Identification and Quantification of a Phytotoxic Metabolite from Alternaria dauci

Article abstract of DOI:10.3390/molecules25174003

Alternaria dauci is the causal agent of Alternaria leaf blight (ALB) in carrot (Daucus carota) crops around the world. However, to date, A. dauci has received limited attention in its production of phytotoxic metabolites. In this investigation, the bioassay-guided isolation of the extract from liquid cultures of A. dauci resulted in the isolation of two metabolites identified as α-acetylorcinol (1) and p-hydroxybenzoic acid (2), based on their spectroscopic data and results from chemical correlation reactions. Testing of both metabolites in different assays showed an important phytotoxic activity for p-hydroxybenzoic acid (2) when tested in the leaf-spot assay on parsley (Petroselinum crispum), in the leaf infiltration assay on tobacco (Nicotiana alata) and marigold (Tagetes erecta), and in the immersion assay on parsley and parsnip (Pastinaca sativa) leaves. Quantification of the two metabolites in the crude extract of A. dauci kept at different times showed that p-hydroxybenzoic acid (2) is one of the first metabolites to be synthesized by the pathogen, suggesting that this salicylic acid derivative could play an important role in the pathogenicity of the fungus.

Full text of DOI:10.3390/molecules25174003

molecules
Article
Identification and Quantification of a Phytotoxic
Metabolite from Alternaria dauci
Martha Leyte-Lugo * , Pascal Richomme , Pascal Poupard and Luis M. Peña-Rodriguez 1,*
1
,
2
3
1
Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, 97205 Mérida, Yucatán, Mexico
UPRES EA921SONAS, SFR 4207 QUASAV, Université d’Angers, 49045 Angers, France;
2
pascal.richomme@univ-angers.fr
UMR 1345 IRHS, SFR 4207 QUASAV, INRAE, Institut Agro, Université d’Angers, 49045 Angers, France;
3
pascal.poupard@univ-angers.fr
*
Correspondence: mleyte@correo.xoc.uam.mx (M.L.-L.); lmanuel@cicy.mx (L.M.P.-R.);
Tel.: +52-5529-631088 (M.L.-L.); +52-9998-990767 (L.M.P.-R.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Francisco Leon
Received: 20 August 2020; Accepted: 1 September 2020; Published: 2 September 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Alternaria dauci is the causal agent of Alternaria leaf blight (ALB) in carrot (Daucus carota)
crops around the world. However, to date, A. dauci has received limited attention in its production of
phytotoxic metabolites. In this investigation, the bioassay-guided isolation of the extract from liquid
cultures of A. dauci resulted in the isolation of two metabolites identified as
p-hydroxybenzoic acid ( ), based on their spectroscopic data and results from chemical correlation
reactions. Testing of both metabolites in different assays showed an important phytotoxic activity for
p-hydroxybenzoic acid ( ) when tested in the leaf-spot assay on parsley (Petroselinum crispum), in the
α-acetylorcinol (1) and
2
2
leaf infiltration assay on tobacco (Nicotiana alata) and marigold (Tagetes erecta), and in the immersion
assay on parsley and parsnip (Pastinaca sativa) leaves. Quantification of the two metabolites in the
crude extract of A. dauci kept at different times showed that p-hydroxybenzoic acid (2) is one of the
first metabolites to be synthesized by the pathogen, suggesting that this salicylic acid derivative could
play an important role in the pathogenicity of the fungus.
Keywords: phytotoxins; Alternaria leaf blight; Daucus carota; p-hydroxybenzoic acid; α-acetylorcinol
1
. Introduction
The Alternaria genus is well-known for its pathogenicity against many economically important
crops worldwide [ ]. It has been reported that this genus produces toxic metabolites, commonly
known as phytotoxins, that play a significant role during the infection process [ ]. One Alternaria
1,2
3–6
species that has received limited attention, particularly in terms of its production of phytotoxic
metabolites, is Alternaria dauci (Kuhn) Groves & Skolko, recognized as the causal agent of the Alternaria
Leaf Blight (ALB) disease in cultivated carrots (Daucus carota) around the world [
initially in the leaves, as small greenish-brown lesions of different shapes and sizes; these lesions
become larger and turn dark-brown, and are often surrounded by a chlorotic halo [ ]. The main
7]. The disease appears
8
effects associated with the disease are the reduction in leaf photosynthetic activity and carbohydrate
production, followed by separation of necrotic foliage from the taproot. These effects contribute to a
significant reduction in the harvesting efficiency of infected carrots [7].
Previous studies on phytotoxin production by A. dauci have reported the identification of some
0
non-host-selective phytotoxins (non-HST s), including alternariol, alternariol monomethyl ether,
and zinniol [5,7–11]. Recently, it was demonstrated that zinniol is not responsible for the phytotoxic
effect caused by the organic extract of A. dauci [12]; these results, which strongly suggested that
the phytotoxic effect of the organic extract of cultures from A. dauci was caused by other lipophilic
Molecules 2020, 25, 4003; doi:10.3390/molecules25174003
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4003
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metabolites, were confirmed with the identification of aldaulactone as a new phytotoxin involved in the
aggressiveness of A. dauci against carrot cells [ ]. Given these findings, and as part of our continuing
5
interest in the isolation and identification of phytotoxic metabolites produced by A. dauci and their role
in the infection process, we wish to report here on the bioassay-guided purification and identification
of
α-acetylorcinol (1) and p-hydroxybenzoic acid (2) as two additional lipophilic phytotoxic metabolites
produced by the fungal pathogen.
2
. Results and Discussion
The bioassay-guided chromatographic purification of the medium polarity (ethyl acetate) fraction
from the extract of A. dauci, using the leaf-spot assay on parsley leaves as a guide, resulted in the
isolation of two phytotoxic metabolites identified as -acetylorcinol ( ) and p-hydroxybenzoic acid (
Figure 1). The resorcinol derivative showed a single component, with a molecular ion peak at m/z
66 corresponding to a molecular formula of C H O , when analyzed by gas chromatography–mass
α
1
2)
(
1
1
9
10
13
3
1
spectrometry (GC-MS). Its spectroscopic ( H and C NMR) data coincided with those reported for
-acetylorcinol ( ), the principal metabolite isolated from Cochliobolus lunata [13]. Treatment of with
iodomethane produced the expected dimethyl ether derivative, which confirmed the presence of two
hydroxyl groups in the structure. Alternatively, the IR spectrum of p-hydroxybenzoic acid ( ) showed
α
1
1
2
−
1
−1
absorption bands corresponding to hydroxyl (3465 cm ) and carbonyl (1673 cm ) groups, and its
GC-MS analysis showed a single component with a molecular ion peak at m/z 138, corresponding to a
molecular formula of C H O . Its spectroscopic data coincided with those reported for 2, obtained by
7
6
3
the oxidative degradation of methylparaben [14,15].
Figure 1. Phytotoxic secondary metabolites isolated from the medium polarity fraction of A. dauci;
α-acetylorcinol (1) and p-hydroxybenzoic acid (2).
The phytotoxic activity of metabolites
1 and 2 was further evaluated using the leaf infiltration
assay in tobacco (Nicotiana alata), and marigold (Tagetes erecta) leaves (Figure 2) and the leaf immersion
assay in parsnip (Pastinaca sativa) and parsley (Petroselinum crispum) (Figure 3). In the leaf infiltration
assay, p-hydroxybenzoic acid (
2
) caused a significant phytotoxic effect in both tobacco and marigold
-acetylorcinol
) only caused a minor effect when tested at the highest concentration (22.0 mM) in tobacco leaves
Figure 2c), and proved to be non-phytotoxic when tested on marigold leaves (data not shown).
) showed varying levels of phytotoxic activity when tested in the
immersion assay using parsnip (necrotic effect at 22.0 mM) and parsley (phytotoxic activity at 14.5 and
2.0 mM) (Figure 3a–c,e,f), while -acetylorcinol ( ) caused a minimal effect when tested on parsnip
leaves at all concentrations, and particularly at 14.5 and 22.0 mM (Figure 2a,b,d,e), while
α
(1
(
Similarly, p-hydroxybenzoic acid (
2
2
α
1
(Figure 3d) and none when tested on parsley (Figure 3g).
The benzoic acid derivative p-hydroxybenzoic acid (2) has been isolated from many natural
sources [16,17], including plant species such as oil palm (Elaeis guineensis) [18], grapes (Vitis vinifera) [19],
betel palm (Areca catechu) [20], Cuban royal palm (Roystonea regia) [21], medlar (Mespilus germanica) [22],
and black-jack (Bidens pilosa) [23]. Furthermore, it is interesting to mention that p-hydroxybenzoic

Molecules 2020, 25, 4003
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acid (2) has been reported to accumulate in Agrobacterium rhizogenes-induced hairy root cultures of
carrots (D. carota) [24] and to be produced when carrot cell cultures are exposed to fungal elicitors [25];
this suggests that this benzoic acid derivative might play an important defensive role in carrot
cultures, either by itself or as an intermediate in the biosynthetic pathway of other metabolites [25].
However, the fact that p-hydroxybenzoic acid (
concentrations [26,27] might explain the phytotoxic activity found in this investigation.
Reports on the production of by fungi are limited, but it has been reportedly obtained
from Alternaria spp [ ], including A. tagetica [28]. Additional reports described its production
2) has been reported as a toxic metabolite at high
2
2
in cultures of the phytopathogens Epichloë bromicola (infecting Elymus tangutorum) [29] and Diaporthe
gulyae (isolated from stem cankers of sunflower) [30]. The many biological activities reported for
hydroxybenzoic acid (2) include phytotoxic activity against Rumex crispus [31] and as a growth inhibitor
of Dactylis glomerate [32]; additionally, it has been reported to have antibacterial [15], antioxidant [33],
antifungal [23], antialgal [34], antimutagenic [35], and estrogenic properties [36].
The resorcinol derivative
from various Alternaria spp [ ], including A. tenuissima and A. brassicicola [37
species including Cochliobolus lunata [13], Stagonospora apocyni [39], Verticillium sp. (an endophyte of
Rehmannia glutinosa) [40], and Cladosporium perangustm FS62 [41]. The phytotoxic activity of against
weed species has been recognized [39], and has also been reported to show antifungal activity against
Trichophyton rubrum and Aspergillus fumigatus [40].
α-acetylorcinol (
1) has also been reported as a secondary metabolite
2
,
38], as well as from fungal
1
1
Tagetes erecta
d
e
Control
Abaxial view
Figure 2. Leaf infiltration assay. Phytotoxic damage of
acid (
) p-hydroxybenzoic acid (22.0 mM), (
and ( ) p-hydroxybenzoic acid (22.0 mM). The sterile uninoculated medium was used as a control.
The circle indicates the phytotoxic effect.
α-acetylorcinol (1) and p-hydroxybenzoic
2
) on tobacco (N. alata) and marigold (T. erecta) leaves: (
a
) p-hydroxybenzoic acid (14.5 mM),
) p-hydroxybenzoic acid (14.5 mM),
(b
c)
α-acetylorcinol (22.0 mM), (d
e

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Figure 3. Leafimmersionassay. Phytotoxicdamageof
leaves excised from parsnip (P. sativa) and parsley (P. crispum) plants: (
) p-hydroxybenzoic acid (14.5 mM), ( ) p-hydroxybenzoic acid (22.0 mM), (
) p-hydroxybenzoic acid (14.5 mM), ( ) p-hydroxybenzoic acid (22.0 mM), and (
22.0mM). Thesterileuninoculatedmediumwasusedasacontrol. Thecircleindicatesthephytotoxiceffect.
α
-acetylorcinol(
1
)andp-hydroxybenzoicacid(
) p-hydroxybenzoic acid (7.25 mM),
-acetylorcinol (22.0 mM),
-acetylorcinol
2)on
a
(b
c
d) α
(e
f
g) α
(
As a result of the isolation of two metabolites, particularly p-hydroxybenzoic acid (2), it was
decided to investigate the production of the two metabolites during the culturing of the fungus.
The HPLC chromatographic profile of the medium polarity fraction of the extract of A. dauci showed
the peaks corresponding to α-acetylorcinol (1) and p-hydroxybenzoic acid (2) at t 6.5 and 7.4 min,
R
respectively (Figure 4). Additionally, the HPLC chromatographic profile of the A. dauci crude extract
showed the presence of several minor components identified as the diketopiperazines cyclo-(pro-val)
(3
), cyclo-(leu-tyr) (4), cyclo-(pro-leu) (
5), cyclo-(pro-phe) (6), cyclo-(val-leu) (7), cyclo-(val-phe) (8),
and cyclo-(leu-phe) (9) (Figure 5), previously reported from a number of microorganisms and recognized
as an important group of bioactive secondary metabolites [42].
The HPLC analyses of the medium polarity fractions obtained from extracts of cultures of A. dauci
kept at different times showed the presence of
Table 1; Figure S1), its production reached a maximum (42.9
the production of -acetylorcinol ( ) in cultures of A. dauci was first detected after 48 h, with its
maximum production (160.2 3.8 g/mg) occurring after 96 h of culturing the fungus (Table 1;
Figure S1). These results indicate that p-hydroxybenzoic acid ( ) is one of the first metabolites produced
2
within 6 h of initiating the culture of the pathogen
(
±
1.0 g/mg) after 72 h. Alternatively,
µ
α
1
±
µ
2
by A. dauci in the liquid culture conditions used in this investigation. These findings are of particular
interest as it has been reported that fungal metabolites produced during spore germination or in the
early stages of fungal growth play a significant role in plant–pathogen interaction [43]. Furthermore,
the phytotoxic effect of p-hydroxybenzoic acid (
2) against different plant species, in different assay
models, supports it being involved in fungal pathogenicity.

Molecules 2020, 25, 4003
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Figure 4. HPLC chromatographic profile of the medium polarity fraction from the extract of
A. dauci at 96 h of culture. The chromatogram shows the peaks corresponding to
α
-acetylorcinol (
1),
p-hydroxybenzoic acid (2), and the diketopiperazines (3–9) at (a) 210 and (b) 257 nm.
Figure 5. Minor diketopiperazines identified in the crude extract of A. dauci; cyclo-(pro-val) (
3
),
),
cyclo-(leu-tyr) (
4), cyclo-(pro-leu) (5), cyclo-(pro-phe) (6), cyclo-(val-leu) (7), cyclo-(val-phe) (
8
and cyclo-(leu-phe) (9).

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Table 1. Quantification of phytotoxins
different times of culture.
1 and 2 in organic extracts from cultures of A. dauci kept at
Content (µg/mg Extract) (Mean ± SD)
Time of Culture (h)
α-Acetylorcinol (1)
p-Hydroxybenzoic Acid (2)
6
-
-
-
31.9 ± 0.2
32.0 ± 0.5
33.6 ± 1.0
41.7 ± 1.3
42.9 ± 1.0
41.0 ± 0.2
30.5 ± 0.3
1
2
4
8
2
6
2
4
7
9
90.8 ± 3.4
124.3 ±1.7
160.2 ± 3.8
154.0 ± 4.9
1
68
3
. Materials and Methods
3
.1. General Experimental Procedures
The IR spectra were obtained on a Thermo Scientific Nicolet 6700 spectrometer (Waltham, MA,
USA), dissolving the samples in CHCl . Nuclear Magnetic Resonance (NMR) spectra were recorded
3
1
on a Varian/Agilent spectrometer (AR Premium COMPACT; Santa Clara, CA, USA) at 600 ( H) and
50 MHz ( C); chemical shifts are listed as δ values. Gas chromatography–mass spectrometry analyses
13
1
were carried out using an Agilent (Santa Clara, CA, USA) 6890N gas chromatograph coupled to
an Agilent 5975C INERT mass spectrometer detector, using an electron impact (70 eV) ionization
source. HPLC analyses were carried out using a Waters Alliance®® (Milford, MA, USA) HPLC system
equipped with a 2998-photodiode array detector (PDA). Control of the equipment, data acquisition,
processing, and management of chromatography was performed by the Empower 2 software program
(Waters) (version 3). Analytical HPLC was performed using a Phenomenex–Luna (Torrance, CA,
USA) RP–C18 column (150 4.6 mm, 5 m particle size), with a flow rate of 1.0 mL/min. Column
×
µ
chromatography (CC) was carried out on Silica gel 60 Å (70-230 mesh ASTM) (E.M. Merck. Darmstadt,
Germany). Thin-layer chromatography (TLC) analyses were carried out on silica gel 60 F254 plates
(
E.M. Merck. Darmstadt, Germany) using phosphomolybdic acid as a visualizing reagent. Deionized
®®
water for HPLC was obtained using a Simplicity
Water Purification System (Millipore Sigma).
All reagents and solvents were obtained from Sigma-Aldrich-Fluka Chemicals (St Louis, MO, USA).
3
.2. Fungal Material
The strain of A. dauci (Kiihn) Groves and Skolko (FRA017; isolated at IRHS and deposited in
COMIC (collection number COMIC C0003), France) was maintained in water/glycerol (20:80) at room
temperature. A portion of the mycelium was used to inoculate V8 agar-containing Petri dishes,
◦
where the fungus was allowed to grow for 15 days at 25 C under natural light/darkness conditions.
The plates were then flooded with sterile distilled water and gently rubbed with a sterile bent plastic
rod to release spores. Fernbach flasks containing 1 L of Czapek-Dox liquid medium were inoculated
4
with a conidial suspension adjusted to 1
×
10 conidia/mL of A. dauci; cultures were kept under
◦
shake (100 rpm) conditions, at 25 C, for 96 h. Czapek-Dox liquid medium contained the following
compounds: 1.5 g of potassium nitrate (Sigma-Aldrich ), 0.5 g of potassium chloride (Fermont),
.5 g of magnesium sulfate (Sigma-Aldrich ®), 0.01 g of ferric sulfate (Sigma-Aldrich ), 1.0 g of
monobasic potassium phosphate (J.T. Baker ), 6 g of sucrose (commercial brand), and 2.0 g of casein
Sigma Aldrich®®) in 1 L of distilled water, with a final pH of 6.0 [10,44].
®®
®
®®
0
®
®
(
3
.3. Extraction, Isolation, and Purification of Phytotoxic Metabolites
The mycelium was separated from the culture filtrate by filtration through cheesecloth.
The aqueous filtrate was then extracted with ethyl acetate (3
was evaporated in vacuo to produce 432.5 mg of extract (ca. 45 mg/L). A suspension of the organic crude
×
, 1:1, υ/υ) and the organic layer

Molecules 2020, 25, 4003
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extract (420 mg) in 50 mL of a mixture of H O/MeOH (95:5,
υ
/υ
) was successively partitioned with
2
hexane (3
×
, 1:1,
υ
/
υ
) and ethyl acetate (3
; 132.6 mg) fractions. Fraction
2.5 cm) using a gradient elution with mixtures of hexane-dichloromethane-methanol to yield
B16). Column chromatographic purification (30 1.0 cm) of
phytotoxic fractions B10 (14.2 mg) and B11 (9.8 mg), using a gradient elution with mixtures of hexane
and ethyl acetate, allowed the isolation of -acetylorcinol ( , 10.2 mg) and p-hydroxybenzoic acid
2, 3.4 mg), respectively, in pure form.
×
, 1:1,
υ
/
υ
), to produce the hexane (
A; 43.8 mg), ethyl acetate
(B; 202.7 mg), and aqueous (
C
B
was purified by column chromatography
(
30
×
sixteen semi-purified fractions (B1
–
×
α
1
(
α
-Acetylorcinol (
1
): Amber oil; UV (MeOH) λmax 208 and 279 nm; IR (CHCl3) νmax 3377, 1697, 1600,
−
1 1
13
and 1148 cm ; H NMR (600 MHz, CD OD)
CD OD) δ 209.6, 159.8, 137.8, 108.9, 102.3, 51.7, 28.9 [13]; EIMS m/z 166 [M] (100), 123 (C H O , 95).
δ
6.16 (m, 3H), 3.54 (s, 2H), 2.12 (s, 3H). C NMR (150 MHz,
3
+
3
7
7
2
p-Hydroxybenzoic acid (2): White powder; UV (MeOH) λmax 207 and 253 nm; IR (CHCl3) νmax 3465,
−
1 1
2
922, 1673, 1593, 1251 cm ; H NMR (600 MHz, CD OD)
δ
7.87 (dd, J = 8.4, 1.8 Hz, 2H), 6.81 (dd,
3
13
J = 8.4, 1.8 Hz, 2H). C NMR (150 MHz, CD OD)
δ
170.6, 163.1, 132.9, 123.4, 115.9 [14 15]; EIMS m/z
,
3
+
1
38 [M] (58), 121 (C H O , 100), 93 (C H O, 25).
7
5
2
6
5
3
.4. Preparation of O,O-Dimethyl-α-acetylorcinol
A solution of -acetylorcinol ( , 7.5 mg) and K CO (100 mg) in dry acetone (1 mL) was treated
α
1
2
3
with iodomethane (0.5 mL) and the reaction mixture was allowed to stir overnight at room temperature.
The mixture was diluted with water and the resulting aqueous suspension was extracted with
dichloromethane; the organic layer was dried over Na SO , filtered, and the solvent evaporated in
2
4
vacuo to yield 8.6 mg (98%) of crude product identified as O,O-dimethyl-
UV (MeOH) λmax 210 and 275 nm; IR (CHCl3) νmax 2938, 1712, 1596, 1463, 1200, and 1157 cm ; H NMR
α-acetylorcinol: Amber oil;
−1 1
(
600 MHz, CDCl3)
.15 (d, J = 0.6 Hz, 3H); EIMS m/z 194 [M] (90), 151 (C H O , 100).
δ
6.37 (t, J = 2.4 Hz, 1H), 6.35 (d, J = 2.4 Hz, 2H), 3.78 (s, 6H), 3.61 (d, J = 0.6 Hz, 2H),
+
2
3
3
9
11
2
.5. Quantification of α-Acetylorcinol and p-Hydroxybenzoic Acid in Organic Fractions
.5.1. Culturing of a Dauci for Quantitative Analyses
4
A 1 mL spore suspension (adjusted to 1
×
10 conidia/mL of A. dauci) was used to inoculate 100 mL
of Czapek-Dox culture medium contained in an Erlenmeyer flask. Cultures were allowed to grow at
5 C for different lengths of time (6, 12, 24, 48, 72, 96, and 168 h). The mycelium was separated from
◦
2
the liquid medium culture by filtration through cheesecloth, and the aqueous filtrate was extracted
with ethyl acetate (3 , 1:1, ). The organic layer was dried over sodium sulfate, filtered, and the
solvent evaporated in vacuo to produce the corresponding organic extract (ca. 2.0 mg/100 mL).
×
υ/υ
3
.5.2. Preparation of Standard Solutions for HPLC Analyses
Stock standard solutions of -acetylorcinol ( ) and p-hydroxybenzoic acid (2) were prepared at
α
1
a concentration of 1 mg/mL in acetonitrile. Five standard working solutions (0.1 to 0.5 mg/mL) of
each phytotoxin were prepared by serial dilution of the standard stock solutions with acetonitrile and
used for the preparation of the corresponding calibration curves. All solutions were filtered through
0.45 mm membrane filters before injection. Under the conditions described, the retention times (tR)
of -acetylorcinol ( ) and p-hydroxybenzoic acid ( ) were found to be 6.5 and 7.4 min, respectively.
α
1
2
The location of each phytotoxic metabolite in the chromatographic profiles was confirmed by the
co-injection of extracts with the pure metabolites.
3
.5.3. Sample Preparation for HPLC Analyses
Samples of A. dauci organic extracts (ca. 2 mg) were suspended in 300
µL of a 9:1 mixture of
water-methanol. The resulting suspension was extracted with ethyl acetate (3
×
, 1:1, ), and the
υ/υ

Molecules 2020, 25, 4003
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organic layer was dried over sodium sulfate and filtered. The solvent evaporated to produce the
corresponding ethyl acetate fractions, which were prepared at a concentration of 1 mg/mL in acetonitrile.
All solutions were filtered through 0.45 mm membrane filters before injection.
3
.5.4. Instrumentation and Conditions for HPLC Quantification of Phytotoxic Metabolites
Quantitative analyses by HPLC were performed using a Waters Alliance®® HPLC instrument
equipped with a quaternary pump, degasser, autosampler, and 2998-photodiode array detector (PDA).
Control of the equipment, data acquisition, processing, and management of the chromatograph was
carried out using the Empower 2 software program (Waters) (version 3). All analyses were carried out on
a reverse-phase Luna RP–C18 column (150
included a mobile phase of A: Acetonitrile and B: 0.1% (v/v) formic acid in water, following a gradient
elution program of 10 50% A and 90 50% for 0 40 min; a flow rate of 1.0 mL/min, room temperature,
and an injection volume of 10 µL. The UV detector was set to wavelengths of 210 and 257 nm.
×
4.6 mm, 5 µm particle size). Chromatographic conditions
−
−
−
3
.5.5. Method Validation
The HPLC-PDA method was validated according to the International Conference on Harmonization
Guidelines (ICH: Validation of Analytical Procedures) [45]. Standard calibration curves for quantifying
the two phytotoxins were obtained by plotting concentration (mg/mL) against response. Five and six
different concentrations for each phytotoxin, in the range of 0.1–0.5 mg/mL for
.05–0.5 mg/mL for p-hydroxybenzoic acid ( ), were prepared in triplicate. The linearity of these curves
was evaluated by the least-squares fit and the correlation coefficient. The detection wavelengths were
10 nm for -acetylorcinol ( ) and 257 nm for p-hydroxybenzoic acid ( ). The limit of detection (LOD)
α-acetylorcinol (1) and
0
2
2
α
1
2
and limit of quantification (LOQ) were determined based on the standard deviation (σ) of the response
and the slope (S) (Table S1):
LOD = 3.3 × σ/S
LQD = 10 × σ/S
The precision was evaluated using repeatability (intraday) and reproducibility (interday). Intraday
and interday were evaluated by analyzing six replicate injections of the two phytotoxins on one and
two different days. The relative standard deviation (RSD) was calculated for each determination and
taken as a measure of precision.
Percent recovery was calculated in order to evaluate the accuracy of the method. The sample
matrix (A. dauci organic fraction) was spiked with known amounts of phytotoxins at different
concentrations levels:
.1, and 0.2 mg/mL). Each sample was analyzed in sextuplicate (Table S2).
Data are expressed as the means SD for the number of samples at each time (n = 3). Analysis
α-Acetylorcinol (1) (0.2, 0.3, and 0.4 mg/mL) and p-hydroxybenzoic acid (2) (0.05,
0
±
of variance (ANOVA) was used to analyze the content of phytotoxins in A. dauci organic extract
cultured at different times. Tukey’s post-hoc test was used to determine the significant differences;
p < 0.05 was considered statistically significant. Prisma Graph-Pad software (version 4.0) was used for
statistical analysis.
3
.6. Phytotoxic Assays
3
.6.1. Leaf-Spot Assay
The assay was carried out as previously described [46]. Parsley (Petroselium crispum) leaves were
excised and washed with sterilized water. A 20 µL drop of the sample (5.0 mg/mL for extracts and
fractions and 7.25 mM for purified phytotoxins), non-inoculated medium (negative control), or sterile
distilled water (blank), was placed over a lightly scratched area made with a sterile, trimmed paintbrush
on the adaxial face of the leaf. The leaves were kept in a high-humidity chamber under natural light at
◦
2
5 C for 72 h. Three leaves, with two applications each, were used for each treatment.

Molecules 2020, 25, 4003
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3
.6.2. Leaf Infiltration Assay
The assay was carried out as previously described [47
,
48]. Ten microliters of extract, fractions
(5.0, 7.5, and 10 mg/mL), or purified phytotoxins (7.25, 14.5, 22.0 mM) was infiltrated abaxially into
leaves of tobacco (Nicotiana alata) and marigold (Tagetes erecta) (both 4–6 weeks old) plants. Distilled
water and non-inoculated medium were used as controls. All evaluations were made in triplicate.
Lesion formation was documented after 24 h.
3
.6.3. Leaf Immersion Assay
The assay was carried out as previously described [49]. The basal part of the stem, of parsnip
(Pastinaca sativa) and parsley (P. crispum) leaves, was placed in a glass tube containing 1 mL of a solution
prepared by combining 100 L of the test solution (5.0, 7.5, and 10 mg/mL for extracts and fractions,
µ
and 7.25, 14.5, and 22.0 mM for purified phytotoxins) and 900
µ
L of water. The leaf was kept in a
◦
high-humidity chamber under natural light conditions at 25 C. Distilled water and non-inoculated
medium were used as controls. All evaluations were made in triplicate. The phytotoxic effect was
recorded after 48 h.
4
. Conclusions
Two metabolites,
α
–acetylorcinol (1) and p-hydroxybenzoic acid (2), have been identified in the
cultures of A. dauci grown in Czapek-Dox medium. The results of the evaluation of the phytotoxic
activity of the two metabolites against different plant species, in different assay models, showed that
while
α
–acetylorcinol (
1
) was weakly phytotoxic, p-hydroxybenzoic acid (2) caused a phytotoxic effect
in different plant species. Furthermore, the production of
2
during the early stages of fungal growth
suggests that this metabolite might play an important role in the plant–pathogen interaction between
A. dauci and D. carota.
Supplementary Materials: The following are available online. Table S1: Regression equations, linear ranges,
LOQs, LODs, and precision for quantitative analysis; Table S2: Average recovery of the phytotoxins quantified
in A. dauci organic fractions; Figure S1: Production kinetics of (a) α-acetylorcinol (1) and (b) p-hydroxybenzoic
1
13
acid (
2
) in cultures of A. dauci; Figures S2 and S3: H and C NMR spectrum (600 and 150 MHz, CD OD,
3
1
respectively) of
α-acetylorcinol (
1
); Figure S4: IR spectrum of
C NMR spectrum (600 and 150 MHz, CD OD, respectively) of p-hydroxybenzoic acid (
); Figure S8: H NMR spectrum (600 MHz, CDCl ) of O,O-dimethyl-
Figure S9: IR spectrum of O,O-dimethyl-α-acetylorcinol.
α-acetylorcinol (
1
); Figures S5 and S6: H and
1
3
2
); Figure S7: IR spectrum
3
1
of p-hydroxybenzoic acid (
2
α-acetylorcinol;
3
Author Contributions: M.L.-L. performed the experiments for the isolation, structure elucidation, and biological
evaluation, and prepared the manuscript; P.R. contributed to the determination of biological activities and revised
the manuscript; P.P. contributed to plant species and sample collection, and revised the manuscript; L.M.P.-R.
contributed to the project organization, experimental guidance, and manuscript writing. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by R
égion Pays de la Loire and SEP-CONACYT-ANUIES-ECOS-NORD
(
México-France) collaborative Project No. 276520.
Acknowledgments: The authors wish to thank Karlina García-Sosa, Carlos Alberto Mejia Hernández, Berenice
Negrete Romero, Dimitri Bréard, and Guillaume Viault for technical support.
Conflicts of Interest: The authors declare no conflict of interest.
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á
í
íguez, L.M.
(
Sample Availability: Samples of the compounds are not available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).