Elicitation of Differential Responses in the Root-Knot Nematode

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Agricultural and Environmental Chemistry

Elicitation of Differential Responses in the Root-Knot Nematode Meloidogyne incognita to Tomato Root Exudate Cytokinin, Flavonoids, and Alkaloids Hillary Kipchirchir Kirwa, Lucy K Murungi, John J. Beck, and Baldwyn Torto J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05101 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Journal of Agricultural and Food Chemistry

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Elicitation of Differential Responses in the Root-Knot Nematode Meloidogyne incognita to Tomato

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Root Exudate Cytokinin, Flavonoids, and Alkaloids

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Hillary K. Kirwa,†,‡ Lucy K. Murungi,‡ John J. Beck,§ and Baldwyn Torto*,†

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†Behavioural

and Chemical Ecology Unit, International Centre of Insect Physiology and Ecology (icipe),

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P.O Box 30772-00100, Nairobi, Kenya ‡Department

of Horticulture, Jomo Kenyatta University of Agriculture and Technology, P.O Box 62000-

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00200, Nairobi, Kenya

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§Chemistry

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Research Service, U.S. Department of Agriculture, 1700 SW 23rd Drive, Gainesville, Florida 32608,

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United States

Research Unit, Center for Medical, Agricultural and Veterinary Entomology, Agricultural

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AUTHOR INFORMATION

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*Corresponding Author

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(Tel.: +254 20 863 2000. Fax: +254 20 863 2001. Email: [email protected])

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1 ACS Paragon Plus Environment


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ABSTRACT

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Root exudates of plants mediate interactions with a variety of organisms in the rhizosphere including

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root-knot nematodes (RKNs, Meloidogyne spp.) We investigated the responses of the motile stage J2s of

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M. incognita to non-volatile components identified in the root exudate of tomato. Using stylet thrusting,

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chemotaxis assays and chemical analysis, we identified specific metabolites in the root exudate that

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attract and repel J2s. LC-QTOF-MS analysis of bioactive fractions obtained from the root exudate,

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revealed a high diversity of compounds; five of which were identified as the phytohormone zeatin

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(cytokinin), the flavonoids quercetin and luteolin, and alkaloids solasodine and tomatidine. In stylet

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thrusting and chemotaxis assays, the five compounds elicited concentration-dependent responses in J2s

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relative to 2% dimethyl sulfoxide (negative control) and methyl salicylate (positive control). These

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results indicate that J2 herbivory is influenced by root exudate chemistry and concentrations of specific

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compounds, which may have potential applications in RKN management.

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KEYWORDS: Agricultural pest, attractant, repellent, Solanum lycopersicum, stylet thrusting,

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chemotaxis.

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INTRODUCTION

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The global production of high value agricultural crops is faced with numerous challenges that include

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pests and diseases. Plant parasitic nematodes are among pests in the rhizosphere estimated to cause crop

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losses worth over $157 billion annually.1,2 In Africa, root-knot nematodes (RKNs) belonging to

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Meloidogyne spp. are the most damaging group of all plant parasitic nematodes causing up to 100%

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yield losses in vegetable crops such as tomato, pepper, cucumber, carrots and cabbage, particularly in

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smallholder farming systems.3–5

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The life cycle of RKNs involves six stages, an egg which develops into a first stage juvenile J1

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inside the egg, then a second stage juvenile J2, which emerges from the egg to seek a host. The J2 stage

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is the motile and consequently the infective stage. It invades the roots of plants and establishes a

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permanent feeding site in the root, whereby the affected root cells then develop into giant cells referred

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to as galls. Once established in the galls, J2s become sedentary, molt twice into J3, J4, before

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development into mature adults.6 Therefore J2s are the most important RKN life stage and

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understanding how they interact with roots is key to developing better targeted control methods.

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To control RKNs, smallholder farmers in Africa rely on cultural and biological approaches including

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crop rotation, organic soil amendments, intercropping, use of dead-end crops, resistant varieties, and

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processed plant products.7,8 However, these management methods are not always effective. In

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commercial holdings, farmers mainly use nematicides such as methyl bromide to control plant parasitic

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nematodes; however, because of environmental concerns, their use has been phased out. In the absence

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of more effective control options, semiochemicals involved in the host seeking process of J2s could

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serve as useful alternatives for RKN control.

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Previous studies have shown that J2s of M. incognita are attracted to carbon dioxide9–12 as well as volatiles released by roots of various crops.13,14 For instance, roots of the solanaceous plants tomato and



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pepper release volatiles including methyl salicylate, sabinene, α-pinene, limonene, and tridecane.13 Of

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these components, methyl salicylate is the most attractive to M. incognita J2s. The volatiles 2-isopropyl-

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3-methoxypyrazine and 2-(methoxy)-3-(1-methylpropyl)pyrazine detected from the Amaranthaceae

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plant spinach, also attract J2s.14 Additionally, for pepper, host attraction depends upon the cultivar. For

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instance, thymol, identified in the pepper cultivar AVRDC PP0237, was found to suppress J2 response

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to this cultivar.13 In addition to the volatile root components noted above,13,14 Yang and colleagues15

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reported differential responses of J2s to the less volatile compounds 2,6-di-tert-butyl-p-cresol, L-

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ascorbyl 2,6-dipalmitate, dibutyl phthalate, and dimethyl phthalate identified in the root exudates of

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three tomato cultivars. While many of these compounds have been established as common industrial

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contaminants, the compounds were reported as suppressing egg hatch and increasing J2 mortality.15

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Nevertheless, while plant species and cultivar root exudate chemistry can differ, research to elucidate

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these differences may help to identify compounds with potential applications for RKN control.

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The current study aimed to test responses of J2s of M. incognita to specific identified root exudate

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components of an RKN-susceptible tomato cultivar, ‘Cal-J’ using a bioassay-guided approach. Using

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bioactivity directed fractionation, we collected and fractionated root exudate from this tomato cultivar,

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identified specific components from bioactive fractions by liquid chromatography-time of flight-mass

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spectrometry (LC-QTOF-MS), and evaluated their biological activity using stylet thrusting and

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chemotaxis assays with J2s.

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MATERIALS AND METHODS

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Plants. Seeds of the tomato cultivar ‘Cal-J’ were purchased locally (Simlaw Seeds Company,

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Nairobi, Kenya). The seeds were sown in a rectangular plastic basin (67 cm x 40 cm x 5cm) (Kenpoly

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Manufacturers Limited, Nairobi, Kenya), containing sterilized sand (autoclaved at 121 oC for 40 min) in


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a screenhouse maintained at 23 ± 2 °C, 60-70% relative humidity (RH) at the International Centre of

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Insect Physiology and Ecology (icipe), Duduville campus, Nairobi, Kenya (1° 13' 18.96"S,

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36° 53' 47.94"E). Two-week old seedlings were transplanted into 2 L plastic pots (17 cm top diameter

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x13 cm base diameter x15 cm depth) containing a mixture of sterilized sand and loamy soil (2:1), and

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watered daily with nutrient solution containing micro- and macro nutrients.13,16 Plants used for the

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experiments were 4-5 weeks old.

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Root-Knot Nematodes. The inoculum of M. incognita was obtained from a nematode population

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culture maintained on tomato cultivar ‘Cal-J’ in the screenhouse at 23 ± 2 °C, 60-70% RH at icipe. Egg

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masses were extracted from galled tomato roots under a Leica M125 stereomicroscope (Leica

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microsystems, Buffalo Grove, IL) and placed in 6-well culture plates containing distilled water to allow

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hatching and emergence of J2s.13,17 The freshly emerged J2s were counted under the stereomicroscope

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and used in the behavioral assays.

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Collection of Root Exudate. Four- to five-week-old tomato plants were brought to the laboratory

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from the screenhouse, carefully uprooted from the soil and washed under running tap water to remove

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the sand and soil debris, followed by rinsing twice with distilled water. A bunch of 500 plants with intact

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cleaned roots were placed into a 4 L rectangular container (21 cm x 14 cm x 15 cm), filled with 1.5 L of

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distilled water that was replenished every 24 h and maintained at 23 ± 2 °C for 48 h for collection of

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root exudate. The container was covered with aluminum foil up to the stems of the tomato plants to

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avoid contamination of the exudate from the leaves and photodegradation. Root exudate was collected in

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triplicate with each replicate comprising 500 plants and 3 L of water. The root exudate was filtered using

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a Whatman No. 1 filter paper to remove particulate matter. Filtrates were freeze-dried in a benchtop

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freeze drier, weighed, then divided into two equal parts for use in either bioassays or chemical analysis.



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Bioactivity of Root Exudate. To determine the effect of root exudate on RKNs, a stock solution of

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5 µg/µL of the freeze-dried root exudate was prepared in distilled water and serially diluted to obtain

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three concentrations of 1.25, 2.5 and 5 µg/µL. Distilled water and methyl salicylate (MeSA) (100

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ng/µL)12 served as the negative and positive controls, respectively. The bioactivity of the respective

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exudate and the controls were tested on freshly emerged J2s in two different experiments.

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Stylet Thrusting Bioassay. To determine the response of J2s to the root exudate, we recorded the

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number of stylet thrusts/min when juveniles were in contact with different concentrations of the exudate

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and the positive and negative controls under the stereomicroscope. Prior to measuring stylet thrusting

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response, 20 µL of each exudate solution were pipetted into a previously formed ring of 30 µL of 23%

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pluronic gel18 (Sigma-Aldrich, St Louis, MO) on a microscope glass slide (Figure 1). A 20 µL

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suspension that contained approximately 50 J2s were added into the ring.19 A cover slip was then placed

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to cover the ring and then slight pressure was applied to the cover slip to ensure an airtight fit and to aid

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the spread of the nematodes to contact the pluronic gel. The set up was left for the nematodes to settle

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for 15 min and J2 behavior, the stylet thrusting, were observed and the number of thrusts/min recorded.

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The stylet thrusting response was observed on 15 J2s, chosen singly per slide (replicate) under a Leica

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DM 2500 compound microscope (Leica microsystems, Buffalo Grove, IL) at 200x magnification. Three

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replicates per treatment were carried out each using fresh root exudate.

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Chemotaxis Bioassay. We tested nematode preference to the exudate in a dual choice sand assay (60

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mm length x 14 mm diameter) divided into three sections A, B and C (Figure 2). The respective sections

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A and B were each filled with 5 g of sterilized clean moist sand and section C with 2 g (Figure 2). The

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sand in sections A and B was mixed with either exudate or the positive control on the stimulus side, and

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distilled water on the control side. The exudate was tested at three different concentrations; 1.25, 2.5,

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and 5 µg/µL, and 20 µL of each sample were mixed with the 5 g of sand in the treated section


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(equivalent to 5.0, 10.0 and 20.0 µg/g sand respectively), with a similar volume, 20 µL of distilled water

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mixed with moist sand in the control sections. J2s (200), were introduced into the release point (Figure

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2C). After 24 h, a modified Baermann extraction17 was used to recover J2s from the respective tubes.

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The number of J2s in each arm of the tube was counted under a stereomicroscope. Three replicates each

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comprising 200 nematodes were carried out per treatment.

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Chemical Analysis of Root Exudate. The freeze-dried crude exudate (1 g) from the tomato cultivar

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‘Cal-J’ was dissolved in 10 mL 10% aqueous methanol and analyzed on a Nexera X2 Series HPLC

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system (Shimadzu, Kyoto, Japan), equipped with a prominence SPD-M30A diode array detector (190-

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700 nm). The LC column used was a 250 mm x 10 mm i.d., 5 µm, ACE 5 RP-18 (Advance

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Chromatography Technologies, Aberdeen, Scotland) set at an oven temperature of 30 °C. The mobile

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phase A (0.01% formic acid in water) and B (acetonitrile) was used at a flow rate held constant at 1

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mL/min and a total run time of 50 min. The following gradient program was employed: 0 min, 5% B;

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0−10 min, 5−20% B; 10−15 min, 20% B; 15−23 min, 20-70% B; 23-30 min, 70% B; 30−38 min, 70-

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100% B; 38−45 min, 100% B; 45− 48 min, 100-5% B; 48-50 min, 5% B. Four fractions were collected:

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fraction 1 (3-12 min); fraction 2 (12-23 min); fraction 3 (23-30 min); and, fraction 4 (30-45 min). All the

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fractions were concentrated in vacuo using a rotary evaporator to give: fraction 1 (100 mg), fraction 2

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(12 mg), fraction 3, (0.7 mg) and fraction 4 (0.5 mg). For each of the fractions, three concentrations

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were prepared and tested for their bioactivity on J2s following procedures described above for stylet

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thrusting and chemotaxis.

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After fractionation and drying of the fractions, concentrations of 1µg/µL of the freeze-dried root

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exudate including the crude and fractions were prepared in water/methanol (90:10, v/v), vortexed for 1

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min, ultrasonicated for 5 min, and centrifuged at 12000 rpm for 5 min. The supernatant was then passed

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through Whatman 0.2 µm pore size syringe filters. The exudate and fractions were analyzed using



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ACQUITY I-class system ultra-performance liquid chromatography (UPLC) (Waters Corp., Milford,

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MA) interfaced with electrospray ionization (ESI) to a Synapt G2-Si QTOF-MS operated in full scan

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MSE in positive mode. The column used was 2.1 x 100 mm, 1.7 µm, BEH RP-18 (Waters Corp.,

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Wexford, Ireland) and was heated at 40 °C. The autosampler tray was kept at 5 °C. The mobile phase

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comprised of 0.01% formic acid in water (solvent A) and acetonitrile (solvent B) and followed a

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gradient system.20,21 Data acquisition was achieved with MassLynx version 4.1 SCN 712 (Waters). The

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mass spectrum was generated for specific peaks and potential assignments done using monoisotopic

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masses with tolerance of 20 ppm. Compounds in the fractions were identified by comparison of MS/MS

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spectra with literature data and online database (ChemSpider, Metlin). The identities of zeatin, luteolin,

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quercetin, solasodine, and tomatidine were confirmed by co-injection with authentic samples.

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Bioactivity of Identified Compounds. Bioassays were carried out using the commercially available

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root exudate compounds zeatin, luteolin, quercetin, tomatidine and solasodine. The compounds were

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prepared in distilled water containing 2% DMSO to make stock solutions of 1000 ng/µL. For assays,

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solutions of the compounds were prepared by serially diluting four-fold the stock solution to yield the

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concentrations 250, 62.3, 15.6, and 4 ng/µL. Bioactivity of each compound was tested on J2s in stylet

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thrusting and chemotaxis (4000.0, 1000.0, 249.2, 62.4 and 16.0 ng/g sand) assays, as previously

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described, in three replicates.

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Chemicals. Analytical grade methanol (≥ 99.9%), acetonitrile (≥ 99.9%), formic acid (98-100%),

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water (LC-MS Chromasolv), trans-zeatin (≥ 97%), luteolin (≥ 98%), quercetin (≥ 95%), solasodine (≥

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95%), tomatidine hydrochloride (≥85%) DMSO (≥ 99.9%), and methyl salicylate (MeSA) (≥ 99%),

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were purchased from Sigma-Aldrich (St Louis, MO).

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Statistical Analyses. The data obtained from J2 stylet thrusting response was log-transformed prior to analysis of variance to normalize the data and stabilize the variance. Means were separated using


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Tukey’s HSD test. For chemotaxis assays, numbers of responding J2s to the different treatments were

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recorded as means and expressed as percent response [(n/N) x 100]. N corresponds to the total number

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of responding J2s, while n is the number of J2s responding to a given treatment. The data obtained from

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the chemotaxis assays was analyzed by Chi-square goodness of fit to assess attraction and/or avoidance

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of M. incognita to the different concentrations of root exudate, fractions and the pure standards tested

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individually compared to controls (distilled water, 2% DMSO and MeSA).13 Non-respondents were not

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included in the analysis. All tests were performed at 5% significance level using R software version

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3.2.3.22

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RESULTS AND DISCUSSION

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Bioactivity of Tomato Crude Root Exudate. Overall, stylet thrusting elicited in J2s by the root

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exudate of ‘Cal-J’ at all the concentrations tested, were significantly higher (F (4,220) = 227.1, P < 0.001)

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than the negative control (distilled water) (Figure 3A). Stylet thrust intermittently increased with

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concentration although there was a slight decrease at high concentration. This could be due to saturation

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of the sensory receptors, thereby generating a negative feedback signal through the central nervous

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system of the nematode to decrease the stylet thrust. This result is in agreement with a study where

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exogenously applied neuromodulators had differing effects on soybean cyst nematode, Heterodera

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glycines and M. incognita J2 behavior.23 For example, serotonin a known activator of stylet thrusting

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elicited a concentration- and time-dependent stylet thrusting which reached a plateau at high

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concentration.23,24 Similarly, in the chemotaxis assays, attractiveness of J2s to the root exudate was

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concentration-dependent and significantly higher (77.2%, χ2 = 26.1, df = 1, P < 0.001) than the negative

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control. For instance, at 10 µg/g sand, the attractiveness of the root exudate compared favorably with the

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positive control, MeSA, 20 ng/g sand, (73.4%, χ2 = 21.94, df = 1, P < 0.001) (Figure 3B). These results



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are in agreement with a previous study which found that small lipophilic molecules in tomato and rice

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root exudates, and the water soluble neurotransmitter, resorcinol, induced stylet thrusting in M.

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graminicola and M. incognita.19 The results also indicated that specific components or blends of these

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components in the root exudate influenced J2 host seeking behavior.

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Bioactivity of Root Exudate Fractions. All the four fractions obtained by chromatography of the

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root exudate elicited significantly higher and concentration-dependent stylet thrusting in J2s than the

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negative control (fraction 1: F(4, 220) = 783.6, P < 0.001; fraction 2: F(4,220) = 637.2, P < 0.001 fraction 3:

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F(4,220) = 512.6, P < 0.001; fraction 4: F(4,220) = 698.3, P < 0.001) (Figure 4A). Interestingly, whereas

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stylet thrusting responses elicited by fractions 1 and 2 compared favorably with the positive control, they

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were lower for fractions 3 and 4. These results indicate that all the individual fractions contained

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compounds which may be necessary for J2 host detection; it is possible that attraction may be enhanced

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by background root volatile components 14, which would require additional research. However, of the

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four fractions, the more potent components appeared to be present in fractions 1 and 2. HPLC analysis

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identified fractions 1 and 2 as the more polar fractions, suggesting J2 utilization of polar compounds for

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host seeking. The results of the chemotaxis assays confirmed this observation, whereby differential

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significant concentration-dependent responses were recorded for fraction 1 (10 µg/g sand, 64.1%, χ2 =

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8.43, df = 1, P < 0.01) and fraction 2 (20 µg/g sand, 70.7, χ2 = 17.12, df = 1, P < 0.001). Fractions 3 and

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4, elicited chemotaxis responses which were not significantly different from that elicited by the negative

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control, except at the concentration of 5 µg/g sand for fraction 4 (59.9%, χ2 = 5.88, df = 1, P < 0.05)

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(Figure 4B). These results indicate that host finding in J2s is complex and may involve semiochemicals

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derived from different compound classes detected at specific concentrations or ratios.

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LC-QTOF-MS Identification of Compounds in Bioactive Fractions. Chemical analysis of the bioactive fractions using LC-QTOF-MS identified a complex blend of polar compounds in fractions 1


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and 2 (Figure 5). Among these, five compounds, including the phytohormone zeatin (cytokinin), 1,

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flavonoids luteolin, 3, and quercetin, 4, and alkaloids tomatidine, 5, and solasodine, 6, present in

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fractions 1 and 2 (Table 1), were identified based on retention time, mass fragmentation and, confirmed

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with authentic standards by co-injections (Figure 5). In addition, tomatine, 2, was tentatively identified

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based on mass fragmentation pattern and literature data. Zeatin, 1, eluted at 1.97 min and had a

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molecular ion peak [M+H]+ at m/z 220.1202 with two key characteristic fragment ions at m/z 202.1093

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[M - H2O]+ and 136.0629, a characteristic adenine derivative ion C5H6N5+.25 Tomatine, 2, eluted at 5.54

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min and had a molecular ion [M+H]+ peak at m/z 1034.5571, with the aglycone tomatidine fragment at

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m/z 416.3510 and fragment ions at m/z 902.5131, 740.4576 and 578.4060 that could be due to

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consecutive losses of a xylose and two glucose moieties.20,26,27 Luteolin, 3, eluted at 6.02 min and was

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identified based on a molecular ion [M+H]+ peak at m/z 287.0562, with key characteristic fragment ions

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at m/z 153.0191 and m/z 135.0449, in addition to m/z 269.0459, m/z 257.0455 and m/z 213.0558,

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corresponding to dehydration of product ion to [M+H-H2O]+, followed by two sequential losses of CO:

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[M+H-H2O-CO]+ and [M+H-H2O-2CO]+ respectively.28 Quercetin, 4, eluted at 6.05 min and was

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identified based on a molecular ion [M+H]+ peak at m/z 303.0519, with characteristic fragment ions at

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m/z 153.0192 and m/z 137.0244 that were in tandem with authentic standard fragment ions. In addition,

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fragment ions corresponding to a loss of H2O and 2 CO were detected at m/z 285.0406 and m/z 229.0511

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respectively (Table 1).28 Tomatidine, 5, eluted at 6.32 min and was identified based on a molecular ion

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[M+H]+ peak at m/z 416.3542, and a fragment ion [M - H2O]+ peak at m/z 398.3445.29 Solasodine, 6,

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eluted at 6.33 min and was identified based on a molecular ion [M+H]+ peak at m/z 414.3357, with a

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fragment ion [M - H2O]+ peak at m/z 396.3250 (Table 1).30 Additional compounds were tentatively

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identified based on mass spectra and diagnostic fragment ions: a glucoside derivative of caffeic acid

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eluting at 1.57 min with a molecular ion [M+H]+ peak at m/z 343.1087, and m/z 180.0872 corresponding



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to loss of a sugar moiety, and at m/z 163.0228, representing loss of H2O; quercetin glucoside eluting at

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2.75 min, with a molecular ion [M+H]+ peak at m/z 465.1038, and m/z 303.1014 (quercetin aglycone)

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due to loss of a sugar moiety; and luteolin glucoside at 3.12 min, with a molecular ion [M+H]+ peak at

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m/z 449.1125, and m/z 287.1003 (luteolin aglycone) due to loss of sugar moiety.31 Additional studies are

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needed to confirm the identities of these glucosides and to identify the unidentified components present

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in the bioactive fractions and to determine their roles in J2 host detection. These results further

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corroborate that a complex blend of semiochemicals mediate host location in J2s. Among these include

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phytohormones, flavonoids, alkaloids and glucosides. Interestingly, none of the previously identified

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less volatile tomato root exudate compounds established by GC-MS,15 which we consider as artefacts,

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was detected in the current study utilizing LC-QTOF-MS analysis of the most bioactive fractions

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(fractions 1 and 2). The approximate concentrations of identified compounds released were 0.003, 0.008,

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0.011, 0.013 and 0.025 ng/plant/h for zeatin, luteolin, quercetin, tomatidine and solasodine respectively.

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Responses of M. incognita to Synthetic Compounds Identified in Bioactive Fractions. All the

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compounds identified elicited significantly higher stylet thrusting than the negative control (2% DMSO),

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with the rate increasing as the concentration of the compounds increased (zeatin, 1, F(6, 308) = 546.2, P

Elicitation of Differential Responses in the Root-Knot Nematode

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