The fungus Lasiodiploda theobromae is one of the main causal agents of trunk canker and dieback of grapevine. The objective of this work was to evaluate the efficiency of photodynamic inactivation (PDI) of L. theobromae with synthetic and natural photosensitizers (PSs) and irradiation with either sunlight or artificial PAR light. Although growth of the mycelium could not be completely prevented with natural sunlight irradiation, phenothiazine dyes (methylene blue, MB; toluidine blue O, TBO), riboflavin and a cationic porphyrin (Tetra-Py⁺-Me) caused complete inhibition under continuous irradiation with artificial light. Free radicals were the main cytotoxic agents in the PDI with MB, indicating the predominance of the type I mechanism. PDI with MB or Tetra-Py⁺-Me may represent a promising approach for the sanitation of vine material in greenhouse nurseries, in order to reduce the risk of infection upon grafting.

Biological material, photosensitizers and ROS scavengers

The strain of Lasiodioplodia theobromae LA-SV1 used in this study was isolated from grapevine in Peru (Rodríguez-Gálvez et al., 2015). The culture was maintained in oatmeal agar (OA, 30 gL^-1 oatmeal, 15 gL^-1 agar) at room temperature (~25 °C) in the dark. Prior to each experiment, an active growing culture was prepared by placing a 6-mm mycelium plug from a pre-culture to the surface of a new OA plate, and incubated for 6 days at 28 °C.

The PS Riboflavin (Merck KGaA, Darmstadt, Germany), toluidine blue O (TBO; Merck KGaA, Darmstadt, Germany) and methylene blue (MB; AppliChem GmbH, Darmstadt, Germany) were used as received from the supplier and the cationic porphyrin Tetra-Py⁺-Me was prepared and purified according to the literature]. The 1H NMR and UV-vis spectra were consistent with the literature. Purity was confirmed by thin layer chromatography and 1H NMR. The molecular structures of the PS are illustrated in Figure 1. ¹H NMR (DMSO-d₆): −3.12 (s, 2H, NH), 4.73 (s, 12H, CH₃ ), 9.00 (d, J = 6.5 Hz, 8H, Py-o-H), 9.22 (s, 8H, b-H), 9.49 (d, J = 6.5 Hz, 8H, Py-m-H).

Stock-solutions of TBO (10 mmol L^-1), MB (10 mmol L^-1) and Tetra-Py⁺-Me (0.5 mmol L^-1) were prepared using dimethylsulfoxide (DMSO; Merck KGaA, Darmstadt, Germany) as solvent. The stock-solution of riboflavin (26.6 mmol L^-1) was prepared in distilled water. All solutions were protected from light with aluminum foil to prevent photodegradation and stored at 4 °C. Prior to each experiment, the working solutions were homogenized by sonification for 15 min at room temprature.

Stock-solutions (1.0 mol L^-1) of D-Mannitol and sodium azide (Merck KGaA, Darmstadt, Germany), used as free radical scavenger and ¹O₂ quencher, respectively, were prepared in distilled water, sterilized by filtration and stored at 4 °C.

PDI of L. theobromae under natural sunlight

PDI assays with solar light (natural daylight) were conducted only with the PSs TBO, MB and riboflavin. The inactivation of L. theobromae was assessed as the inhibition of mycelium growth in double-layered solid medium. The PSs (1.0 and 2.0 mmol L^-1 TBO, 1.0 and 2.0 mmol L^-1 MB or 2.66 and 5.32 mmol L^-1 riboflavin) were incorporated in soft OA (0.5 % agar). Four-mL overlays were poured over solid OA in 9 cm diameter Petri dishes. Cultures were inoculated in the centre of the plate.

The cultures were incubated for 7 days at room temperature, exposed to daylight. Plate lids were replaced daily to avoid shading from moisture accumulating in the inner side. Mycelia were measured along two perpendicular lines, to determine the average radial growth. Each assay included a light control (LC), in which L. theobromae was exposed to the same light conditions as the test but without PS, and dark controls (DC) in which cultures were exposed to each PS but the experiment was conducted in the dark. Three independent assays, each including 5 replicates for each experimental condition were conducted. The results are presented as average ± standard deviation.

PDI of L. theobromae under artificial light

PDI assays with artificial PAR light (380-700 nm) were conducted with the PSs TBO (1.0 mmol L^-1), MB (1.0 mmol L^-1), riboflavin (2.66 mmol L^-1) and the cationic porphyrin Tetra⁺-Py-Me (50 µmol L^-1) as a reference PS. The PSs were incorporated in the soft-agar overlay, and inoculation was performed as described previously. The cultures were incubated at room temperature for 7 days, under an array of 13 fluoresce lamps (OSRAM 21 18W) delivering PAR light (380-700 nm) continuously (24 h/day), with an irradiance of 25 W m^-2. Plate lids were replaced daily and mycelia growth was monitored daily. Light and dark controls were also included. A positive control, control (+), in which the fungus was cultivated in the dark and without any PS, was also included for comparison. Three independent assays, each including 5 replicates for each experimental condition were conducted. The results are presented as average ± standard deviation.

Effect of PDI on biomass production and mechanism of photosensitization

The effect of photosensitization on fungal biomass production was assessed in assays conducted in liquid cultures irradiated continuously (7 days) with PAR light (380-700 nm) at an irradiance of 25 W m^-2. Oatmeal broth was distributed in 250 mL flasks (50 mL) and amended with MB (50 µmol L^-1) or Tetra-Py⁺-Me (5.0 µmol L^-1). Sets of 5 replicates for each experimental condition were inoculated with plugs of actively growing mycelium and incubated at room temperature. Light controls (LC) without PS were irradiated in parallel with the tests. Dark controls, containing the same PS concentration used and positive controls (control (+), incubated in the dark without PS) were protected from light with aluminum foil. In order to determine the type of photosensitization mechanism (type I or type II) involved in photodynamic inactivation of L. theobromae, an identical experiment was conducted in which parallel sets of test cultures containing each PSs were amended with either 100 µmol L^-1 of D-mannitol or 100 µmol L^-1 of sodium azide. After 7 days of incubation, mycelia were collected on pre-weighted sterile gauze by vacuum filtration and dried for 48 h at 50 ºC. The filters containing the dry mycelium were weighted and biomass, expressed as dry weight, was determined as the difference in weight. Three independent assays, each including 5 replicates for each experimental condition were conducted. The results are presented as average ± standard deviation.

Statistical analysis

Significant differences on radial growth and biomass production between different experimental conditions were assessed by univariate analysis of variance (ANOVA) with the IBM SPSS Statistics 25 package with a 5% significance threshold. Normality and homogeneity of variances were checked by the Kolmogorov-Smirnov and the Levene tests, respectively.