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Evaluation of the fungicide treatment with copper
oxide and potassium phosphonate solutions for the
sustainable management of P. pinaster trees
infected with B. xylophilus
Adrián López-Villamor ( Adrianvilamor@gmail.com )
Misión Biológica de Galicia (CSIC)
Marta Nunes da Silva
Universidade Católica Portuguesa
Marta W. Vasconcelos
Universidade Católica Portuguesa
Research Article
Keywords: Copper oxide, minerals, pinewood nematode, Pinus pinaster, potassium phosphonate
Posted Date: March 8th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2608869/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Fungicides induce changes in the plants promising to increase tolerance of
Pinus pinaster
against the
pathogenic pinewood nematode (PWN). To test this hypothesis,
P. pinaster
plants were inoculated with
the PWN, treated with copper oxide (CO) or potassium phosphonate (PP), and evaluated post-inoculation
for: i) the extent of foliar symptoms; ii) nematode density inside stem tissues; iii) proxies for oxidative
damage and antioxidant activity, iv) mineral concentration; and v) bacterial diversity. The mortality of
infected plants reached 12.5% regardless of the treatment, but plants treated with fungicides, particularly
with PP, had signicantly lower PWN density (up to 0.61-fold). Plants treated with PP had substantially
higher concentrations of anthocyanins at 14 dai than those treated with CO and non-Treated plants (by
1.47-fold), possibly contributing to the lower PWN colonization and degree of foliar symptoms observed.
CO and PP led to increased lipid peroxidation at 28 dai (by 1.84- and 1.77-fold), and PP showed higher
avonoids concentration than CO (by 1.37- and 0.49-fold), corroborating its higher potential in increasing
plant antioxidative response during infection.
Fungicides also induced signicant changes in micronutrient accumulation in plant tissues, resulting in a
decrease in Zn and P concentrations in plants treated with either fungicide as compared to infected non-
treated plants. Finally, CO treatment increased the diversity of the bacterial communities, while PP
decreased microbial biodiversity. Altogether, results suggest that treatment with CO and PP increases
tolerance against
B. xylophilus
by promoting the plant antioxidant system, changing the accumulation of
essential minerals, and modulating plant-associated bacterial diversity.
Introduction
Pinus
spp. is the most widely used genus in industrial forest plantations worldwide (Mbabazi 2011).
Maritime pine (
P. pinaster
) is particularly relevant to the European timber industry. Several Western
European countries, such as Portugal, Spain, France, and some North African countries (Chupin et al.
2015), produce maritime pine. Despite its economic and social importance, in recent years, its production
primarily decreased due to signicant losses of forest area and wood volume due to re and also to the
spread of the pinewood nematode (PWN) (Abad et al. 2016), which causes the pine wilt disease (PWD),
an exotic pathology of invasive and especially virulent behaviour that causes the rapid death of infested
trees. Given the severity of this disease, which has been and continues to be devastating in many
countries, and the lack of effective control measures, developing new phytosanitary prevention and
control strategies is of utmost urgency. Currently, the control of the PWD relies on the use of pheromone
traps against the insect vector
Monochamus galloprovinciallis
(Álvarez et al. 2016, Firmino et al. 2017)
and breeding for resistance, which have high costs, are time-consuming, and can only be applied to new
plantations (Kurinobu 2008, Nose and Shiraishi 2008, Carrasquinho et al. 2018, Menéndez-Gutiérrez et al.
2018).Currently, research efforts against the PWD involve the use of biological control agents, such as
ectomycorrhizal fungi that improve plant defences (Nakashima et al. 2016, Chu et al. 2019), or the use of
elicitors, such as methyl-jasmonate (MeJA) and salicylic acid (SA), which activate systemic acquired
resistance (SAR) or induced systemic resistance (ISR), resulting in higher tolerance to the pathogen (Bari
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and Jones 2009, Salas-Marina et al. 2011, Kolosova and Bohlmann 2012, Nakashima et al. 2016, Klessig
et al. 2018, Chu et al. 2019, Tripathi et al. 2019, Park et al. 2020 ).
Using fungicides can also activate the resistance of plants against pests and diseases (Daniel and Guest
2005, Prasad et al. 2017), but their effectiveness against the PWN has yet to be discovered. Copper oxide
(CO) is a well-known fungicide used in organic farming against mildew (Cabús et al. 2017), which has
shown high antibacterial activity against several parasitic microorganisms, including nematodes (Soli et
al. 2010, Burke et al. 2016; La Torre et al. 2018). Copper is a vital mineral and critical component of plant
defensive pathways, resulting from its antimicrobial properties and function as a co-factor of several
essential enzymes (Borgatta et al. 2018, Elmer et al. 2018, Strayer-Scherer et al. 2018, Mir et al. 2021).
This compound has shown
in vitro
nematicidal activity against
B. xylophilus
and other plant pathogenic
nematodes (Tan et al. 2013, Mohamed et al. 2019). Likewise, potassium phosphonate (PP), a phosphonic
acid salt, is a systemic fungicide that activates SAR and local acquired response (LAR). It promotes the
activity of the enzyme phenylalanine-ammonium lyase (PAL), a key regulator of secondary plant
metabolites, such as phenols, lignin, phytoalexins, suberin, and compounds derived from cinnamic acid
(Astaneh et al. 2018, Kahromi and Khara 2021). Thus, this fungicide could help control the PWD, as the
accumulation of metabolites such as soluble phenolics and lignin correlate with higher resilience against
the PWD (Moreira et al. 2009, Nunes da Silva et al. 2015, Zas et al. 2015).
When the nematode enters the plant host, it moves and reproduces within the resin canals, causing
general oxidative damage that results in visible leaf necroses (Kuroda 2008, Yamada 2008). To
counteract the detrimental effects of these oxidative molecules, plants activate several antioxidant
enzymes, such as superoxide dismutase, peroxidases, and catalase. Therefore, the elicitation of plant
defences before infection through the application of fungicides like CO and PP could improve the
tolerance of
P. pinaster
against oxidative damage caused by PWN, owing to their ability to promote plant
antioxidant system. In addition, micro and macronutrients also play an essential role in plant tolerance
against biotic and abiotic stresses (particularly in resistance to pests and diseases) (Kirkby and Römheld
2004, Bala et al. 2018, Mukherjee et al. 2019, Chan et al. 2021). Therefore, changes in plant mineral
composition resulting from fungicide application could affect their response to these stresses (Hossain et
al. 2013, dos Santos Silva et al. 2020, Motta-Romero et al. 2021, Parent and Quinche 2021). Although this
phenomenon occurs in other species, the impact of CO and PP on pine plants’ mineral accumulation and
its potential repercussion on plant susceptibility to the PWN is unknown.
Concurrently, plant-associated bacterial communities also play an essential role in the absorption of
certain minerals, plant growth promotion, and defence against pathogens (Doornbos et al. 2012,
Burketova et al. 2015, Zhang et al. 2018, Gu et al. 2020, Wang et al. 2020). Although the bacterial
communities associated with
P. pinaster
and PWN contribute to the development of PWD (Proença et al.
2010, 2017, Roriz et al. 2011, Vicente et al. 2011), the effect of fungicides like CO and PP on the
modulation of bacterial diversity has not been evaluated yet.
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Thus, this study aimed to assess the effectiveness of CO and PP as tools to induce the tolerance of pine
plants against the PWN through the evaluation of i) nematode progression in plant tissues; ii) foliar
symptoms and photosynthetic pigments; iii) proxies for plant defensive capability and oxidative damage
(anthocyanins, carotenoids, total polyphenolics, avonoids, and lipid peroxidation), iv) plant-associated
bacterial populations, and v) plant mineral prole (B, Cu, Fe, Zn, K, and P).
Materials And Methods
Plant material and experimental design
One hundred and seventeen two-year-old
P. pinaster
plants were grown in the greenhouses of Misión
Biológica de Galicia-CSIC (MBG-CSIC, Pontevedra, Spain; 42.4054° N, 8.6426° W). Seeds from French-
Landes provenance region were planted in 2L containers lled with peat and perlite (3:1, v:v). The average
height and diameter of the plants used were 124 ± 14 and 0.89 ± 0.05 cm, respectively. These plants were
transferred to Centro de Biotecnologia e Química Fina-Universidade Católica Portuguesa (CBQF-UCP,
Porto, Portugal; 41.1539° N, -8.6733° W), where the experiments took place. These were carried out from
April 9th to May 14th 2019 under natural environmental conditions.
Treatments
Seven days before the infection with
B. xylophilus
, the two selected fungicides were applied to a group of
66 pine plants; 33 plants were sprayed with a suspension of 0.2% copper oxide (CO) (commercial product,
Nordox copper 75WG Copper oxide 75% ecological) and 33 were treated with a suspension of 0.4%
potassium phosphonate (PP) (commercial product, Alexin 75 LS) (both concentrations recommended by
the manufacturer). Finally, a group of 33 plants were used as non-infected control plants and 18 plants
served as non-treated control plants.
Nematode culture and plant inoculation
Seven days after of fungicide treatment, a virulent strain of
B. xylophilus
(strain 17AS) was used for plant
inoculation. The nematodes were maintained in mycoboxes with
Botrytis cinerea
(Pers) mycelia growing
in barley seeds at 25 ºC for 14 days. Nematodes were extracted from the culturing medium using the
Baermann funnel technique (Baermann 1917) for 24 h at 25 ºC, and their concentration adjusted so that
a solution with 2 000 nematodes in 750 µL of sterilized water was obtained. Inoculation was performed
as described by Futai (2003). Briey, at approximately 20 cm from the top of each plant, leaves were
removed from a 3 cm portion of the stem, and transversal cuts were made using a sterile blade. A piece
of absorbent paper was placed around the wound, the nematode suspension was pipetted, and paralm
was used to seal the inoculation site. Thirty-three plants of each treatment, i.e., non-treated, treated with
CO, and treated with PP, were inoculated with 2.000 PWNs, while an additional group of 18 non-treated
plants was inoculated with deionized water (mock-inoculation), resulting in 4 treatments: mock-
inoculated non-treated control plants (niCTR), inoculated non-treated controls (iCTR), inoculated CO-
treated plants (iCO) and inoculated PP-treated plants (iPP).
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Scoring of foliar symptoms and sampling
Eight plants from each treatment were used to evaluate the progression of the disease through visual
analysis of leaf foliar symptoms at ve different time-points: 7, 14, 21, 28 and 35 days after inoculation
(dai). These consisted of wilting and defoliation and were visually assessed on a 0–4 scale: 0 = 0–10%
symptomatic leaf tissue; 1 = 11–33%; 2 = 34–66%; 3 ≥ 67%; and 4 = total leaf wilting or defoliation
(Sánchez et al. 2005).
Plant sampling was performed at the same time-points (7, 14, 21, 28 and 35 dai). The leaves of 5 plants
randomly selected from each treatment were separated from the stems, ground to a ne powder with
liquid nitrogen, and used for chlorophyll, lipid peroxidation, total soluble phenols and total avonoids
content and minerals quantication, whereas stems were used for whole-stem nematode quantication
and the microbiological analysis.
Nematode quantication
The leaves of plants used for nematode quantication (n = 5) were removed, and stems were cut into
small portions (
ca
. 0.5 cm). Nematodes were extracted from stems using the Baermann funnel technic
for 24 at 25 ºC, and quantied using a nematode counting dish under a transmitted light stereo
microscope, as described by Nunes da Silva et al. (2015).
Primary and secondary metabolites
For total chlorophyll and carotenoids quantication, the Sims and Gamon (2002) method was used. In
brief, 0.1 g of leaf tissue was mixed with 10 mL of cold acetone/Tris buffer solution at 1 M (80:20, v:v, pH
= 7.8) and incubated at 4 ºC for 24–72 hours, after which samples were centrifuged at 13.000 rpm for 5
minutes. Using the NanoPhotometer™ UV/VIS spectrometer (Implen GmbH, Germany) absorbances were
recorded at 470, 537, 647 and 663 nm, and the concentration of pigments was calculated as follows,
taking into consideration the sample fresh weight:
Anthocyanin = 0.08173
A
537 – 0.00697
A
647 – 0.002228
A
663
Chla = 0.01373
A
663 – 0.000897
A
537 – 0.003046
A
647
Chlb = 0.02405
A
647 – 0.004305
A
537 – 0.005507
A
663
Carotenoids = (A470 – (17.1 x (Chla + Chlb) – 9.479 x Anthocyanin) / 119.2
For the quantication of soluble phenols and avonoids, 50 mg of lyophilized leaf tissue was extracted
with 1.5 mL of 80% aqueous methanol (v:v) in an ultrasound bath for 20 minutes. The extract was
recovered after centrifugation at 15.000 g for 15 minutes.
Total soluble phenolics were determined according to the Folin-Denis’ method (Marinova et al. 2005). To
100 µL of methanolic extract, 4.5 mL of ultrapure water and 500 µL of Folin-Denis’ reagent were added.
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The mixture was vigorously mixed and the reaction allowed to occur for 5 minutes, after which 5 mL of
sodium carbonate at 7% (w:v) was added. After incubation at room temperature in the dark for 1 hour, 2
mL of ultrapure water was added to each sample. The absorbances were recorded at 750 nm using a
NanoPhotometer™ UV/VIS spectrometer (Implen GmbH, Germany) and the concentration of total soluble
phenolics determined using a gallic acid calibration curve.
For avonoids determination, the aluminium chloride method (Zhishen et al. 1999) was used. To 100 µL
of methanolic extract were added 2 mL of ultrapure water and 150 µL of NaNO2 at 5%. The mixture was
incubated for 5 minutes at room temperature. Afterwards, 150 µL of AlCl3 at 10%, 1 mL of 1M NaOH and
1.2 mL of ultrapure water were added. The absorbances were recorded at 510 nm using a
NanoPhotometer™ UV/VIS spectrometer (Implen GmbH, Germany) and avonoids concentration was
determined using a catechin calibration curve.
Quantication of lipid peroxidation
Determination of lipid peroxidation was performed through malondialdehyde (MDA) quantication,
following a modied version of the protocol described by Li (2000). In brief, to 0.1 g of leaf sample were
added 10 mL of 0.5% thiobarbituric acid in 20% trichloroacetic acid. Each sample was homogenised
through vigorous agitation for 30 seconds and incubated in a water bath at 100 ºC for 30 minutes. After
the incubation period, the reaction was terminated by transferring the samples into ice. Samples were
centrifuged for 10 minutes at 5.000 rpm and the supernatant was ltrated. The absorbance was
measured at 450, 532 and 600 nm and MDA was quantied through the equation:
MDA (µmol. L− 1) = 6.45 x (
A
532 –
A
600) – 0.56 x
A
450
Extraction and isolation of plant-associated bacterial
populations
Analysis of plant bacterial population was carried out at the end of the assay (35 days after infection), in
mock-inoculated non-treated control plants (niCTR), in non-treated infected control plants (iCTR) and
infected plants treated with the two different fungicides (iCO and iPP). The stems of three plants
randomly selected from each treatment were separated into small portions, which were sterilized by
submerging in 75% ethanol for 15 seconds, and the excess ethanol was removed by washing in deionized
water (Xie and Zhao 2008). The extremities of each stem segment were removed in aseptic conditions,
and each segment was cut horizontally and placed in Nutrient Agar (NA) medium with the vascular tissue
facing down. After incubation at 26 ºC for 3 days, morphologically distinct bacterial colonies were
identied and isolated until pure cultures were obtained. For each treatment, three plants were used, and
for each plant, 6 stem portions and 2 replicates were analysed.
Molecular identication of the bacterial populations
For the molecular identication of the bacterial cultures obtained as described before, the total genomic
DNA of each bacterial isolate was extracted using the heat-shock method as performed by Calheiros et al.
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(2010). Colonies were added to 200 µL of sterile ultra-pure water, homogenized through vigorous stirring
and incubated at 95 ºC for 10 minutes. Samples were then transferred into ice for 5 minutes, vortexed,
and centrifuged at 15.000 rpm for 5 minutes in a microcentrifuge (Heraeus Pico 17, Thermo Scientic,
USA). The concentration and integrity of the extracted DNA was evaluated spectrophotometrically using a
NanoPhotometer™ UV/VIS spectrometer (Implen GmbH, Germany).
16S rRNA genes were amplied by PCR using 12.5 µL of NZYTaq II 2x Green Master Mix (NZYTech,
Portugal) with 0.5µM of primers 27F (5'-GAGTTTGATCCTGGCTCA-3') and 1493R (5'-
TACCTTGTTACGACTT-3'), and 5 µL of bacterial DNA in a total volume of 25 µL. The PCR reactions were
performed on a thermocycler DOPPIO (VWR, USA) using the parameters: 1 cycle of initial denaturation at
95 ºC for 120s, 25 cycles of denaturation at 95 ºC for 30 seconds, annealing at 54 ºC for 30 seconds and
extension at 72 ºC for 1 minutes and nally one cycle of a nal extension at 72 ºC for 5 minutes. The
nal product was analysed by electrophoreses in a 1% agarose gel in Tris-EDTA (TAE) buffer with DNA
stains Gel Red™ (Biotium, Inc., USA) for 45 minutes at 120 V and 400mA. PCR products of all 47 bacterial
isolates were sequenced by STAB VIDA, Lda. (Lisbon, Portugal) and identied using the Basic Local
Alignment Search Tool (blastN, National Center for Biotechnology Information, USA).
Mineral determination by ICP-OES
For mineral determination at 28 dai, three plants were randomly selected from each treatment. Leaf
samples (
ca
. 0.2 g) were mixed with 5 mL of 65% HNO3 in a Teon reaction vessel and heated in a
SpeedwaveTM MWS-3þ (Berghof, Germany) microwave system. The digestion procedure was conducted
in ve steps, consisting of different temperature and time sets: 130 ºC/10 min, 160 ºC/15 min, 170 ºC/12
min, 100 ºC/7 min, and 100 ºC/3 min (Santos et al. 2015). The resulting clear solutions of the digestion
procedure were then adjusted to 50 mL with ultrapure water for further analysis. Mineral determination
was performed using the inductively coupled plasma optical emission spectrometer (ICP-OES) Optima
7000 DV (PerkinElmer, USA) with radial conguration. For each sample, two technical replicates were
prepared.
Statistical analysis
Results were analysed using GraphPad Prism v.8 (GraphPad Software, USA). Effect of plant treatments
(T) and time-point (Tp) and their interaction (T x Tp) on the number of nematodes, anthocyanin,
carotenoids, chlorophyll-A and chlorophyll-B, lipid peroxidation, total soluble phenolics and avonoids
and mineral concentration in leaf tissues were analysed considering T, Tp and their interaction as xed
factors. The signicant differences between the treatments with fungicides were determined using
Missed-effects model (REML), which uses the restricted likelihood method and a probability value P <
0.05 as the threshold level of signicance. The correlation between the different variables measured at 28
dai were determined using Pearson’s correlation matrix.
Results
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Measurement of disease symptoms and nematodes
quantication
The results of the foliar damage are shown in Fig.1. Non-infected control plants (niCTR) did not present
foliar symptoms. Foliar damage was only observed in infected plants at 21 days after infection (dai),
where 62.5% of the non-treated infected control plants (iCTR) presented damage on stage 1 (11–33%)
and 2 (34–66%), while only 25% of infected CO-treated (iCO) and PP-treated plants (iPP) presented foliar
damage of stage 1. At 28 dai, 72.5% of iCTR plants presented leaf damage, of which 12.5% were in stage
3 (≥ 67%), whereas iCO only presented 62.5% of foliar damage between stage 1 and 2, and iPP presented
37.5% of foliar damage of stage 1 and 3. At the end of the assay (35 dai), 12.5% of iCTR plants had died,
72.5% presented foliar damage between the stage 3 and 2 and only 25% did not present any foliar
damage. Similarly, 12.5% of iCO and iPP plants had died and 62.5% presented foliar damaged for iCO
and iPP (between stage 1 and 3), and 25% did not present any foliar damage. The percentage of leaf
damage was highly correlated with nematode numbers inside stem tissues (Table3).
In inoculated plants without fungicide application, nematodes signicantly increased from 1528 ± 236 (at
7 dai) to 28760 ± 3540 (at 35 dai), i.e., 18.8-fold (Fig.2, Table1). In contrast, in fungicide-treated plants,
the number of nematodes was signicantly lower in all treatments. At 35 dai the number of nematodes
inside stem tissues of iCO (16667 ± 14612) and iPP (11227 ± 10307) were 0.42- and 0.61-fold
signicantly lower than in iCTR (28760 ± 3540).
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Table 1
Effect of time-point (Tp, 7, 14, 21, 28 and 35 days after inoculation) and plant
treatments (T, infected non-treated control trees (iCTR), infected trees treated with
copper oxide (iCO) or potassium phosphonate (iPP)) and their interaction (T x Tp) on
the number of nematodes, anthocyanin, carotenoids, chlorophyll-A and chlorophyll-B,
lipid peroxidation, total soluble phenolics and avonoids. Signicant P values (< 0.05)
are indicated in bold.
Response variable Factor
F
ratio
P
value
Number of nematodes (nematodes.plant− 1)Treatment (T) 8.72 0.0005
Time-point (Tp) 31.72 < 0.0001
T ´ Tp 2.54 0.0188
Anthocyanin (mmol.g− 1 leaf) Treatment (T) 7.11 0.0017
Time-point (Tp) 15.00 < 0.0001
T ´ Tp 18.90 < 0.0001
Carotenoids (mmol.g− 1 leaf) Treatment (T) 0.63 0.5389
Time-point (Tp) 2.75 0.0489
T ´ Tp 7.89 < 0.0001
Chlorophyll-A (mmol.g− 1 leaf) Treatment (T) 1.35 0.2952
Time-point (Tp) 7.03 0.0005
T ´ Tp 2.55 0.0211
Chlorophyll-B (mmol.g− 1 leaf) Treatment (T) 1.21 0.3336
Time-point (Tp) 1.58 0.2103
T ´ Tp 11.16 < 0.0001
Total soluble phenolics (mg.g− 1 leaf) Treatment (T) 3.69 0.0308
Time-point (Tp) 18.05 < 0.0001
T ´ Tp 5.14 < 0.0001
Total avonoid content (mg.g− 1 leaf) Treatment (T) 14.12 < 0.0001
Time-point (Tp) 8.70 0.0005
T ´ Tp 3.91 0.0009
Malondialdehyde (µmol.g− 1 leaf) Treatment (T) 18.10 0.0002
Time-point (Tp) 10.75 0.0002
T ´ Tp 0.90 0.5269
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Primary and secondary metabolites
In general, both treatments (T) and time-points (Tp) were signicant for anthocyanin while only time-
points and the interaction T x Tp were signicant for carotenoid content (Table1). Anthocyanin (Fig.3A)
accumulation in non-treated infected control plants (iCTR) showed a progressive and signicant decrease
from 7 to 28 dai (by 0.43- fold), slightly increasing at 35 dai (reaching 9.80 ± 1.94 µmol. g− 1 leaf). In
infected CO-treated (iCO), the concentration of anthocyanins gradually increased until 35 dai (by 1.32-
fold), greatly increasing at 28 dai (reaching 16.82 ± 4.72 µmol. g− 1 leaf). At 15 dai, PP-treated plants (iPP)
presented the highest anthocyanin concentrations (37.80 ± 10.89 µmol. g− 1 leaf). The anthocyanin was
negatively and highly correlated with nematode numbers inside stem tissues and the leaf damage
(Table3). Despite the signicative inuence of treatments and time-points in anthocyanins concentration
in leaf tissues, no signicant differences were observed in the other leaf metabolites analysed
(chlorophyll-A and chlorophyll-B, Table1 and Fig.1S).
Both treatments and time-points signicantly affected total soluble phenols and avonoids content
(Table1). Moreover, a positive correlation was observed between soluble phenols and avonoids content
and foliar damage and nematode density, whereas avonoids content soluble phenols were found to be
negatively correlated to anthocyanins (Table3). Non-treated infected control plants (iCTR) showed a
gradual decrease in phenols and avonoids accumulation along time (by 0.70- and 0.49-fold,
respectively), but a signicant increase was observed at 28 dai in phenols (by 1.65-fold) (Fig.4). iCO
showed a gradual decreased in phenols accumulation throughout the experimental period (by 0.71-fold),
while avonoid accumulation showed a gradual increase until 21 dai (by 1.39-fold), followed by a
decrease until the end of the experiment (by 0.76-fold). Contrastingly, iPP showed an increasing trend in
soluble phenols and avonoids concentrations, with the highest concentration being recorded at 28 dai
(by 1.49- and 2.28-fold, respectively), decreasing at 35 dai (by 0.67 and 0.40-fold, respectively) (Fig.4).
Lipid peroxidation
In general, infected plants (both with and without fungicide treatment) displayed a progressive and
signicant increase in MDA levels until 28 dpi, slightly decreasing at 35 dai (Fig.5). From 1 to 35 dpi,
MDA signicantly increased in all treatments (by 1.42-fold in iCTR, 1.21-fold in iCO and 1.57-fold in iPP),
comparing with niCTR.
Mineral composition
Plant treatments signicantly affected the concentration of micronutrients (B, Cu, Fe and Zn) and
macronutrients analysed (K and P) (Table2). The highest B concentration was found in niCTR) (36.9 ±
3.7 µg.g− 1), whereas iCO had the lowest (24.1 ± 7.2 µg.g− 1) (Fig.6A). The average concentrations of Cu
were similar between niCTR, iCTR and iPP (around 3 ± 0.4 µg.g− 1), while iCO presented the highest
average concentration (189.7 ± 39.1 µg.g− 1; Fig.6B). Fe concentration was higher in non-infected control
plants (niCTR; 0.16 ± 0.02 µg.g− 1), and lowest in iCO (0.11 ± 0.02 µg.g− 1 leaf; Fig.6C). Moreover, iCTR
presented the highest Zn concentration (84.88 ± 11.17 µg.g− 1), while iCO had the lowest (51.46 ± 8.94
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µg.g− 1; Fig.6D). The infected trees (iCTR, iCO and iPP) presented a similar average concentration of K
(around 7570 ± 2183 µg.g− 1), while niCTR presented the highest concentration (12.2 ± 1.3 µg.g− 1;
Fig.6E). Finally, iCO presented the lowest concentration of P (2.4 ± 0.55 µg.g− 1), while iCTR had the
highest (6.1 ± 2.3 µg.g− 1 ; Fig.6F).
Table 2
Effect of plant treatments at 28 days after inoculation (T, non-
infected non-treated control plants (niCTR), infected non-treated
control trees (iCTR), infected trees treated with copper oxide (iCO) or
potassium phosphonate (iPP)) on the concentration (µg.g-1) of
micronutrients (MiN) and macronutrients (MaN) in leaf tissues.
Signicant P values (< 0.05) are indicated in bold.
Response variable Factor
F
ratio
P
value
MiN B (µg.g− 1 leaf) Treatment (T) 5.05 0.0092
Cu (µg.g− 1 leaf) Treatment (T) 136.8 < 0.0001
Fe (µg.g− 1 leaf) Treatment (T) 6.88 0.0023
Zn (µg.g− 1 leaf) Treatment (T) 7.917 0.0011
MaN K (µg.g− 1 leaf) Treatment (T) 8.06 0.0010
P (µg.g− 1 leaf) Treatment (T) 8.67 0.0007
Bacterial endophytic population
The groups with the highest bacterial diversity at the end of the experimental period were iCO with four
genera and iCTR with three different genera, whereas niCTR showed two different genera and iPP only
one (Fig.7).
The main genera found were
Pseudomonas
, represented by two species,
P. uorescens
present in all
groups and
P. monteilii
(only present in iCO). The second more abundant genus found was
Klebsiella
,
represented by only one specie (
K. oxycota
) present in all groups except in iPP. Other genera found was
Bacillus
, represented by only one specie (
B. cereus
) in iCTR and the genus
Pantoea
, represented by
P.
agglomerans
present only in iCO.
Table 3 Person's correlation matrix between the different variables measured at 28 days after inoculation:
% FDamage (percentage of foliar damage), Nnemat (number of nematodes), Antho (anthocyanins),
phenols (total phenols), Flavo (total avonoids), LPerox (lipid peroxidation), micronutrients (B, Cu, Fe and
Zn) and macronutrients (K and P).
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Discussion
Fungicide application, particularly with PP, ameliorates the
development of the pine wilt disease
When pine plants are infected, two infection stages take place. In the rst stage, most nematodes remain
close to the inoculation site and surrounding cortical tissues (Suzuki 1984). The second phase occurs
after transpiration rates are impaired, and is characterized by a substantial reduction in oleoresin ow. At
this stage, there is an exponential increase in PWN population and disease symptoms progression
(Suzuki 1984, Myers 1988). In the current work, the increase in the number of nematodes and the
beginning of the appearance of foliar symptoms occurred at 21 dai, indicating the successful infection
and multiplication of the PWN inside plant tissues over time. In previous work, with one-year-old (40–50
cm height)
P. pinaster
plants maintained in a growth chamber (16 h light/8 h darkness photoperiod at
25/18°C), PWN infection progressed at higher rates between 7 and 14 days after inoculation (Nunes da
Silva et al. 2021). This divergence could result from differences in plant age and size between the two
experiments. Compared with non-treated plants, fungicide treatment decreased nematode density by up
to 0.58- and 0.39-fold (for CO and PP, respectively) at the end of the experimental period.
Anthocyanins and carotenoids are involved in plant protection against photosystem damage (Elkhouni et
al. 2018). It appears that the increase of these metabolites occurs at the beginning of the infection and
the end of the assay in infected non-treated control plants (iCTR) as an effort by plants to lessen the
harmful effects caused by PWN (Nunes da Silva et al. 2021, López-Villamor et al. 2022). Regarding plants
treated with fungicides, iPP had a substantial peak of anthocyanin concentration at 14 dai. In
comparison, iCO had a higher concentration at 28 dai, suggesting a higher antioxidant response to the
PWN corroborated by the lower presence of foliar damage at that time (Bali et al. 2018, Gökbayrak and
Gözel 2022, López-Villamor et al. 2022). In fact, despite preventing nematode reproduction in plant
tissues, CO and PP fungicides did not prevent cellular damage, leading to increased MDA accumulation
and greater foliar damage. These effects could result from the abrupt induction of plant defence
mechanisms upon PWN infection (Agrawal et al., 2002). The peroxidation of unsaturated lipids in cell
Page 13/31
membranes results from xylem parenchyma and cortex cell necrosis, and the destruction of phloem
caused by PWN in susceptible species of the genus Pinus (Apel and Hirt 2004, Yamada 2008). The
accumulation of MDA, a secondary compound of lipid peroxidation reactions, indicates cell damage
induced by free radicals (Santos et al. 2012). Polyphenols, avonoids, anthocyanins and carotenoids are
also secondary metabolites in plant tolerance to biotic stress (Kawaguchi 2006, Kuroda et al. 2011,
Kusumoto et al. 2014, Gaspar et al. 2017). Total soluble phenols and avonoid concentration gradually
decreased until the end of the experimental period in iCTR and iCO, increasing progressively until 28 dai in
iPP. At 28 dai, polyphenol concentration correlated signicantly with increased colonization by PWN
(Table3). In fact, in iCTR there was both a higher nematodes density and a more signicant
accumulation of phenols, as compared with plants treated with fungicides (Fig.4A). This observation is
consistent with a positive correlation between nematode migration and polyphenols concentration,
previously reported in a migration assay of PWN through wood tissues of two-year-old branches from 10
years old plants (Zas et al. 2015).
Minerals play an important role in tolerance against the
PWN
Mineral concentration in leaf tissues was analysed at 28 dai, coinciding with the point of most signicant
biotic stress indicated by the high accumulations of polyphenols and MDA (Fig.4A and 5), increased
progression of disease symptoms and higher PWN density inside plant tissues (Figs.1 and 2).
Minerals and other nutrients have important specic functions in plant physiology, are essential for plant
metabolism, and play an important role in defending plants against biotic and abiotic stress ((Waraich et
al. 2011, Ahanger and Ahmad 2019, Pathak et al. 2020). Many of these minerals (Cu, Fe, Mn and Ni) are
constituents of enzymes, specically metalloproteins; others (Mn and Zn) participate in the activation of
enzymes, and some (B and Zn) are constituents of plant cell walls and membranes (Asher et al. 1991,
Römheld and Marschner 1991, Bergmann and Caesar 1994, Welch and Shuman 1995, Mengel and Kirkby
2001, Kirkby and Römheld 2004, Epstein and Bloom 2005, Marschner 2011). For this reason,
Fungicide application can alter the mineral composition of plant tissues (Shahid et al. 2018, dos Santos
Silva et al. 2020, Motta-Romero et al. 2021). Zou et al. (2000) observed a signicant relationship between
the tolerance of Masson pine (
P. massoniana
) against the PWN and leaf mineral content, and this
correlation changed at different stages of plant development. Likewise, differences in plant mineral
content have been reported following infection by
B. xylophilus
. In the current work, there was a decrease
in the concentration of B in infected plants (Fig.7A), compared with non-infected control plants (niCTR).
B is involved in carbohydrate metabolism, and when B is limited, the pentose phosphate pathway
becomes predominant in carbohydrate degradation, leading to the formation of phenolic compounds
(and tryptophan) through the shikimic acid pathway (Chen et al. 2014). However, the accumulation of
phenols and the increased activity of polyphenol oxidase in plant tissues lead to the formation of highly
reactive intermediates, such as quinones (Ruíz et al. 1998, Ölçer and Kocaçalışkan 2007). These
compounds, and also photo-activated phenols, are highly effective in the production of superoxide
Page 14/31
radicals, which may damage membranes through lipid peroxidation. Therefore, the decrease in B caused
by PWN infection seems to trigger oxidative stress, causing the accumulation of polyphenols and MDA.
Cu is somewhat similar to Fe in that it forms highly stable chelates that allow the transfer of electrons
(Printz et al. 2016). For this reason, it plays a role comparable to Fe in redox processes (Yruela 2005).
Several Cu-containing proteins play critical roles in photosynthesis, respiration, superoxide radical
detoxication, and lignication (Droppa and Horváth 1990, Burkhead et al. 2009, Broadley et al. 2012). Cu
deciency causes the accumulation of phenols (Pilon et al. 2006, Hänsch and Mendel 2009); therefore,
this mineral is vital to increase plant tolerance to several diseases (Schulten and Krämer 2017).
Fungicides such as copper sulphate, in which the main active ingredient is Cu, have shown
in vitro
nematicidal activity, causing high mortality and decreasing the mobility of
B. xylophilus
(Tan et al. 2013).
In the current study, iCTR and iPP had the lowest Cu concentrations (Fig.6B). The high negative
correlation with foliar damage and the number of nematodes and the high positive correlation with
avonoids (Table3) found in this trial supports that the lower Cu concentration may be related to the
higher foliar damage (Fig.1C). Accordingly, iCO presented higher concentrations of Cu (Fig.6B) and lower
foliar damage (Fig.1C).
Cytochrome oxidase, catalase, and peroxidase are Fe-dependent enzymes (Hänsch and Mendel 2009),
and their activities often decrease under conditions of Fe deciency (Mengel and Kirkby 2001). A drastic
decrease in peroxidase activity with subsequent accumulation of phenolic substances has been reported
under Fe-limiting conditions (Römheld and Marschner 1981). Here, the lowest concentrations of Fe
(Fig.6C) were observed in infected plants when compared to the non-infected control plants (niCTR), and
the former were also the ones with the highest accumulation of phenols and lipid peroxidation (Table3).
This suggests that Fe plays a vital role in the regulation of enzymes that could prevent the oxidation of
tissues caused by PWN.
Zn has an essential role in plant metabolism, including effects on carbohydrate metabolism, protein
synthesis, hormonal regulation, and membrane integrity (Mengel and Kirkby 2001). Here we found a high
correlation (Table3) between higher Zn levels in non-treated infected control plants (iCTR) (Fig.7C),
which also had higher concentrations of phenolic compounds (Fig.4A) and nematodes numbers (Fig.2).
In
Kandelia obovata
a strong correlation was also found between Zn concentrations and the
accumulation of phenolic compounds (Chen et al. 2019). On the other hand, Georgieva et al. (2002)
suggested that plants grown at higher Zn levels have higher nematode levels, but this was probably due
to adverse effects on nematode antagonists. Therefore, the increase in Zn may be related to the higher
number of PWNs isolated in the non-treated infected control plants (iCTR) as opposed to the lower
concentration of this micronutrient and, therefore, a lower number of nematodes observed in fungicide-
treated plants.
Potassium (K) plays an essential role in plant metabolism, supporting plants' tness under biotic and
abiotic stresses (Wang et al. 2013). K-decient plants generally are more susceptible to biotic diseases
than plants with an adequate K supply (Mukherjee et al. 2019). In the present assay, non-infected control
Page 15/31
plants (niCTR) had the highest concentrations of K (Fig.6E), when compared to the infected plants, which
were the ones with the highest accumulation of phenols and lipid peroxidation (Table3), corroborating
that K plays an essential role in defence regulation following PWN infection. Manghi et al. (2021) showed
that plants could not use PP as a source of K and P. Hence its concentration does not increase in the
analysed tissues (Manghi et al. 2021).
Phosphorus (P) plays a vital role in plant immunity and is one of the most relevant minerals related to
plant growth (Chan et al. 2021). The presence of high concentrations of Cu and PP negatively inuences
P absorption (Rawat et al. 2018, Manghi et al. 2021). In addition, P deprivation induces the synthesis of
secondary metabolites with antimicrobial activity, such as avonoids and glucosinolates (Pant et al.
2015), and defence hormones, such as salicylic acid (SA) and especially jasmonic acid (JA) (Khan et al.
2016, Castrillo et al. 2017, Morcillo et al. 2020), which modify the immunity of plants. Therefore, the low
levels of this mineral observed in iCO and iPP (Fig.6F), compared to the high levels in iCTR, along with
the lower levels of damage and nematode density (Figs.1 and 2) observed in fungicide-treated plants,
might result from the induction of defence hormones caused by P limitation.
Fungicide treatment induces changes in the bacterial
community of P. pinaster
La Torre et al. (2018) demonstrated that CO can combat bacterial diseases and that PP activates
different plant defence pathways, SAR and LAR (INTAGRI 2017). These pathways produce exudates that
can alter the plant microbiome (Lebeis et al. 2015, Liu et al. 2017, 2020, Mannaa et al. 2020). Since PWD-
associated endophytic bacteria may be necessary to develop or suppress infection caused by the
nematode (Alves et al. 2018, Kim et al. 2019), the present work aimed to study the modications of PWN-
associated bacterial communities after fungicide treatment. We observed that the non-infected control
plants had two species,
K. oxytoca and P. uorescens
.
K. oxytoca
was present in all treatments except in
the plants treated with PP, while all plants had
P. uorescens
.
K. oxytoca
is an endophytic plant growth-
promoting strain (Hallmann et al. 1997); this nitrogen-xing bacterium exists in rice roots (Nguyen Т et al.
1989). Roriz et al. (2011) suggested that it may be associated with the Portuguese region because
previous works did not identify this bacterium in
P. pinaster
. On the other hand,
P. uorescens
, a plant
growth-promoting rhizobacteria (PGPR), increases primary productivity by promoting growth and
triggering induced systemic tolerance in plants (Hol et al. 2013, Panpatte et al. 2016). Apart from
P.
uorescens
, niCTR also presented
Bacillus cereus
, previously described in Masson pine, by having a
nematocidal activity (Li et al. 2020). iCO plants had
Pseudomonas monteilii
, present in the
Pseudomonas
putida
group (Anzai et al. 2000), described to promote plant growth and induce systemic tolerance to root
rot fungi.
P. putida
can induce systemic resistance against PWN in seedlings and pine callus (Pandya et
al. 2014, Kim et al. 2019, Urooj et al. 2020). iCO also presented
Pantoea agglomerans
, a species
belonging to the family Erwiniaceae and adapted to live epiphytically in several plant tissues (Zhang and
Qiu 2015, Luziatelli, Gatti, et al. 2020). Some strains of this species are agronomically important for their
biocontrol activity (Dutkiewicz et al. 2016) and their ability to produce the auxin indole-3-acetic acid
(Luziatelli, Ficca, et al. 2020). Although all these genera have been previously described as related to both
Page 16/31
pine plants and the PWN (Proença et al. 2010, 2017, Roriz et al. 2011, Vicente et al. 2011), this work
identied some species for the rst time, such as
B. cereus
(niCTR),
P. montielli
and
P. agglomerans
(iCO).
Christian et al. (2016) showed that fungicides modulate bacterial types and diversity in white snakeroot
(Ageratina altissima), but it is the rst report in pine. Therefore, the application of fungicides modies the
diversity of the
P. pinaster
microbiome favouring the proliferation of species that improve resistance
against the PWN, particularly in plants treated with CO, which presented critical bacterial species related
to plant growth promotion (
K. oxytoca
and
P. uorescens
) and resistance to diseases, including the PWD
(
P. agglomerans
and
P. monteilii
).
Conclusions
This study describes for the rst time several physiological and microbial changes occurring in
P
.
pinaster
plants infected with
B. xylophilus
after exogenous application of CO and PP. All infected plants
(treated and no-treated) presented the same percentage of dead plants (12,5%) at the end of the assay,
but fungicide-treated plants had fewer nematodes and foliar symptoms than infected control plants, thus
decreasing the progression of PWD. Fungicides promoted defence mechanisms of pine plants against
the PWN by increasing the concentrations of anthocyanins, carotenoids, and avonoids in plant tissues
at specic stages following infection. Infected plants treated with CO and PP had high MDA
concentrations, which may correlate with a stronger redox activity. Moreover, fungicide application
induced changes in the plant bacterial community, promoting benecial bacteria for the defence of
P.
pinaster
against the PWN. It also altered the concentration of several essential minerals, such as Cu and
P, associated with plant growth, defence and immunity. This integrated study supports the use of Cu and
P-based fungicides as a biocontrol method against PWN, for example, nursery conditions, due to their
benecial role in the accumulation of crucial plant metabolites and the promotion of plant defences.
Declarations
Funding
This study was supported by the project "POINTERS - Interactions between nematodes and host pine
trees: the discovery of sustainable alternatives for the management of the pine wilt disease", funded by
the Competitiveness and Internationalization Operational Program (POCI-01-0145- FEDER-031999) and
by Fundação para a Ciência e a Tecnologia under its OE component (PTDC/ASP-SIL/31999/2017). This
work was also supported by National Funds from Fundação para a Ciência e a Tecnologia through the
scientic collaboration under the FCT Project UIDB/50016/2020 and for ALV scholarship funded by the
Regional Government GAIN-Xunta de Galicia (Ref.: 530 IN606B).
Conict of interest
The authors declare that they have no conict of interest.
Author contribution statement
Page 17/31
ALV and MV: designed the experiment; ALV and MN: infected the plants; ALV: developing the experiment,
treated the plants with CO and PP, did the sampling, performed the biochemical, microbiological, mineral
analysis and data analysis; ALV: coordinated the nal writing and all authors made helpful suggestions;
MV: obtained the funding, helped plan the experiments and revised the manuscript. All authors
contributed to the writing and discussion of the manuscript.
Data Availability
The datasets generated during and/or analysed during the current study are available from the
corresponding author on reasonable request.
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Figures
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Figure 1
Foliar symptoms (%) at (a) 7-14 days, (b) 21 days, (c) 28 days and (d) 35 days after inoculation in mock-
inoculated non-treated control plants (niCTR), infected non-treated control trees (iCTR), and infected trees
treated with copper oxide (iCO) or potassium phosphonate (iPP)
Figure 2
Page 27/31
Number of nematodes (Nematodes.plant-1) in infected non-treated control trees (iCTR), and infected trees
treated with copper oxide (iCO) or potassium phosphonate (iPP) (at 7, 14, 21, 28 and 35 days after
inoculation). Values represent the mean of 5 biological replicates ± standard error of the mean.
Signicance levels of treatments and time-point for number of nematodes: ***. P < 0.001; **. P < 0.01; *. P
< 0.05; ns. not signicant
Figure 3
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(a) Anthocyanin (µmol.g-1 leaf tissue) and (b) carotenoids (µmol.g-1 leaf tissue) in infected non-treated
control trees (iCTR),and infected trees treated with copper oxide (iCO) or potassium phosphonate (iPP)
(at 7, 14, 21, 28 and 35 days after inoculation). Values represent the mean of 5 biological replicates ±
standard error of the mean. Signicance levels of treatments and time-point for anthocyanin and
carotenoids: ***. P < 0.001; **. P < 0.01; *. P < 0.05; ns. not signicant
Figure 4
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(a) Total soluble phenols (mg.g-1 leaf) and (b) total avonoids content (mg.g-1 leaf) in infected non-
treated control trees (iCTR), and Infected trees treated with copper oxide (iCO) or potassium phosphonate
(iPP) (at 7, 14, 21, 28 and 35 days after inoculation). Values represent the mean of 5biological replicates
± standard error of the mean. Signicance levels of treatments and time-point for phenols and avonoids:
***. P < 0.001; **. P < 0.01; *. P < 0.05; ns. not signicant
Figure 5
Malondialdehyde (MDA) (µmol.g-1) in infected non-treated control trees (iCTR), and infected trees treated
with copper oxide (iCO) or potassium phosphonate (iPP) (at 7, 14, 21, 28 and 35 days after inoculation).
Values represent the mean of 5 biological replicates ± standard error of the mean. Signicance levels of
treatments and time-point for MDA: ***. P < 0.001; **. P < 0.01; *. P < 0.05; ns. not signicant
Page 30/31
Figure 6
Concentration of the boron (B), copper (Cu), iron (Fe), zinc (Zn) micronutrients (a-d) and of potassium (K)
and phosphorus (P) macronutrients (e-f) in mock-inoculated non-treated control plants (niCTR), infected
non-treated control trees (iCTR), and infected trees treated with copper oxide (iCO) or potassium
phosphonate (iPP) at 28 days after infection. Values represent the mean of 5 biological replicates ±
Page 31/31
standard error of the mean. Signicance levels of treatments and time-point for B. Cu. Fe. Zn. K and P:
***. P < 0.001; **. P < 0.01; *. P < 0.05; ns. not signicant
Figure 7
Bacterial populations in mock-inoculated non-treated control plants (niCTR), infected non-treated control
trees (iCTR), infected trees treated with copper oxide (iCO) or potassium phosphonate (iPP) at 35 days
after inoculation
Supplementary Files
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Figure1Sa.png
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