Sampling site and sample collection
Twenty-five soil samples were collected from healthy banana rhizosphere soil in an area affected with Fusarium wilt of banana in Zhanjiang City, Guangdong Province in November of 2015. From a healthy plantain garden where bananas has been grown for more than 10 years, rhizosphere soil was collected at 10–30 cm of the root of healthy plants. Soil samples were collected and placed inside a sterile plastic bag, sealed, and preserved in an ice box. In the laboratory, the roots and stones were removed and stored at 4 °C before the actinomycetes were isolated.
Pathogenic fungi strains includes Fusarium oxysporum f. sp. cubense Race1 (FOC.1), F. oxysporum f. sp. cubense Race 4 (FOC.4), Curvulatia fallaxis (CFO), Colletotrichum gloeosporides (CG), and Alternaria tenuissima Maa (MAA), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), and Staphylococcus aureus (S. aureus)were provided by the Institute of Environment and Plant Protection. Taq PCR Master Mix and bacterial genomic DNA rapid extraction kit were purchased from Sigma-Aldrich Co. (USA).
“Gause’s no. 1 culture”: 20.0 g Soluble starch, 0.5 g NaCl, 1.0 g KNO3, 0.5 G K2HPO 4·3H2O, 0. 5 g MgSO 4·7H2O, 0.01 g FeSO 4·7H2O, 20 g agar, 1000 mL H2O
Isolation and purification of actinomycetes
Actinomycetes were isolated by serial dilution method on Gause’s no. 1 culture [13, 19, 21, 24, 25], which was sterilized at 121 °C for 20 min, static, and cooled to room temperature. In total, 3 mL of 1% dichromate solution after filter sterilizer was added to the culture (per liter); 0.5 mL of 5% mycetin was set aside for reverse plate. Soil sample at 10 g was dissolved in 100 mL aseptic water. The suspension was mixed well and then diluted 10 times to prepare 10− 4, 10− 5, and 10− 6 suspension. Next, 0.1 mL of suspension was added to the Gause’s no. 1 culture. The gradient was set to three iterations and incubated at 28 °C for 4 days. After a single colony was purified, it was transferred to Gause’s no. 1 culture for 4 days [14, 15, 26]. The serial number was preserved.
Screening of antagonistic Actinomycetes
The antimicrobial activity of the isolated actinomycetes was screened by double-layer agar method. After culturing for 5 days, actinomycetes were cut into pieces with a perforator (Φ = 8 mm) and moved to the center of Gause’s no. 1 culture. They were then grown at 28 °C for 2 days for colonization. Fusarium wilt of banana fungal pathogen was prepared for the fungi suspension (6 × 108 CFU/mL) with aseptic water and evenly sprayed on the plate with a 2-day culture of actinomycetes. Suspension was poured into 15 mL Nutrient Agar (NA) culture (the formula: 10.0 g protein, 3.0 g beef powder, 5.0 g sodium chloride and 15.0 g agar were dissolved in 1 L distilled water, pH was adjusted to 7.3) at 40 °C. Each treatment was repeated thrice. A plate without actinomycetes was used as the control. The diameter of the inhibition circle was measured at 28 °C for 24 h [16, 23, 25,26,27,28].
Inhibitory effect of antagonistic Actinomycetes on the growth of pathogenic Fungi and Bacteria
Before the inhibitory experiment, the screened antagonistic actinomycete was cultured for 5 days, and pathogenic fungal cake was prepared by sterile culture (Φ = 8 mm). The fungi were placed at the center of a PDA flat [14, 15]. After culturing for 2 days at 28 °C, five kinds of pathogenic fungal cakes were inoculated symmetrically 2 cm apart from both sides of the actinomycetes by using the same method. A fungal disc was placed in the center of the plate. A fungal mycelia disc alone in the center of the plate served as a control. The diameters of the inhibition zones were measured after incubation for 7 days at 28 °C. Each treatment was repeated thrice, and the width of the inhibition band was utilized to measure the inhibitory effect of this strain on pathogenic fungi. E. coli, S. aureus, and B. subtilis, were made into bacterial suspension (1 × 108 cfu/ml) with sterile water. The paper soaked with actinomycetes was evenly placed in a 9 cm plate coated with 200 mL bacterial solution at 37 °C and cultured for 24 h. The diameter of the bacteriostat was measured. Meanwhile, paper soaked in normal saline was used as the control. We aimed to analyze the inhibitory effect of the screened antagonistic actinomycete on pathogens and bacteria.
Pot culture experiments
Pot culture experiments were performed under greenhouse conditions, i.e., 28 °C, 70% humidity, and sufficient natural light. The fermentation broth of actinomycete FS-4 was inoculated in sterilized Gause’s no. 1 culture and incubated with shaking (150 rpm) at 28 °C for 7 days. The fermentation broth was filtered then diluted 50-fold, and 50 ml of the liquid was inoculated into the banana seedlings to establish four treatment groups, as follows: CK1 (non-inoculated FOC.4 and application of sterile water); CK2 (inoculated FOC.4 and application of sterile water); CK3 (inoculated FOC.4 and polymycin); and A (inoculated FOC.4 and fermentation broth of actinomycete FS-4). Banana seedlings with consistent growth rate of 5–6 leaves were selected and soaked in pathogen suspension at a concentration of 107 cfu /mL. Then, they were transplanted into a plastic bowl containing 700 g soil and filled with 50 mL pathogen suspension at the rhizosphere soil of banana seedlings. The banana seedlings were cultivated in a greenhouse. Each experiment was repeated thrice. The watering treatment liquid A and the positive control CK3 were applied to the roots of the banana seedlings. The control treatment of the wilt disease was water treated with water for several days. The disease index and the disease prevention effect of banana seedlings transplanted during 49 days were calculated by the following formula. The average fresh biomass and incidence of banana seedlings were determined on the 49th day.
$$ \mathrm{Disease}\ \mathrm{index}=\frac{\sum \left(\mathrm{number}\ \mathrm{of}\ \mathrm{disease}\mathrm{d}\ \mathrm{plants}\times \mathrm{representative}\ \mathrm{value}\right)}{\mathrm{the}\ \mathrm{sum}\ \mathrm{of}\ \mathrm{the}\ \mathrm{number}\ \mathrm{of}\ \mathrm{plants}\times \mathrm{the}\ \mathrm{representative}\ \mathrm{value}\ \mathrm{of}\ \mathrm{the}\ \mathrm{most}\ \mathrm{serious}\ \mathrm{disease}}\times 100\% $$
$$ \mathrm{Disease}\ \mathrm{prevention}\ \mathrm{effect}\ \left(\%\right)=\frac{\mathrm{control}\ \mathrm{disease}\ \mathrm{index}-\mathrm{treatment}\ \mathrm{of}\ \mathrm{disease}\ \mathrm{index}\ }{\mathrm{control}\ \mathrm{disease}\ \mathrm{index}}\times 100\% $$
Classification and identification of strains
Observation of morphological characteristics
Sterilized glass was inserted into the Gause’s no. 1 culture inoculated with actinomycetes at 45 °C. Coverslips were taken out after incubation at 28 °C for 21 days [14, 21]. Aerial hyphae, hypha, and spore characteristics were observed with a transmission electron microscope.
Physiological and biochemical characteristics
The target actinomycetes were cultured at 28 °C for 21 days. The color of hyphae and airborne hyphae and the existence of soluble pigments were observed [15, 21].
Phylogenetic characteristics
Genomic DNA of FS-4 was isolated using the bacterial genomic DNA rapid extraction kit (Sigma-Aldrich Co., Ltd., USA) and the bacterial 16S rDNA was amplified using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). PCR amplifications were performed with a Trio PCR System (Biometra, Germany), in a total volume of 25 μl consisting of template DNA (2.0 μL), 2 × Taq PCR Master Mix (12.5 μl), ddH2O (8.5 μl), Upstream primer (1 μL) and Downstream primer (1 μL). The conditions for thermal cycling were as follows: denaturation of the DNA at 94 °C for 5 min; and 35 cycles at 94 °C for 30 s, primer annealing 30 s at 55 °C for template DNA, and DNA elongation at 72 °C for 90 s.
PCRs were performed in the Trio PCR System (Biometra, Germany). The PCR system and conditions were as described by Himaman et al. [29]. The PCR amplification products were visualized by 1.0% (w/v) agarose gel electrophoresis. The amplified PCR products were sequenced by a Sanger-based, automated sequencer (Applied Biosystems).
The product was purified and sequenced. GenBank was used to search for sequence similarity, and the sequence of the pattern strain with high similarity was selected. The homology was compared by using the neighbor-joining method in MEGA5.0. A phylogenetic tree was constructed to determine the taxonomic position of the actinomycetes [19, 22].
Optimization of the conditions for improving the bacteriostatic activity of the Actinomycete FS-4 fermentation broth
After cultivation in Gause’s no. 1 culture at 37 °C at 120 r/min, precipitation for 24 h at 4 °C, and autoclaving at 121 °C and 0.1 MPa, treatment with actinomycetes FS-4 antagonistic bacterial fermentation broth was performed for 20 min.
Effects of Different Carbon and Nitrogen Sources
Under the same culture conditions, 2% glucose, saccharose, soluble starch, corn flour, and lactose were used to replace the soluble starch in the basic fermentation culture. Then, 0.1% of ammonium sulfate, monarkite, nitrate potash, yeast powder, and peptone instead of KNO3 were respectively added to the basic fermentation culture. The best carbon and nitrogen sources were determined thrice according to the method described in Section 2.4.
Effect of Inorganic Salt Content
The concentrations of sodium chloride, potassium hydrogen phosphate, and magnesium sulfate were set at 0.025, 0.050, 0.075, 0.100, and 0.125%. The best inorganic salt content was determined thrice according to the method described in the section on the screening of indicator strains.
Effects of Initial pH, Fermentation Temperature, and Time
The initial pH of the culture was set at 5–9, temperature was set at 22 °C–34 °C, and time was at 48–144 h. The optimum initial pH value, fermentation temperature, and time were determined thrice according to the method described in the section on the screening of indicator strains.
Optimization of fermentation conditions by response surface analysis
On the basis of the single-factor experiment, sucrose, peptone, and fermentation time were selected as the investigation factors, and response surface analysis was carried out according to the method described in the section on the screening of indicator strains.
Statistical analysis
Design-Expert V8.0.6 software was used to design the Box–Behnken experiment. All the experiments were repeated thrice, and ANOVA was carried out by SPSS 19.0.