Samples Material
The pathogen used in this study was obtained from Guangxi Academy of Sciences (Fusarium oxysporum f. sp. cubense (FOC) 1, Fusarium oxysporum f. sp. cubense (FOC) TR4, Fysarium fujikuroi, Fusarium proliferatum, Penicillium crustosum, Cladosporium sphaeroporum, Escherichia coli, Listeria monocytogenes, Ralstonia solanacearum moderately, Staphylococcus aureus, Pseudomonas fluorescens). Citrus fruits were collected during January 2021 in the town of Nanning. Bacillus was isolated from decaying Citrus and stored in the microbial collection of the Laboratory preservation Center at Guangzhou. Penicillium sp. strain was grown at 25℃ in Potato Dextrose Agar (Potato Dextrose Agar, PDA: bought from Coolaber). Bacillus strain was grown at 37℃ in LB medium (0.5% yeast extract, 1% peptone, 1% sodium chloride).
The Bacillus biological control agents isolation and identification
The Bacillus biological control agents (ACBL) were isolated from the citrus peel during January 2021. They were obtained from 3 citrus producing regions in Nanning, China. The Bacillus isolation was carried out according to Babadoost et al. (2002). The epidermis of fruit disinfected by 75% ethanol was cut into pieces and placed in LB medium to enrich the bacteria. Using the decimal dilution technique and a sterile water solution sterile distilled water. Plating was performed in triplicate using LB culture media (Yeast extract peptone dextrose: 0.5% yeast extract, 1.0% peptone, 1.0% sodium chloride, 1.5% agar dissolved in 1.0 liter of distilled water). The bacteria with inhibitory effect on Penicillium sp. were screened by plate confrontation tests (Ji et al. 2013). The strain about the most obvious inhibition zone was sequenced for verification. Briefly, the web-based tool from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) was used to analyze strain types. The strain utilized the universal primers 5′-AGAGT TTGATCCTGGCTCAG-3′ and 5′-GGTTACCTTACGACTT − 3′. Using the wr8 sequence as the target, 27 strains were selected, which were different from the wr8 type. Multiple sequence alignments of wr8 and other strain sequences were generated using ClustalW version 1.83. A phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA 6.0 with 1000 bootstrap replications.
Antifungal efficacy of Bacillus velezensis wr8 against Penicillium sp.
PDA medium was used to grow fungal colonies. After 5 days of culture, agar discs (1 mm in diameter) with mycelia were excised and then transplanted to the center of PDA dishes (90 mm in diameter) under sterile conditions. The growth radius of pathogenic bacteria is 1 cm, Bacillus velezensis wr8 was inoculated. The diameters of the colonies were measured to analyze the growth of Penicillium sp..
Screening of the Bacillus strains for antifungal activity in vitro against Penicillium sp.
Antifungal activity of secondary metabolite against Penicillium sp. was evaluated by agar well diffusion method (Puskarova et al. 2017). Penicillium sp. spore concentration was adjusted to 1 × 106 conidia mL− 1, Then mix 1ml spores into PDA soft agar and we poured evenly on the plate. Agar well diffusion (9 mm) containing 100µL secondary metabolite were gently the agar plates. The plates were incubated at 25°C for 3 days, and the diameter of the inhibition zone was observed. Bacillus strain was grown at 37℃ in LB medium (0.5% yeast extract, 1% peptone, 1% sodium chloride) and Obtaining secondary metabolites by 1 × 106 conidia mL− 1, 0.22um membrane filtration.
Inhibitory activity of Bacillus velezensis wr8 against citrus pathogenic Penicillium sp.
According to the methodology of Ferraz et al. (2016), the safe 64 oranges were washed, superficially disinfected with 0.2% (v/v) sodium hypochlorite for 3 min and rinsed in sterile water to eliminate the sodium hypochlorite. Then, the fruits were wounded at two equidistant points, and 20 µL of Penicillium sp. conidial suspension (1 × 106 conidia mL− 1) was inoculated in the wounded area, which was treated 24 h later with 20 µL wr8 secondary metabolites (1 × 107 mL− 1). The control group was treated with the equal amount of sterile water instead of wr8 suspension. Disease severity was assessed on the 15th days after inoculation by observation blue mold lesions region size phenotype.
Inhibition Spectrum of Bacillus velezensis wr8
Antibacterial spectrum of Bacillus velezensis wr8 was examined using agar well diffusion assay (Perumal et al. 2019). In brief, LB agar plates were overlaid with 9 mL of LB soft agar (0.8% agar) inoculated with 1% of each indicator strain, which was previously grown until the suspensions reached 1 × 106 conidia mL− 1. Wells were made on the seeded plates using a sterile Pasteur pipette tip (9 mm in diameter) and culture 100 µl sterile supernatan t of Bacillus velezensis wr8 was added into each well, by 0.22um filter membrane filter. The plates were incubated overnight at 37℃ and then inhibition zones were examined. Antifungal spectrum of Bacillus velezensis wr8 was detected by plate confrontation method.
Purification of Bacillus velezensis wr8 secondary metabolites
For Bacillus velezensis wr8, incubated 1 L bacterial suspension at 220 r·min− 1 for 24 h and centrifuged to remove cells. After the sterile supernatant was lyophilized and dissolved in methanol at the rate of 1:50 (w/v) and stirred for 6 h. Methanol was evaporated by rotary evaporator. Each Bacillus culture was filtered and a 0.22 µm Millipore membrane after incubation to remove Bacillus cells according to a protocol adapted from Valarini et al. (1995). The active dialyzed fraction was then further purified by AKTA prime plus system (Amersham-Bioscience, Uppsala, Sweden) calibrated with 20 mmol·L− 1 Tris-HCl buffer, at a flow rate of 0.8 mL·min− 1. The active CEC fraction was introduced to preequilibrated (ddH2O with 2% acetonitrile and 0.1% TFA) RP-HPLC purifier and elution was conducted using a linear gradient from 95% solvent A (ddH2O with 2% acetonitrile and 0.1% TFA) and 5% solvent B (100% acetonitrile with 0.1% TFA) for 10 min to 100% solvent B for 50 min. All fractions were procured at 280 nm absorbance and lyophilized. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed by using a MALDI-TOF spectrometer (Bruker Daltonics, Bremen, Germany) in positive ion mode, to determine the molecular mass of pure bacteriocin.