Plant material and inoculum preparation
The tobacco cultivar HonghuaDajinyuan (HD), which is highly sensitive to black shank, was used as material. The seeds (Gifted by tobacco companies) were sown in seedling trays, and the seedlings were transferred to bigger pots (diameter=30 cm) at 5-6 true-leaved stage, keeping one seedling per pot, and grown in the greenhouse (25±3℃), Yunnan Agricultural University, Kunming, Yunnan Province, China. The plantlets were used within two weeks of transplanting.
P. nicotianae strain maintained in our laboratory were routinely recultured on potato dextrose agar (PDA: 200 g of potato, 20 g of glucose and 15 g of agar in 1000 ml water) at 27°C in the darkness for 15 days standby16,17.
Effect of VB1 at different concentrations on mycelial growth and sporangium production of P. nicotianaein vitro
VB1 stock solution was prepared in sterile distilled water, and the solution was filtered through a microfiltration membrane. The effect of VB1 on mycelial growth of P.nicotianae was evaluated on PDA plates according to the method of Zhang et al18,19. A 7-mm P. nicotianae agar disk from actively growing 15-day mycelium of the pathogen was transferred into a new PDA plate with different concentrations of VB1 (0, 1, 2, 5, 10, 20 and 50 mM). The mycelial disk was placed in the center of the plate (diameter 90 mm). After 15 days of incubation at 28℃, the colony diameters were measured by the cross method. Each of the VB1 concentrations was replicated on four plates and the experiment was repeated thrice.
According to the reported method20, with slight modifications, the effect of VB1 on the sporangia of P. nicotianae was studied. Briefly, 0.1% KNO3 was used to prepare induction solutions containing different concentrations of VB1 (0, 1, 2, 5, 10, 20, 50 mM), and 7 mM agar of P.nicotianae agar disk from actively growing 15-day mycelium was transferred to a petri dish supplemented with induction solution (10ML), followed by culturing at 28°C for 48h. In an aseptic operating environment, the agar medium at the lower part of the bacterial disk was cut off in parallel to make the thickness about 1 mm, and placed on the slide to observe the number of sporangia under a 10x20 optical microscope, and take pictures and record. All treatments consisted of four replicates and the experiment was repeated thrice. By studying the effect of VB1 on the mycelial growth and sporangium production of P. nicotianae, an optimal VB1 induction concentration was screened for the next experiments.
Induction of ‘HD’ tobacco resistance against P. nicotianae by VB1
Tobacco seedlings of uniform size were sprayed with either distilled water (DW) or VB1 at concentrations of 20 mM. After that, the treated seedlings in each treatment group were separately covered with plastic bags to maintain high humidity and incubated in a climate-controlled room. A second spray was given seven days later. Each treatment was conducted in triplicates and each replicate contained 10 plants. Leaves were detached from the same layer of plants for assays at 0, 6, 24, 72 and 120 h, respectively21.
Three days after treatment of either DW or VB1(20mM) on the leaves, P. nicotianae was inoculated using the stem base trauma inoculation method of mycelium blocks (cut the base 2mm wound of tobacco stem with a scalpknife, inoculated with 5g P.nicotianae per plant, and then moisturized with sterile cotton)22. The inoculated seedlings were then maintained in a greenhouse. Experimental design and inoculation with P.nicotianae, two months old tobacco seedlings were treated as follows: (a)control:distilled water treated/distilled water treated (DW); (b)distilled water treated/inoculated with P.nicotianae (DW+P.nic); (c)VB1 treated/distilled water treated(VB1+DW); (d)VB1 treated/inoculated with P.nicotianae(VB1+P.nic). Six plants were used for each treatment. The experiment was repeated three times. Leaves were collected from plants at different intervals ( 1, 5, 10 dpi) and immediately frozen in liquid nitrogen, and stored at -80℃ until use for enzyme assays and gene expression analysis.
Protein extraction, H2O2 content and enzyme activity assays
Protein content, H2O2 content,and the activities of main antioxidant enzymes including CAT, POD and PAL were determined using protein assay kit, H2O2 content Assay kit, CAT Assay Kit, POD Assay Kit, PAL Assay Kit (Suzhou Greys Biological Technology Co., Ltd, Suzhou, China) , respectively, following the protocol provided by the manufacturer.
Leaf samples (0.1g fresh weight) were ground in a chilled mortar with liquid nitrogen, and then homogenized with 1 mL of cold 0.1 M Tris-HCl buffer, pH 7.0 containing 0.25% (v/v) triton-X and 3% (w/v) polyvinylpolypyrrolidone (PVPP). The extracts were then centrifuged at 12,000 rpm for 10 min at 4℃, and the supernatants were used for determining H2O2 content, enzyme activities and total protein content.
Protein and H2O2 content
The protein content was determined by the biuret method. In a strong alkaline solution, the biuret and CuSO4 form a purple complex. The color of the purple complex was proportional to the protein. The absorbance value was detected at a wavelength of 540nm. For H2O2 content assay, frozen sample (0.1g) was ground and homogenized with 1mL of chilled 100% acetone and then centrifuged at 10,000 rpm for 10 min at 4℃. By measuring the absorbance of the titanium-peroxide complex at 415 nm. The absorbance values were calibrated against a standard curve and expressed as µmol per gram of fresh weight (µmol g-1 FW).
CAT, POD and PAL activities
For CAT activity assay, the sample was treated with excess hydrogen peroxide, and the absorbance of the remaining H2O2 was measured at 510nm. One unit of CAT activity was defined as 1 µmol of H2O2 used in 1 min. The activity was represented as units per gram fresh weight (U g-1 FW). According to the reaction principle of peroxidase catalyzed by H2O2, the activity of peroxidase was determined by monitoring the increase in absorbance at 470 nm. One unit of POD activity was defined as the amount of enzyme that provided a change of 0.5 in absorbance per min per gram fresh weight (U g-1 FW). For PAL activity assay, according to PAL-catalyzed cleavage of L-phenylalanine into trans-cinnamic acid and ammonia, The maximum absorption value of trans-cinnamic acid was 290nm, and the PAL activity is calculated by measuring the increase rate of absorbance value. One unit was defined as the amount of enzyme that caused an increase in absorbance for 0.05. PAL activity was identified as units per gram fresh weight (U g-1 FW).
Total phenolic content and lignin detection
Leaf samples (0.1 g fresh weight) were frozen immediately in liquid nitrogen, ground to a fifine powder with a chilled mortar and pestle and then homogenized with 1.5 mL of 60% ethanol. The homogenate was centrifuged at 12,000 rpm for 10min at room temperature. The total phenol content of the extract was determined by Folin phenolic method. Under alkaline conditions, the phenolic substances reduced tungstomolybdic acid to produce blue compounds. The absorbance value was read at 760nm to determine the total phenol content. The absorbance values were calibrated against a standard curve and expressed as µg per Milliliter (µg ML-1). For the determination of lignin content, the leaf samples (1.5mg dry weight) were ground into a fine powder with a mortar and pestle, then homogenized with 1.5mL 80% ethanol and centrifuged at 12,000 rpm for 10min at room temperature. The total phenol content of the extract was determined by acetylation method, and the phenol hydroxyl group in the lignin was acetylated. The absorbance value was read at 280nm to determine the lignin content. The absorbance values were calibrated against a standard curve and expressed as milligram per gram of dry weight (mg g-1 DW).
SA and scopoletin measurements
The contents of SA and scopoletin in the leaf of tobacco seedlings were measured by high performance liquid chromatography (HPLC). SA content was determined according to previous studies23,24, With some little modifcations. Leaf samples (0.2 g fresh weight) were ground to a fine powder with mortar and pestle and then homogenized with 1 mL of 70% methanol, Extracted overnight at 4°C. After centrifugation at 8000g for 10min, the supernatant was extracted with 0.5ml 70% methanol for two hours. After centrifugation, the supernatant was extracted, combined with twice supernatant. The supernatant was adjusted with 50% (w/v) trichloroacetic acid (TCA) to produce a final concentration of 5% (w/v) TCA, and subsequently filtrated through a 0.45 µm membrane.
Scopoletin content was determined according to the method of Lerat et al25, Leaf samples of 0.1g fresh weight were ground into a fine powder with a mortar and pestle, and then dissolved in a flask with 20mL 50% methanol. The mixture was ultrasonically extracted at room temperature for 20 min, then centrifuged at 3000 rpm for 5 min and filtered through a 0.45µm aqueous phase membrane.
The chromatographic separation was performed on a C18 reverse-phase column (250mm×4.6mm,5μm) using jingdao LC-20AT high performance liquid chromatograph. The compound in the sample (10µL) separated from the mobile phase contained methanol and 0.1% acetic acid water. The flow rate was 0.8mL min-1, the column temperature was controlled at 35℃, and the alifting time was 40min. The SA UV detection wavelength was 306nm and the scopoletin detection wavelength was 340nm. Each sample was conducted by HPLC with three independent replicates.
Analysis of genes expression by quantitative real-time PCR
Transcription of defense-related genes was determined, and the expression levels of PR protein, SA pathway, ET pathway and HR pathway genes listed in Table 1 were detected26. Total RNA was extracted with MagenHiPure HP Plant RNA Mini Kit (R4165-02, Magen, China) from tobacco leaf tissue. The RNA samples were measured for the quality and quantity by measuring a ratio of 260/280 nm absorption and their integrity was evaluated by visualizing the bands on a 1% agarose gel electrophoresis. cDNA for RT-qPCR was synthesized from 2 ug total RNA using the abm 5×All-in-One RT MasterMix (with AccuRT Genomic DNA Removal Kit) kit according to the manufacturer's instructions.
The PCR conditions were as follows: an initial incubation at 95℃ for 3 min, followed by 45 cycles of 95℃ for 3 s and 60℃ for 30 s and then by a melting curve cycle. The threshold period (CT) and melting curve of each gene were analyzed. The relative mRNA amount was calculated by the 2-△△Ct method. Three biological replicates were performed for each experiment.
All of the data were analyzed using the SPSS 20.0. Significant differences between each experimental value between treatments were analyzed at p < 0.05 by Student’s t-test. Data are presented as means±SE. The graphs were generated using the origin 2018.