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.