2.1 Plants, bacterial strains, fungi, oomycete, and growth conditions
All Arabidopsis lines used in this study were as follows: Col-0 (Arabidopsis wild type); wrky33 (Zheng et al., 2006);pen3-3 , pdr12-2 , the PEN3 and PDR12 gene double mutant line pen3-3/pdr12-2 (He et al., 2019).Arabidopsis seeds were surface sterilized (soaked in 70% ethanol solution for 10 min and rinsed 5-8 times in sterile water) and then incubated at 4℃ dark conditions. After 4 d of vernalization, seeds were transferred to a half-strength Murashige and Skoog (1/2 MS) plate using a pipette and incubated for about a week. Seedlings were then transferred into black pots containing sterilized vermiculite: nutrient-rich soil (in a 2:1 volume ratio) for cultivation at an ambient temperature of 22℃ under a 16-h light/8-h dark photoperiod and 70% relative humidity. The PGPR strain B. cereus AR156 was grown on Luria-Bertani (LB) agar plates for 24 h at 28℃. Single colonies were picked and inoculated into liquid LB medium and incubated at 28℃ and 200 r for 24 h. Subsequently, the bacterial solution was collected by centrifugation at 4500 rpm and resuspended with 0.85% NaCl to a concentration of about 5 × 107 CFU mL-1. Pst DC3000 was grown in KB agar medium containing 50 mg·L-1 rifampicin and 50 mg·L-1 kanamycin for 2 d. Single colonies were picked and inoculated into KB liquid containing 50 mg·L-1 rifampicin and 50 mg·L-1 Kanamycin grown overnight at 28℃.Pst DC3000 organisms were collected by centrifugation and resuspended with 10 mM MgCl2 (containing 0.02% (v/v) Silwet L-77) and adjusted to 5 × 107 CFU mL-1 for use. The gray mold fungus was grown on potato dextrose agar (PDA) medium at 25℃ for about 10 d. Conidia were then collected and resuspended in 10 mM MgCl2, filtered through three layers of gauze to remove mycelium, and the number of conidia was counted using a hemocytometer plate and adjusted to 1 × 106 conidia mL-1 (Aziz et al., 2003). P. capsici LT263 was grown in dark culture on V8 juice agar medium at 25℃ for 3 d. 2-mm disks of 4-day growth medium were cut along the edges with a scalpel and incubated in V8 liquid medium for 2 ; the culture solution was discarded, the mycelium was washed three times with sterilized tap water, and incubated in sterile tap water for 12 h until mature sporangia were induced. Zoospores were obtained by incubating at 4℃ for 20 min followed by 2 h incubation at 25℃.
2.2 Biocontrol bacterial treatment and pathogen infection assays Four-week Arabidopsis was pre-treated with B. cereus AR156 as previously described (Jiang et al., 2015). For bacterial treatments, 10 mL of 5 × 107CFU·mL-1 cell suspension of AR156 was irrigated on the soil around the roots of Arabidopsis in each pot, with an equal volume of sterile 0.85% NaCl as a control. The pathogens were inoculated after 5 d of pretreatment with B. cereus AR156. The leaves were challenged with a 10-il droplet containing approximately 500P. capsici zoospores, with 10 µL droplets of B. cinerea at 1 × 106 conidia mL–1 or by sprayingPst DC3000 at 1 × 108CFU·mL-1 to evaluate disease symptoms. The biomass ofP. capsici and B. cinerea was determined as previously reported and further improved slightly. Briefly, leaf discs (1 cm in diameter) around spore droplets were collected from different leaves of each treatment using a punch. Genomic DNA was extracted by the CTAB method and pathogen biomass was quantified by real-time PCR. To calculate colonization of Pst DC3000 on Arabidopsisleaves, samples were collected with a puncher at 0 and 3 days (dpi) after inoculation with Pst DC3000. Arabidopsis leaf discs were surface sterilized in 70% ethanol for 30 seconds and washed five times with sterile distilled water. The samples were then ground and gradient diluted with 0.9 ml of 10 mM MgCl2. The samples were then ground and gradient diluted with 0.9 ml of 10 mM MgCl2. Subsequently, the dilutions of 100 μL were applied uniformly to a KB agar medium containing 50 mg·L-1 rifampicin and 50 mg·L-1kanamycin and incubated at 28℃. Colonies were counted after 48 h and the density of Pst DC3000 in the leaves was calculated.