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Nematicidal Activity of (E,E)-2,4-decadienal and (E)-2-decenal from Ailanthus altissima against Meloidogyne javanica Pierluigi Caboni, Nikoletta G. Ntalli, Nadhem Aissani, Ivana Cavoski, and Alberto Angioni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf2044586 • Publication Date (Web): 05 Jan 2012 Downloaded from http://pubs.acs.org on January 17, 2012
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Nematicidal Activity of (E,E)-2,4-decadienal and (E)-
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2-decenal from Ailanthus altissima against
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Meloidogyne javanica
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Pierluigi Caboni*,§, Nikoletta G. Ntalli§, Nadhem Aissani§, Ivana Cavoski§, Alberto
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Angioni§
7 8 9
§
Department of Pharmaceutical Chemistry and Technology, University of Cagliari,
via Ospedale 72, 09124 Cagliari, Italy
10 11 12 13 14
* To whom correspondence should be addressed
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Department of Pharmaceutical Chemistry and Technology, Via Ospedale 72, 09124
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Cagliari (Italy)
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Tel. +390706758617. Fax +390706758612.
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e-mail:
[email protected]
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Running title: Nematicidal activity of Ailanthus altissima methanol extracts
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Abstract
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Methanol extracts of various plant parts of Ailanthus altissima were tested against the
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root knot nematode Meloidogyne javanica. Extracts of bark (ABE), wood (AWE),
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roots (ARE), and leaves (ALE) from A. altissima were investigated against freshly
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hatched second stage juveniles (J2). AWE was the most active extract with EC50/3d of
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58.9 mg/L while the ALE, ARE and ABE did not show nematicidal activity. The
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chemical composition of the extracts of A. altissima was determined by gas
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chromatography mass spectrometry and (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-
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decenal, hexanal, nonanal and furfural were the most prominent constituents. (E,E)-
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2,4-decadienal and (E)-2-decenal, furfural showed the highest nematicidal activity
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against M. javanica EC50/1d = 11.7, 20.43 and 21.79 mg/L respectively, while the
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other compounds were inactive at the concentrations tested. The results obtained
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showed that AWE and its constituents (E,E)-2,4-decadienal and (E)-2-decenal could
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be considered as a potent botanical nematicidal agents.
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KEYWORDS: GC-MS, tree of heaven, unsaturated aldehydes, Meloidogyne
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javanica, botanical pesticide, reactive carbonyl species, (E,E)-2,4-decadienal,(E)-2-
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decenal.
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INTRODUCTION
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Crops infestation by root knot nematodes (RKN; Meloidogyne spp.) causes annually
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US$ 70 billion of crop losses in fruit and vegetables production (1). Among potential
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strategies to control these pests natural nematicides isolated from plants or
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microorganisms are successfully used as bio-control agents to reduce non-target
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exposure to hazardous pesticides and to face resistance development (2, 3). The
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control of nematodes on cucumber, tomato, carrot has been done primarily by
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fumigants such us metam sodium and 1,3-dichloropropene or by the means of a
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biological control using bacteria such us Bacillus firmus and Bacillus chitinosporus,
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or by using botanical extracts such us sesame stalk or oil, neem cake and crab shell
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meal. Nematicidal application is performed before planting or during crop growth.
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The pressure to find viable alternatives to the soil fumigant methyl bromide,
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withdrawn in 2005 according to the Montreal Protocol on Substances that Deplete the
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Ozone Layer, has been intensified in the recent years.
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Plant secondary metabolites that have no apparent role in lie processes of plant
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structure play an important role in plant-insects interactions (4) and therefore such
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compounds called allelochemicals have insecticidal, hormonal, and antifeedant
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activities against pests (2). Reynolds reported that M. incognita and other generalists
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nematodes with a wide host range may rely almost exclusively on general plant clues
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with right blend and concentration of semiochemicals (1). Plants may use some
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known nematicidal cues like 2-undecanone, furfural, benzaldehyde, thymol, limonene,
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neral, geranial and carvacrol for defending themselves against the attacker in the
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underground (5-13). On the other hand, some nematodes can be used by plants for the
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indirect defense against plant herbivores (14). Another role of secondary metabolites 3
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is that of allelopathy, the inhibition of one plant’s growth by another through the
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production and release of toxic chemicals into the environment (14). Unsaturated
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aldehydes are known to be formed in large amounts in plant tissues in responses to
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wounding and the subsequent action of the lipoxygenase enzyme system (LOX)
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involving lipid oxidation (15). According to Wenke belowground volatiles produced
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by plants facilitate interactions between roots and soil organisms (16) while
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Hildebrand (17) suggested that LOX-mediated products including aliphatic aldehydes,
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ketones, and alcohols are involved in plant defense.
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The nematode cuticle is flexible and the exoskeleton resilient allowing locomotion,
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protection and permitting growth by molting. The cuticle is composed of cross-linked
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collagens, insoluble proteins called cuticlins, glycoprotein and lipids. The cuticle
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collagens are encoded by a large gene family and mutation of individuals genes can
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result in a range of defects from abnormal morphology, to larval deaths confirming a
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crucial and essential role of the cuticle structures. Activated small molecular weights
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carbonyls are an important class of intermediates known as reactive carbonyl species.
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(RCS). In our search for new naturally occurring compounds we found that 2-
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undecanone constituent of the methanol extract of Ruta chalepensis and furfural from
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Melia azedarach exhibited strong nematicidal activity against J2 larvae of
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Meloidogyne incognita and M. javanica (5, 6). These compounds are by products of
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cellular metabolism including lipid peroxidation, glycation and are activated by
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α,β,γ,δ insaturation and/or β oxo-substitution. Among the damage caused by RCS is
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DNA damage, proteosome degradation as well as cellular and extracellular protein
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alteration. The latter has been recently linked to skin and collagen deterioration (18).
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RCS especially mono and di-carbonyl compounds can react with proteins to form a 4
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variety of adducts through a Maillard reaction. These compounds are known as
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advanced glycation end-products.
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A. altissima, commonly known as “tree of heaven" is a deciduous tree of the
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Simaroubaceae family. When the leaves and flowers are crushed emit a foul smelling
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odour. A. altissima is native to northeast and central China and was introduced in
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Europe as street tree at the end of the 18th century. A. altissima grows rapidly and is
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capable of reaching heights of 10-15 meters and for this reason it has become an
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invasive species capable to colonise disturbed areas. Characteristics of this plant
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include: the versatility of the reproductive methods, the tolerance to unfavourable
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conditions and the potential presence of allelochemicals (14). The tree of heaven was
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been already used in traditional medicine in many parts of Asia including China,
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while the bark and leaves are being used for their bitter-tonic, astringent, vermifuge
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and antitumoral properties (19). Different phytochemical studies reported the presence
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in the plant of chemical compounds such us quassinoids, alkaloids, lipids and fatty
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acids, volatile and phenolic compounds, flavonoids and coumarins (19). Kraus et al.
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reported that the ailanthone extracted with methanol from A. altissima seeds turned
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out to be a potent antifeedant and insect growth regulator (20).
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In this work we studied the composition of methanol extracts from various part of A.
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altissima by GC/MS. We also investigated the nematicidal activity of these extracts
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and their components against root knot nematode M. javanica to find potential
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botanical alternatives to the currently used synthetic nematicidal agents or model
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compounds for the development of chemically synthesized derivatives with enhanced
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activity and environmental compatibility.
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MATERIALS AND METHODS
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Chemicals. Standards of (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-decenal, (E)-2-
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octenal, nonanal, hexanal, acetic acid, furfural, 2,3-butanediol, hexanoic acid, 5-
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hydroxymethylfurfural, heptanal of purity greater than 98%, as well as Tween 20 and
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dimethylsulfoxide were obtained from Sigma-Aldrich (Milano, Italy). Methanol and
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water were HPLC grade.
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Extraction and Chemical Characterisation. Plant materials. Leaves, bark, wood of
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A. altissima were collected before flowering in April 2011 at Cagliari (Italy), and
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were dried in the absence of light at room temperature. Then they were sealed in
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paper bags and stored at room temperature kept in the dark, until use. Voucher
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specimens were deposited in the Department of Pharmaceutical Chemistry and
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Technology, University of Cagliari for species identification.
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A. altissima Methanol Extracts. Dried leaves, bark, roots and wood plant parts (100 g)
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were grinded and extracted with methanol (1:10 w/v) in a sonicator apparatus for 15
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min, filtered through a Whatman no. 40, and centrifuged for 15 min at 13000 rpm.
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The extract was analysed for components identification by means of GC-MS.
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GC-MS Analysis. The chromatographic separation and identification of main
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components of methanol extracts of Ailanthus altissima was performed on a Trace GC
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Ultra Gas Chromatograph (Thermo Finnigan, MA, USA) coupled with a Trace DSQ
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mass spectrometry detector, a split-splitless injector, and an Xcalibur MS platform.
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The column was a CP-WAX 57CB from Varian (60 m, 0.25 mm i.d. and 0.25 µm film
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thickness; Varian Inc., U.S.A.). The injector and the transfer line were at 200 °C. The
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oven temperature was programmed as follows: 50 °C (hold 1 min) then raised to 220
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°C (3 °C/min), and isothermally hold for 13 min. Helium was the carrier gas at 6
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constant flow-rate of 1 mL/min; 1 µL of each sample was injected in the splitless
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mode (60 s). Mass spectrometry acquisition was carried out using the continuous (EI
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positive) scanning mode from 40 to 500 amu. A. altissima methanol extracts
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components were identified by a) comparison of their relative retention times and
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mass fragmentation with those of authentic standards; b) computer matching against a
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NIST98 commercial library. Quantitative analysis of each component was carried out
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with the external standard method.
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Effect of A. altissima extracts on J2 motility. Effects of ALE (Ailanthus Leaves
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Extract), ABE (Ailanthus Bark Extract) and AWE (Ailanthus Wood Extract) on M.
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javanica J2 motility were tested at the test concentration range of 15.6 to 250 mg/L for
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EC50s calculation. Pure compounds contained in the extracts were tested individually
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against M. javanica at the concentration range of 1 to 50 mg/L for EC50s calculation.
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The compounds used for the paralysis experiment were: (E,E)-2,4-decadienal, (E)-2-
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decenal, (E)-2-undecenal, (E)-2-decenal, nonanal, heptanal, hexanal, furfural, and 5-
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hydroxymethylfurfural. Stock solutions were prepared in methanol to overcome
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insolubility, whereas tween 90 in distilled water was used for further dilutions. Final
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concentrations of methanol in each well never exceeded 1% v/v, since preliminary
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experiments showed that this concentration was not toxic to nematodes. Distilled
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water as well as a mixture of water and tween (0.3% v/v) (carrier control) served as
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controls. In all cases, working solutions were prepared containing double the test
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concentration and mixed in CellstarR 96-well cell culture plates (Greiner bio-one) at a
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ratio of 1:1 v/v with suspensions of 15 J2 added to each well. Multiwell plates were
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covered to prevent evaporation and maintained in the dark at 28◦C.
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Juveniles were obtained by an Italian population of M. javanica reared for two months
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on tomato (Solanum lycopersicum) in a glasshouse at 25+2 °C. Juveniles were
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observed with the aid of an inverted microscope (Zeiss, 3951, West Germany) at 10×
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after 1, 24 and 72 h and were ranked into two distinct categories: motile or paralysed.
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Moreover, at that point, nematodes were moved to plain water after washing in tap
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water through a 20-µm pore screen to remove the excess of extracts. Numbers of
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motile and paralysed J2 were assessed by pricking the juvenile body with a needle and
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they were counted.
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Statistical Analysis. Treatments of paralysis experiments were replicated five times,
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and each experiment was performed twice. The percentages of paralysed J2 in the
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microwell assays were corrected by elimination of the natural death/paralysis in the
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water
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corrected%=[(mortality% in treatment-mortality % in control)/(100 - mortality % in
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control)}] 100, and they were analyzed (ANOVA) combined over time. Because
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ANOVA indicated no significant treatment by time interaction, means were averaged
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over experiments. Corrected percentages of paralysed J2 treated with A. altissima
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extracts or pure compounds were subjected to nonlinear regression analysis using the
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log-logistic equation proposed by Seefeldt et al. (22): Y = C + (D - C)/{1 + exp[b
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(log(x) - log(EC50)]}, where C = the lower limit, D = the upper limit, b = the slope at
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the EC50, and EC50 = A. altissima extract or pure compound concentration required for
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50% death/paralysis of nematodes after elimination of the control (natural
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death/paralysis). In the regression equation, the A. altissima extract or pure compound
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concentration (% w/v) was the independent variable (x) and the paralysed J2
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(percentage increase over water control) was the dependent variable (y). The mean
control
according
to
the
Schneider
Orelli
formula
(21),
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value of the five replicates per test concentration and immersion period was used to
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calculate the EC50 value.
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Scanning Electronic Microscopy Analysis. The physical mechanism that (E,E)-2,4-
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decacadienal and furfural used to interact with the external nematode cuticle was
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observed by SEM in the environmental mode (1-20 Torr). Freshly hatched nematodes
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were treated for 24 hours by immersion in a 100 µL solution containing 100 mg/L of
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the test compounds. Thereafter, a topographic visualization by using a FEI Quanta
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200 microscope (FEI) was performed.
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RESULTS AND DISCUSSION
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Mass spectrometry coupled to gas chromatography is a useful analytical platform for
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the chemical characterisation of plant extracts because it allows the identification of a
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large number of plant metabolites. A major disadvantage of this technique is that
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analytes used must be derivatized to improve the volatility to the injection port. In this
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work Ailanthus altissima methanol extracts were directly injected in the GC/MS
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neither employing derivatization nor any purification steps. Using a CP-WAX 57CB
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Carbowax column we were able to separate polar and medium polar plant secondary
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metabolites such us (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-decenal, (E)-2-
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decenal, hexanal, nonanal, acetic acid, furfural, 2,3-butanediol, hexanoic acid, 5-
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hydroxymethylfurfural (Table 1). We also tried to chemically characterize the
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methanol extract by LCMS TOF but aldehydes were not detected due to their
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volatility and low ionization efficiency during atmospheric pressure ionization.
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Among unsaturated aldehydes identified in A. altissima extracts, (E,E)-2,4-decadienal,
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(E)-2-decenal and furfural were the most active against J2 with EC50/1d of 11.70 and
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20.43 mg/L respectively, while (E)-2-octenal, nonanal, heptanal and hexanal did not
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provoke paralysis on J2s (Table 2). Interestingly, 1 hour post J2 immersion in test
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solutions EC50s were calculated even lower (7.5 and 11.75 mg/L for (E,E)-2,4-
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decadienal and (E)-2-decenal), but this activity was characterised as nematostatic
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rather than nematicidal since to some extend J2 regained their movement later.
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Moreover, no fumigant activity of plant methanol extracts or pure compounds was
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detected. The activity of (E,E)-2,4-decadienal, (E)-2-decenal against M. javanica is
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rather high if compared with the nematicidal activity of fosthiazate (EC50/1d=15.9
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mg/L). 10
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Αccording to the GC-MS analysis (Figure 1), AWE afforded (E,E)-2,4-decadienal,
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(E)-2-decenal, hexanal, nonanal, acetic acid, furfural, 2,3-butanediol, 2-decenal, 2-
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undecenal, hexanoic acid, and 5-hydroxymethylfurfural (Table 1), while ALE, ARE
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and ABE afforded nonanal, acetic and hexanoic acid and 2,3-dihydro-3,5-dihydroxy-
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6-methyl-4H-pyran-4-one. As a result of the GC-MS analysis fourteen plant
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metabolites, accounting for 82.6 % of the methanol extract were identified. Not taking
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into account plant compound bioavailability nor synergetic effect when AWE was
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tested against M. javanica a clear dose response relationship was established and
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significant paralysis of J2 was evident after three days of exposure with an EC50/3d
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value calculated at 58.9 mg/L (Table 3). This value is rather low considering the
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activities of Ruta chalepensis methanol extracts against M. incognita exhibiting an
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EC50 value of 1001 mg/L after 1 day of J2 immersion in test solutions (5) as well as
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Plantago lanceolata with an EC50 of 43.7% after 2 days of immersion (22).
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This is the first report of the irreversible nematicidal activity of unsaturated aldehydes
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as constituents of A. altissima against M. javanica. According to our results (E,E)-2,4-
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decadienal, (E)-2-decenal were the principal nematicidal constituents of AWE.
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Interestingly the other aldehydes or ketones were not found nematicidal against RKN
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(data not shown). Our results clearly indicate that α,β,γ,δ-unsaturated C10 aldehydes
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are generally more potent nematicidal than their shortest C chain counterparts versus
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M. javanica in vitro experiments. Kim reported that α,β-unsaturated aldehyde 2-
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decenal showed the highest nematicidal activity at 200 mg/L against the pine wood
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nematode (Bursaphelenchus xylophilus) if compared with other non unsaturated C8-
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C10 aldehydes (24). On the other hand, Andersen et al. reported that C9 unsaturated
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aldehydes i.e. (E)-2-nonenal and (E,Z)-nonadienal showed the strongest antifungal
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activity against Alternaria alternata if compared with shortest chain aldehydes 11
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concluding that the effectiveness is due to the their increased propensity to react with
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thiols and amino groups of the target fungi (25).
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Understanding the mode of action of the α,β and α,β,γ,δ-unsaturated C10 aldehydes is
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of practical importance for developing new formulations and delivery systems for
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nematode control. We observed that nematodes treated with aldehydes and ketones
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were paralyzed in a straight shape, in a similar way as reported by Kim that treated
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nematodes with plant essential oils (24), while Kong et al reported that pine wood
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nematode treated with muscle activity blockers levamisole or morantal tartrate were
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paralyzed in semi-circular and coiling shapes respectively (26). Moreover, we have
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recently reported of the circular shape paralysis of J2s after immersion with the
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organoposphorous fosthiazate (5).
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Aliphatic aldehydes and in a lesser extent ketones are relatively reactive compounds.
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The carbonyl carbon is an electrophilic site and reacts with primary amines and thiols
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resulting in the formation of substituted imines called Schiff bases and hemiacetals
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respectively. Aldehydes bringing one or two insaturations become even more reactive
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being easily the site of nucleophilic attack. Taking into account the reactivity of
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α,β,γ,δ-unsaturated aldehydes and E-SEM experiment photographs of the external
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nematode cuticle following treatment with (E,E)-2,4-decadienal and furfural at 100
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mg/L led us to hypothesized of the reaction of α,β,γ,δ-aldehydes with the nematode
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cuticle through a Michael addiction. This reaction consists of a nucleophilic addition
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of a cuticle amino or thiol group to an α,β-unsaturated carbonyl. This interaction leads
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to evident cuticle damage and leakage of the internal fluid nematode material (Figure
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2). Similar nematode cuticle damages were reported for Panagrellus redivivus caused
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by a unique fungal structure on the vegetative hyphae of Coprinus comatus. The latter
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was also able to produce potent nematicidal toxins such us 5-mehylfuran-3-carboxylic 12
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acid and 5-hydroxy-3,5-dimethylfuran-2(5H)-one (27, 28). Moreover, Luo et al.
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observed that the fungus Stropharia rugosoannulata produced a severe mechanical
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damage on the cuticle of nematodes Panagrellus redivivus and Bursaphelenchus
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xylophilus through finger-like projections called acanthocytes (29).
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The exact mechanism underlying the interaction of unsaturated aldehydes with the
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nematode cuticle is still unclear. Currently, we are investigating the interaction of
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aldehydes with the nematode cuticle trough a proteomic approach to well understand
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protein glycation.
272 273
ABBREVIATIONS USED
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GC-MS, gas chromatography-mass spectrometry; AWE, A. altissima wood
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methanolic extract; ALE, A. altissima leaves methanolic extract, ABE, A. altissima
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bark methanolic extract, ARE, A. altissima roots methanolic extract.
277 278
ACKNOWLEDGEMENTS
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Special thanks to Dr. Marco Oggianu for performing ESEM analysis and Barbara
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Liori for helpful suggestions.
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xylophilus
(Nematoda
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Table 1. Composition of AWE, ALE, ARE, and ABE Extracts Determined by GCMS Analysis, Listed in Order of Elution. Identification Was Done by Comparison of Mass Spectra of Commercial Standards with the Respective Data of NIST Library in Total Ion Current (TIC) and the Literature, anc=not calculated.
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compound
tR
EIMS
(min)
m/z (amu), (abundance)
mol. wt.
AWE
ALE
ABE
ARE
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
____________________________________________________________________________________________________________________ hexanal
10.88 56 (100%); 72(63%); 82(47%)
100.2
179
-
nonanal
22.71 57 (100%); 82 (51%); 70 (49%)
142.2
61.1
53
(E)-2-octenal
24.27 70 (100%); 55 (82%); 83 (70%)
126.1
44.7
acetic acid
25.59 60 (100%); 69 (10%)
60.0
furfural
25.84 95 (100%); 94 (91%); 59 (13%)
-
-
76
58
-
-
-
108
271
419
280
96.1
1.38
-
-
-
[R-(R*,R*)]-2,3-butanediol 28.73 60 (100%); 75 (80%)
90.1
7.36
-
-
-
[S-(R*,R*)]-2,3-butanediol
30.20 57 (100%); 75 (60%); 72 (20%)
90.1
14.7
-
-
-
2-decenal
32.89 70 (100%); 55 (77%); 83 (65%)
154.2
33.2
-
-
-
2-undecenal
36.99 70 (100%); 83 (73%); 55 (62%)
168.3
37.7
-
-
-
(E,Z)-2,4-decadienal
37.52 81 (100%); 83 (28%); 67 (17%)
152.2
68.6
-
-
-
(E,E)-2,4-decadienal
39.20 81 (100%); 83 (19%); 67 (18%)
152.2
124
-
-
-
hexanoic acid
40.55 56 (100%); 73 (79%); 87 (24%)
116.2
48
4.0
11.9
13.3
144.1
nc
n.c.
n.c.
2,3-dihydro-3,5-dihydroxy- 54.52 97 (100%); 126 (79%); 69 (31%) 6-methyl-4H-pyran-4-one
n.c.
5-hydroxymethylfurfural 61.59 144 (100%); 101 (48%);73 (28%) 126.1 70 55 ____________________________________________________________________________________________________________________ 19
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Table 2. EC50 and R2 Values of Individual Compounds against Meloidogyne javanica Calculated at 1 hour and 1 day after Immersion in Test Solutionsa
1 hour
1 day
______________________ R2
EC50 compound
________________________
(mg/L)
EC50
R2
(mg/L)
(E,E)-2,4-decadienal
7.53
0.98a
11.70
0.97
(E)-2-decenal
11.75
0.98
20.43
0.93
(E)-2-undecenal
>25
-
>25
-
(E)-2-octenal
n.a.
-
>25
-
nonanal
>50
-
n.a.
-
heptanal
n.a.
-
n.a.
-
hexanal
n.a.
-
n.a.
-
furfural
>25
-
21.79
0.98
5-hydroxy-methylfurfural
>25
-
>25
-
fosthiazate
>25
-
15.9
0.98
_______________________________________________________________________________ a
If R2 values are not presented and the EC50 values have not been calculated, they were outside the test concentration range and they are estimated higher than the upper concentration level (25 mg/L). n.a. not active in the range 25-1000 mg/L.
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Table 3. EC50 and R2 Values of Ailanthus altissima Extracts (ALE, ABE, AWE and ARE) against Meloidogyne javanica Calculated after 3 Days of Immersion in Test Solutionsa and Respective Concentration of Each Extract Provoking 100 % Paralysis.
3 days
4 days
___________________________
________________________
EC50 extract a
R2
(mg/L)
100% mortality
100% mortality
(mg/L)
(mg/L)
_______________________________________________________________________________ AWE
58.9
0.91
500
31.2
ABE
>250
-
625
312
ALE
>250
-
>2500
>2500
ARE
>250
-
>2500
>2500
________________________________________________________________________________________________________________________________________
a
A. altissima methanolic extracts of: AWE = wood; ALE = leaves; ABE = bark: ARE = roots.
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List of figure captions of the paper entitled:
Figure 1. GC-MS chromatograms of A. altissima methanol extracts. Peaks A). (1) hexanal, (2) nonanal, (3) (E)-2-octenal, (4) acetic acid, (5) furfural, (6) (E,Z)-2,3-butanediol, (7) (E,E)-2,3butanediol, (8) (E)-2-decenal, (9) (E)-2-undecenal, (10) (E,Z)-2,4-decedienal, (11) (E,E)-2,4decedienal, (12) hexanoic acid, (13) 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (14) 5hydroxymethylfurfural. Peaks B). (1) nonanal, (2) acetic acid, (3) 2-nonanol, (4) 1-octanol, (5) hexanoic acid. Peaks C) (1) nonanal, (2) acetic acid, (3) hexanoic acid (4) 2,3-dihydro-3,5dihydroxy-6-methyl-4H-pyran-4-one (5) 5-hydroxymethylfurfural. Peaks D) (1) nonanal, (2) acetic acid, (3) decanal, (4) hexanoic acid. Figure 2. E-SEM topographical images from immersion treatment of J2 with water A) and B), C) and D) furfural at 100 mg/L, while E) and F) were treatment with (E,E)-2,4-decadienal at 100 mg/L. Degradation of the external cuticle of M. incognita juveniles is evidenced by the leakage of internal fluids D) E) and F).
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Figure 1 100 90
A wood
8
80
14
11
70
13
1 60
12
2
50
10 7
40
9
6
30
4 3 5
20 10 0 10
15
20
25
30
35 40 Time (min)
45
50
55
60
65
45
50
55
60
65
100 90
B root
80 70
2
60 50
5 40 30
3 20
4
1
10 0 10
15
20
25
30
35 40 Time (min)
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4
100 90
C leaves
80 70 60 50
5
2
40 30
3
20
1
10 0 10
15
20
25
30
35 40 Time (min)
45
50
55
60
65
45
50
55
60
65
3
100 90 80
D bark 70 60
2
50 40
1
30
4
20 10 0 10
15
20
25
30
35 40 Time (min)
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Figure 2
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A
B
C
D
E
F
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Degradation of the external cuticle of M. javanica with evident leakage of its internal fluids after treatment with (E,E)-2,4-decadienal at 100 mg/L
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