Flavescence Dorée Strain-Specific Impact on Phenolic Metabolism Dynamics in Grapevine (Vitis vinifera) throughout the Development of Phytoplasma Infection

Flavescence dorée phytoplasma (FDp) is a phytopathogenic bacterium associated with Grapevine yellowS disease, which causes heavy damage to viticultural production. Epidemiological data revealed that some FDp strains appear to be more widespread and aggressive. However, there is no data on mechanisms underlying the variable pathogenicity among strains. In this research, we employed chromatographic and spectrophotometric techniques to assess how two strains of FDp influence the levels of grapevine phenolic compounds, which are frequently utilized as indicative markers of stress conditions. The results pointed to the upregulation of all branches of phenolic metabolism through the development of infection, correlating with the increase in antioxidative capacity. The more aggressive strain M54 induced stronger downregulation of phenolics’ accumulation at the beginning and higher upregulation by the end of the season than the less aggressive M38 strain. These findings reveal potential targets of FDp effectors and provide the first functional demonstration of variable pathogenicity between FDp strains, suggesting the need for future comparative genomic analyses of FDp strains as an important factor in exploring the management possibilities of FDp.


Determination of total phenolics content
For the determination of total phenolics content in grapevine leaves extracts, method according to Singleton et al. (1999) was used.Extracts (10 μL) were mixed with 790 μL of deionized water and 50 μL of Folin-Ciocaulteu (FC) reagent and the mixture was homogenized on a vortex mixer.Then, 150 μL of 1.88 M Na2CO3 were added and the mixture was homogenized and incubated for 30 min at 45 °C in an incubator.After the incubation, 200 μL of the sample were transferred in four technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 765 nm on a FLUOstar Optima microplate reader (BMG LABTECH, Ortenberg, Germany).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the total phenolics content in the samples was calculated indirectly as mg of gallic acid equivalents (GAE) per g of DW based on the calibration curve of standard gallic acid solutions of known concentrations (5-0.05mg/mL).

Determination of flavonoid content
For the determination of total flavonoid content in grapevine leaves extracts, method according to Zhishen et al. (1999) was used.Extracts (70 μL) were mixed with 280 μL of deionized water and 21 μL of 5% NaNO2 solution and the mixture was homogenized on a vortex mixer and incubated for 5 min at room temperature (RT).Then, 21 μL of 10% AlCl3 was added, and the mixture was homogenized and incubated for 6 min at RT.After incubation, 140 μL of 1 M NaOH and 168 μL of deionized water were added.Afterwards, 200 μL of each sample were transferred in three technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 510 nm on a FLUOstar Optima microplate reader (BMG LABTECH, Ortenberg, Germany).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the total flavonoid content in the samples was calculated indirectly as mg of quercetin equivalents (QE) per g of DW, based on the calibration curve of standard quercetin solutions of known concentrations (1-0.0125mg/mL).

Determination of catechin content
Catechin (flavan-3-ol) content in grapevine leaves extracts was evaluated using the method modified from Rusak et al. (2021).The extract was diluted with 70% ethanol (v/v) to 10 mg/mL and 280 μL of each diluted extract was mixed with 420 μL of DMACA (0.1% pdimethylaminocinnamaldehyde in 1 M HCl, in methanol) solution.After mixing the samples on a vortex mixer, 200 μL of each sample were transferred in three technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 595 nm on a FLUOstar Optima microplate reader (BMG LABTECH, Ortenberg, Germany).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the total catechin content in the samples was calculated indirectly as mg of catechin equivalents (CE) per g of DW based on the calibration curve of standard catechin solutions of known concentrations (1-0.01 mg/mL).

Determination of proanthocyanidin content
Proanthocyanidin (condensed tannin) content was evaluated according to the method in Šamec et al. (2014).Away from light, 420 μL of vanillin (4%, w/v; dissolved in methanol) was mixed with 70 μL of the extract and 210 μL of HCl was added.The mixture was homogenized on a vortex mixer and incubated for 15 min at RT.After the incubation, 200 μL of each sample were transferred in three technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 495 nm on a FLUOstar Optima microplate reader (BMG LABTECH, Ortenberg, Germany).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the total catechin content in the samples was calculated indirectly as mg of catechin equivalents (CE) per g of DW, based on the calibration curve of standard catechin solutions of known concentrations (1-0.01 mg/mL).

Determination of anthocyanin content
Content of anthocyanins in grapevine leaves extracts was evaluated using the method modified from Tušek et al. (2016).A volume of 150 μL of each extract was mixed with 450 μL of 70% (v/v) ethanol and 50.4 μL of concentrated HCl.The samples were briefly mixed on a vortex mixer and incubated for 60 min in a Thermomixer (Eppendorf, Hamburg, Germany) at 80 °C and 300 rpm.After the incubation, 200 μL of each sample were transferred in three technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 520 nm on a FLUOstar Optima microplate reader (BMG LABTECH, Ortenberg, Germany).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the total anthocyanin content in the samples was calculated indirectly as cyanidin-3-glucoside equivalents (C3GE) based on the molar extinction coefficient of 34300 M -1 cm -1 adapted for the measurement using the microplate reader and a molecular weight of 449.2 g mol -1 .

Determination of flavonol and hydroxycinnamic acid content
Flavonols and hydroxycinnamic acids content was estimated according to the method described in Howard et al. (2003).Extract (10 μL) was mixed with 40 μL of 70% (v/v) ethanol, 50 μL of HCl (1 g/L in 96% ethanol) and 910 μL of HCl (2 g/L in H2O).After homogenizing the sample on a vortex mixer, 200 μL of each sample were transferred in four technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 320 nm for hydroxycinnamic acids and 360 nm for flavonols on a MultiSkan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, USA).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the flavonol content in the samples was calculated indirectly as mg of quercetin equivalents (QE) per g of DW based on the calibration curve of standard quercetin solutions of known concentrations (1-0.00625mg/mL).The hydroxycinnamic acid content in the samples was calculated indirectly as mg of caffeic acid equivalents (CAE) per g of DW based on the calibration curve of standard caffeic acid solutions of known concentrations (2-0.05mg/mL).

Determination of phenolic acid content
Phenolic acids content was estimated using the method described in Šamec et al. (2014), with some modifications.Extract (70 μL) was mixed with 140 μL of 0.5 M HCl and 140 μL of Arnow reagent (10% NaNO2, 10% Na2MoO2 in H2O), 140 μL of 8.5% NaOH and 210 μL of H2O.After homogenizing the sample on a vortex mixer, 200 μL of each sample were transferred in three technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 495 nm on a FLUOstar Optima microplate reader (BMG LABTECH, Ortenberg, Germany).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the phenolic acid content in the samples was calculated indirectly as mg of caffeic acid equivalents (CAE) per g of DW based on the calibration curve of standard caffeic acid solutions of known concentrations (2-0.1 mg/mL).

Determination of tannin content
For the determination of tannins content, method according to Sangeetha & Vedasree (2012) was used.Away from light, 840 μL of deionized H2O, 50 μL of FC reagent, 100 μL of 3.5% Na2CO3 and 10 μL of the extract was mixed and homogenized on a vortex mixer followed by an incubation for 30 min at RT.After the incubation, 200 μL of each sample were transferred in three technical replicates on a 96-well plate and the colour intensity was quantified by measuring the absorbance at 700 nm on a MultiSkan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, USA).As blank, 70% ethanol was used instead of the extract.The measured absorbance of the blank was subtracted from the absorbance of the samples and the total catechin content in the samples was calculated indirectly as mg of catechin equivalents (CE) per g of DW based on the calibration curve of standard catechin solutions of known concentrations (2-0.05mg/mL).
Relative content of total phenolics and groups of phenolic compounds of M38-and M54infected grapevine leaves at three time points, in relation to the corresponding controls.Values are ratios and represent mean ± standard deviation of three replicates with three technical replicates each.Different letters indicate a significant difference between different time points for the corresponding samples (ANOVA, Duncan test, p ≤ 0.05).