Accepted Manuscript

Title: Exogenous phenol increase resistance of Ulmus minorto Dutch elm disease through formation of suberin-likecompounds on xylem tissues

Authors: Juan A. Martın, Alejandro Solla, M. RosarioDomingues, Manuel A. Coimbra, Luis Gil

PII: S0098-8472(08)00062-2DOI: doi:10.1016/j.envexpbot.2008.05.004Reference: EEB 1899

To appear in: Environmental and Experimental Botany

Received date: 3-7-2007Revised date: 27-3-2008Accepted date: 3-5-2008

Please cite this article as: Martın, J.A., Solla, A., Domingues, M.R., Coimbra, M.A.,Gil, L., Exogenous phenol increase resistance of Ulmus minor to Dutch elm diseasethrough formation of suberin-like compounds on xylem tissues, Environmental andExperimental Botany (2007), doi:10.1016/j.envexpbot.2008.05.004

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Title page

(i) Title: Exogenous phenol increase resistance of Ulmus minor to Dutch elm

disease through formation of suberin-like compounds on xylem tissues

5

(ii) Authors: Juan A. Martína, Alejandro Sollab,*, M. Rosário Dominguesc, Manuel

A. Coimbrac, Luis Gila

(iii) Institute addresses:aAnatomía, Fisiología y Genética Forestal, ETSI de Montes, Universidad 10

Politécnica de Madrid, Paseo de las Moreras s/n, 28040 Madrid, Spain bIngeniería Técnica Forestal. Universidad de Extremadura, Avenida Virgen del

Puerto 2, 10600 Plasencia, SpaincDepartamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal

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(iv) *Corresponding author: Tel: +34 927427000; fax: +34-927425209; e-mail:

asolla@unex.es

Manuscript

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Abstract

The survival of some elms to Dutch elm disease (DED) epidemics could be related with

the application of disinfectant products based on simple phenols. To test this hypothesis,

the protective effect of different phenolic treatments in Ulmus minor trees was evaluated 5

through inoculations with Ophiostoma novo-ulmi, the current DED pathogen. During

spring 2004 and spring 2005, 4-year-old elms were: (i) watered with a 0.02% solution

of the phenolic fraction of phenolic oil, (ii) watered with a 0.02 and 0.2% solution of a

phenol-cresol mixture, and (iii) trunk injected with a 0.2% solution of phenol-cresol

mixture. In May, trees were artificially inoculated with O. novo-ulmi. At the end of the 10

2004 and 2005 vegetative periods, phenol treated trees showed significantly lower

wilting values than control trees. One week of bud break delay was observed in trees

watered with the 0.2% solution of phenol-cresol mixture. Fourier transform-infrared

spectroscopy and electrospray ionisation mass spectrometry evidenced enhanced levels

of suberin-like compounds in phenol treated trees with respect to non-treated trees. The 15

deposition of suberin in xylem tissues, as a response to phenol treatments, might be

considered as one of the mechanisms of resistance of elms to O. novo-ulmi.

Keywords: Ulmus minor Mill.; Ophiostoma novo-ulmi Brasier; Dutch elm disease;

Phenol; Suberin20

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1. Introduction

Dutch elm disease (DED) is a wilt disease of elms caused by the fungi Ophiostoma

ulmi (Buisman) Nannf. and the more aggressive O. novo-ulmi Brasier, which spread

systemically within the xylem vessels causing their cavitation and occlusion. The DED 5

fungi are transmitted from diseased to healthy trees by elm bark beetles, mainly in the

genus Scolytus and Hylurgopinus (Webber, 2004), or through root grafts. Gagnon

(1967) reported differences in the distribution of phenolic compounds between healthy

and O. ulmi-infected American elms (U. americana L.). He observed that phenols

formed in parenchyma cells in response to infection were extruded with the protoplasm 10

of these cells into the vessels, participating in vessel occlusion. Later studies on induced

responses of Ulmus sp. against O. ulmi have confirmed the accumulation of polyphenols

in axial parenchyma cells (Tippet and Shigo, 1981; Rioux and Ouellette, 1991) and the

production of several sesquiterpene phenolics (Duchesne et al., 1992), which were

identified as fungitoxic phytoalexin-like compounds (mansonones). Moreover, the 15

phenolic profile of elm leaves after O. ulmi inoculation was reported to differ among

Ulmus species of different susceptibility to DED (Heimler et al., 1994).

Most mature European and North American elms have died due to two massive DED

pandemics during the 20th century. However, recent surveys undertaken in Spain

revealed the presence of some elms and elm stands that are still alive within areas 20

ravaged by the disease. This is the case of the Rivas-Vaciamadrid (Madrid) Ulmus

minor Mill. stand, which is probably the best elm stand remaining in Spain. Studies

focused on explaining survival of the trees of this stand to DED confirmed the presence

of O. ulmi, O. novo-ulmi and Scolytus multistriatus Marsh. (Martín et al., 2006; Solla et

al., 2008). The spread of DED through this elm stand is abnormally slow, although a 25

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genetic characterization of these elms through nuclear DNA markers revealed that most

trees were a unique susceptible clone (Gil et al., 2004). Disinfectant products based on

simple phenolic compounds, such as Zotal® (phenolic oil : emulsifier, 4 : 25, v/v), were

commonly applied directly to the cattle or to the soil in Rivas-Vaciamadrid and in other

Spanish grazing areas to prevent insect bites or hoof infections since the beginning of 5

the 20th century. We hypothesize that the application of phenols within Rivas-

Vaciamadrid elm stand could have enhanced tree resistance against O. novo-ulmi

infections. The main objectives of the present work were to (i) test a possible protective

effect of different phenol treatments in U. minor trees against O. novo-ulmi, (ii) study

the toxicity of phenols on U. minor, and (iii) detect possible metabolic differences in 10

xylem samples between treated and untreated U. minor trees that might explain the

increased resistance.

2. Materials and methods

15

2.1. Plant material

Two susceptible U. minor clones from Spain (UPM121, and UPM168) were used in

this work (Table 1). These clones were selected since previous studies confirmed their

taxonomy (Gil et al., 2004) and their high susceptibility to O. novo-ulmi (Solla et al., 20

2005). In December 2000, the clones were propagated by root cuttings at the Forest

Breeding Centre in Puerta de Hierro, Madrid. During the first season, the ramets grew in

2 L pots containing perlite and peat (1:1, v/v) and were irrigated to field capacity. In

December 2001, the ramets were transplanted to 15 L pots containing a mixture of fine

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sandy loam texture soil and peat (4:1, v/v). The ramets were weekly watered to field

capacity and placed outside. When the first treatments were applied the ramets were 4

years old and their height 1.56 m ± 0.12 (mean ± SE).

Four additional U. minor clones from Spain (UPM016, UPM092, UPM129, and

UPM175) were used in the toxicity study (Table 1). In December 1999 the clones were 5

propagated by root cuttings at the same Centre. After being cultivated one year under

the same conditions as described above, the ramets were transplanted to 30 L pots

containing a mixture of fine sandy loam texture soil and peat (4:1, v/v). The ramets

were weekly irrigated to field capacity and placed outside. When the first treatments

were applied the ramets were 5 years old and their height 1.93 m ± 0.11 (mean ± SE).10

All pots were placed following a random spacial distribution under a shading mesh

providing 25% of full sunlight throughout the experiment.

2.2. Treatments

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In 2004, UPM121 and UPM168 ramets were divided as follows: (i) non-treated; (ii)

phenol treated; (iii) non-treated and O. novo-ulmi inoculated; and (iv) phenol treated

and O. novo-ulmi inoculated (Table 2). The treatment period was from February 12 to

July 29. Phenol concentrations of 0.02 and 0.2% were selected having taken into

account (i) previous research on DED chemotherapy (Zentmyer et al., 1946), in which 20

phenols were tested at concentrations from 0.05 to 0.2%, and (ii) the concentration of

phenols in the disinfectant product Zotal®, estimated to be round about 0.06-0.1%. Non-

treated plants (groups i and iii; T1 plants) were watered once a week with 0.5 L of water

per plant. Phenol treated plants (groups ii and iv) were divided again in T2, T3, T4 and

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T5 subgroups. T2 plants were watered weekly with 0.5 L of 0.02% of the phenolic

fraction of phenolic oil (kindly provided by Laboratorios Zotal S.A., Sevilla, Spain).

Phenolic oil is made of naphtha (55%), washing oil (20%), phenol (20%) and o-cresol

(4%). In order to remove naphtha and washing oil, the product was mixed with water

(1:1, v/v), shaken for 3 days (170 rpm) and the upper layer discarded. The remaining 5

liquid was redissolved in water, so each plant received weekly about 0.1 mL of a phenol

and o-cresol mixture (5:1 v/v). T3 and T4 plants were weekly watered with 0.5 L of a

mixture of phenol (Panreac, Barcelona, Spain) and o-cresol (Merck, Hohenbrunn,

Germany) (5:1, v/v), at 0.02% (T3) and 0.2% (T4). Thus, T3 and T4 plants received

weekly about 0.1 and 1 mL of phenol mixture, respectively. pH values of solutions used 10

for T1, T2 T3 and T4 plants were 7.5, 7.4, 7.2 and 6.0, respectively. Finally, on April

15, 2004, T5 trees were injected 0.1 mL of a mixture of phenol and o-cresol (5:1, v/v) at

0.2 % into the main trunk. To accomplish this, a transverse cut was made with a flame-

sterilized scalpel blade into the sapwood of the trunk 3 to 5 cm above the soil surface on

both north and south sides of each T5 tree. In each cut a 0.1 mL drop of the solution was 15

applied by hypodermic syringe; the solution was immediately absorbed by the xylem.

Each treatment (T1 to T5) comprised 16 plants (8 ramets per clone). Eight plants per

treatment (4 ramets per clone) were inoculated with Ophiostoma novo-ulmi for

symptom assessment (T1i-T5i plants). The remaining 8 plants per treatment (4 ramets

per clone) were not inoculated in 2004 and were used as controls for sample removal 20

and subsequent chemical analysis (T1c-T5c plants).

On May 1, 2004, inoculations were carried out following the procedure described in

(Solla et al., 2005), using a highly virulent strain of O. novo-ulmi subsp. americana

labelled OR-VR1. The isolate was collected in 1996 from an U. minor tree in Verín

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(Orense, Spain) and had an in vitro growth of 4.3 mm per day on 2% malt extract agar

(20ºC) (Solla et al., 2008). The fungus was conserved at 4ºC in the dark. At 3-months

intervals it was subcultured and its in vitro growth rate verified to detect any possible

loss of virulence. The virulence of the strain was also checked through inoculations

undertaken on elms in 2003 (Martín et al., 2005a) and in 2004 (Martín et al., 2005b).5

From February 10 to July 28, 2005, Tc plants were treated again using the same dose

and frequency as in 2004. On T5c plants, trunk injections were carried out on April 25.

All Tc plants were inoculated on May 9, using the same isolate and methodology as in

2004 (Table 2).

10

2.3. Toxicity test

During 2004 and 2005, UPM016, UPM092, UPM129 and UPM175 ramets were

tested for toxicity to a phenol and o-cresol solution (5:1, v/v) mixture. Ramets were

divided as follows: non-treated plants (TO1), plants watered weekly with 1 L of 15

solution at concentrations of 0.02, 0.2 and 2% (TO2, TO3 and TO4, respectively), and

trunk-injected plants at concentration of 2% (TO5). The watering periods and trunk

injection dates were the same as described above (Table 2). Four trees were used for

each treatment and concentration, each clone represented by a single ramet.

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2.4. Phenology notations, symptom evaluation, and toxicity

Phenology of elms was studied from March to May during 2004 and 2005. Using 20

buds per tree, their development was assessed weekly as follows (Santini et al., 2004):

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1= dormant buds; 2= swollen buds, but scales closed; 3= bud scales open and

extremities of the first leaf visible at the apex of the buds; 4= extremities of all leaves

out; and 5= two leaves or more completely expanded. Bud break date was defined as the

day when half of the buds had reached phase 3. Plant height was measured on dormant

trees before the treatments and at the end of the 2004 and 2005 growing seasons. On the 5

inoculated plants, disease severity was evaluated at the beginning of September 2004

and September 2005, about 120 days after inoculation by recording the percentage of

leaf wilting in the crown (Solla et al., 2005). Each month during 2004 and 2005,

treatment toxicity was evaluated by the percentage of leaf wilting and dieback.

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2.5. Extraction of ethanol soluble material

On July 30, 2004, coinciding with the end of the watering treatments, three 3-cm-

long twig samples were removed from each non-inoculated UPM121 and UPM168

ramets (T1c-T5c plants). The twigs were 2 years old and were located in the upper third 15

of the crown (northern side, if available). The samples were immediately frozen in

liquid N2 and stored at –80 ºC until analysed. After bark removal, the xylem of the

outermost growth ring of the twig samples was extracted and pulverized. In order to

trace differences in secondary metabolite composition between xylem tissues, the wood

powder obtained was extracted in 85% ethanol at 85 ºC for 10 min. This ethanol 20

solution was filtered to eliminate wood residues and was subjected to rotary evaporation

to remove the ethanol. The resultant ethanol extract residue was freeze-dried and was

analysed by Fourier transform-infrared spectroscopy and electrospray ionisation mass

spectrometry.

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2.6. Fourier transform-infrared (FT-IR) spectroscopy

Samples were analysed by FT-IR spectroscopy as described in (Martín et al., 2005b).

The FT-IR instrument used was a Brucker IFS-55 FT-IR spectrometer equipped with a 5

GoldenGate single-reflectance ATR accessory. The spectra were acquired by

accumulating 64 interferograms at a resolution of 4 cm-1 at the absorbance mode from

4000 to 600 cm-1. Four replicate spectra per sample were obtained and averaged. One

ethanol extract per tree was used.

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2.7. Electrospray ionization mass spectrometry (ESI-MS and ESI-MS/MS)

As a complementary method, samples from trees showing extreme metabolic profiles

through the FT-IR analysis, belonging to T1c, T3c, and T5c, were analysed by ESI-MS.

Since the FT-IR analysis of xylem tissues among treatments revealed differences in the 15

hydrophobic fraction (suberin-like compounds), the freeze-dried ethanol extracts were

washed in 1 mL water and the residue obtained after centrifugation for 15 min at 15,000

g (water insoluble material) was used for analysis. ESI-MS mass spectra were obtained

using a Q-TOF2 hybrid tandem mass spectrometer (Micromass, Manchester, UK). The

water insoluble material was dissolved in methanol, water, and formic acid (50:49:1, 20

v/v/v), and introduced at a flow rate of 10 L min-1 into the electrospray source. MS/MS

spectra of selected ions were obtained by collision induced dissociation (CID), using

argon as the collision gas.

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2.8. Data processing and statistical analyses

Percentages of wilting were normalized using the arcsine transformation (x/100)1/2.

Normality of transformed data was confirmed using the Shapiro-Wilks statistic.

Parameters for each clone were analysed using a multifactorial ANOVA, considering 5

treatment and clone as factors. LSD tests were applied to compare means (α= 0.05).

Analyses were carried out under Statgraphics Plus v5.1 software (Statgraphics

Centurion, Herndon, VA).

The FT-IR spectra were transferred through JCAMP.DX format into the data

analysis software package developed by Barros (1999). All spectra were auto-scaled 10

(divided by the standard deviation and centred). Principal component analysis (PCA)

was applied to the spectral range 3500-700 cm-1, which includes all the main spectral

peaks, in order to reduce the dimensionality of the spectral data. The PCs were

displayed graphically as scores scatter plots for observing any clustering within the data

set. In PCA, coefficients by which the original variables must be multiplied to obtain 15

the PCs are called loadings. Numerical values of a loading of a given wavenumber

indicate the extent to which this wavenumber is related with the localization of the

sample with respect to the corresponding PC axis. Thus, loadings plots were used to

detect the wavenumbers responsible for the separation among clusters. In order to

monitor chemical differences between samples from control and treated trees, a digital 20

subtraction of their average spectra was applied (McCann et al., 1997). ESI-MS and

ESI-MS/MS data were processed using MassLynx v4.0 software (Micromass,

Manchester, UK).

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3. Results

Phenology and symptom values among different clones within each treatment were

non-significant (P > 0.10); in consequence, the following mean values refer to all clones 5

included within each treatment. Bud break occurred on April 5, 2004 and on April 13,

2005 on T1c, T2c, T3c and T5c trees. During both years, bud break occurred one week

later on T4c trees. No wilting or dieback was observed on Tc trees during 2004. Bud

break occurred on April 5, 2004 and on April 13, 2005 on plants tested for toxicity

belonging to TO1, TO2 and TO5. During both years, bud break occurred two weeks 10

later on TO3 and TO4 trees. No wilting or dieback was observed on TO1, TO2, TO3

and TO5 trees. However, trees watered with a 2% phenol solution (TO4) were 100%

wilted on May 1, 2004, with a mean dieback value of 87%. These trees did not re-sprout

during the following year. Except for trees watered with a 2% phenol solution, plant

growth did not significantly vary among toxicity treatments (P > 0.10). During 2004, 15

plant growths of TO1, TO2, TO3, TO4 and TO5 trees were 28.2 ± 4.7, 22.0 ± 2.8, 23.9

± 4.0, 7.5 ± 1.9, and 27.4 ± 2.7 cm (mean ± SE), respectively. During 2005, plant

growths of the same groups were 22.4 ± 6.7, 20.0 ± 3.2, 19.2 ± 4.7, 0 ± 0, and 24.8 ±

5.6 cm (mean ± SE), respectively.

In 2004, untreated and then O. novo-ulmi inoculated trees (T1i) showed greater 20

wilting percentages than trees watered with 0.02% of the phenolic fraction of phenolic

oil and then inoculated (T2i), trees watered with a 0.02% phenol mixture and then

inoculated (T3i), and trees injected with a 0.2% phenol mixture and then inoculated

(T5i) (Table 3). Plant growth of T1c, T2c, T3c, T4c and T5c trees were 24.8 ± 3.6, 21.9 ±

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1.2, 20.2 ± 1.4, 17.5 ± 0.9 and 17.3 ± 1.4 cm (mean ± SE), respectively. In 2005,

untreated and then inoculated trees (T1c) showed greater wilting percentages than trees

treated with phenols and then inoculated (T2c-T5c) (Table 3). During that growing

season, wilting values ranged from 0% (T5c) to 100% (T1c).

The absorption FT-IR spectra (4000 to 600 cm-1) obtained from the xylem ethanol 5

extract of U. minor trees showed sharp absorbing peaks in the 2800-2900 cm-1 region.

These peaks, not observed directly in the xylem spectra, presented variations according

to the different phenolic treatments (results not shown). A PCA of these ethanol extracts

was done in order to highlight these differences. According to the PCA scores scatter

plot of the FT-IR spectra, T1c and T4c samples showed positive scores in PC1 axis, and 10

T2c, T3c, and T5c samples showed negative scores. The PC1 loadings plot showed a

prominent positive peak at 3245 cm-1, and several negative sharp peaks whose

corresponding assignments are specified in Table 4. Digital subtractions of the FT-IR

average spectra of xylem samples from treated trees and xylem samples from control

trees provided spectra with a similar pattern (results not shown). Subtracting the average 15

spectrum of T1c samples to the average spectrum of T2c, T3c and T5c samples a

spectrum was obtained with major absorbance peaks at 2951, 2852, and 1755 cm-1. This

resulting spectrum is very similar to suberin one (comparable with Fig. 4 of Rocha et

al., 2001).

In the ESI-MS spectra of the ethanol extracted material from T1c, T3c and T5c20

samples it was possible to identify the [M+Na]+ ions of esters of hydroxylated fatty

acids and diacids at m/z 591, 619, 635, 647, 711, 739 and 771, whose tentative

assignments are listed in Table 5. Although present in all fractions, the relative

abundance of these ions was higher in the phenol treated tree samples.

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The assignments of the two most abundant ions, at m/z 619 and 647, were confirmed

by ESI-MS/MS. The fragmentation of the ion at m/z 619 originated a major fragment

ion at m/z 499, formed by the C-C bond -β cleavage of two carboxylic acid terminals

(Fig. 1a) in a fragmentation typical to occur in fatty acid derivatives (Nizigiyimana et

al., 1997; Harvey, 2005). The presence of ions at m/z 335 and 313 should be attributed 5

to the sodium and proton adducts, respectively, of the fragment originated by C-C bond

2-3 cleavage in the ω-hydroxyfatty acid in the vicinity of the ester bond. This type of

C-C bond cleavage has been reported to occur by fragmentation of fatty acid derivatives

(Griffiths, 2003). The fragmentation observed in the MS/MS spectrum of ion at m/z 619

allowed to infer the presence of a hexadecanoic diacid residue (DIA16:0) esterified with 10

a ω-hydroxy-eicosanoic acid residue (ω-OH20:0) (Fig. 1a). The fragmentation of the

ion at m/z 647 originated a major fragment ion at m/z 527, formed by the C-C bond -β

cleavage of the two carboxylic acid terminals, similarly as observed for the ion at m/z

619. The presence of ions at m/z 335 and 391 was attributed to the sodium adducts of

the fragments originated by C-C bond 2-3 cleavage in the ω-hydroxyfatty acid, in 15

the vicinity of the ester bond formed by DIA16:0 and DIA20:0, respectively, with the

ω-hydroxyl groups of ω-hydroxyfatty acids. Thus, the MS/MS spectrum of ion at m/z

647 corresponded mainly to a mixture of DIA16:ω-OH22 and DIA20:ω-OH18 (Fig.

1b,c).

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4. Discussion

The wilting values shown by the control plants (T1i) in 2004, 24% in average, are too

low if compared to values obtained from previous inoculations undertaken in

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experimental field plots (65 and 48% for UPM121 and UPM168, respectively) (Solla et

al., 2005). Limited symptom expression has been probably obtained due to limited plant

growth and root development inside the pots (Zürcher, 1986). However, this study

provides evidence of increased resistance of elms to DED after phenol application

through soil watering and trunk injections, in accordance to previous research 5

(Zentmyer, 1942; Zentmyer and Horsfall, 1943; Zentmyer et al., 1946; Went, 1954).

Application of several organic compounds, through injection or watering, prevented O.

ulmi development in young U. americana trees or reduced the severity of symptoms of

elms already infected. The following four phenolic compounds were selected as the

most effective chemicals for preventive (applied before inoculation) or curative (applied 10

after inoculation) purposes: pyrogallol, 8-hydroxyquinoline sulphate, hydroquinone and

p-nitrophenol (Zentmyer, 1942; Zentmyer and Horsfall, 1943; Zentmyer et al., 1946). In

vitro experiments showed complete inhibition of O. ulmi growth by 8-hydroxyquinoline

(Zentmyer, 1943). A slight increase of resistance in p-nitrophenol treated elms was

observed in a subsequent study, but reactions in bark tissues advised against its use due 15

to the possible phytotoxicity of this chemical (Went, 1954). Since the 1970’s, research

on DED chemotherapy has focused on benzimidazole and related fungitoxicants

because they were demonstrably systemic and active at low concentrations (Stennes,

2000), although not always effective.

In the 2004 samples, the metabolic distinction of T2c, T3c and T5c trees, compared to 20

T1c, could be related with the lower external symptoms observed in T2i, T3i and T5i

trees. Besides the intense but unspecific positive PC1 loading peak at 3245 cm-1, which

is characteristic of O-H stretching vibrations (Pandey and Pitman, 2003), the negative

loading peaks specified in Table 4 evidenced the presence of suberin-like compounds in

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xylem tissues (Cordeiro et al., 1998; Lopes et al., 2000; Lopes et al. 2001; Rocha et al.,

2001). Consequently, differences found within the PC1 axis are likely due to formation

of suberin precursors within the xylem tissues of plants subjected to T2c, T3c, and T5c.

Comparison of the subtraction spectrum and the spectrum of polymeric suberin

available from literature (Rocha et al., 2001) supports this hypothesis (results not 5

shown).

The ESI-MS spectra of the ethanol extracted material also support the occurrence of

several suberin-like precursors, whose tentative structures were proposed in Table 5.

Additionally, it was possible to identify by ESI-MS/MS the presence of three esters.

These suberin-like compounds, although present in all tissues analysed by ESI-MS, 10

were shown to be present in a relative higher abundance in the samples obtained from

phenol treated trees. These suberin polymers major components are aliphatic domains

containing -hydroxy fatty acids, and dicarboxilic acids.

Suberin is a polymeric material composed of aromatic (lignin-like phenolics) and

aliphatic (lipid) constituents (Kolattukudy, 1981; Holloway, 1983). Functionaly, suberin 15

contributes to both cell wall strength and resistance to water loss during plant growth

and development. Suberin is also considered to be an important component of induced

defense responses against wounding and pathogen attack in many plant species (Pearce,

1996), including elms (Rioux and Ouellette, 1991; Martín et al., 2005a, b). Deposition

of suberin was also observed under environmental stress, such as excess of salinity and 20

boron (Reinhardt and Rost, 1995; Ghanati et al., 2005). The shade and watering

conditions of the experiment reproduced the natural habitat of juvenile U. minor trees,

preventing excess of light and water stress. To our knowledge, these environmental

conditions should not have had any influence on the observed suberin accumulation.

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The presence of induced suberin in phenol-treated trees may be one of the mechanisms

of resistance to O. novo-ulmi, hindering fungal spread and allowing the expression of

other active defense mechanisms that probably contributed to a reduction of symptoms.

We ignore which mechanism or mechanisms were involved in the increased

resistance of the treated plants, but three main and non-exclusive hypotheses emerge: (i) 5

phenols had a fungitoxic or fungistatic effect on O. novo-ulmi, (ii) phenol treatments

induced “environmental resistance” against O. novo-ulmi, and (iii) phenols triggered

tree defence mechanisms. Recent studies revealed that monophenols significantly

inhibited the in vitro growth and in vitro sporulation of O. ulmi and O. novo-ulmi

(authors, unpublished results). Similar results have been shown with polyphenols in 10

other Ophiostomatoid pathogenic fungi (Sallé et al., 2005). In the present study, the

presence of monophenols or their transformed products (Korte et al., 2000) within the

sap flow could have hindered fungal sporulation and development, resulting in lower

external symptoms.

Environmental resistance to the DED fungi has been reported in elms exposed to 15

water stress, soil with low water retention and certain meteorological conditions (Kais et

al., 1962; Smalley and Kais, 1966; Sutherland et al., 1997; Solla and Gil, 2002).

Normally, any stress factor causing a reduction of the normal growth of elms, or any

alteration of earlywood or latewood formation, will generate resistance (Beckman,

1958; Brener and Beckman, 1968; Solla and Gil, 2002; Solla et al., 2005). No toxic 20

effects or growth reduction were observed with the phenol-treated plants, at least at the

concentrations which increased resistance. However, phenology delay was observed in

elms watered with phenol concentrations equal to or above 0.2% (T4c, TO3, and TO4),

which probably implies a delay in earlywood formation (results not available). A

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delayed earlywood formation was found to be related with increased levels of resistance

to O. ulmi in U. americana elms treated with sodium trichlorophenylacetate (Brener and

Beckman, 1968). Research is in progress to study the effect of various monophenols on

U. minor earlywood and latewood formation. More caution should be taken with plant

growth measurement in order to relate results to the growth-differentiation balance 5

hypothesis (Herms and Mattson, 1992). This hypothesis states that any environmental

stress that slows growth more than photosynthesis will increase the resource pool

available for defense. Biomass weigh will be a more accurate parameter to estimate

possible growth costs caused by increased resource allocation to defense (e.g. suberin

formation). However, if twigs are sampled for biochemical analyses, the weight of these10

samples should be taken into account.

Concerning the third hypothesis, exogenous monophenols can be absorbed by higher

plants through the roots (Glass and Bohm, 1971; Arziani et al., 2002). Several authors

have suggested that the accumulation of phenolics may induce a systemic accumulation

of secondary metabolites, which could serve as precursors for compounds implicated in 15

expression of resistance (Fried, 1981; Perumalla and Health, 1991; Cvikrová et al.,

2006). Suberin formation in xylem tissues could have been resulted from a systemic

response to phenol accumulation in roots or stem tissues. No differences on pH values

between the water control and the phenol solutions at 0.02% were observed (both values

about 7.4), although the phenol pH value of the solution at 0.2% was 6. Since major 20

differences in suberin accumulation with respect to the control samples were observed

for the solutions at 0.02%, it can be assumed that the pH of the solutions did not induce

any differential expression of enzymes active in suberin biosynthesis. The possible role

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of exogenous monophenols in systemic responses on elms is uncertain and will require

more specific studies.

The results presented here provide new insights into the possible role of external

factors in the resistance of U. minor to O. novo-ulmi. The survival of some elm stands

or individual elm trees in the field may be related with the long-term use of disinfectant 5

products containing phenols around standing trees. The deposition of suberin in xylem

tissues may be one mechanism of increased resistance in these elms. The use of

disinfectant products containing phenols may also influence the fungus transmission by

modifying the preferences of bark beetles during feeding or breeding. Differences in

beetle preference have been related to differences in elm chemicals acting in the 10

processes of host finding and host acceptance. Several simple phenolics have been

reported as feeding stimulants for Scolytus mulstistriatus (Meyer and Norris, 1974),

although some other phenolics extracted from the bark of nonhost trees were reported as

feeding deterrents (Gilbert et al., 1967; Norris, 1977). Considering the repellent

properties against insects of the disinfectant phenols used in this experiment, it is 15

conceivable that exogenous phenols applied in Rivas-Vaciamadrid could have delayed,

to some extent, the fungus transmission through the existing bark beetles. In a previous

work, in which the bark components of elms were studied, a higher concentration of

triterpenes were found in elms of Rivas-Vaciamadrid than in elms of other stands of

Madrid (Martín et al., 2004). 20

The possible effect of phenols on the transmission of O. novo-ulmi through root

grafts could be considered as an additional explanation of the delayed disease

development at the Rivas-Vaciamadrid area. Phenols applied to the soil are expected to

produce higher fungitoxic activity and increased suberin accumulation in roots than in

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stems, resulting in a delayed spread of the DED fungi among trees. However, since root

transmission of the disease normally involves the previous death of aerial tissues, the

protective effect of phenols within the elm stand is suspected to act mainly at the aerial

level. Further experiments are necessary to test these hypotheses and to validate the

results here obtained with a more diversified plant material, including other elm clones 5

and species, and using several fungal strains. Caution must be taken with the

environment, however, since the use of phenol and o-cresol as pesticides is nowadays

not allowed by the European legislation. This restriction would limit the use of these

phenols for the development of a control method against O. novo-ulmi.

10

Acknowledgments

We thank M. Burón, D. González (Universidad Politécnica de Madrid, Spain), and A.

Ferreira (Universidade de Aveiro, Portugal) for their technical support, and M. Venturas

(Universidad Politécnica de Madrid) for the English revision. The collaboration of Dr. I. 15

Delgadillo, Dr. S. Rocha, and Dr. A. Barros (Universidade de Aveiro) is gratefully

acknowledged. This work was supported by an agreement established between DGB

(Ministerio de Medio Ambiente) and ETSI Montes (Universidad Politécnica de

Madrid), and by the INIA Project RTA2005-00151-00-00 (Ministerio de Educación y

Ciencia).20

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Table 1

Plant material specifications

Original trees Ramets for phenol

treatments

Ramets for toxicity

study

Species Code Origin from Spain Environment Altitude (m) n Age (years) n Age (years)

U. minor UPM121 Tres Cruces, Madrid Garden 640 40 4 - -

UPM168 Madridejos, Toledo Field 650 40 4 - -

UPM016 Poveda, Almería Garden 840 - - 5 5

UPM092 Dehesa de la Villa, Madrid Garden 660 - - 5 5

UPM129 Pedrizas, Málaga Field 820 - - 5 5

UPM175 Santa Olalla, Toledo Field 460 - - 5 5

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Table 2

Treatment specifications of Ulmus minor trees with water, phenols, and Ophiostoma novo-ulmi. Each treatment included 8 trees.

Date (treatment)

Group Code Feb 12 to Jul 29, 2004

(watering once a week)

Apr 15, 2004

(2 trunk injections)

May 1, 2004

(inoculation)

Feb 10 to Jul 28, 2005

(watering once a week)

Apr 25, 2005

(2 trunk injections)

May 9, 2005

(inoculation)

(i) T1c Water - - Water - O. novo-ulmi

(ii) T2c 0.02% phenolic oil - - 0.02% phenolic oil - O. novo-ulmi

T3c 0.02% phenol mixture - - 0.02% phenol mixture - O. novo-ulmi

T4c 0.2% phenol mixture - - 0.2% phenol mixture - O. novo-ulmi

T5c Water 0.2% phenol mixture - Water 0.2% phenol mixture O. novo-ulmi

(iii) T1i Water - O. novo-ulmi - - -

(iv) T2i 0.02% phenolic oil - O. novo-ulmi - - -

T3i 0.02% phenol mixture - O. novo-ulmi - - -

T4i 0.2% phenol mixture - O. novo-ulmi - - -

T5i Water 0.2% phenol mixture O. novo-ulmi - - -

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Table 3

Mean wilting percentages of Ulmus minor trees treated with water and phenols, and

inoculated with Ophiostoma novo-ulmi. Each treatment included 8 trees.

Treatment codea September 2004 September 2005

T1c 0% ab 59% b

T2c 0% a 18% a

T3c 0% a 21% a

T4c 0% a 20% a

T5c 0% a 4% a

T1i 24% c - c

T2i 10% b -

T3i 5% ab -

T4i 14% bc -

T5i 9% b -

a For treatment specifications, see Table 2.

b Within each date, wilting values with different letters were significantly different at P=0.05. 5

c Plants not available in 2005.

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Table 4

Summary of the frequencies and proposed structural assignments of the most

characteristic negative loading bands of PC1, main factor of distinction

between spectra from ethanol extracted samples from plants subjected to

different phenol treatments.5

Frequency (cm-1) Assignmenta

2921 C-H aliphatic stretch

2852 C-H aliphatic stretch

1745 C=O stretch (ester groups)

1630 C=C stretch (aliphatic)

1594 Aromatic C=C skeletal stretching

1508 Aromatic C=C skeletal stretching

1458 C-H asymmetric deformation (aliphatic)

1371 C-H symmetric deformation (aliphatic)

1240 C-O-C stretch

1155 C-O-C asymmetric stretch

1089 C-H, C-O, C-O-H deformation

958 C-H bend (RHC=CHR’)

865 C-H bend (C=C)

720 CH bend (RHC=CHR’)

a References: Séné et al., 1994 ; Pandey and Pitman, 2003 ; Rocha et al., 2001

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Table 5

Tentative assignments of the main ions found in the region of m/z 540-790 ESI-MS spectrum

of ethanol extracts of xylem samples from T2c, T3c and T5c trees.

[M + Na]+ Tentative assignmentMolecular

formula

591.5 OH16:0 + DIA18:0 C34O6H64

619.5 OH18:0 + DIA18:0 C36O6H68

635.5 dihydroxyFA20:0 + DIA16:0 or OH20:0 +

hydroxyDIA16:0

C36O7H68

647.5 OH16:0 + DIA22:0

OH20:0 + DIA18:0

C38O6H72

711.6 Glycerol + OH16:0 C38O10H72

739.6 Glycerol + OH20:0 + 10-methoxy-9,18-

dihydroxyoctadecanoic acid

Glycerol + OH18:0 + 10-methoxy-9-

hydroxyoctadecanedioic acid

C41O9H80

C40O10H76

771.6 Glycerol + 9,10,18-trihydroxyoctadecanoic acid +

10-methoxy-9-hydroxyoctadecanedioic acid

C38O12H72

5

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Figure captions

Fig. 1. Scheme of ESI-MS/MS fragmentation proposed for ions (a) at m/z 619, and (b, c) at

m/z 647.

5

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Na+

O

O

(CH2)9 (CH2)12

O

OH

O

OH

m/z 335

cleavages

cleavage

m/z 499

Na+

O

O

(CH2)9

O

OH Na+

O

O

(CH2)9 (CH2)12

(a) DIA16:wOH20

Na+

O

O

(CH2)9 (CH2)14

O

OH

O

OH

(b) DIA16:wOH22

m/z 335

cleavages

cleavage

m/z 527

Na+

O

O

(CH2)9

O

OHNa+

O

O

(CH2)9 (CH2)14

Na+

O

O

(CH2)13 (CH2)10

O

OH

O

OH

(c) DIA20:wOH18

m/z 527

cleavages

cleavage

m/z 391

Na+

O

O

(CH2)13

O

OHNa+

O

O

(CH2)13 (CH2)10

Figure(s)