DOI: 10.5061/dryad.905qfttjk
title: Photodynamic inactivation of Lasiodiplodia theobromae: lighting the way towards an environmentally friendly phytosanitary treatment

description: 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.
description: Biological material,
 photosensitizers and ROS scavengers
 The strain of
 <i>Lasiodioplodia theobromae</i> 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<sup>-1</sup>
 oatmeal, 15 gL<sup>-1</sup> 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<sup>+</sup>-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.
 <sup>1</sup>H NMR (DMSO-d<sub>6</sub>): −3.12 (s,
 2H, NH), 4.73 (s, 12H, CH<sub>3</sub> ), 9.00 (d,
 <i>J</i> = 6.5 Hz, 8H, Py-<i>o</i>-H), 9.22 (s,
 8H, b-H), 9.49 (d, <i>J </i>= 6.5 Hz, 8H,
 Py-<i>m</i>-H). Stock-solutions of TBO (10 mmol
 L<sup>-1</sup>), MB (10 mmol L<sup>-1</sup>) and
 Tetra-Py<sup>+</sup>-Me (0.5 mmol L<sup>-1</sup>)
 were prepared using dimethylsulfoxide (DMSO; Merck KGaA, Darmstadt,
 Germany) as solvent. The stock-solution of riboflavin (26.6 mmol
 L<sup>-1</sup>) 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<sup>-1</sup>) 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 <i>L.
 theobromae</i> under natural
 sunlight PDI assays with solar light (natural
 daylight) were conducted only with the PSs TBO, MB and riboflavin. The
 inactivation of <i>L. theobromae </i>was assessed as the
 inhibition of mycelium growth in double-layered solid medium. The PSs (1.0
 and 2.0 mmol L<sup>-1</sup> TBO, 1.0 and 2.0 mmol
 L<sup>-1</sup> MB or 2.66 and 5.32 mmol
 L<sup>-1</sup> 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 <i>L. theobromae</i>
 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 <i>L.
 theobromae</i> under artificial
 light PDI assays with artificial PAR
 light (380-700 nm) were conducted with the PSs TBO (1.0 mmol
 L<sup>-1</sup>), MB (1.0 mmol L<sup>-1</sup>),
 riboflavin (2.66 mmol L<sup>-1</sup>) and the cationic
 porphyrin Tetra⁺-Py-Me (50 µmol L<sup>-1</sup>) 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<sup>-2</sup>. 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<sup>-2</sup>. Oatmeal broth was
 distributed in 250 mL flasks (50 mL) and amended with MB (50 µmol
 L<sup>-1</sup>) or Tetra-Py⁺-Me (5.0 µmol
 L<sup>-1</sup>). 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 <i>L. theobromae</i>,
 an identical experiment was conducted in which parallel sets of test
 cultures containing each PSs were amended with either 100 µmol
 L<sup>-1</sup> of D-mannitol or 100 µmol
 L<sup>-1</sup> 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.