Ph. liquidambaris strain B3 and the plant pathogen F. proliferatum Ff-1 caused rice spikelet rot disease, were obtained from the Jiangsu Key Laboratory for Microbes and Functional Genomics, China. Ph. liquidambaris strain B3 was labeled with a green fluorescent protein (GFP) by the vector plasmid pCT74 and colonized in root of rice as root endophyte. Ph. liquidambaris strain B3 was stored and activated as previously described (Sun et al. 2020). Fungal hyphae were collected and washed twice with sterile distilled water and suspend again in sterile distilled water as fungal inoculum. The plant pathogen F. proliferatum Ff-1 was stored and activated on PDA (200g − 1 potato extract, 20 g− 1 glucose, pH 7.0) for 5 d at 28°C. Add sterile distilled water and use sterile spreading rod to scrape spore suspension (106 spores mL− 1) as plant pathogen inoculants.
Plant material and growth conditions
The rice seed used was a common cultivar “Nanjing 5055” grown in Jiangsu Province, China. All rice seeds were surface disinfected with 70% (v/v) ethyl alcohol for 5 min, followed by 10% (v/v) sodium hypochloride (NaClO) for 3 min and washed with sterile distilled water (SDW) to remove excess NaClO and germinated in the dark at 28°C. The 10- day- old rice seedings were transferred to float tray (8/15 cm, diameter/height) filled with sterilized soil substrate composed by the rice soil and vermiculite (2:1) and mixed thoroughly. For the inoculated group (E+), each rice seedling root was inoculated with 2 mL of the fungal inoculum by injector, and the control treatment (E-) was treated with 2 mL SDW.
In order to study the influence of Ph. liquidambaris B3 different inoculation time on plant immune response, experiments were carried out, containing five treatment: (a) infected with F. proliferatum Ff-1 after inoculation with Ph. liquidambaris B3 three days (BF, B3 pre-inoculation), (b) infected with F. proliferatum Ff-1 and inoculated with Ph. liquidambaris B3 at the same time (FB, B3 instant-inoculation), (c) infected with F. proliferatum Ff-1 alone (F), (d) inoculated with P. liquidambaris B3 alone (B), (e) noninoculated control(CK). The float trays were randomly placed at a control incubator (28°C for 16-h light and 8-h dark) with nutrient solution every 4 d. Fresh plant was collected at 0, 2, 4, 6, 10 d for experiment after inoculated. In addition, pathogenicity experiments were carried out to prove that Ff-1 can colonize and cause disease in rice seedlings (Fig. S1).
Endophytic and pathogenic colonization
Fresh leaves and roots were collected and rinsed thoroughly with SDW to extract the rice DNA
using an AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen Biosciences, CA, USA). Ph. liquidambaris colonization of rice root was quantified with a specific B3 ITS primer (Bf1/ Br1) set as previously described (Zhu et al. 2020). The quantitative detection of F. proliferatum in rice leaves was determined using specific primer (Ff1/ Fr1) as previously described (Zhu et al. 2020).
Determination of chlorophyll content
Fresh leaves were collected, and 0.1 g samples were immediately ground in liquid N. Chlorophyll was extracted with 20 mL 80% acetone at 4°C in the dark for 24 h. The solution was centrifuges at 12,500 gat 4°C for 10 min. The supernatants were separated and analyzed for chlorophyll content, and the absorption value was measured at 645 nm and 663 nm as per Hunt et al. (2005). Each sample was amplified in triplicate in each experiment.
Defense- enzyme assay
Fresh leaves and roots were collected, and 0.1 g samples were immediately ground in liquid N, and extracted with 1 mL 0.1M phosphate buffer solution (pH 7.4, 8.0 g L − 1 NaCl, 0.2 g L − 1 KCl,1.44 g L − 1 Na2HPO4, and 0.24g L − 1 KH2PO4), and centrifuged at 12,500 rpm at 4°C for 10 min. Enzyme activities of superoxide dismutase (SOD) and polyphenol oxidase (PPO) was measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. One unit of SOD activity was defined as the quantity when the inhibition rate is 50%. One unit of PPO activity was defined as an absorbance change of 0.01 per minute. Enzyme activities of chitinase and β-1,3-glucanase were detected with the 3,5- dinitrosalicylic acid (DNS) method according to Sridevi et al (2008). Colloidal chitin and laminarin (Sigma-Aldrich, Missouri, USA) were used as the substrates in chitinase and β-1,3-glucanase determined, respectively. One unit of enzyme activity (U) was defined as the amount of reducing sugar released per hour from chitinase and β-1,3-glucanase. Each sample was amplified in triplicate in each experiment.
Defense-related substance assay
The total phenol content in rice plants was determined using the Folin-phenol method (Zhao et al. 2016). Fresh plant samples were fully grinded and extracted in 90% ethanol solution and filled 25 mL to volume. 2 mL ethanolic extract and 2 mL foline-phenol solution were added in 10 mL centrifuge tube, then add 2 mL 10% Na2CO3 within 3 min and mix thoroughly in the dark for 1 h for detected at 700 nm. The control was determined with distilled water as extract solution. Each sample was amplified in triplicate in each experiment.
H2O2 and NO assay
Fresh rice tissues (0.1 g) were immediately ground in liquid N, and extracted with 0.9 mL 0.1M phosphate buffer solution and centrifuged at 12500 rpm at 4°C for 10 min. The H2O2 and NO contents were measured with assay kit (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China), respectively.
Histochemical marker staining assay
Trypan blue dye and Evans blue staining was used to observe plant cell death according to Morcillo et al. (2019) described, with some modification. In brief, fresh plant leaf tissues were collected at 5 d after F. proliferatum infection and placed into 5 mL centrifuge tubes containing trypan blue or evans blue and boiled for 3 min, then stained at room temperature for 2 h for leaves. The leaves were treated with 95% ethanol until they were completely colorless.
The detection of O2•− by NBT staining and DAB staining for H2O2 was performed as described by Wohlgemuth et al. (2002) and Bindschedler et al. (2006). In brief, fresh rice leaves were collected at 5 days after F. proliferatum infection and placed in 0.5 mg mL− 1 NBT or 1 mg mL− 1 DAB staining at 37 ° C for 2 h in darkness. Then the samples boil in a water bath for 10 min and treat with 95% ethanol until the leaves were completely colorless.
Quantification of plant hormone SA
At 0, 2, 4, 10 d after treated, the leaves and roots of plant were samples for SA content determined according to Yuan et al. (2016). Briefly, 0.1 g samples were immediately ground in liquid N, and added 0.5 mL methyl alcohol vortex shocked for 1 min followed by ultrasonicated for 5 min and extracted at 4°C for 24 h. The methyl alcohol extracted was centrifugated at 14,000 rpm for 10 min at 4°C and the precipitation was resuspension by methyl alcohol. The methyl alcohol supernatant was collected after twice centrifugation and then 10 µL of 0.2 M NaOH was added followed by quickly rot-evaporate to remove methyl alcohol. Resuspend the precipitate with 250 µL 5% trichloroacetic acid and added 0.8 mL of ethyl acetate: cyclohexane (1:1, v/v). Collected the supernatants after extract twice and added 60 µL 0.2 M NaAc (pH 5.5) followed by quickly rot-evaporate, and dissolve the residue by 600 µL of methyl alcohol for detection.
The SA concentration was determined using HPLC (Agilent Technology, Germany) after extraction, purification, and filtration (0.22 µm) according to our previous method (Wang et al. 2011). Chromatographic conditions: Agilent C18 column (250 × 4.6 mm, 5 µm); mobile phase: methanol: 2% acetic acid water: distilled water (50:40:10, v/v/v), the injection volume 20 µL, flow rate 0.7 mL min− 1, column temperature 25°C, detection wavelength 290 nm. The SA peak in the fresh sample was determined by comparing the retention time and area with the control standard.
RNA extraction and quantitative RT-PCR analysis
Real-time quantitative PCR (RT-qPCR) was performed to determine the expression levels of the genes of SA in plant. Plants were harvested at 0, 2, 4, 10 d after treated. Total RNA was extracted from plant samples using TRIzol reagent (Invitrogen, CA, USA), according to the manufacturer’s recommendations. The primers used in the study are listed in Table S1.
qRT-PCR was performed using the StepOne Real-time PCR system (Applied Biosystems) with SYBR Green I fluorescent dye (Takara, Dalian, China). The qRT-PCR reactions were conducted in 20 µL, which included 10 µL 2× SYBR Premix Ex Taq (Takara, Dalian, China), 0.4 µL of each primer, 1 µL of DNA template, and 7.6 µL ddH2O. The reactive step of qPCR followed: 94°C for 1 min, followed by 45 cycles of 94°C for 15 s, 60°C for 45 s and 72°C for 30 s. Relative expression levels for each gene were calculated by the 2−ΔΔC method (Zhu et al. 2020).
The chemical treatment was performed in the 10-day-old seeding by adding 10 mM H2O2, 5 mM DMTU (ROS scavenger, Hangzhou, China), or both them to rice leaves. The applied dose levels of the chemicals were referred to previous studies with some modified (Liu et al. 2015). The experiment contains 5 treatments: (1) infected with F. proliferatum Ff-1 and inoculated with Ph. liquidambaris B3 at same time (FB); (2) FB and 5 mM DMTU (FB + D); (3) infected with F. proliferatum Ff-1 and 10 mM H2O2 (F + H); (4) infected with F. proliferatum Ff-1,10 mM H2O2 and 5 mM DMTU (F + H + D); (5) infected with F. proliferatum Ff-1 (F). At 7 d after treated, the leaves were collected for further detection.
All experiments performed in the study were at least three times. All statistical analyses were performed using SPSS 18.0 (SPSS, Inc., Chicago, USA) and the final data were expressed as the mean with standard error (SE). When three or more groups were compared, a one-way ANOVA was performed followed by a Tukey’s multiple-comparison test. The data were considered significantly different at P ≤ 0.05. Graphs and images were assembled using Adobe Photoshop CS6 (CA, USA).