Plant materials
Virus-free potato tubers cvs Spunta, Nicola, Selatar, and Diamant, used in this study, were kindly provided by the Potato Brown Rot Project, Ministry of Agriculture and Land Reclamation, Egypt.
Source of the viral isolate
Potato virus Y (PVYNTN) isolate, used in this study, was previously isolated from infected potato plants (Mahfouze et al. 2012) and kindly provided by the Virology Laboratory, Agric. Microbiol. Dept., Fac. of Agric., Ain Shams Univ., Cairo, Egypt. It was propagated and maintained on Datura metel L. plants. The PVYNTN inoculum was prepared from infected potato plant leaves by extraction of infectious sap in the presence of 0.1 M potassium phosphate buffer pH 7 containing 0.02 M sodium sulfite (Nasr-Eldin et al. 2018).
Source of Streptomyces isolates
Streptomyces netropsis (DSM 40093) (SCF7), S. ambofaciens (SCF11), and S. actuosus (SCF20) were kindly obtained from Agric. Microbiol. Dept., Fac. of Agric., Ain shams Univ. (Cairo, Egypt) and subcultured and maintained on starch nitrate (SN) agar medium (Waksman and Lechevalier 1961). Streptomyces spp. were grown in a liquid medium and incubated on a shaking incubator at the optimum growth conditions (30 °C and 200 rpm for 5 days). The liquid cultures were filtered through two layers of Whatman No. 2 filter paper (0.5 pore size) and the filtrates were also sterilized through the Millipore filters (0.45 μm) (Megahed et al. 2013).
Greenhouse experimental design
Greenhouse experiment was carried out during the winter growing season of 2017 under conditions (25 °C ± 5) at the Faculty of Agriculture, Ain shams Univ. (Cairo, Egypt) for studying the effect of different CSFs on the induction of resistance in potato cultivars against PVYNTN. Potato cvs. Spunta, Nicola, Selatar, and Diamant were cultivated in plastic pots (20 cm in diameter) containing 3 kg of clay soil as one potato tuber/each pot. The experiment was conducted in five treatments (12 replicates/potato cultivar) as follow: (foliar spraying of plants with SCF7 + PVYNTN); (foliar spraying with SCF11 + PVYNTN); (foliar spraying with SCF20 + PVYNTN); (foliar spraying of plants with SN broth medium only + inoculation of viral extraction buffer, healthy control); and (foliar spraying of plants with SN broth medium only +PVYNTN, infected control). The foliar spraying of SCF was applied two times; the first application with SCF was done individually 2 weeks after cultivation, using 5 ml of each SCF (7 days pre virus inoculation, DPVI). After 7 days, potato leaves of the different cultivars were mechanically inoculated by PVYNTN and 7 days after virus inoculation (DAVI), the plants were foliar sprayed for the second time by 5 ml of SCF. All plants were daily observed and development of the symptoms was recorded. All experiment was repeated twice.
Evaluation of SAR
Recording of % of infection and disease severity
The progress of PVYNTN disease was evaluated visually and % of infected plants was recorded relative to time of external symptoms appeared. Disease index was recorded based on a scale proposed by Nasr-Eldin et al. (2018) of 0–10 as follows: 0 = no symptoms; 2 = mild mosaic; 4 = mild necrosis; 6 = mild mosaic with mild necrosis; 8 = severe mosaic with necrosis; 10 = severe apical necrosis. The disease severity (DS) values were calculated 7, 14, and 21 days after viral inoculation DAVI using the following formula according to Yang et al. (1996). Area under disease progress curve (AUDPC) was calculated.
$$ \mathrm{DS}=\frac{\sum \left(\mathrm{disease}\ \mathrm{grade}\ \mathrm{X}\ \mathrm{number}\ \mathrm{of}\ \mathrm{plants}\ \mathrm{in}\ \mathrm{each}\ \mathrm{grade}\right)}{\left(\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{plants}\times \mathrm{Xhighest}\ \mathrm{disease}\ \mathrm{grade}\right)} $$
Quantitative detection of PVYNTN concentration by DAS-ELISA
Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was used to detect the PVYNTN infection and to check the virus concentration in potato leaves after 7 and 21 DAVI (Clark and Adams 1977) at the Virology and Phytoplasma Department, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt. DAS-ELISA was repeated in triplicate/each treatment specific for cultivar.
Detection of PR-1b gene by real-time RT-PCR
For studying the gene expression of PR-1b gene in potato plants at 7 and 21 DAVI, total RNA was extracted from the collected fresh potato cvs leaves specific for each treatment. Leaves were grinded thoroughly under a liquid nitrogen using a mortar. The formed powder was placed into a separate micro-centrifuge tube and the total RNA extraction was performed by using TRIzol™ Reagent, Invitrogen, USA. The extracted RNA was stored at − 30 °C for further study.
Complementary DNAs (cDNAs) were carried out, using the high capacity cDNA reverse transcription Kits (Applied Biosystems, USA) according to the manufacturer’s protocol. All reagents and supplies used in this step, were nuclease-free. Reverse transcription master mix (2×) was prepared. To create a 1× mix reaction, 2 μg of total RNA per 20-μl reaction was added individually to the 2× RT master mix and the reaction volume was set to 20 μl. The reactions were loaded into the thermal cycler (Biomtera, T personal, USA) and thermos cycling condition was programmed as following: step 1 (25 °C for 10 min), step 2 (37 °C for 120 min), step 3 (85 °C for 5 min), and step 4 (4 °C on hold).
The yield concentration of cDNAs were measured in Nanodrop spectrophotometer (Thermo scientific, USA) and stored at − 30 °C for further study. PCR amplifications were performed using real-time PCR (7500, Applied Biosystems) in total volume 25 μl using the above cDNA as templates. In addition to the samples to be analyzed, a negative control reaction without cDNA was included, Reference gene (Coxa) was included for each sample for potato plant (cox gene) (designed with the potato cytochrome oxidase gene sequence) with the following primer sequence: forward, 5-CGT CGCATT CCA GAT TAT CAA-3 and reverse, 5-AA CTACGG ATA TAT AAG AGC CAA AAC TG-3 according to Wilmer et al. (2006). Primers for PR-1b were designed as described by Baebler et al. (2009) were 5-GTATGAATAATTCCACGTACCATATGTTC-3 (forward primer) and 5-GTGGAAACAAGAAGATGCAATACTTAGT-3 (reverse primer). The reaction mixture components of different genes expression consist of: 12.5 μl of SYBR Green (Applied Biosystems, USA) RT-PCR Master Mix (1×), 1.0 μl of forward primer and reverse primer (10 pM), 8.0 μl of RNAse-free H2O, and 2.5 μl of cDNA templates. Thermo-cycler program was performed according to Oufir et al. (2008) as follow: 5 min at 94 °C, 40 cycles of (60 s at 95 °C, 60 s at 58 °C, 60 s at 72 °C). The fluorescence data was acquired during the 72 °C extension and the specificity and identity of the RT-PCR products were verified by performing a melt curve analysis: A melting step (dissociation stage) was added that ramps from 72 to 99 °C, raised by 1 °C in each step, and waited for 30 s on the first step and 5 s each step afterward. The standard curve method was used for relative gene expression quantification, and the transcript accumulation of each gene was normalized to Cox Reference gene. Expression level of PR1-b gene was performed at two time interval, 7 and 21 DAVI. The experiment was repeated twice. The results were the mean of three replicate/treatment. Relative expression levels were calculated as the ratio of expression of each gene against that of the Cox gene. The normalized ΔCT data were used to calculate the relative gene expression fold change using a selected calibrator (reference sample) according to Livak and Schmittgen (2001).
Fold Change (RQ) = 2-ΔΔCT
Where, ΔΔCt = Δ CT (test sample) − Δ CT (calibrator sample)
Δ CT (test sample) = CT (target of interest) − CT (reference gene in sample) and
Δ CT (calibrator sample) = CT (target in control) − CT (reference gene in control).
During calculation of the fold change in gene expression, the undetectable or low expression values of PR-1b gene in different treatments (Ct values were above 30 or undetermined) were excluded from analysis. As the gene was never expressed in PVYNTN infected plants, therefore the Δ CT value of the PVYNTN infected potato plants was equal to the value of ΔΔCt during calculation of fold change.
GC-MS fractionation of SCF20 ethyl acetate extract
The components of S. actuosus (SCF20) crude filtrate were extracted, using ethyl acetate as a solvent. The solvent was added to the filtrate in the ratio of 1:1 (v/v) and was shaken vigorously for 20 min. The ethyl acetate phase that contained the secondary metabolites was separated from the aqueous phase using separating funnel. Ethyl acetate layer was concentrated by evaporating to dryness at 50 °C using a rotary evaporator and the residue was injected through gas chromatography-mass spectroscopy (GC-MS) analysis (Ahmed 2007). GC-MS analysis was performed to identify the chemical compounds found in the crude extract of S. actuosus. Thermo scientific technologies Trace 1310 with capillary column TG-5 (30 m × 250 μm × 0.25 μm) system were used and the test was performed at the Regional Center For Mycology and Biotechnology, AL Azhar University, Cairo, Egypt. Mass detector used in split mode, and helium gas with flow rate of 1.5 ml/min was used as a carrier. Injector was operated at 230 °C and oven temperature for initial setup was 60 °C for 2 min, ramp 10/min to 300 °C for 8 min. Mass spectra were taken at 70 eV, during running time 45 min.
Statistical analysis
Post-hoc analysis (Dunnett’s test) for one-way independent ANOVA (SPSS 23) was employed to test the significant difference in AUDPC between PVY infected control and each of the different treatments for separately. Post-hoc analysis (Bonferroni test) was conducted to test the significant difference in AUDPC among different potato verities. Linear contrast (one-way ANOVA) analysis was conducted to test the difference in virus concentration for different treatments after 7 and 21 DAVI for each potato cultivar separately.