10.5061/DRYAD.905QFTTJK
Cunha, Angela
0000-0002-9118-3521
University of Aveiro
Garcia, Marina
University of Aveiro
David, Bruna
University of Aveiro
Sierra-Garcia, Isabel
University of Aveiro
Alves, Artur
0000-0003-0117-2958
University of Aveiro
Esteves, Ana Cristina
University of Aveiro
Photodynamic inactivation of Lasiodiplodia theobromae: lighting the way
towards an environmentally friendly phytosanitary treatment
Dryad
dataset
2021
FOS: Biological sciences
Fundação para a Ciência e Tecnologia
https://ror.org/00snfqn58
FCT UID/MAR/LA0017/2019
Fundação para a Ciência e Tecnologia
https://ror.org/00snfqn58
FCT UID/QUI/00062/2019
Fundação para a Ciência e Tecnologia
https://ror.org/00snfqn58
Fundação para a Ciência e Tecnologia
https://ror.org/00snfqn58
PTDC/AGR-PRO/2183/2014 - POCI-01-0145-FEDER-016788
2021-04-09T00:00:00Z
2021-04-09T00:00:00Z
en
38164 bytes
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CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.