at 121 °C for 30 min. Five activated F. graminearum disks (diameter 0.7 cm) were transferred to flasks containing aseptic sorghum grains and incubated at 26 °C for 5–6 days with manual shaking once per day. The experiment was repeated twice.
In sequence, aseptic soil (peat soil: vermiculite, 2:1, v/v), of which the organic matter was 25.6 g/kg, available nitrogen 148.5 mg/kg, available phosphorous 50.6 mg/kg, rapidly available K 208.4 mg/kg with pH value 7.3, was placed in a pot (diameter 15 cm and 10 cm high), and then 20 sorghum grains inoculated with F. graminearum were placed on the soil surface, followed by 0.5 cm soil, five seeds, and finally 1.5 cm soil. Then, NPK (15-15-15) compound fertilizer (fertilizer:water for 1:20) was used to irrigate the root once every 2 weeks. Saturated crude lipopeptide extract of BM21 diluted 4000 times with tap water was used to irrigate each pot at time 0 h and again after 7 days with three replicates. An equal volume of sterile water was applied in the control. The seedlings were incubated in a greenhouse at 23 °C ± 5 °C with a photoperiod of 12 h/12 h (light/dark) for 20 days. The experiment was repeated twice. The specific treatments applied were as follows: (1) 0.2 mL lipopeptide extract applied as a root drench to each seedling after emergence; (2) 0.6 mL lipopeptide extract applied as a root drench to each seedling; (3) 1 mL lipopeptide extract applied as a root drench to each seedling; (4) 2 mL 64% Mancozeb WP (5 × 10−3 g plant−1) applied as a root drench to each plant; and (5) non-treated control. The pots were placed in a greenhouse at 25 ± 3 °C. The seedlings were watered daily using overhead irrigation to maintain soil moisture. Disease incidence was evaluated 20 days after sowing and was quantified as the percentage of diseased plants (Li et al. 2018). Disease reduction (%) was calculated as
$$ \frac{\mathrm{disease}\ \mathrm{incidence}\ \mathrm{of}\ \mathrm{untreated}\ \mathrm{control}-\mathrm{disease}\ \mathrm{incidence}\ \mathrm{of}\ \mathrm{the}\ \mathrm{treatment}}{\mathrm{disease}\ \mathrm{incidence}\ \mathrm{of}\ \mathrm{the}\ \mathrm{untreated}\ \mathrm{control}}\times 100\% $$
Data analysis
All experiments were conducted twice under similar conditions. Data were analyzed by analysis of variance (ANOVA), using IBM SPSS Statistics 19.0 (IBM Corporation, Armonk, NY, USA). Significant differences among treatment means were distinguished using Duncan’s multiple range test (P < 0.05).
