Fungal phytopathogens
Seven fungal phytopathogens (Colletotrichum gloeosporioides CFCC80308, Fusarium oxysporum CFCC82468, Alternaria tenuissima CFCC84533, Cytospora chrysosperma CFCC89600, Botryosphaeria dothidea CFCC82975, Mucor sp. CFCC80870, and Absidia sp. CFCC80375) were provided by China Forestry Culture Collection Center (CFCC). These fungi were maintained on potato dextrose agar (PDA) slants at 4 °C and grown on PDA plates at 28 °C when used.
Isolation of rhizobacteria
Rhizobacteria were isolated from the rhizosphere soil of healthy poplar plants in Haidian and Changping districts of Beijing City, China. Five grams of soil sample was transferred to 45 ml of sterilized water and shaken at 200 rpm for 20 min prior to use (Kurabachew and Wydra 2013). Gradient dilutions of the soil suspension were spread onto Luria-Bertani (LB) agar plates, and the plates were incubated at 30 °C in darkness. A single bacterial colony that is well separated from the other colonies were selected and re-streaked onto a new LB agar plate with an inoculating loop. Then, the plate was incubated at 30 °C for 24 h, and then stored at − 80 °C in LB with 30% glycerol (v/v).
In vitro screening of rhizobacteria against C. gloeosporioides
The rhizobacteria isolates were screened for antifungal activity against C. gloeosporioides using the dual culture assay described by Huang et al. (2014). The inhibition distance was defined as the width of the inhibition zone between the bacterial and fungal strains. The strain showed the highest antifungal activity was selected for further study.
Morphology, culture conditions, and physiological-biochemical characteristics of selected strain
Morphology, culture conditions, and physiological-biochemical characteristics were performed according to previously published methods of Dong et al. (2001) and Furuya et al. (2011).
Phylogenetic analysis of selected strain
The 16S rRNA gene was amplified and sequenced, using a single intact colony of selected strain according to a previously described method (Huang et al. 2014). The primers 63f (5′-CAGGCCTAACACATGCAAGTC-3′) and 1387r (5′-GGGCGGWGTGTACAAGGC-3′) were used for amplification. Polymerase chain reaction (PCR) conditions were as follows: 94 °C for 4 min, 35 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1.5 min, and a final extension at 72 °C for 10 min. The PCR products were sequenced by Invitrogen Corporation (Beijing, China). The sequences were blasted in the GenBank database, using a blastn search and aligned using Clustal W (ver. 1.82). Phylogenetic analysis was performed using MEGA (ver. 5.0) and a neighbor-joining phylogenetic tree was constructed by bootstrap analysis with 1000 replicates.
Evaluation of the effects of sterile culture filtrate
The strain selected from the in vitro test was grown in 100 ml modified medium no. 3 (g L−1; glucose 10.0, peptone 5.0, soybean meal 5.0, KH2PO41.0, MgSO4·7H2O 0.5, NH4Cl 3.0, Na2HPO41.0, and yeast extract 0.5; pH 7.0–7.2) in a 500-ml Erlenmeyer flask at 28 °C with 200 rpm. Culture broth was incubated for 3 days and centrifuged with 10,000 rpm at 4 °C for 20 min. Sterile culture filtrate (SCF) was obtained by passing the supernatant through a sterile membrane filter (0.22 μm; Pall, Ann Arbor, USA). The antifungal effects of SCF against 7 fungal phytopathogens were assayed by measuring the mycelial growth inhibition rates (Song et al. 2012). Briefly, 2 ml of SCF was mixed with 20 ml of PDA at 40–45 °C in a culture plate; no SCF was added for the control. After the medium was solidified, freshly growing fungal mycelial plugs (6 mm) were collected and inoculated onto the center of the plate using a sterile steel borer. The plates were incubated at 28 °C. The diameters of the fungal colonies were measured when those of the control colonies covered more than three quarters of the diameter of the dish according to the crisscross method. All experiments were repeated 3 times. The percentage of growth inhibition was calculated using the following formula (Yang et al. 2011):
$$ P\left(\%\right)=100\times \left[\left(C--d\right)--\left(T--d\right)\right]/\left(C--d\right) $$
where P is the inhibitory rate, C is the diameter of a control colony, T is the diameter of a treated colony, and d is the diameter of the mycelial plug.
Evaluation of the effects of crude protein and crude lipopeptide extracts
Crude protein and crude lipopeptide extracts were obtained respectively from SCF according to a previously described method (Huang et al. 2014). Then, the extracts were sterilized by passing through a sterile membrane filter (0.22 μm; Pall). The antifungal effects of crude protein and crude lipopeptide extracts against 7 fungal phytopathogens were assayed by measuring the mycelial growth inhibition rates using the procedure described above.
Evaluation of the effects of volatile compound(s)
The antifungal effects of the volatile compound(s) (VOCs) against the 7 fungal phytopathogens were tested by co-cultivating the fungi and bacteria on two-compartment plates (Yuan et al. 2012). One half of each plate was filled with PDA, while the other half contained TYB medium. A 6-mm diameter fungal plug was placed on the PDA side, and the candidate antagonistic strain was inoculated on the other side 24 h later. Plates not inoculated with the test strain served as the controls. Then, the plates were sealed with parafilm and incubated at 28 °C. The diameters of fungal colonies and percentages of growth inhibition were measured as described above. All experiments were repeated three times.
Characterization of cell wall-degrading traits
The cellulase activity and β-1, 3-glucanase activity of the test strain was determined, using a method described previously (Essghaier et al. 2009). The activities of β-1,3-glucanase, chitinase, protease, and lipase were determined using aniline blue pachyman agar plate, 0.2% colloidal chitin plate, skim milk agar plate, and 1% Tween 80 agar plate, respectively (Dong et al. 2001; Gao et al. 2009; Ren et al. 2013). Briefly, a single colony of test strain collected from an overnight culture was seeded onto the center of the plates; then, the plates were monitored for the appearance of a clear zone around the developing bacterial colonies at 28 °C.
Evaluation of biocontrol activity of the candidate strain under greenhouse conditions
To evaluate the biocontrol activity of the candidate strain towards poplar anthracnose caused by C. gloeosporioides, a pot assay was performed using 1-year-old poplar seedlings (Populus ×euramericana cv. ‘74/76’) with 7–10 expanded leaves (Huang et al. 2014). Each treatment consisted of 15 plants with 3 replicates (5 plants per replicate). Three treatments were set up as follows: SZ, the candidate strain and C. gloeosporioides sprayed on leaves at the same time; CK1, only sterile distilled water sprayed on leaves; and CK2, leaves only sprayed with C. gloeosporioides. For the phytopathogen inoculation, a spore suspension (1.0 × 105 spores ml−1) of C. gloeosporioides was sprayed on both sides of the leaves (2 ml per leaf) using an artist’s airbrush (YD12-F111, Yudi, Zhejiang, China). Sterile distilled water and the suspension (1.0 × 109 cfu ml−1) of the candidate strain were sprayed on the leaves using the same method as for C. gloeosporioides. The diseased leaves rate (DLR), disease severity (DS), disease index (DI), and greenhouse control efficacy (GCE) were investigated 12 days after C. gloeosporioides inoculation, respectively. The DS was expressed as a percentage of lesion area over the total surface area per leaf and divided into 6 ratings: 0, no lesion; 1, lesion area ≤ 5%; 2, lesion area 5–25%; 3, lesion area 25–45%; 4, lesion area 45–65%; and 5, lesion area > 65%. The DLR, DI, and GCE were calculated using the following formulas:
$$ {\displaystyle \begin{array}{l}\mathrm{DLR}\ \left(\%\right)=100\times \left(\mathrm{No}.\mathrm{of}\ \mathrm{affected}\ \mathrm{leaves}/\mathrm{No}.\mathrm{of}\ \mathrm{total}\ \mathrm{leaves}\right)\\ {}\mathrm{DI}=\left[100\times \sum \left(\mathrm{No}.\mathrm{of}\ \mathrm{affected}\ \mathrm{leaves}\times \mathrm{corresponding}\ \mathrm{DS}\right)\right]/\left(\mathrm{No}.\mathrm{of}\ \mathrm{total}\ \mathrm{leaves}\times 5\right)\\ {}\mathrm{GCE}\ \left(\%\right)=100\times \left[\left(\mathrm{DI}\ \mathrm{of}\ \mathrm{CK}2--\mathrm{DI}\ \mathrm{of}\ \mathrm{CK}1\right)--\left(\mathrm{DI}\ \mathrm{of}\ \mathrm{SZ}\ \mathrm{treatment}--\mathrm{DI}\ \mathrm{of}\ \mathrm{CK}1\right)\right]/\left(\mathrm{DI}\ \mathrm{of}\ \mathrm{CK}2--\mathrm{DI}\ \mathrm{of}\ \mathrm{CK}1\right)\end{array}} $$
Statistical analysis
The mean values and standard deviations were calculated, and statistically compared using analysis of variance (ANOVA) and Duncan’s multiple range tests (p ≤ 0.05) using SPSS software version 20.0 (SPSS Inc., Chicago, Illinois).