Plant-Mediated Interactions between Two Cereal Aphid Species

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Agricultural and Environmental Chemistry

Plant-mediated interactions between two cereal aphid species: infestation of phytotoxic aphid Schizaphis graminum in wheat promotes aphid performance but attracts more parasitoids Yong Zhang, Jia Fan, Yu Fu, Frederic Francis, and Julian Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06150 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 24, 2019

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Journal of Agricultural and Food Chemistry

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Plant-mediated interactions between two cereal aphid species:

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infestation of phytotoxic aphid Schizaphis graminum in wheat

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promotes aphid performance but attracts more parasitoids

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Yong Zhang 1, Jia Fan1, Yu Fu1, Frédéric Francis2, Julian Chen1*

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1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant

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Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P.R. China

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2 Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of

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Liège, Gembloux, B-5030, Belgium

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*Corresponding

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[email protected]

author:

Julian

Chen.

Tel.:

12 13 14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment

+86-10-62813685;

Email:


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Abstract

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Here, we investigated changes in physiological characteristics in wheat affected

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by phytotoxic aphid Schizaphis graminum and non-phytotoxic aphid Sitobion avenae

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feeding. We also determined whether shared host-mediated interspecific interactions

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occur between S. graminum and S. avenae. S. graminum but not S. avenae feeding

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induced significant chlorophyll loss and hydrogen peroxide accumulation in wheat.

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Gene expression analysis and GC/MS metabonomic results indicated that S. graminum

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infestation induced stronger salicylic acid-mediated defense responses than S. avenae

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did and significantly increased the contents of several amino acids in wheat leaves.

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Feeding on wheat preinfested with S. graminum significantly increased the

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reproduction of both aphids and shortened the development time of S. graminum.

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However, olfactometer bioassays showed that parasitoid wasp Aphidius gifuensis was

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more attracted to the odors of S. graminum-infested wheat than to those of control and

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S. avenae-infested wheat. This study demonstrates that S. graminum and S. avenae

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feeding induced different defense responses and changes in plant nutrition quality.

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Additionally, plant-mediated interactions occurred between these cereal aphids.

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Key words: Schizaphis graminum, Sitobion avenae, defense responses, nutrition

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quality, aphid performance, Aphidius gifuensis

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1 Introduction

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Plants have evolved a set of defense responses against insect herbivore attacks,

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including direct and indirect defenses. 1,2 In direct defense, secondary metabolites and

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defensive proteins induced by insect herbivores, such as toxins and proteinase inhibitors

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in plants, negatively affect the physiology of the attackers, decreasing their growth,

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survival or reproductive rate. 3 In indirect defense, plants produce and release a blend

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of volatiles, also called herbivore-induced plant volatiles (HIPVs), in response to

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herbivore damage that attracts predators, parasites, and other natural enemies. 4,5

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Jasmonic acid (JA) and salicylic acid (SA) are two important defense6,7

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associated signaling molecules involved the induction of plant defense responses.

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Insects with different feeding guilds tend to induce various plant defense responses.

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The JA-mediated defense pathway is mainly triggered by leaf-chewing herbivores as

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their feeding usually results in mechanical damage to plant tissues. 8 Hemipterans, such

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as aphids and whiteflies, have highly modified piercing-sucking mouthparts that can

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penetrate extracellular components and eventually feed on phloem sap from sieve

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elements. 9 Although stylets puncture most plant cells during probing, the damage to

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cells is minimal compared to that caused by leaf-chewing herbivores. Most research has

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demonstrated that hemipteran feeding results in the induction of SA-dependent defense

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responses, which is similar to plant responses to microbial pathogen infestation.

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Additionally, several studies have found that feeding by some herbivores can alter host

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plant nutrition conditions for their own benefit. For example, the green peach aphid

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Myzus persicae feeding altered Arabidopsis plant nitrogen metabolism by increasing 3


8, 10


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host nitrate reductase activity and subsequently free amino acids in plant phloem sap.

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11 Amino

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inadequate diet for aphids; phloem sap has a high sucrose content but a low

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concentration of essential amino acids or other sources of nitrogen.

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some aphid species, such as the Russia wheat aphid Diuraphis noxia 13 and M. persicae

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14,

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resulting in positive effects on aphid performance.

acids are essential for aphid development and fecundity, and phloem sap is an

12

Infestation of

could also significantly increase the contents of amino acids in plants, directly

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In natural ecological systems, different insect species usually attack the same plant

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simultaneously, inducing various plant defense responses and physiological changes in

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their host plants. Therefore, either positive or negative plant-mediated interactions

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occur indirectly among different species.15, 16 For example, the whitefly Bemisia tabaci

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infestation results in reduced attraction of predatory mites Phytoseiulus persimilis to

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lima bean plants preinfested with the two-spotted spider mite Tetranychus urticae by

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inhibiting spider mite-induced JA-dependent defense responses. 17 Leaf chewer Pieris

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brassicae develops faster and reaches a larger body size on Brassica oleracea

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previously infested by the phloem-sucking insect Brevicoryne brassicae than on

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uninfested plants.

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performance of T. urticae on tomato by suppressing effective JA defense responses. 19

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Previous feeding of an omnivorous predator Macrolophus pygmaeus induces JA

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defense responses in tomato and sweet pepper plants, which decreases the performance

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of two herbivores, T. urticae and the western flower thrips Frankliniella occidentalis.

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20, 21

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The russet mite Aculops lycopersici promotes the reproductive

Although several studies have investigated the interactions among different 4


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herbivores mediated by induced plant defenses, there is little information available on

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the physiological changes in wheat after cereal aphid feeding and interspecific

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interactions on the same plants.

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Worldwide, the grain aphid Sitobion avenae and the greenbug Schizaphis

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graminum are two important, destructive cereal pests that directly draw phloem sap and

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transmit various plant viruses; additionally, these pests usually feed on wheat together

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in a mixed population pattern. 22 Aphids are classified as nonphytotoxic or phytotoxic

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based on the extent to which they directly damage plant tissues by activating plant

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defenses or by other physiological alterations.

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important phytotoxic aphids, S. graminum infestation often induces obvious foliar

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chlorosis in leaves, eventually resulting in plant death in susceptible hosts. 25 Similar to

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most aphid species, S. avenae are nonphytotoxic, and no typical plant damage

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symptoms are induced immediately during feeding processes. 23 Our main hypothesis

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is that S. graminum feeding induces different plant defenses and physiological changes

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than S. avenae does. Plant-mediated interactions may also exist between S. graminum

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and S. avenae. In our study, we first detected changes in the contents of total chlorophyll

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and hydrogen peroxide (H2O2) in wheat after infestation with two cereal aphids and

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performed reverse transcription quantitative real-time polymerase chain reaction (RT-

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qPCR) and gas chromatography-mass spectrometer (GC/MS)-based metabolomics to

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examine the induction of defense responses and modification of metabolite profiles

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after aphid feeding. Then, we investigated the effects of preinfestation of S. graminum

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on aphid performance and the attraction of their natural enemy, the parasitic wasp

23, 24

As one of the most economically

5



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Aphidius gifuensis.

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2 Materials and methods

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2.1 Plants and aphids

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Seeds of aphid-susceptible winter wheat, Triticum aestivum var. Beijing 837

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26,

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were immersed in 4% sodium hypochlorite (Amresco, OH, USA) for 30 min to sterilize

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the surface. Next, the seeds were washed 3 times and germinated in distilled water for

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3-4 days at a temperature of 25 ± 1°C in sterilized petri dishes. Seedlings with similar

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sizes were carefully transferred to plastic plots with organic soil and were maintained

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in a climate chamber with a temperature of 20 ± 1°C, a relative humidity of 40-60%

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and a photoperiod of L: D = 14 h: 10 h.

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A clone of S. graminum and S. avenae was initially established from a single aphid

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collected from a wheat field in Langfang city, Hebei province, northern China, and was

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reared on wheat plants (Beijing 837 variety) for more than 8 yrs (25-30 generations

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every year) in an indoor environment (L: D = 16 h: 8 h; 20 ± 1℃).

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2.2 Aphid infestation treatments

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At the two-leaf stage, 20 wingless adult S. graminum or S. avenae or a mixture of

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10 S. graminum and 10 S. avenae were transferred to the first wheat leaf and restricted

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in a plastic ecological cage (2.7 × 2.7 × 2.7 cm) to prevent the escape of aphids. The

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edge of the ecological cage was covered with a sponge to avoid causing mechanical

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wounds on the leaf. Each pot contained one wheat plant, which was grown in a climate

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incubator with 20 ± 1°C and a photoperiod of 14: 10 h (L: D). After approximately 30

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min, the aphids had begun settling and feeding, and this time was recorded as 0 h. Plants 6


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with ecological cages but no aphids were used as control groups.

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2.3 Detection of total chlorophyll content and H2O2 in leaves after aphid feeding

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Total chlorophyll content in wheat leaves after aphid infestation was detected

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using a Chlorophyll Assay Kit (Solarbio, Beijing, China) according to the

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manufacturer’s instructions. H2O2 staining in wheat leaves was performed according to

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the histochemical methods described by Wang et al.

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Twenty wingless adult S. avenae and S. graminum were transferred to wheat plants at

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the two-leaf stage and restricted in ecological cages as previously described. After 48 h

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of feeding, leaf segments were cut off using a sterilized scissor and then immediately

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transferred to 1 mg mL-1 3’-diaminobenzidine (DAB) solution (10 mmol L-1 Na2HPO4,

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pH 3.8) under vacuum infiltration for 5 min. After incubation in a dark chamber for 8

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h, the leaves were decolorized in boiling 95% ethanol for 10 min and then hyalinized

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in saturated chloral hydrate. The leaves were stored in 50% glycerol solution and

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photographed with an Olympus SZX-16 microscope (Olympus Corporation, Japan).

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H2O2 contents were assayed using the methods recorded by Ferguson et al. 28

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2.4 Total RNA isolation and cDNA synthesis

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with some modifications.

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Aphids were gently removed from wheat leaves using a brush, and approximately

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2.5 × 2.5 cm leaf tissues from the aphid feeding (20 wingless adults) sites of each plant

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were collected using sterilized scissors. Then, the samples were transferred to liquid

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nitrogen immediately and stored at -80°C until use. Total RNA was extracted from

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leaves using TRIzol® Reagent (Invitrogen, CA, USA) following the protocols provided

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by the manufacturer. The quality and quantity of RNA were assessed by a NanoDrop™ 7



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2000 Spectrophotometer (Thermo Scientific, CA, USA). A total of 1 μg RNA was

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reverse transcribed into cDNA with TranScript One-step gDNA Removal and cDNA

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Synthesis Supermix Kit (TransGen Biotech, Beijing, China) following the

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manufacturer’s instructions, and cDNA templates were stored at -20°C until used for

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RT-qPCR.

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2.5 Real-time qPCR

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The relative expression of genes involved in the JA and SA defense signaling

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pathways of wheat was detected after 12, 24, 48, and 72 h of aphid feeding using RT-

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qPCR, and 0 h without aphid infestation was used as a control. Target genes detected

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for the JA pathway included Ω-3 fatty acid desaturase (FAD) and lipoxygenase (LOX),

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which are involved in JA biosynthesis. The genes tested for the SA pathway were SA

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synthesis enzyme phenylalanine ammonia lyase (PAL) and induced SA marker

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pathogenesis-related 1 protein (PR1). Actin and 18S RNA were used as reference genes.

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The primers were designed and synthesized according to previous studies.

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qPCR was performed using the same protocols and conditions as previously described.

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30

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consisted of 3 technical replicates.

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2.6 Sample preparation and GC/MS analysis

29, 30

RT-

There were 3 biological replicates for each treatment, and each biological replicate

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After 48 h of S. graminum and S. avenae feeding, sixty micrograms of accurately

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weighed leaf sample was transferred to a 1.5 mL tube. Two small steel balls were added

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to the tube. Three hundred sixty microliters cold methanol (Sigma-Aldrich, MO, USA)

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and 40 μL 2-chloro-l-phenylalanine (0.3 mg mL-1) (Sigma-Aldrich) dissolved in 8


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methanol as an internal standard were added to each sample, and the samples were

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incubated at -80°C for 2 min. Then, the samples were ground at 60 HZ for 2 min. The

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mixtures were ultrasonicated at ambient temperature for 30 min. Two hundred

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microliters of chloroform (Sigma-Aldrich) was added to the samples, and the mixtures

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were vortexed. Next, 400 μL water was added. Samples were vortexed again and then

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ultrasonicated at ambient temperature for 30 min. The samples were centrifuged at

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13800 g for 10 min at 4°C. The QC sample was prepared by mixing aliquots of all

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samples to create a pooled sample. An aliquot of 500 μL supernatant was transferred to

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a glass sampling vial for vacuum drying at room temperature. Then, 80 μL 15 mg mL-

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1

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mixture was vortexed vigorously for 2 min and incubated at 37°C for 90 min. A total

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of 80 μL BSTFA (with 1% TMCS) and 20 μL n-hexane were added to the mixture,

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which was vortexed vigorously for 2 min and then derivatized at 70°C for 60 min. The

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samples were incubated at ambient temperature for 30 min before GC-MS analysis.

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Leaves without aphid feeding were set as control groups. Six replicates were used for

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each treatment.

methoxyamine hydrochloride (Sigma-Aldrich) in pyridine was added. The resultant

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Derivatized samples were analyzed on an Agilent 7890B gas chromatography

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system coupled to an Agilent 5977A MSD system (Agilent Technologies Inc., CA,

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USA). A DB-5MS fused-silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent

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J & W Scientific, Folsom, CA, USA) was utilized to separate the derivatives. Helium

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(> 99.999%) was used as the carrier gas at a constant flow rate of 1 mL min-1 through

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the column. The injector temperature was maintained at 260°C. The injection volume 9



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was 1 μL by the split mode (split ratio is 4:1). The initial oven temperature was 60°C,

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which was increased to 125°C at a rate of 8°C min-1, to 210°C at a rate of 4°C min-1, to

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270°C at a rate of 5°C min-1, to 305°C at a rate of 10°C min-1, and finally held at 305°C

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for 3 min. The temperatures of MS quadrupole and ion source (electron impact) were

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set to 150 and 230°C, respectively. The collision energy was 70 eV. Mass data were

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acquired in full-scan mode (m/z 50-500), and the solvent delay time was set to 5 min.

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QCs were injected at regular intervals (every 9 samples) throughout the analytical run

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to provide a set of data from which repeatability could be assessed.

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2.7 Effects of previous infestation of S. graminum and exogenous SA application

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on aphid reproduction and development time

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For exogenous application of molecules, SA (Sigma-Aldrich, MO, USA) was

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initially dissolved in ethanol to 1 mol L-1 and then diluted into 5 mmol L-1 solution by

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distilled water. SA replaced by distilled water was used as a control (carrier solutions).

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At the two-leaf stage, the leaves of plants were sprayed with either 1 mL SA or control

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solutions using 10 mL sterilized hand sprayers in a separate room to avoid

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contamination. After the sprays dried, one adult wingless S. avenae or S. graminum was

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transferred to each first leaf and restricted in an ecological cage as previously mentioned.

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Each pot contained one wheat plant. Plants with the same treatment were reared in one

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climate incubator at 20 ± 1°C and a photoperiod of 14: 10 h (L: D). Leaves were treated

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according to the “2.2 Aphid infestation treatments” section as preinfestation treatments.

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After infestation with 20 S. avenae or S. graminum for 48 h, all aphids were gently

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removed using a brush, and one wingless adult S. avenae or S. graminum was 10


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transferred to the feeding site and restricted in the ecological cage. The number of

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newborn nymphs was recorded every day until 7 days, and all nymphs were removed

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after each record. A total of 12 replicates were acquired for each treatment.

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After wheat leaves were treated with SA or preinfestation of aphids for 48 h, 1-2

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alate adult aphids were transferred to leaves and restricted in ecological cages as

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described in the previous section. After 24 h, all adults were removed, leaving only one

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newborn nymph. Aphid nymphs were checked every 8 h to record the time they first

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produced nymphs. The period of each aphid from birth to first nymph production was

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recorded as the development time. Fifteen replicates were used for each treatment.

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2.8 Choice test

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The aphid choice test was performed as described by Zhang et al. 36 In brief, after

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the infestation of wingless adult S. graminum for 48 h, all aphids were removed. Then,

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aphid-infested and clean intact leaves (without aphids) were carefully inserted into a

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transparent plastic column (24 cm in width, 5 cm in height) from holes on opposite

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sides. Thirty winged adult S. avenae or S. graminum were collected in a 1.5 mL

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centrifuge tube and then transferred to a filter placed in the middle of the plastic column

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device. Two 14-W cool white fluorescent lights (Philips, Netherland) were placed on

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top of the plastic column. The number of aphids on each leaf was recorded after 48 h

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of release. Eight replicates were used for each test.

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2.9 Y-tube olfactometer bioassays

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A Y-tube olfactometer bioassay was performed to investigate the preference of

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parasitoid wasp A. gifuensis towards aphid-infested and control wheat plants. The 11



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treatment and control plants were placed into two 2.5 L glass containers. A Y-tube

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olfactometer (stem, 10.0 cm; arms, 10.0 cm at 60° angle; internal diameter: 2.0 cm) was

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placed in the observed chamber. Two 14-W cool white fluorescent lights (Philips,

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Netherland) were placed on top of the observation chamber to eliminate lighting bias.

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The air was first filtered through activated charcoal filter, followed by distilled water.

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Then, the air was blown into each arm at a rate of 150 mL min-1, which was controlled

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by two vacuum pumps and airflow meters (Beijing Institute of Labor Instrument,

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Beijing, China).

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Tested female parasitoids were used only once, and each individual was

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introduced into the Y-tube olfactometer at the entrance of the stem. The Y-tubes were

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replaced by clean tubes, and odor sources were also changed after 10 individuals had

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been tested. When parasitoids crossed 5 cm of the arm of the Y-tube olfactometer within

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5 min, it was a successful choice; otherwise, they were scored as “nonresponders”. For

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each treatment, a total of 50 female individuals were recorded. We presented the wasps

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with the following choices: S. graminum-infested plant vs. clean plant; S. avenae-

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infested plant vs. clean plant; S. graminum-infested plant vs. S. avenae-infested plant;

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S. graminum-infested plant vs. both S. graminum and S. avenae-infested plants; S.

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avenae-infested plant vs. both S. graminum and S. avenae-infested plants.

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2.10 Statistics analysis

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All data were analyzed using SPSS Statistics 20.0 software (SPSS Inc., Chicago,

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IL., USA), and the differences between or among groups were examined using

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independent samples t-test or one-way analysis of variance (Duncan). P values less than 12


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0.05 were considered statistically significant. The percentages of aphids that settled on

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plant leaves in the choice test were arcsine-square-root transformed before analysis. A.

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gifuensis preferences between odors were analyzed using a chi-square test (χ2 test). For

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RT-qPCR, the fold change in the expression of genes involved in the JA and SA

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signaling defense pathways after treatment compared to those in the control was

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calculated using the 2–ΔΔCT method

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normalized data were used for orthogonal partial least squares discriminant analysis

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(OPLS-DA) by SIMCA 14.1 (Umea AB, Umea, Sweden).

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3 Results

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3.1 Chlorophyll content in wheat leaves after S. avenae and S. graminum feeding

31

and was converted to log2 values.

32

The

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As shown in Figures 1a and 1b, S. graminum infestation induced obvious leaf

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chlorosis at 48 and 72 h, but S. avenae feeding had no significant effects on leaf

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symptoms. The results in Figure 1c also suggested that the total chlorophyll content in

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wheat leaves after S. avenae infestation was not significantly different from that after

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the control. However, the total chlorophyll content was significantly decreased to 2.03

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± 0.11 mg g-1 FW after 24 h of S. graminum (F 2,6 = 41.932, P < 0.001) feeding and was

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further reduced to 1.06 ± 0.12 mg g-1 FW at 72 h, which is significantly lower than that

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after the control (2.98 ± 0.13 mg g-1 FW) and S. avenae infestation (2.79 ± 0.12 mg g-1

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FW) treatments (F 2,6 = 54.705, P < 0.001).

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3.2 H2O2 detection

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DAB staining results showed that S. graminum feeding induced obvious H2O2

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accumulation at 48 hpi, but no distinct staining was found in the control leaf and at the 13



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feeding sites of S. avenae (Figure 2a). In Figure 2b, the H2O2 content was significantly

288

higher after S. graminum feeding at 12 hpi (74.87 ± 10.51 μmol g-1 FW; F 2,6 = 14.487,

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P = 0.005) than after the control and peaked at 48 h (206.13 ± 24.62 μmol g-1 FW; F 2,6

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= 31.094, P = 0.001). In addition, the H2O2 content induced by S. graminum feeding

291

was significantly higher than that induced by S. avenae feeding between 12 h and 48 h.

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The levels of H2O2 were also significantly upregulated after 48 h of S. avenae feeding

293

(101.42 ± 12.31 μmol g-1 FW; F 2,6 = 31.094, P = 0.001) but decreased at 72 h. There

294

was no significant difference between the S. avenae feeding and control groups.

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3.3 Expression of defense-related genes after infestation with two cereal aphids

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Changes in the transcript levels of some key genes involved in the JA and SA

297

defense pathways were detected at different time points after S. avenae and S.

298

graminum infestation. The results in Figure 3a show that S. graminum infestation

299

significantly increased the expression level of the JA defense-related gene LOX at 12 h

300

(0.44 ± 0.13-fold) and 24 h (0.74 ± 0.18-fold; F4,10 = 6.802, P = 0.007). After 48 h, the

301

expression levels decreased, showing no significant difference from the control. After

302

S. avenae feeding for 12 h, the expression level of LOX significantly increased (0.31 ±

303

0.029-fold; F

304

upregulated to the peak values at 24 h (0.59 ± 0.11-fold). However, its expression then

305

decreased to levels that were not significantly different from those of the control at 48

306

h and 72 h. The expression of FAD was significantly upregulated by S. graminum

307

infestation at 24 hpi (0.26 ± 0.061-fold) and 72 hpi (0.19 ± 0.054-fold; F 4,10 = 6.157, P

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= 0.009). At 24 and 48 hpi, FAD expression in leaves infested with S. avenae

4,10

= 13.068, P = 0.001) compared with that after the control and was

14


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significantly increased to 0.21 ± 0.035-fold and 0.21 ± 0.051-fold (F 4,10 = 6.862, P =

310

0.006), respectively, but decreased to a level at 72 hpi that was not significantly

311

different from the control (Figure 3b).

312

The expression of PAL in leaves infested with S. graminum significantly increased

313

and reached peak values (2.08 ± 0.34-fold) at 12 hpi. Then, PAL expression decreased

314

at 72 hpi but was still higher than that of the control (0.88 ± 0.043-fold; F 4,10 = 19.647,

315

P < 0.001). S. avenae feeding also induced significant increases in PAL expression at

316

12 (0.70 ± 0.12-fold), 24 (1.23 ± 0.077-fold) and 48 hpi (0.85 ± 0.043-fold; F

317

46.002, P < 0.001) (Figure 3c). The expression of PR1, a marker gene of the SA

318

pathway, was significantly upregulated immediately at 12 h post-S. graminum

319

infestation and peaked at 24 hpi (7.89 ± 0.051-fold). The expression level was

320

significantly greater than that of the control at any time point (F

321

0.001). S. avenae feeding also significantly increased PR1 expression at 24 hpi and

322

peaked at 72 hpi (3.01-fold; F 4,10 = 194.768, P < 0.001) (Figure 3d).

4,10

4,10

=

= 511.342, P
1

336

and p-value of Student’s test < 0.05) are shown in Table S1.

337

Several metabolites of interest with significant changes are also listed in Table 1.

338

For example, S. graminum infestation upregulated some free amino acid contents in

339

wheat leaves, such as phenylalanine (2.876-fold), proline (2.807-fold), glutamine

340

(11.110-fold), and tryptophan (2.442-fold). In contrast, no amino acids were

341

significantly upregulated after S. avenae feeding for 48 h, and glycine and lysine

342

contents were significantly decreased. SA, a plant defense signaling molecule in wheat

343

leaves, was significantly increased after S. graminum and S. avenae infestation at 48 h,

344

and the fold change in the upregulation of SA levels induced by S. graminum feeding

345

(23.198-fold) was higher than that induced by S. avenae feeding (7.231-fold).

346

3.5 Effects of exogenous application of SA on aphid reproduction and

347

developmental time

348

The expression level of PR1 in leaves was upregulated 1.39 ± 0.22-fold at 12 h

349

and 3.22 ± 0.38-fold at 24 h after SA application, showing a significant increase

350

compared to that after control application (F 2,6 = 37.684, P < 0.001) (Figure 6).

351

The effects of defense molecules in response to SA treatment on aphid performance

352

were also examined. Both the total reproduction (Figure 7) and development time 16


Page 16 of 51

Page 17 of 51


353

(Table 2) of S. graminum and S. avenae after feeding on wheat leaves treated with SA

354

solution did not differ significantly from those after feeding on wheat leaves treated

355

with control solution (P > 0.05, t-test).

356

3.6 Effects of previous S. graminum infestation on aphid performance

357

The total reproduction and development time were recorded to detect the effects

358

of aphid preinfestation on the performance of aphids and other species. The total

359

reproduction of S. graminum significantly increased after feeding on leaves infested

360

with conspecifics compared with that after feeding on control leaves (at 24 h: t22 = 2.815,

361

P = 0.01; at 48 h: t 22 = -2.4, P = 0.025) (Figure 8a). Additionally, the total reproduction

362

of S. avenae significantly increased after feeding on leaves previously infested with S.

363

graminum (at 48 h: t 22 = 3.386, P = 0.003) (Figure 8b).

364

The development time of S. graminum significantly decreased after feeding on

365

leaves preinfested with conspecifics for 48 h. However, preinfestation of S. graminum

366

had no significant effects on the development time of S. avenae at 24 and 48 hpi (Table

367

3).

368

3.7 Aphid choice test

369

In the choice test, the percentages of winged S. graminum that landed on leaves

370

preinfested with aphids were not significantly different from the percentages of those

371

that landed on control leaves (t

372

percentages of S. avenae that landed on preinfested leaves were not significantly

373

different from the percentages of those that landed on control leaves (t 14 = -0.018; P =

374

0.986) (Figure 9).

14

= -0.234; P = 0.818) at 48 h. Additionally, the

17



375

3.8 Choices of parasitic wasps in Y-tube olfactometer

376

In dual-choice assays, the female adults of wasps A. gifuensis showed a significant

377

preference for the volatiles from wheat plants after S. graminum (χ2 = 17.391, P < 0.001)

378

and S. avenae (χ2 = 5.378, P = 0.020) infestation when the volatiles from uninfested

379

plants were offered as an alternative (Figure 10). The wasps were significantly more

380

attracted to the volatiles from plants infested with S. graminum than those from plants

381

infested with S. avenae (χ2 = 5.628, P = 0.019). Furthermore, the wasps preferred the

382

odor blend from plants infested with S. graminum and the S. avenae mixed population

383

over that from plants infested with S. avenae (χ2 = 5.149, P = 0.023), indicating that the

384

presence of S. graminum attracts more parasitoids.

385

4 Discussion

386

As two important cereal pests, S. avenae (nonphytotoxicity) and S. graminum

387

(phytotoxicity) caused different plant damage symptoms in wheat plants. First, total

388

chlorophyll content in wheat leaves significantly decreased after S. graminum

389

infestation, resulting in obvious foliar chlorosis, but S. avenae infestation had no effects

390

on plant symptoms and chlorophyll content. The involvement of oxidative stress has

391

been proposed in the induction of hypersensitive cell death and senescence in plants.

392

33,34

393

in wheat leaves after S. graminum infestation than in those after S. avenae infestation.

394

Based on this finding, S. graminum feeding may cause leaf chlorosis in wheat.

In the present study, we first found that the H2O2 content was significantly higher

395

Our study also demonstrated that SA defense-related genes and SA levels in wheat

396

leaves were significantly upregulated after aphid feeding, suggesting that either S. 18


Page 18 of 51

Page 19 of 51


397

graminum or S. avenae infestation mainly induced the SA-dependent defense pathway

398

in wheat. In accordance with these findings, aphid attacks, such as M. persicae mainly

399

elevate SA levels and initiate the SA signaling cascade in plants. 35, 36 Furthermore, S.

400

graminum activated stronger SA pathway defense responses in wheat than S. avenae

401

did. Aphid saliva has proven vital roles in activating plant defense. For example,

402

salivary proteins between 3 and 10 kD secreted by M. persicae could elicit plant defense

403

responses in Arabidopsis thaliana. 37 Moreover, infiltration of S. avenae watery saliva

404

induced defense responses in wheat. 30 The different levels of plant damage symptoms

405

and defense responses induced by these two cereal aphids could be a result of divergent

406

secretory salivary proteins.

407

activities of polyphenol oxidase (PPO) in S. graminum saliva were much higher than

408

those in the saliva of S. avenae, and exogenous application of PPO increased the

409

expression level of related genes of the plant defense signaling pathway in wheat

410

seedlings.

411

chlorosis-inducing pathogenic toxins such as ToxA, are hypothesized to rapidly elicit

412

plant damage in wheat. 42 However, the underlying mechanisms remain unknown and

413

should be further investigated.

41

38, 39,40

A previous study also reported that the enzymatic

Additionally, some salivary proteins in S. graminum saliva, similar to

414

Although aphid feeding induced SA-dependent defense, wheat seedlings treated

415

with SA solution did not exhibit changes in the total reproduction and development

416

time of both S. avenae and S. graminum. Compared with JA responses, SA responses

417

may not be effective in plant resistance to aphids.

418

SA defenses in tobacco had no measurable influence on the population growth of M.

8,15

Tobacco mosaic virus-induced

19



Page 20 of 51

419

persicae. 43 In contrast, exogenous application of JA on Arabidopsis, sorghum, legume

420

and tomato plants had a negative effect on aphid population growth.

421

SA signaling pathways involve an antagonistic interaction. 45 Aphids and other species

422

in Hemiptera may be able to manipulate host plants for their own benefit by inducing

423

the SA pathway and utilizing JA-SA negative crosstalk to suppress more detrimental

424

JA defenses. 46 Several studies have also demonstrated that SA-dependent defenses act

425

against phloem feeders.

426

probably depend on the plant species. 49

47, 48

15,44

The JA and

Therefore, the roles of JA and SA in insect resistance

427

In addition to the induced plant defense responses, S. graminum feeding increased

428

the levels of several amino acids in wheat leaves, such as proline, phenylalanine, and

429

glutamine, thereby improving the nutrition quality of the host plant. These results are

430

consistent with a previous study showing that S. graminum and D. noxia feeding

431

significantly increased the concentrations of amino acids, especially essential amino

432

acids, in the phloem of wheat and barley. 50 Several upregulated amino acids induced

433

by S. graminum feeding in our study were also proven to be involved in the activation

434

of plant defense responses. For example, proline metabolism is involved in the ROS

435

burst and the hypersensitive response triggered by an avirulent pathogen; 51 moreover,

436

exogenous proline solutions induced a series of resistance responses, including SA and

437

ROS accumulation in leaves. 52 Phenylalanine is involved in the biosynthesis of SA in

438

plants via benzoic acid that phenylalanine first converts into trans-cinnamic acid with

439

the catalysis of PAL.

440

changes in nutritional quality, such as the levels of amino acids induced by insect

53, 54

These results suggest that both defense responses and

20


Page 21 of 51


441

herbivores in host plants, should be considered in the research of insect-plant

442

interactions. Given that aphids are specifically feeding on the phloem

443

assessing metabolite changes in the phloem would be interesting in future studies.

10,

directly

444

To further detect potential plant-mediated interactions between S. graminum and

445

S. avenae, we measured the total reproduction and development time of aphids after

446

they fed upon leaves previously infested with S. graminum. Although S. graminum

447

triggered strong SA defense, the total reproduction of S. graminum and S. avenae

448

increased significantly after feeding on leaves preinfested with S. graminum.

449

Additionally, the development time of S. graminum became shorter after feeding on

450

leaves previously infested with conspecifics. In our study, nutritional enhancement of

451

host plant induced by preinfestation of S.graminum may benefit aphid performance.

452

Plant phloem sap, the diet of aphids, is grossly deficient in essential amino acids which

453

are important for aphid development 12. Stronger SA defense responses and higher H2O2

454

contents activated by S. graminum feeding may cause leaf senescence-like changes in

455

susceptible wheat and result in increases in amino acid levels derived from protein

456

degradation in the host, contributing to better aphid performance. Glutamine is

457

considered the major nitrogen form translocated from senescent leaves to sink organs

458

in rice and other plants. 55, 56 The substantial increase in the levels of glutamine induced

459

by S. graminum feeding in our study supports this hypothesis. Machado-Assefh et al.

460

also found that dark-induced senescence in Solanum tuberosum leaves promoted aphid

461

ingestion and reduction in the prereproductive time of M. persicae. 57 The green spruce

462

aphid, Elatobiurn abietinum, infestation induced chlorosis in spruce trees and resulted 21



Page 22 of 51

58

463

in an increase in the amino acid levels, eventually promoting aphid performance.

464

Nutritionally advantageous changes induced by aphid preinfestation would also explain

465

why S. graminum can outbreak within a short period of time.

466

Plants release volatiles upon attacks by herbivores as indirect defense responses

467

that are repellent to herbivores or attract their natural enemies. 59, 60 A preliminary study

468

conducted by Zhu et al. demonstrated that infestation of wheat seedlings with the bird

469

cherry oat aphids Rhopalosiphum padi induced the release of several volatile

470

compounds, such as methyl salicylate and 6-methyl-5-hepten-2-one, which reduced the

471

preference of winged aphid individuals for wheat plants. 60 However, in our study, the

472

proportions of S. graminum and S. avenae settling on wheat leaves with S. graminum

473

preinfestation showed no significant differences with regard to the proportion landing

474

on clean leaves, suggesting that induced defenses by S. graminum feeding had no

475

effects on the aphid host plant choice. It is possible that the enhancement of nutrition

476

quality by S. graminum reduced and even masked the repellent effects of induced

477

indirect defenses of wheat plants on aphids. Additionally, visual perception of color

478

stimuli strongly affects aphid orientation. Most studies have shown that aphids

479

preferentially orient towards yellow surfaces.

480

chlorophyll loss and leaf yellowing and/or chlorosis induced by S. graminum feeding

481

also interfere with the repellent effects of plant indirect defenses on aphids.

482

61, 62

Therefore, it is assumed that

According to previous studies, both S. avenae and S. graminum are preferred the 63, 64

483

hosts of A. gifuensis.

Plant volatiles induced by aphid feeding play a key role in

484

the process of host location of aphid parasitoids.

65, 66

22


We found that wheat plants

Page 23 of 51


485

infested with either S. avenae or S. graminum were more attractive to the natural enemy

486

parasitoid wasp A. gifuensis than were noninfested plants. However, our research also

487

showed that A. gifuensis was significantly more attracted to the odors of plants infested

488

with S. graminum than to those of plants infested with S. avenae, increasing the

489

possibility of being parasitized by aphids. Induced volatiles vary quantitatively and

490

qualitatively according to the herbivore species feeding on the plants. 67, 68 One reason

491

for this variation may be that S. graminum feeding induced stronger SA-dependent

492

defense responses than S. avenae did, and plants released more volatiles that can attract

493

their natural enemy. It is also possible that plants on which S. graminum fed produce

494

volatile components different from those produced by plants infested with S. avenae.

495

Additionally, the parasitoids tested here may prefer these volatiles. Therefore, further

496

comparative studies on the volatile components released by wheat plants after S.

497

graminum and S. avenae feeding might aid in improving the efficiency of A. gifuensis

498

in the field and provide an important basis for pest management control.

499

In summary, our findings showed that infestation of S. graminum, a phytotoxic

500

aphid, triggered higher H2O2 accumulation and stronger SA defense responses in wheat

501

than nonphytotoxic aphid S. avenae did. Furthermore, S. graminum feeding induced a

502

significant increase in some amino acid concentrations, enhancing the nutrition quality

503

of plants. Plant-mediated interactions occurred between these two cereal aphids;

504

specifically, the performance of both S. graminum and S. avenae increased after feeding

505

on leaves preinfested with S. graminum. However, S. graminum feeding may also

506

“betray” these aphids by attracting more natural enemies. Therefore, further research is 23



507

needed to detect the volatile components induced by S. graminum infestation that

508

attracted parasitoids and the role of aphid saliva in the induction of leaf chlorosis by S.

509

graminum feeding.

510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 24


Page 24 of 51

Page 25 of 51


529

Abbreviations Used

530

HIPVs, herbivore-induced plant volatiles; JA, jasmonic acid; SA, salicylate acid; H2O2,

531

hydrogen peroxide; RT-qPCR, reverse transcription quantitative real-time polymerase

532

chain reaction; GC/MS, gas chromatography-mass spectrometer; DAB, 3’-

533

diaminobenzidine; FAD, Ω-3 fatty acid desaturase; LOX, lipoxygenase; PAL,

534

phenylalanine ammonia lyase; PR1, pathogenesis-related 1 protein; ROS, reactive

535

oxygen species; PPO, polyphenol oxidase.

536

Funding

537

This study was funded by the National Natural Science Foundation of China (31871979,

538

31371946), National Key R & D Plan in China (2017YFD0201701, 2016YFD0300701),

539

and the Cooperation Project between Belgium and China from MOST (2014DF32270).

540

Notes

541

The authors declare no competing financial interest.

542

Acknowledgements

543

The authors thank technician Ms. Yanxia Liu for aphid rearing. The authors are also

544

grateful to Dr. Hongjian Ding from Arkabsas Regional Laboratory of FDA, who kindly

545

pre-reviewed the manuscript before submission.

546

Supporting Information

547

Descriptions of Table S1.

548

Table S1. Changes in metabolite profiles of wheat leaves after 48 h of S. graminum and

549

S. avenae feeding.

550 25



551

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749

Figure Captions Figure1. Damage symptoms (a, b) and changes of total chlorophyll content (c) of wheat leaves after Sitobion avenae (a) and Schizaphis graminum (b) feeding at different time points. The values were shown as Mean ± SE. Different letters indicate significant differences among treatments (P < 0.05, Anova). Figure 2. Changes of H2O2 content in wheat leaves after S. avenae (Sa) and S. graminum (Sg) infestation at different time points using DAB staining (a) and standard curve method (b). Error bars indicate standard error (SE). Different letters indicate significant differences among Sa, Sg and control groups at the same time point (P < 0.05, Anova). Figure 3. Effects of S. graminum and S. avenae infestation on expression of genes involved in jasmonate (a: LOX; b: FAD) and salicylate-mediated (c: PAL; d: PR1) defense pathway at different time points. The results were shown as Mean ± SE log2 (Fold Change). Gray bars represent S. graminum infestation (Sg); white bars represent S. avenae infestation (Sa). Different letters indicate significant differences among treatments (P < 0.05, Anova). Figure 4. Effects of S. graminum, S. avenae and their mixed population infestation on expression of salicylate-mediated defense marker PR1 at different time points. The results were shown as Mean ± SE log2 (Fold Change). White bars, light gray bars and black bars represent S. avenae infestation (Sa), S. graminum infestation (Sg) and their mixed population infestation (Sa + Sg), respectively. Different letters indicated 35



significant differences among treatments (P < 0.05, Anova). Figure 5. Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots showing the metabolomic changes of wheat leaves with infestation of S. graminum (a), S. avenae (b). A: leaves infested with S. graminum for 48 h; B: leaves infested with S. avenae for 48 h; C: untreated leaves. Figure 6. Relative expression of PR1 in wheat leaves after the application of salicylic acid at 12 h and 24 h. Significant differences among different time points were analyzed by ANOVA test. Error bars indicate standard error (SE). Different letters indicated significant differences among treatments (P < 0.05, Anova). Figure 7. Total reproduction of S. graminun (a) and S. avenae (b) in 7 days after feeding on leaves treated with SA. Significant differences between treatments was analyzed by independent samples t test (P < 0.05). “ns” indicates no significant difference between groups. Error bars indicate standard error (SE). Figure 8. Total reproduction of S. graminum (a) and S. avenae (b) in 7 days after feeding on leaves pre-infested with S. graminum. The total reproduction of S. graminum fed on uninfested leaves was set as control groups. The values were shown as Mean ± SE. An asterisk indicates significant differences between treatments (P < 0.05; t test). Figure 9. Percentages of winged S. graminum and S. avenae landed on wheat leaves with S. graminum pre-infestation and control leaves. Gray bars represent data from leaves infected with S. graminum; white bars represent clean (non-infested) leaves. The values were shown as Mean ± SE. Figure 10. Preference of female Aphidius gifuensis towards the wheat volatiles with or 36


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without aphid attack. “Control” indicates plant without aphid infestation; “Sa” indicates plant infested with S. avenae; “Sg” indicates plant infested with S. graminum; “Sg + Sa” indicates plant infested with both S. graminum and S. avenae. Numbers in bars represent the number of A. gifuensis responding to different odors. “ns” indicates no significant difference between treatment groups and control. An asterisk indicates significant differences between treatments (P < 0.05; χ2 test).

37



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Tables Table 1. Several metabolites measured by GC/MS differentiating wheat leaves with Schizaphis graminum infestation (A), Sitobion avenae infestation (B) and no aphid treatment (C). RT: retention time; VIP: Variable Importance in the Projection. “-”: no significant changes were detected. Metabolites

RT (min)

S. graminum infestation (48 h)

S. avenae infestation (48 h)

Fold Change (A/C)

VIP

P

Fold Change (B/C)

VIP

P

Amino acids Leucine

10.324

1.965

1.161

0.000549

-

-

-

Phenylalanine

18.715

2.876

1.377

1.005E-05

-

-

-

Proline

10.841

2.807

1.128

3.98131E-05

-

-

-

Cysteine

16.953

4.859

1.445

5.86791E-06

-

-

-

Glutamine

22.584

11.110

1.356

0.000206

-

-

-

Tryptophan

32.646

2.442

1.082

0.0169

-

-

-

Ornithine

18.545

5.694

1.038

0.0134

-

-

-

Glycine

11.019

-

-

-

0.534

1.590

0.0277

Lysine

26.297

-

-

-

0.447

1.543

0.0464

15.747

23.198

1.722

0.000272

7.231

1.12705

0.00118

Plant defense hormone Salicylic acid

38


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Table 2. Developmental time of S. graminum and S. avenae after feeding on wheat leaves treated with 5 mmol L-1 SA solution. The results were represented as Mean ± SE. Significant differences among treatments was analyzed by independent samples t test (P < 0.05). Aphid

Developmental time (d) SA treatment

df

t values

P

Control

S. graminum

7.44 ±0.11

7.24 ± 0.08

28

-1.49

0.149

S. avenae

7.73 ± 0.13

7.82 ± 0.10

28

0.54

0.598

39



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Table 3. Developmental time of S. graminum and S. avenae after feeding on wheat leaves pre-infested with S. graminum. The results were represented as Mean ± SE. An asterisk indicates significant differences between treatments (P < 0.05; t test). Aphid

Treatments

Development time

t

df

P

S. graminum

S. graminum pre-infestation (24 hpi)

7.04 ± 0.12

-1.628

28

0.115

Control

7.31 ± 0.11


7.36 ± 0.12

0.652

28

0.520

Control

7.24 ± 0.11


6.89 ± 0.07 *

-3.596

28

0.001

Control

7.38 ± 0.11


7.20 ± 0.10

-1.609

28

0.119

Control

7.44 ± 0.09

S. avenae

S. graminum

S. avenae

40


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Figure graphics Figure 1. a

b

0h

12 h

24 h

48 h

0h

72 h

12 h

24 h

48 h

h

h

h

41


72 h


Figure 2.

42


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Figure 3.

43



Figure 4.

44


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Figure 5.

45



Figure 6.

46


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Figure 7.

47



Figure 8.

48


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Figure 9.

49



Figure 10.

50


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Table of Contents

51


Plant-Mediated Interactions between Two Cereal Aphid Species

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