ECOLOGICAL RELEVANCE OF THE MAJOR ALLELOCHEMICALS IN

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Bioactive Constituents, Metabolites, and Functions

ECOLOGICAL RELEVANCE OF THE MAJOR ALLELOCHEMICALS IN SOLANUM LYCOPERSICUM ROOTS AND EXUDATES Carlos Rial, Elisabeth Gómez, Rosa M. Varela, Jose M. G. Molinillo, and Francisco A. Macias J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01501 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Journal of Agricultural and Food Chemistry

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Ecological Relevance of the Major Allelochemicals in Lycopersicon esculentum

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Roots and Exudates.

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Carlos Rial, Elisabeth Gómez, Rosa M. Varela*, José M.G. Molinillo and Francisco A.

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Macías

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Allelopathy Group, Department of Organic Chemistry, Institute of Biomolecules

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(INBIO). Campus de Excelencia Internacional (ceiA3), School of Science, University of

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Cadiz, C/ República Saharaui nº 7, 11510 Puerto Real, Cadiz, Spain.

10 11

*

Corresponding author (Tel: +34 956012729; Fax: +34 956016193; E-mail: [email protected]).

1 ACS Paragon Plus Environment


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ABSTRACT

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Stigmasterol, bergapten and α-tomatine were isolated from tomato roots. The

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preliminary phytotoxic activity of stigmasterol and α-tomatine were evaluated on wheat

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coleoptile bioassay and α-tomatine was the most active compound. To confirm the

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phytotoxic activity, α-tomatine was tested on Lactuca sativa and two weeds (Lolium

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perenne and Echinochloa crus-galli), and it was active in all cases. The stimulatory

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activity of α-tomatine and stigmasterol on parasitic plant germination was also

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evaluated and α-tomatine was found to be active on Phelipanche ramosa, a parasitic

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plant of tomato. α-Tomatine was identified in root exudates by LC-MS/MS and this

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confirms that α-tomatine is exuded by roots into the environment, where it could act as

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both an allelochemical and as a stimulator of P. ramosa, a parasitic plant of tomato.

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Keywords Tomato, parasitic plant bioassays, LC-MS/MS, phytotoxicity, α-Tomatine.


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INTRODUCTION

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In order to meet the current demand for food, the use of herbicides in crops is

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indispensable to increase productivity and to reduce the losses caused by pathogens.

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However, the intense use of herbicides has led to various environmental and resistance

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problems.1 For these reasons, it is necessary to develop more sustainable, ecological and

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environmentally friendly agriculture.

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In this respect, it is a prerequisite to have a knowledge of the defence mechanisms

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of the crops against weeds and the biocommunicators used by plants to contact the

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environment, for example the germination stimulators used by parasitic plants to

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confirm the presence of a host plant. The study of these interactions is called

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allelopathy2 and this knowledge could provide a useful tool for our common benefit.

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Tomato (Lycopersicon esculentum) is an important crop in Southern Europe, the

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Americas, the Middle East and India. Tomato is a member of the Solanaceae family,

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which contains numerous other plant species of commercial and/or nutritional interest

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(e.g., potato, pepper, eggplant and tobacco). Tomato metabolites are widely studied,

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particularly those from the fruit and the aerial parts of the plant, and numerous

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compounds have been described, including carotenoids,3 phenolic compounds,4

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alkaloids,5 and glycoalkaloids,6 amongst others. These compounds have shown a wide

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range of activities; for instance, carotenoids are involved in the defence against

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oxidative stress,7 the attraction of pollinators and the communication between cells,8

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and phenolic compounds are antimicrobials and antivirals.9 However, the phytotoxic

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activity of tomato metabolites has not been widely reported.

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All of these compounds could be released into the soil by the roots in exudates and,

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once in the soil, they could develop their bioactivities. This hypothesis – coupled with

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the fact that almost all studies have concerned analysis of the content of secondary



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metabolites in fruit and tomato shoots – make the inedible parts of tomato a new and

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unexplored source of natural products. In this context, we aimed to carry out research

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into tomato from another perspective by focusing the study on the metabolites presented

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in the roots, i.e., compounds that could be exuded to the soil. In the soil these

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compounds could act as allelochemicals in the tomato defence mechanism as well as

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chemical signals for parasitic plants to confirm the presence of a host and, thus,

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facilitate their survival, showing an interesting example of adaptive evolution.

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MATERIAL AND METHODS

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Plant material and chemicals. Fifty tomato plants for transplanting of the local

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variety Lycopersicon esculentum Mill. 'Moruno' were purchased from “La huerta del

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abuelo” (Conil de la Frontera, Spain) grown under optimum conditions. Lettuce seeds

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were provided by FITO (Barcelona, Spain). Echinochloa crus-galli and Lolium perenne

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seeds were purchased from Herbiseed (Reading, UK). Organic solvents were UHPLC-

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grade and were purchased from Fischer Chemicals (Geel, Belgium). Water was type I

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obtained from an Ultramatic system from Wasserlab (Barbatáin, Spain). The internal

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standard (IS) protodioscin was isolated from Urochloa ruziziensis.10

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Root exudate preparation for LC-MS/MS analysis. Tomato exudate was collected

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by rinsing the soil of each plant with 100 mL of water once a day during 5 d. The

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exudate was fractionated by reverse phase vacuum column chromatography (VCC) in a

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glass filter plate (Pobel, Madrid, Spain) (70 mm × 90 mm i.d., porosity 4) using

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LiChroprep RP-18 (40–63 µm) silica gel from Merck (Darmstadt, Germany) as the

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stationary phase and mixture of water/acetone as the mobile phase (60:40, fraction A,

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40:60; fraction B; 20:80, fraction C and 0:100, fraction D; v/v; 250mL of each polarity).

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Acetone was evaporated on a rotary evaporator, the water lyophilized and the dry

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material was stored at –20 ºC.


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Root extraction and isolation procedure. Once the exudate was obtained, tomato

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roots were collected and dried in an oven for 2 d at 40 ºC. The dried material (36.61 g)

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was powdered in an industrial mill and extracted with 250 mL of acetone in an

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ultrasonic bath for 20 min four times. After this extraction, the same roots were dried in

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an oven for 24 h at 40 ºC and extracted again following the same procedure with 250

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mL of methanol (MeOH) four times. The extracts were filtered and the solvents were

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evaporated on a rotary evaporator to give 223.8 mg and 1531.4 mg of acetone and

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MeOH extract, respectively.

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The MeOH extract was fractionated by reverse phase VCC following the procedure

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described above using mixtures of water/MeOH as the mobile phase (100:0, 80:20,

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60:40, 40:60, 20:80 and 0:100, v/v, 250 mL of each polarity) to afford six fractions. The

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fraction obtained with water/MeOH 20:80, v/v, (77 mg) was purified by preparative

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layer chromatography (PLC) Silica gel 60 RP-18 F254s (20 × 20 cm) from Merck

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(Darmstadt, Germany) using water/acetone 30:70, v/v, as the mobile phase with 10–15

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mg of sample loaded onto the PLC plate. The procedure was repeated three times to

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yield 1 (26.8 mg).

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The acetone extract was fractionated by normal phase column chromatography

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using Geduran Si 60 (0.063–0.200 mm) silica gel from Merck (Darmstadt, Germany) as

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the stationary phase and a mixture of hexane/acetone as the mobile phase (from 100:0 to

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0:100, v/v, in 20% increments, 250 mL of each polarity) to afford nine fractions.

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Fractions 2 and 3 (6.5 and 3.7 mg, respectively) were separated by HPLC using a Luna

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Silica (2) 100Å (250 x 4.60 mm, 10 µm) analytical column (Phenomenex Torrance,

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CA) with an isocratic mixture of hexane/acetone 80:20, v/v, as the mobile phase and

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refraction index as the detection mode on a Merck-Hitachi L-6200 HPLC system to

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yield 2 (0.6 mg). Fractions 5 and 6 were also separated by HPLC using a LiChrospher



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SI 60 (10 µm, 250 × 10 mm) column from Merck (Darmstadt, Germany) using the same

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procedure as described above to yield 3 (7.5 mg).

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The structures of the isolated compounds were determined from the 1H and

13

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NMR spectra obtained on a INOVA 500 MHz NMR spectrometer from Agilent (Santa

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Clara, CA) and are shown in Figure 1.

C

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Coleoptiles Bioassay. Wheat coleoptile bioassays were carried out following the

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procedure previously described by Rial and co-workers.11 For the sample preparation,

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extracts and compounds were first dissolved in DMSO (0.1% v/v) and diluted in

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phosphate-citrate buffer at pH 5.6 (sucrose 2%). Concentration used for extracts were

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800,400 and 200 ppm and for compounds 1000,300,100,30 and 10 µM.

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Parallel controls were also carried out. The buffer described above was used as

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negative control and the commercial herbicide Logran was used as an internal

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reference.12

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Three test tubes were used per dilution, with five coleoptiles and 2 ml of solution, and

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they were placed in the dark at 25ºC and 6 rpm in a roller tube apparatus for 24 h The

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coleoptiles elongation are expressed as percentage difference from the control. Welch’s

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test was used as statistical analysis. Stimulation was represented as positives values and

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inhibition as negatives.

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Phytotoxicity Bioassays. Lettuce (Lactuca sativa L.) and the weeds Echinochloa

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crus-galli and Lolium perenne were tested in this work following the procedure

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previously described by Rial and co-workers11 with 6 days of growth for all the species.

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α-Tomatine was dissolved and diluted using 2-[N-morpholino]ethanesulfonic acid

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(MES) buffer at 10–2 M (pH 6.0) containing 5 µL/mL of DMSO. Concentrations tested

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were 1000, 300, 100, 30 and 10 µM. Parallel controls were also carried out as described

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before for coleoptile bioassay.


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Germination rate, root length and shoot length were measured using a Fitomed

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system.11 Welch’s test was used as statistical analysis, with significance fixed at 0.01

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and 0.05. Results are presented as percentage differences from the control.

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Parasitic plant bioassay. α-Tomatine and stigmasterol were tested on the seeds of

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three parasitic weed species: Orobanche cumana, Orobanche crenata and Phelipanche

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ramosa following the procedure described by Cala and co-workers.13 They were

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dissolved in acetone and diluted with type 1 water to a concentration range of 100 to 0.1

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µM. The final concentration of acetone was adjusted to 1% (v/v). Each treatment was

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replicated 5 times. Parallel controls were also run. A solution of water:acetone 99:1

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(v/v) was used as negative control and the synthetic strigolactone GR24 was used as an

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internal reference.

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Percentage data were approximated to a normal frequency distribution by means of

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angular transformation (180/п × arcsine (sqrt[%/100])) and subjected to analysis of

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variance (ANOVA) using SPSS software for Windows, version 21.0 (SPSS Inc.,

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Chicago, IL). The evaluation of the significance of mean differences between negative

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control and treatments was made by two-sided Dunnett’s test. Null hypothesis was

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rejected at the level of 0.05.

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LC-MS/MS. Exudates were analyzed on an EVOQ Triple Quadrupole Mass

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Spectrometer from Bruker (Billerica, MA) with an electrospray ionization source (ESI)

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in positive mode. The compound-dependent parameters were optimized by direct

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infusion on the mass spectrometer to achieve maximum multiple reaction monitoring

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(MRM) signal intensities using argon as the collision gas. For α-tomatine precursor ion

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was m/z 1035 and quantifier and qualifier product were m/z 1016 (collision energy

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(C.E.) 71 eV) and m/z 398 (C.E. 45 eV) respectively. For protodioscin precursor ion

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was m/z 1032 and quantifier and qualifier product were m/z 415 (C.E. 27 eV) and m/z



150

869 (C.E. 30 eV) respectively Samples were injected and separated using a Kinetex

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1.7µm C18 100 Å column (100 × 2.1 mm) (Phenomenex, Torrance, CA) maintained at

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40 ºC. The mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B

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(MeOH, 0.1% formic acid) and the flow rate was set to 0.4 mL/min. The optimized

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linear gradient system was as follows: 0−0.5 min, 50% B; 0.5−4 min, to 100% B; 4−7

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min, 100% B; 7−7.5 min, to 50% B; 7.5−10.5 min, 50% B. The autosampler was set to

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5 °C to preserve the samples. The injection volume was 5 µL. The injection needle was

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washed after each injection with water and MeOH. The instrument parameters were as

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follows: spray voltage +4500 V, cone temperature 300 ºC, cone gas flow 15 psi, heated

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probe temperature 400 ºC, heated probe gas flow 15 psi, nebulizer gas flow 55 psi and

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collision pressure 2.0 mTorr.

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Calibration curve. A stock standard solution of 10 mg/L of α-tomatine was

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prepared by dissolving 1.02 mg in 100 mL of MeOH. The external standard calibration

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curve was prepared by the serial dilution of the working standard solution from 1000 to

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50 µg/L (7 levels). Also, a stock solution of the IS protodioscin of 10 mg/L was

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prepared dissolving 0.99 mg in 100 mL of MeOH, and this was added to all samples to

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give a final concentration of 100 µg/L. The concentration of internal standard (IS) was

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optimized to obtain similar signals intensities for the IS and the α-tomatine in the

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sample. A 5-µL aliquot of each standard solution was injected three times onto the

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UHPLC column. The calibration curve was constructed by plotting the peak area ratio

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(y) of standard to IS versus the ratio of their concentrations (x). The curve was fitted to

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a linear function with a weight of 1/nx (R2 > 0.99), being “n” the calibration level. The

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concentration of α-tomatine in the samples were determined by their peak area ratio

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with respect to the IS and by reference to the standard curve. All standards and stock


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solutions were filtered through a polytetrafluoroethylene (PTFE) syringe filter (0.22

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µm) prior to analysis and samples were stored at –80 ºC.

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Sample preparation for LC-MS/MS analysis. Exudates were dissolved with MeOH

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to achieve a ratio of 1/1 g/L. IS was added to each sample to give a final concentration

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of 100 µg·L–1. The concentration of IS was optimized to obtain a similar signal intensity

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for the IS and the α-tomatine in the sample. A 5-µL aliquot of each sample was injected

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three times onto the UHPLC column. All samples were filtered through a PTFE syringe

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filter (0.22 µm) prior to analysis and samples were stored at –80 ºC.

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Data analysis. Data acquisition, calibration curves and the statistical analysis of the

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data from the quantitation was performed with the software MS Data Review (Bruker

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Chemical Analysis).

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RESULTS AND DISCUSSION

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In order to find the metabolites produced by tomatoes in the crops, tomato plants

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were purchased from a local greenhouse and were grown under optimal conditions.

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Tomato roots were extracted with acetone and MeOH and the bioactivities of the

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extracts were tested using the coleoptile bioassay.11 The advantages of this test are its

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rapidity and sensitivity shown on a wide range of bioactive compounds,14–16 including

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plant growth regulators and herbicides. Three dilutions were used in this assay (800,

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400 and 200 ppm) and these were prepared from dried extracts. It can be seen from

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Figure 2 that the MeOH extract was the most active and it inhibited coleoptile growth

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by 78% at 800 ppm and 56% at 200 ppm. The acetone extract showed a lower inhibitory

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effect. In this case only the activity at 800 ppm (63%) was noteworthy. Regarding these

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results, the isolation of components from both the acetone and MeOH extracts was

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carried out. The acetone extract was fractionated by column chromatography and the

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purification of compounds was carried out by high performance liquid chromatography



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(HPLC) to give stigmasterol, 3, as the major compound and bergapten, 2, identified by

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comparison of their spectroscopic data to those previously reported.17,18 The MeOH

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extract was fractionated by vacuum column chromatography. The major compound

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from these fractions was α-tomatine, 119,20 isolated by PLC, the structure of which was

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confirmed by comparison with spectroscopic date previously reported in the literature.21

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Stigmasterol belongs to a family of steroids that has two functions in plants: they

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are necessary for the formation of cellular membranes22 and they stimulate cell

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division.23 α-Tomatine has shown a protective effect in leaves against microorganisms24

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and it inhibits the growth of eight saprophytic fungi and two pathogens of tomato.25 It

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has also been reported that α-tomatine slightly inhibited the stem elongation (7-13%)

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when applied as spray to etiolated 4-d.-old seedlings of sesbania (Sesbania exaltata

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(Raf.) Rybd), sicklepod (Senna obtusifolia L.), mungbean (Vigna radiata L.), wheat

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(Triticum aestivum L.) and sorghum (Sorghum vulgare L.).26

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These two major compounds were assayed in the wheat coleoptile bioassay in an

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effort to determine whether they are responsible for the activity observed in the extracts.

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Bergapten was not tested because the isolated amount was not enough. However, it was

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previously tested and was found to show moderate activity.27 The results obtained in the

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bioassay are presented in Figure 2. Stigmasterol did not show significant activity

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whereas α-tomatine showed very high inhibitory effects. α-Tomatine showed even

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higher inhibitory effects than the commercial herbicide Logran at the lowest

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concentration assayed.

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In view of the results discussed above, α-tomatine was selected to test its

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phytotoxicity on three seeds: Lactuca sativa, which is the most widely used

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dicotyledonous species in allelopathy bioassays, and the weeds Echinochloa crus-

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galli,28 which affects transplanted tomatoes, and Lolium perenne, which directly affects


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planted tomatoes.29 The results obtained in the bioassay are shown in Figure 3. The

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most affected parameter was the root length and the values obtained were higher than

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that for Logran for all species, with inhibition values of 85% at 1000 µM and 45% at

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300 µM. On weeds, α-tomatine showed even higher inhibitory effects and gave values

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of 91% at 1000 µM. Germination was not affected and shoots were only affected at

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1000 µM, with inhibition of 35% on L. perenne. The results obtained are consistent with

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the low inhibition shown by α-tomatine against stem elongation on the other species

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previously tested.26 However, it has shown to be active on inhibiting root growth in the

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species herein tested, which agrees with the fact that it is exuded by roots.

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To confirm that α-tomatine is responsible for the inhibitory activity and to study it

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stability, it was extracted from the sheet of Whatman No.1 filter paper with MeOH

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when the bioassay was finished. 1H-NMR spectroscopy showed that α-tomatine remains

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after the bioassay, confirming that α-tomatine is the active compound and not any other

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degradation product. All of these results confirm that α-tomatine has phytotoxic activity

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even on weeds.

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Parasitic plants are some of the most important agricultural pests. They have an

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organ called the haustorium, which they use to acquire nutrients and water from their

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host, causing significant losses in crops. For instance, species belonging to

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Orobanchaceae parasitize legumes, crucifers, sunflower, hemp, tobacco and tomato.30–32

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Germination occurs when the seeds detect chemical signals, i.e., germination stimulants

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produced and released from the roots of host and nonhost plants.33 Some of these

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stimulants have been identified in root exudates.34–37 The use of stimulants to induce the

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suicidal germination of parasitic plants prior to crop sowing has been proposed to

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reduce the seed banks on the soil. With this aim in mind, α-tomatine and stigmasterol

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were tested in a parasitic plant germination bioassay on Orobanche cumana, Orobanche



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crenata and Phelipanche ramosa seeds and the results are shown in Figure 4. Bergapten

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was not tested because the isolated amount was not enough. Stigmasterol did not show

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activity. α-Tomatine only stimulated the germination of the tomato parasite (P. ramosa)

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and showed 77% germination at 100 µM and 55% at 1 µM. Orobanche spp. were not

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stimulated by α-tomatine. These results demonstrate that P. ramosa could use α-

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tomatine as a signal to confirm the presence of its host.

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In view of the activity shown by α-tomatine and in an effort to determine its

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ecological role, it is necessary to demonstrate that α-tomatine is exuded into the soil by

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the roots. As a consequence, α-tomatine was analysed in tomato exudates by LC-

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MS/MS using multiple reaction monitoring (MRM) with electrospray as the ionization

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source. The proton adduct (m/z 1035) and the most stable fragments (m/z 1016 and 416)

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were used. An intense peak was observed in the exudates obtained by reverse phase

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VCC in fractions A, B and C, being the fraction B the most intense, with the same

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retention time of the α-tomatine standard (2.1 min) (Figure 5). To carry out the α-

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tomatine quantitation the matrix effect is a critical point in LC-MS/MS. To correct this

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effect, it is mandatory to use an internal standard (IS) which must have similar

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characteristic to the analyte. Protodioscin10,38 is a steroidal saponin with a structure and

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molecular weight very similar to α-tomatine, for these reasons, it was selected as the IS

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(tR 2.9 min) to quantitate α-tomatine. The calibration curve obtained was y =0.0939x +

268

0.3124 with a correlation coefficient r2 = 0.9986. The quantitation (LOQ, 10.38 µg/L)

269

and detection (LOD, 3.12 µg/L) limits were determined by nine replicate analyses and

270

considered to be 10 and 3 times the standard deviation of baseline noise. The precision

271

of the method was studied in an intra- and interday assay (n = 9). The method was

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found to be precise with RSD value of 3.47% for area and 0.81% for retention time.

273

Intermediate precision was 4.89% for area and 0.94% for the retention time injected on


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three different days. The concentrations of α-tomatine in each sample are shown in

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Table 1. In total, the concentration of α-tomatine in the exudates was 1723 µg/L and the

276

production was 7 µg/L per day and plant. This concentration makes it possible that α-

277

tomatine is present in the soil in an amount enough to develop the stimulatory activity

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on the parasitic weed and the phytotoxic activity shown in the bioassays if it is

279

accumulated in the soil.

280

The results confirm that α-tomatine is exuded through the root to the soil, where it

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performs the allelopathic activities found in the bioassays. In this respect α-tomatine

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plays a beneficial role in the defence of tomato against weeds but helps parasitic plants

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by acting as a chemical signal to confirm the presence of a host. This molecule therefore

284

showed multipurpose behaviour as described for other metabolites.39 From an

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ecological point of view, it is reasonable that the specific parasitic weed of tomato

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should detect the presence of a compound such as tomatine, which is typical of this

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species.

288

AUTHOR INFORMATION

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Corresponding Author

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*Tel: +34 956012729. Fax: +34 95016.193. Email: [email protected]

291

Notes The authors declare no competing financial interest.

292 293

ACKNOWLEDGMENTS

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This research was supported by the Ministerio de Economía, Industria y

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Competitividad (MINEICO) (Project AGL2013-42238-R). We would like to thank

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FITÓ S.A. (Barcelona, Spain) for supplying lettuce and wheat seeds.

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FIGURE CAPTIONS

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Figure 1. Structure of the compounds isolated from Lycopersicon esculentum roots and

426

the internal standard used for LC-MS/MS quantitation.

427

Figure 2. Bioassay results of extracts from tomato roots and the major isolated

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compounds on the elongation of etiolated wheat coleoptiles. The commercial herbicide

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Logran was used as a positive control. Values are expressed as percentage difference

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Figure 3. Bioassay results of α-tomatine on Lactuca sativa, Lolium perenne and

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control. Values are expressed as percentage difference from control.

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Figure 4. Stimulatory activity of stigmasterol and α-tomatine on the germination of

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Orobanche Cumana, Orobanche crenata and Phelipanche ramosa seeds. * Indicates

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significant differences at P < 0.05.

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Figure 5. UHPLC-MS/MS analysis of α-tomatine and tomato exudates with the IS

438

protodioscin.



Table 1. Concentration of α-Tomatine in Tomato Samples.

a

Sample

α-tomatine (µg/L (RSD %)a)

Fraction A

359.12 (8.06)

Fraction B

755.17 (7.80)

Fraction C

609.33 (1.88)

Fraction D

n.d.

RSD (%), relative standard deviation


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Figure 1.



Figure 2.


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Figure 3.



Figure 4


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Figure 5



Table of Contents Graphic


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ECOLOGICAL RELEVANCE OF THE MAJOR ALLELOCHEMICALS IN

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