Nematicidal Activity of trans-2-Hexenal against Southern Root-Knot

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Nematicidal Activity of trans-2-Hexenal against Southern RootKnot Nematode (Meloidogyne incognita) on Tomato Plants Hongbao Lu, Shuangyu Xu, Wenjuan Zhang, Chunmei Xu, Beixing Li, Daxia Zhang, Wei Mu, and Feng Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04091 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 4, 2017

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Journal of Agricultural and Food Chemistry

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Nematicidal Activity of trans-2-Hexenal against Southern Root-Knot

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Nematode (Meloidogyne incognita) on Tomato Plants

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Hongbao Lu a, b*, Shuangyu Xua, b*, Wenjuan Zhang

a, b

, Chunmei Xu a, b, Beixing Li a, b,

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Daxia Zhang a, b, c, Wei Mua, b, Feng Liu a, b **

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a

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Pests, College of Plant Protection Shandong Agricultural University, 61 Daizong Street,

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Tai’ an, Shandong 271018, PR China.

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b

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271018, PR China.

Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect

College of Plant Protection, Shandong Agricultural University, Tai’ an, Shandong

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c

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University, Tai’an, Shandong 271018, China.

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*

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**

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Prof. Feng Liu. College of Plant Protection, Shandong Agricultural University, 61

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Daizong Street, Tai’an, Shandong 271018, P.R. China.

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Tel. /fax: +86 0538 8242611. E-mail address: [email protected] (F. Liu).

Research Center of Pesticide Environmental Toxicology, Shandong Agricultural

Hongbao Lu and Shuangyu Xu share joint first authorship. Corresponding author:

1

ACS Paragon Plus Environment


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Abstract

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Botanical nematicides have recently received increasing interest, because of the high

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risks of some traditional nematicides to human health and the environment. In this study,

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we evaluated the nematicidal activity of a plant volatile, trans-2-hexenal, against

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Meloidogyne incognita. This compound exhibited higher activity in a fumigation

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experiment than in the aqueous phase in vitro. Both in pot tests and in field trials, trans-2-

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hexenal showed significant efficacy against M. incognita while maintaining excellent

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plant growth, especially at the doses of 1000 and 500 L ha-1, which were superior to that

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of abamectin at 180 g ha-1 via hole application treatment but not significantly different

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from fumigation with 400 kg ha-1 of dazomet. Furthermore, plants treated with 500 L ha-1

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of trans-2-hexenal had fruit yields 20.2% and 45% greater than the control group. Based

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on, trans-2-hexenal may be a potential alternative fumigation agent for controlling M.

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incognita on tomato crops.

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Keywords: trans-2-hexenal; nematicidal activity; soil fumigation; Meloidogyne

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incognita; tomato

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INTRODUCTION

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Plant parasitic nematodes have caused serious damage to crop production worldwide over

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the last few decades and have recently caused even more severe problems. Chitwood et al.

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estimated that the loss of annual global agriculture caused by plant parasitic nematodes

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reached $125 billion worldwide. 1 Among the parasitic nematode species, the southern 2


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root-knot nematode (Meloidogyne incognita) is one of the most damaging agricultural

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nematode species due to its infection of the roots of crops, 2 especially vegetables, causing

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serious yield losses in tropical, sub-tropical and temperate areas.

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lycopersicum L.) is one of the most important greenhouse cash crops in China and is

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cultivated worldwide. 4 M. incognita also causes serious losses in the yield of tomatoes.

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In recent years, these yield losses have dramatically increased due to continuous cropping

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of susceptible vegetables, which have induced a high invasion rate of root-knot

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nematodes. 5

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Tomato (Solanum

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Currently, the main strategy for controlling nematodes is based on using synthetic

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chemical products, including soil fumigants and non-fumigant nematicides. 6 However,

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some chemical products are no longer available because of the high risks to human health

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and the environment. 7 For instance, methyl bromide (MeBr) and aldicarb are the most

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effective treatments for the management of root-knot nematodes. Nevertheless, methyl

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bromide has a detrimental effect on the stratospheric ozone and was set to be totally

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phased out by 2015, 8 and the use of aldicarb has been restricted due to its high toxicity. 9

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Therefore, identifying alternatives for nematode control and developing effective and

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safe application techniques are both urgent strategies for alleviating the nematode-

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induced damage .10 Recently, the preferred alternatives for managing root-knot nematodes

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are biological agents, among which botanical nematicides (i.e., nematicidal chemical

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compounds emitted from plants or microorganisms)

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interest because of their high effectiveness and environmentally friendly characteristics. 15

11-14

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have received increasing


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In addition, the development of biological pesticides is encouraged by the Chinese

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government. 16

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In response to wounds and insect attacks, plants emit volatile organic chemicals, most 17, 18

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of which are aldehydes and alcohols with short chains.

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compounds improve the food flavor, and they are generally considered safer than

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artificial pesticides.

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insecticidal activities.

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alkenals showed more than 95% nematicidal activity against the pinewood nematode.

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Consistent with this, (E, E)-2, 4-decadienal and (E)-2-decenal, which are botanical

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aldehydes from Ailanthus altissima, exhibit strong nematicidal activity against

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Meloidogyne species. 22, 23 Furthermore, Aissani et al. have demonstrated the activity of

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(E)-2-hexenal against Meloidogyne incognita.24

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Some of these volatile

Interestingly, these chemicals can trigger antimicrobial and Seo et al. reported that the aliphatic compounds C6-C10 2E21

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trans-2-Hexenal is a plant volatile compound that is naturally present in many fruits

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and vegetables. 25 trans-2-Hexenal has an extremely potent aroma, is therefore commonly

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used as an artificial flavoring and as a food additive, 26 and has been quantified at 113 µg

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L-1 in orange, in which it is believed to play an important role in the flavor of the juice. 27

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It has been reported that trans-2-hexenal contributes to the aroma of most virgin olive

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oils in Europe. 28 Furthermore, trans-2-hexenal has been reported to have antimicrobial

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activity and has been used to control penicillium expansum in pears and apples. 29, 30 Miao

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et al. reported that trans-2-hexenal (2.62 µL L-1) induced 100% mortality in the second-

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stage juveniles of both M. incognita and Heterodera avenae. 31 However, to date, there 4


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are no reports regarding its control efficacy against nematodes on tomato plants.

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Therefore, we conducted laboratory and field trials to demonstrate the nematicidal

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activity of trans-2-hexenal in managing root-knot nematodes on tomato plants and to

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evaluate its prospects for application in China.

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MATERIALS AND METHODS

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Chemicals. trans-2-Hexenal (99% purity, relative density: 0.849 g mL-1) was purchased

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from Aladdin Industrial Co. (Shanghai, China). Dazomet (98% MG, a.i.) was provided by

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Nantong Shizhuang Chemical Co. (Jiangsu Province, China). Abamectin (3% CS, a.i.)

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was purchased from Qingdao Rainsen Agrochemical Co.(Shandong Province, China).

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Nematodes. A population of M. incognita was obtained from tomato (cultivar

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“Fenguan F1”) roots collected from a greenhouse at Liuyi Farm, near Qihe County,

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Shandong Province, China (116°47'24" E, 36°54'36" N, at an elevation of 21 m above

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mean sea level). The eggs of M. incognita were extracted from the infected roots of

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tomato plants (Solanum lycopersicum L.) into a solution of NaOCl. 32 To obtain second-

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stage juveniles (J2), eggs were spread on a nylon sieve (30 µm) in a petri dish containing

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water and incubated at 25 °C. Emerging J2 individuals were collected daily and stored at

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4 °C for laboratory experiments.

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Nematicidal Activity Bioassays with Fumigation Experiment. Newly emerged 5



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juveniles of M. incognita were used in a fumigation bioassay. Four concentrations of

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trans-2-hexenal (0.05, 0.35, 0.65 and 0.95 µL L-1) were applied, each tested separately,

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and one untreated control was used. The nematodes were collected in distilled water, a

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nematode suspension was made (approximately 100 nematodes per 50 µL), and then 50

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µL of the nematode suspension of M. incognita J2 was mixed with 150 µL of distilled

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water and transferred to the wells of a three-well slide (65 mm × 35 mm × 5 mm).

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Various doses (0.5, 3.5, 6.5 and 9.5 µL) of trans-2-hexenal were applied to a piece of

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filter paper. The filter papers with the test compound were quickly pasted on the inner

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wall of the aquarium (10 L, 20 cm × 20 cm × 25 cm) by using double-sided adhesive tape,

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and the three-well slides containing the nematodes were placed into these aquariums

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concurrently. Then, the aquariums were sealed with Parafilm. The experiments were

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performed at 25 ± 1 °C. The dead J2s were observed with the aid of a stereomicroscope

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(Olympus SZ51, Wuhan Jie measuring instrument co., Ltd, Hubei Province, China) at 24

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h. Nematodes were classified as dead if their bodies were motionless (i.e., straight) even

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after being transferred to clean water for 12 h. The experiment was conducted twice with

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three replicates, using a three-well slide as a single replicate.

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Nematicidal Activity Bioassays in Aqueous Phase. Concentrations of the test

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solution of trans-2-hexenal (60, 90, 120, 150, 180 mg L-1 respectively) were prepared by

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serial dilution with distilled water containing Tween-80 (1000 mg L-1). The test solutions

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were introduced into wells of 24-well tissue culture plates (Nunc, Roskilde, Denmark). In 6


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each well, the concentration of nematodes was approximately 100 juveniles of M.

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incognita per 1000 µL of water. A distilled water-Tween-80 solution (1000 mg L-1) was

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used as a control. The plates were covered and maintained at 25 ± 1 °C. The nematode

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mortality was observed under a stereomicroscope at 24 h, at which point the nematodes

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were transferred to distilled water after rinsing with plain water through a 25 µm screen

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sieve to remove the excess of tested chemicals. Nematodes were classified as dead if their

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bodies were motionless (i.e., straight) even after being transferred to clean water for 12 h.

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The experiments were performed two times, and each treatment was replicated four times.

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Pot Experiments. Experiments were performed to test the nematicidal activity of

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trans-2-hexenal against M. incognita. Nematode-infested soil was obtained from a

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commercial farm near Dongdawu Country, Shandong Province, China (117°7'48" E,

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35°58'48" N). To simulate the fumigation process, 8000 g of infested soil (approximately

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600 J2 per 100 g soil) was placed in a plastic case (42 cm × 30 cm × 13 cm). Volumes of

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trans-2-hexenal were pipetted into the soil, corresponding to the experimental rates of

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250, 500 and 1000 L ha-1, and the plastic cases were sealed with Parafilm and kept in a

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growth chamber at 25 ± 2 °C. The water content of the soil in the treatments was

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approximately 50%. One week after treatment, the plastic film was removed, and 500 g

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of treated or untreated soil were placed in plastic pots (approximately 800 cm3). Tomato

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seedlings (1 month old, Fenguan F1) were transplanted 7 days later and kept at a constant

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temperature of 25 ± 2 °C. As a positive control, dazomet was uniformly broadcast into 7



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the soil at a dose of 400 kg ha-1, and abamectin was applied via hole application at a

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concentration of 180 g ha-1 with 100 mL water per plant when transplanting the tomato

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plant; soil treated with water alone was used as the negative control. The tomato plants

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were uprooted after seven weeks, and the shoot fresh weight, the root galling index and

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the number of nematode eggs were recorded. The root galling index was assessed on a

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scale of 0-10, where 0 = no galls, 1 = 0-10% galled roots, 2 = 10-20% galled roots, 3 =

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20-30% galled roots, 4 = 30-40% galled roots, 5 = 40-50% galled roots, 6 = 50-60%

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galled roots, 7 = 60-70% galled roots, 8 = 70-80% galled roots, 9= 80-90% galled roots,

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and 10 = 90-100% galled roots.

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replicates for each treatment.

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The experiment was performed twice with seven

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Field Experiment. Field trials were conducted in July 2013 and in February 2014. The

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farm was near Qihe County, Shandong Province, China, and it had been planted with

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tomatoes, cucumbers and melons for more than 10 years before the start of the

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experiment. The selected farm had a history of heavy natural infestations of root-knot

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nematode (M. incognita). The soil was loam, composed of 55% sand, 40% silt and 5%

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clay, and it contained 13.9 g·kg-1 of organic matter and had a pH of 8.3.

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The fumigation programs used for treatment were as follows: (i) trans-2-hexenal was

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furrow applied at three doses (i.e., 1000, 500 and 250 L ha-1); (ii) dazomet was furrow

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applied at a dose of 400 kg ha-1 as a reference treatment; (iii) abamectin as a routine

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treatment was applied with hole application at a dose of 180 g ha-1 with 800 mL water; 8


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and (iv) an untreated control was also established.

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All of the doses of trans-2-hexenal were determined according to the bioassay results

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obtained in the laboratory, while the dose of dazomet and abamectin were based on

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routine use in the field. 34, 35

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Each treatment had five repetitions and all the treatments were laid out in a completely

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randomized block design. Each plot consisted of four rows of tomatoes, and the plot area

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was 20 m2.

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Before planting, each plot was tilled using a rotary tiller to a depth > 30 cm with land

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leveling and irrigation. Then, the fumigation was applied on 12 July 2013 and on 17

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February 2014 respectively, at which point the soil moisture was between 60% and 70%.

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All of the operations were ordinary procedures and were performed as follows. The

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furrows of the planting rows were 0.25 m in depth and spaced 0.50 m apart. Then, the

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planting rows were treated with 2 L of trans-2-hexenal solution that was diluted with 0.1%

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Tween-80 or with particles of dazomet via uniform broadcasting to the indicated furrows.

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The planting rows were then quickly bedded and pressed into rows that were 0.80 m wide

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at the base, 0.70 m wide at the top, 0.20 m high, and spaced 0.70 m apart as measured

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from the center of the row. 36 To avoid the loss of the fumigant, immediately after the

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fumigant application, the beds were pressed and covered with a plastic film coating that

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was 0.06 mm thick. At 7 days after treatment, the plastic film was removed in order to

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volatilize excessive fumigants. Before the transplantation, abamectin was applied via hole

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application with 800 mL water. The tomato seedlings were transplanted, at which point 9



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the solution was completely absorbed by the soil. Untreated plots were regarded as the

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control group.

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Six-week-old tomato seedlings (Fenguan F1) were transplanted into the top of the beds

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on 2 August 2013 and 10 March 2014. Each plot contained 60 tomato plants.

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Conventional irrigation and fertilizer were provided according to the requirements of the

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tomatoes. A foliar spray of insecticides and fungicides were applied after transplantation,

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beta-cypermethrin (2% a.i.)·buprofezin (18% a.i.) EC (Beijing Huarong Biological

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Hormone Plant, Beijing, China) was used for managing Trialeurodes vaporariorum, and

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beta-cypermethrin (2% a.i.)·acetamiprid (3% a.i.) WP (Hubei vying bio-chemical

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pesticide co., Ltd, Hebei Province, China) was used to control Bemisia tabaci, In addition

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procymidone 43% SC (Rotam Crop Sciences Ltd., Jiangsu Province, China) and

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flusilazole 10% EW (Lier Chemical co., Ltd, Sichuan Province, China) were used to

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manage Botrytis cinerea and leaf mold disease, respectively.

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During the growing season of tomato, the plant heights and stem diameters of 10 plants

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per plot were measured at 15, 30, 45 and 60 days after transplantation (DAT). The M.

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incognita populations in the soil were determined before fumigation (BF), and at 30, 60

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and 90 DAT. Soil samples 2.5 cm wide and 20 cm deep from 10 tomato plants in each

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plot were extracted from the rhizosphere. 37 M. incognita individuals were separated from

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100 cm3 of soil by using a standard sieving and sugar flotation/centrifugation procedure

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with a 500-mesh sieve (25 µm opening). Then, the number of M. incognita was counted

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under a stereomicroscope. The root galling index was determined at the time of harvest 10


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by digging up the roots of ten tomato plants in each plot and using a 0-10 scale to

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evaluate root damage. 33 The tomato yield was measured twice (i.e., at 13 and 15 weeks

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after transplanting)

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current market standards, and the average weight of the tomatoes was calculated based on

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a survey of ten tomato plants per plot.

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. The number of tomatoes per plant was counted according to

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Data Analysis. Data from the two trials were analyzed for the homogeneity of

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variances. When the variance was homogeneous, the test data were combined. Data were

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subjected to an analysis of variance (ANOVA) followed by the Tukey-Kramer HSD test.

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Differences with P < 0.05 were considered statistically significant. The number of

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nematode eggs per plant was statistically analyzed after transforming to log10 (x + 1), and

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the nematode control effect was calculated according to the following equation: Control

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Effect (%) = [(Root galling index control - Root galling index treated) / Root galling

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index control] × 100 39. All data analysis and determinations of the lethal concentrations

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(LC50 and LC90) were performed using SPSS software (version 13.0 for Windows). The

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data from nematicidal bioassays are expressed as corrected mortality. Corrected mortality

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was calculated according to the Schneider-Orelli’s formula

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mortality percentages were calculated by eliminating of a percentage of mortality in the

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control using the following equation: Corrected % = 100× [(mortality% in treatment –

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mortality% in control) / (100-mortality% in control)]. Then, the LC50 and LC90 values

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were calculated by probit analysis. 11


40

, whereby the corrected


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RESULTS

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Nematicidal Activity Bioassays. Fumigation for 24 h with trans-2-hexenal exhibits

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significant nematicidal activity. The mortality increased with increasing doses of trans-2-

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hexenal. The juveniles of M. incognita showed an LC50 values of 2.79 mg L-1 and an

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LC90 values of 8.67 mg L-1. However, in the aqueous phase, trans-2-hexenal showed

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lower nematicidal activity, with LC50 and LC90 values of 102 mg L-1 and 162 mg L-1

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respectively (Table 1).

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Pot Experiments. In these experiments, the nematode-infested soil was treated with

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trans-2-hexenal at 250-1000 L ha-1 or with dazomet and abamectin, and all of the

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treatments reduced the galling index and the number of nematode eggs per plant (Figures

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1A and B). Treatments involving trans-2-hexenal at 500 or 1000 L ha-1 or dazomet and

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abamectin provided M. incognita control efficacy of 57.1-67.6%. The tomato plants

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treated with trans-2-hexenal at 250 and 500 L ha-1 showed greater plant height (P ≤

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0.0029) than the control plants (Figure 2A), in which the highest plant height (68.8 and

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69.3 cm) was observed in plots treated with 250 L ha-1 trans-2-hexenal and 400 kg ha-1

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dazomet. However, the plant height was lowest in trans-2-hexenal plots at 1000 L ha-1. In

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other treatments, the plant height was moderate and better than in the untreated control.

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Tomato shoot fresh weight showed a trend similar to that of plant height (Figure 2B).

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Field Experiments. The number of nematodes (M. incognita) in 100 cm3 of soil was 12


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evaluated, and the results revealed the excellent nematicidal activity of trans-2-hexenal in

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both the 2013 and 2014 experiments (Table 2). In the 2013 experiment, the populations of

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M. incognita in soil were clearly decreased in the treatments with trans-2-hexenal,

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dazomet, and abamectin, among which trans-2-hexenal at 1000 L ha-1 was the most

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effective at reducing the number of root-knot nematodes. During the growing season, the

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number of nematodes increased gradually, but the numbers in the treated plots remained

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significantly lower than those in the untreated plots (P = 0.0001). The number of

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nematodes in the soil was lower in 2014 than in 2013, but the trend in the variation was

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similar.

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Nematode infestations were evaluated at harvest time by calculating the root galling

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index. These results confirmed the excellent nematicidal activity of trans-2-hexenal in

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both the 2013 and 2014 experiments (Figure 3). Treatments involving trans-2-hexenal,

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dazomet and abamectin were effective in lowering the galling index, in which trans-2-

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hexenal at 1000 L ha-1 was the most effective treatment for reducing galling (2.2 in 2013

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and 2.5 in 2014) and for achieving the best control effect (72.5% in 2013 and 70.5% in

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2014) against M. incognita. The root galling index was highest for tomatoes grown in

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untreated plots (8.02 in 2013 and 8.4 in 2014). Furthermore, the control effect of dazomet

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at 400 kg ha-1 showed no significant difference from trans-2-hexenal at the doses of 1000

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and 500 L ha-1 (P ≥ 0.2084). trans-2-Hexenal at 250 L ha-1 showed a lower control effect

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(54.9% in 2013 and 56.0% in 2014) for M. incognita and was not significantly different

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from abamectin at 180 g ha-1 (P ≥ 0.4084). 13



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The nematicide application significantly influenced the stem diameter and plant height

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of tomato plants, of which ratings also increased compared to the untreated controls

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(Figure 4). The largest stem diameter was obtained in the plots treated with trans-2-

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hexenal at the dose of 250 L ha-1 and dazomet at 400 kg ha-1, the trans-2-hexenal at 500 L

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ha-1 and the abamectin group had an intermediate stem diameter that was greater than that

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in the untreated controls in both the 2013 and 2014 experiments, whereas the high dose of

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trans-2-hexenal (i.e., 1000 L ha-1) slightly decreased the stem diameter of tomato plants

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(Figures 4A and B). Tomato plant height showed the same trend as stem diameter. The

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greatest tomato plant heights were obtained in plots treated with trans-2-hexenal at a dose

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of 250 L ha-1 (142.6 cm, 60 DAT) and in plots treated with dazomet (142.4 cm; Figures

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4C and D).

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In both trials, all of the treatments significantly increased the fruit yield. Compared

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with the untreated controls, the various treatments increased the yield by 2.64 - 45%

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(Table 3). The maximum weight of fruit (182.4 g) was obtained following the treatments

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with trans-2-hexenal at 500 L ha-1, with dazomet 400 kg ha-1 (179.9 g) and with

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abamectin 180 g ha-1 (176.3 g), whereas the lowest weight was achieved in the untreated

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control (163.7 g). Other treatments produced weights of 171.8 and 173.1 g. There were

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no significant differences among the treatments, including the control group, in the

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numbers of tomato fruits (2013, P ≥ 0.0709; 2014, P ≥ 0.2933). The total yield showed a

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similar trend, with the highest yield (82.1 and 99.0 t ha-1) produced in the plots treated

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with trans-2-hexenal at 500 L ha-1, which had yields 20.2% and 45% greater than the 14


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control group.

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DISCUSSION

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trans-2-Hexenal is one of the most widespread and abundant volatile compounds

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naturally occurring in plant tissue.

295

amounts of trans-2-hexenal occur in bananas, 42 and that concentration of this compound

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reaches 295 mg L-1 in apple essence concentrate. 43

41

Previous publications have reported that high

297

In our study, we tested the nematicidal activity of trans-2-hexenal both in the

298

laboratory and in the field. In the vitro tests, trans-2-hexenal showed significantly

299

nematicidal activity both in the fumigation programs and in aqueous solution. Compared

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to fumigation, trans-2-hexenal showed lower nematicidal activity in the aqueous phase,

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in which the LC50 and LC90 values were 102 and 162 mg L-1 respectively. In liquid, the

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respiration rate of nematodes (i.e., the rate of oxygen diffusion into the body) slows down

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due to the low oxygen content. 44 We suggest that the rate at which trans-2-hexenal enters

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nematode bodies may be closely related to the diffusion of oxygen.

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Here, both the pot and field tests demonstrated the potential of trans-2-hexenal as a

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soil fumigant for the management of M. incognita to enhance tomato yields. trans-2-

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Hexenal may be a good nematicide that could match the efficacy of dazomet and

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abamectin. Our results showed that trans-2-hexenal at doses of 250-500 L ha-1 could

309

significantly enhanced the plant height and stem diameter of tomato plants, and these

310

results were consistent with the report that Eruca sativa, which can release (E)-2-hexenal, 15



311

improves the growth of tomato plants. 24 Furthermore, Prost et al. had emphasized the

312

role of oxylipins in the defense against pathogens in plants. 45 In addition, Miao et al.

313

confirmed that trans-2-hexenal at 21 µL L-1 could completely inhibit the mycelia growth

314

of soil-borne fungal pathogens. 31 Therefore, the main mechanisms by which trans-2-

315

hexenal promotes the growth of tomato plants might be its effectiveness for preventing

316

attacks on the roots by nematodes and soil-borne fungal pathogens. Meanwhile, although

317

trans-2-hexenal at a high dose (i.e., 1000 L ha-1) can slightly inhibit the growth of the

318

plant, perhaps due to its high concentration in the soil, extending the time used to

319

volatilize excessive compounds may solve this problem.

320

Our study also found that trans-2-hexenal was effective at reducing the number of

321

nematodes in the soil and that it led to lower root galling index in a clear dose-response

322

relationship, confirming the results of Aissani et al., who reported that trans-2-hexenal

323

has significant nematicidal activity on M. incognita. 24 On the other hand, trans-2-hexenal

324

can also induce the expression of resistance-related genes. Kisimoto et al. reported that

325

trans-2-hexenal can activate the expression of defense genes in Arabidopsis thaliana that

326

lead to the accumulation of antitoxin and lignin, thus inducing resistance to Botrytis

327

cinerea in Arabidopsis thaliana. 46 Further, Hirao et al. have demonstrated that trans-2-

328

hexenal is a wounded signal in plants, and may enhance the methyl jasmonate response.

329

47

330

influence in inhibiting the nematode infestations.

331

Therefore, trans-2-hexenal may contribute to the defense of tomato plants through its

One effective indicator for evaluating the nematicidal activity is the crop yield. Our 16


Page 16 of 36

Page 17 of 36


332

study indicated that each of the treatments had a positive effect on tomato yield, and

333

trans-2-hexenal at the dose of 500 L ha-1 produced the highest yield. These results suggest

334

that trans-2-hexenal is very effective at controlling nematodes on tomato plants.

335

To date, the use of chemical nematicides is an essential practice for protecting plants

336

from nematodes. Dazomet and sulfuryl fluoride are the most common potential

337

alternatives to methyl bromide, which is commonly applied to soils to control nematodes.

338

48, 49

339

cause skin reactions. Previous studies have reported that sulfuryl fluoride has the

340

potential to play an important role in global warming due to its persistence in the

341

environment. 50 Moreover, rats fed with sulfuryl fluoride at a dose of 300 mg L-1 for 13

342

weeks suffered a range of adverse health effects, such as dental fluorosis and neurological,

343

pulmonary, and renal toxicity. 51 In contrast, rats fed with trans-2-hexenal at 1600 mg L-1

344

for 13 weeks showed no untoward hematological effects, no change in renal weight, and

345

no change in ovary weight in female rats. 52 Furthermore, trans-2-hexenal can be applied

346

as a fumigant, and it can kill nematodes more rapidly than the non-fumigation

347

nematicides due to its strong penetration both in the soil and in nematodes. This may

348

avoid the problem of siol adsorption characteristics of the non-fumigant nematicides (e.g.,

349

abamectin). In addition, trans-2-hexenal, as liquid, maybe more convenient to administer

350

than the traditional fumigant nematicides (e.g., chloropicrin, sulfuryl fluoride).

However, dazomet can irritate the eyes, and its degradation product (i.e., MITC) can

351

Overall, trans-2-hexenal is a potential alternative nematicide. However, more detailed

352

studies are needed to address the capability of trans-2-hexenal to induce nematode 17



353

resistance in tomato plants, its chronic toxicity and its persistence in the soil.

354 355

ACKNOWLEDGMENTS

356

This work was supported financially by the Foundation for Outstanding Young Scientist

357

in Shandong Province (BS2011NY012).

18


Page 18 of 36

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358


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26


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Table 1 Toxicity of trans-2-hexenal against Meloidogyne incognita Concentration mg L−1 (95% Cl)-1 a Treatment

LC50

LC90

Slop ± SE

χ2

Fumigation

2.79 b (1.81-4.37)

8.67 (5.23-34.8)

2.61 ±0.255

15.0

In-vitro (water)

102 (96.9-107.2)

162 (152-176)

6.38 ±0.479

1.34

a

95% confidence interval.

b

Data were obtained from two experiments with three replicates each.

27



Page 28 of 36

Table 2 Effects of fumigation programs on number of nematodes (Meloidogyne incognita) in the soil in 2013 and 2014 Treatments

dose per ha

Nematodes in 100 cm3 soilb BFa

30 DAT

60 DAT

90 DAT

2013 trans-2-hexenal

1000 L

128.4c ab

36.4 d

79.8 e

166.6 f

trans-2-hexenal

500 L

128.2 ab

89.4 c

178.8 c

259.8 d

trans-2-hexenal

250 L

120.6 c

125.6 b

273.2 b

380.2 b

dazomet

400 kg

129.2 a

41.2 d

140.6 d

220.2 e

abamectin

180 g

124.2 b

36.4 d

174.8 c

274.2 c

control

-

132.2 a

144.8 a

331.2 a

432.4 a

trans-2-hexenal

1000 L

103.6 b

42.2 d

62.2 e

137.2 f

trans-2-hexenal

500 L

105.8 ab

53 c

96.2 c

189.4 d

trans-2-hexenal

250 L

98.2 c

71 b

112.4 b

237.2 b

dazomet 400 kg

400 kg

108.2 a

47 d

87.4 d

162 e

abamectin 180 g

180 g

99.2 c

29 e

95 cd

227.2 c

control

-

104.6 ab

163 a

259.6 a

302.2 a

2014

a b

BF = Before fumigation, DAT = days after transplantation. Nematodes (M. incognita ) in 100 cm3 soil were collected at BF, 30, 60, 90 DAT using a standard Screening and

separation method in two growing seasons. c

Data are arithmetic means of five replications and means were separated with Tukey-Kramer HSD test. In each column,

values followed by the same letter are not significantly different (P > 0.05).

28


Page 29 of 36


Table 3 Effects of fumigation programs on the number of tomatoes per plant, the average weight of the tomatoes and the yield in two field trials Dose

Number of tomatoes

Average weight of

Yield

per ha

per plant

the tomatoes (g)

(t ha-1)

trans-2-hexenal

1000 L

12.8 aa

171.8 ab

70.1 bc

trans-2-hexenal

500 L

13.6 a

182.4 a

82.1 a

trans-2-hexenal

250 L

12.7 a

173.1 ab

75.7 ab

dazomet

400 kg

13.5 a

179.9 a

78.8 a

abamectin

180 g

13.5 a

176.3 a

77.8 a

Control

-

12.1 a

163.7 b

68.3 c

trans-2-hexenal

1000 L

12.7 a

173.9 b

73.3 bc

trans-2-hexenal

500 L

13.8 a

183.2 a

99.0 a

trans-2-hexenal

250 L

12.9 a

177.3 ab

83.8 b

dazomet

400 kg

13.7 a

180.9 ab

98.3 a

abamectin

180 g

13.4 a

179.8 ab

85.7 ab

Control

-

12.4 a

165.0 c

68.1 c

Treatments

2013 experiment

2014 experiment

a

Data are arithmetic means of five replications and means were separated with Tukey-Kramer HSD test. In each

column, values followed by the same letter are not significantly different (P > 0.05).

29



Figure captions Figure 1 Effects of treatments with trans-2-hexenal, dazomet and abamectin on the root galling index and the control effect (A) and on the number of nematode eggs (B) on the roots of tomatoes grown in Meloidogyne incognita - infested soil. Data are means ± SD of the experiment that was performed twice with seven replicates, and the means were separated with the Tukey-Kramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). The number of nematode eggs per plant was transformed to log10 (x + 1) before the statistical analysis. trans-2-Hexenal at 1000 L ha-1, T-1000; trans-2-hexenal at 500 L ha-1, T-500; trans-2-hexenal at 250 L ha-1, T-250; abamectin at 180 gha-1, AV-180; dazomet at 400 kg ha-1, DZ-400; untreated control, CON. Figure 2 Effects of trans-2-hexenal, dazomet and abamectin on plant height and shoot fresh weight in the laboratory experiments (A: plant height, B: shoot fresh weight). Data are means ± SD of seven replicates of two repetitions of the experiment, and the means are separated with the Tukey-Kramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). trans-2-Hexenal at 1000 L ha-1, T-1000; trans-2hexenal at 500 L ha-1, T-500; trans-2-hexenal at 250 L ha-1, T-250; abamectin at 180 gha-1, AV-180; dazomet at 400 kg ha-1, DZ-400; untreated control, CON. Figure 3 Effects of trans-2-hexenal, dazomet and abamectin on the galling index and the control effect on tomatoes in the field experiment. Data are means ± SD of five replicates, and the means are separated with the Tukey-Kramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). trans-2-Hexenal at 1000 L ha-1, T-1000; 30


Page 30 of 36

Page 31 of 36


trans-2-hexenal at 500 L ha-1, T-500; trans-2-hexenal at 250 L ha-1, T-250; abamectin at 180 gha-1, AV-180; dazomet at 400 kg ha-1, DZ-400; untreated control, CON. Figure 4 Effects of trans-2-hexenal, dazomet and abamectin on stem diameter and plant height of tomato plants in the field. Data are means ± SD of five replicates, and the means are separated with the Tukey-Kramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). (A, C) represent 2013. (B, D) represent 2014. trans-2Hexenal at 1000 L ha-1, T-1000; trans-2-hexenal at 500 L ha-1, T-500; trans-2-hexenal at 250 L ha-1, T-250; abamectin at 180 gha-1, AV-180; dazomet at 400 kg ha-1, DZ-400; untreated control, CON.

31



Figure 1 Effects of treatments with trans-2-hexenal, dazomet and abamectin on the root galling index and the control effect (A) and on the number of nematode eggs (B) on the roots of tomatoes grown in Meloidogyne incognita - infested soil. Data are means ± SD of the experiment that was performed twice with seven replicates, and the means were separated with the Tukey-Kramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). The number of nematode eggs per plant was transformed to log10 (x + 1) before the statistical analysis. trans-2-Hexenal at 1000 L ha-1, T-1000; trans-2-hexenal at 500 L ha-1, T-500; trans-2-hexenal at 250 L ha-1, T-250; abamectin at 180 gha-1, AV-180; dazomet at 400 kg ha-1, DZ-400; untreated control, CON. 336x96mm (300 x 300 DPI)


Page 32 of 36

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Figure 2 Effects of trans-2-hexenal, dazomet and abamectin on plant height and shoot fresh weight in the laboratory experiments (A: plant height, B: shoot fresh weight). Data are means ± SD of seven replicates of two repetitions of the experiment, and the means are separated with the Tukey-Kramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). trans-2-Hexenal at 1000 L ha-1, T-1000; trans-2-hexenal at 500 L ha-1, T-500; trans-2-hexenal at 250 L ha-1, T-250; abamectin at 180 gha-1, AV180; dazomet at 400 kg ha-1, DZ-400; untreated control, CON. 208x92mm (300 x 300 DPI)



Figure 3 Effects of trans-2-hexenal, dazomet and abamectin on the galling index and the control effect on tomatoes in the field experiment. Data are means ± SD of five replicates, and the means are separated with the Tukey-Kramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). trans-2Hexenal at 1000 L ha-1, T-1000; trans-2-hexenal at 500 L ha-1, T-500; trans-2-hexenal at 250 L ha-1, T250; abamectin at 180 gha-1, AV-180; dazomet at 400 kg ha-1, DZ-400; untreated control, CON. 305x220mm (300 x 300 DPI)


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Figure 4 Effects of trans-2-hexenal, dazomet and abamectin on stem diameter and plant height of tomato plants in the field. Data are means ± SD of five replicates, and the means are separated with the TukeyKramer HSD test (P = 0.05). The same letters are not significantly different (P ≤ 0.05). (A, C) represent 2013. (B, D) represent 2014. trans-2-Hexenal at 1000 L ha-1, T-1000; trans-2-hexenal at 500 L ha-1, T500; trans-2-hexenal at 250 L ha-1, T-250; abamectin at 180 gha-1, AV-180; dazomet at 400 kg ha-1, DZ400; untreated control, CON. 222x180mm (300 x 300 DPI)



TOC graphic 119x45mm (300 x 300 DPI)


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Nematicidal Activity of trans-2-Hexenal against Southern Root-Knot

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