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Use of lentinan to control sharp eyespot of wheat, and the mechanism involved Zhongxiao zhang, Hongyan Wang, Kaiyun Wang, LIli Jiang, and Dong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04665 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 3, 2017

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Journal of Agricultural and Food Chemistry

Title Page

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Title: Use of lentinan to control sharp eyespot of wheat, and the mechanism involved

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Authors: Zhongxiao Zhang a, Hongyan Wang a,d, Kaiyun Wang a, LiliJiangb,c*, DongWang c

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a

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P. R. China

Department of Plant protection, Shandong Agricultural University, Tai’an, Shandong 271018,

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b

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Shandong 271000, P. R. China

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c

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China

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d

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250100, P. R. China

Shandong Institute of Pomology, Shandong Academy of Agricultural Science, Tai’an,

Department of Agronomy, Shandong Agricultural University, Tai’an, Shandong 271018, P. R.

Cotton Research Center, Shandong Academy of Agricultural Sciences, Ji’nan, Shandong

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Corresponding authors:

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Dr. Lili Jiang, Shandong Institute of Pomology, Shandong Academy of Agricultural Science,

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Tai’an, Shandong 271000, P. R. China. Tel: +8605388266607, Email:[email protected];

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[email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Lentinan (LNT), a complex polysaccharide with a β-(1→3)-linked backbone of

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D-glucose residues, has been reported to inhibit plant diseases. Our objective was to

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explore the efficacy and action mechanism of LNT used as a seed dressing to control

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sharp eyespot of wheat. Seed dressing promoted wheat growth. At control

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germination rates of 50%, 8 g LNT/100 kg seeds of the Jimai 22, Shannong 23, and

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Luyuan 502 cultivars significantly increased seed germination to 54%, 52%, and 51%,

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respectively. Seven days after emergence, the heights and root activity of wheat

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treated with LNT were significantly greater than those of controls. These effects were

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dose dependent. At this time, the plant heights of Jimai 22, Shannong 23, and Luyuan

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502 cultivars were 9.52, 8.52, and 10.52 cm, respectively, significantly higher than the

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controls. LNT prevented the development of wheat sharp eyespot. In the highly

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susceptible Jimai 22 cultivar, sharp eyespot development was reduced by 33.7%,

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31.9%, and 30.4% at 7, 14, and 21 days after germination. LNT somewhat increased

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phenylalanine ammonia-lyase, peroxidase, and superoxide dismutase activity; reduced

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the malondialdehyde content; increased chlorophyll a and b levels; and enhanced root

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vigor of wheat. These effects peaked 7 days after germination. LNT increased

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transcription of the genes encoding alternative oxidase (AOX), β-1,3-glucanase

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(GLU), the salicylic acid signaling pathway-related gene NbPR1a, and the sharp

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eyespot resistance–related gene RS33. A significant dose–effect relationship was

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evident in terms of AOX transcription; we thus speculate that AOX may be the target

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gene.


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Keywords: Wheat sharp eyespot; Lentinan; Seed treatment; Induced resistance



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INTRODUCTION

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Wheat is the third most important cereal crop worldwide after rice and corn. In

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2014, 224 million ha of wheat were grown worldwide, and 729 million metric tons

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were harvested.1 Rhizoctonia cerealis, the causal agent of wheat sharp eyespot, has

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become endemic in many countries (except those of South America). Wheat damage

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caused by the disease is becoming increasingly severe.2 Traditional agrochemicals

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play critical roles in disease control but compromise the environment and induce

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pesticide resistance. Plant activators have attracted much interest; these materials

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induce system-acquired resistance (SAR), triggering plant immunity and countering a

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broad spectrum of diseases.3,4

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β-1,3–1,6 glucans are major inducers of plant immunity,5which can afford

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broad-spectrum disease resistance by increasing the oxidative burst6,7 and activate

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downstream defenses such as enzymes involved in the phenylpropanoid pathway and

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pathogenesis-related proteins.8,9 Still, they can enhance resistance to viral10and fungal

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infections.11Previous reports indicated that β-1,3–1,6 glucan could protect tobacco and

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Arabidopsis from tobacco mosaic virus infection12 associated with enhanced H2O2

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production at infective sites, upregulation of defense-related genes, enhanced callose

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and phenol deposition, and increased hypersensitive response-like cell death.13In rice

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(Oryza sativa) suspension cells, β-1,3 glucancan stimulate chitinase and

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phenylalanine ammonialyase activity(PAL)14, and these enzymes are important for

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plant disease resistance. Anusuya (2015) found that foliar spray of β-d-glucan


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nanoparticles (0.1%, w/v) can elicit marked increase in the activity of defense

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enzymes and significantly reduce the rot incidence offering 77% protection.15

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Lentinan(LNT) is a neutral polysaccharide within the fruiting body of Lentinus

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edodes featuring a β-(1→3)-linked backbone of D-glucose residues to which two

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β-(1→6)-D-glucosyl residues are attached to one in five of the main-chain D-glucose

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residues(Fig.1).16,17 LNT exhibits antimicrobial and antibacterial activity and also

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inhibits infection by both naked and enveloped viruses, primarily during early-stage

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infection.18 LNT both blocks virus entry and increases host resistance by enhancing

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chitinase and β-1,3-glucanase activity. Wang et al. (2013) found that LNT improved

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the resistance of apple to Penicillium infection.19 However, the induction effect of

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LNT on wheat disease and the induce mechanism are not clear. In this study, the

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biochemical and molecular approaches were applied to investigate the potential of

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LNT as a seed dressing controlling sharp eyespot of wheat and the possible

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mechanisms in wheat.

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MATERIALS AND METHODS

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Fungal isolates, culture media and fungicides. Rhizoctonia cerealis strains used

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in this study were provided by the Microbe Provincial Key Laboratory of the

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Shandong Agricultural University. The strains were used for subsequent experiments

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after activation and purification.

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Potato dextrose agar (PDA) medium, prepared with 200 g potato, 20 g dextrose and

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20 g agar for 1 L, was used for activation, cultivation and fungicide toxicity tests of R.



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cerealis. For liquid cultivation, the same media was prepared, only excluding the

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addition of agar.

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The fungicides used, fludioxonil(98%), thifluzamide(96%), validamycin(64%),

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carbendazim(98%), were all technical grade. Stock solutions (1×104µg/mL) were

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prepared with 3% hydrochloric acid solution for carbendazim and acetone for others,

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and stored at 4°C.

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Extraction and purification of LNT. L. edodes, bought from Yutai, Shandong

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Province, was decocted with water into decoction. And the LNT was extracted by

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water extraction and alcohol precipitation. LNT was purified as follows: to remove

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protein by Sevag’s method20, to remove pigment by active carbon adsorption, then

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throughD101 macroaperture resin column (Solarbio, Beijing, China), ADS7 polymer

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adsorbents column (Solarbio, Beijing, China), DEAE A25 cellulose and Sephadex

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G200 column (Solarbio, Beijing, China) in turn. The polysaccharide contents of LNT

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were measured by Vitriol–anthrone taking anhydrous glucose as standard control. At

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last the purified LNT was obtained, and the polysaccharide content (w/w) was 91%.

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In vitro toxicity test. A series of LNT dilutions was prepared based on results of

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preliminary toxicity experiments. 1 mL of each diluted solution was mixed with 50

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mL of sterilized PDA medium before pouring in plates. Mycelial plugs of activated

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strains with 5 mm in diameter were used to inoculate the center of the PDA plates.

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After 8 d incubating at 25°C in the dark, the colony diameter of each plate was

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measured in two perpendicular directions, subtracting the original inoculation plug (5

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mm)21. The 50% effective concentration (EC50) was estimated from a regression curve


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between the percentage of inhibition and the log10-transfomed concentration of the

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fungicide22.

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Effects of LNT seed dressing on wheat germination and seedling growth. We

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explored the effects of LNT on the Jimai 22 and Shannong 23 wheat cultivars which

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are sensitive to sharp eyespot and on the resistant Luyuan 502 cultivar. Wheat seeds

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were surface-disinfected for 3 min in 1% (w/v) NaOCl followed by 3 min in 70% (v/v)

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ethanol and rinsed three times in sterile distilled water.23 Then we coated seeds (100

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kg) with 4 or 8 g LNT or same volume of sterile water (control).

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Germination test: Sterilized wet sand (60–70 mesh) was placed in glass dishes with

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diameter 15 cm, each of which received 25 seeds (four dishes in every treatment) and

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grown at 20 ± 2°C under a relative humidity of 70 ± 5% and a light dark (L:D) ratio

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of 14 h:10 h.24 All experiments were performed in triplicate. All germination rates

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were recorded when the control germination rates were 50% and 100% and 3 days

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later.

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Seedling growth test: Nutrient and particle uniform soil, with moisture content

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about 60%, was packed into plastic pots, sown 10 seeds each (in triplicate), and

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cultivated in a greenhouse at 20±2°C, a relative humidity of 70 ± 5%, and an L:D

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ratio of 14 h:10 h. Plant heights were recorded 7, 14, and 21 days after emergence25.

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Effects of LNT on sharp eyespot. Wheat seeds dressed with LNT at 4 and 8 g/100

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kg or clean water (control) were grown in soil of uniform particle size, with a

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moisture content of about 60%. Ten seeds were sown in each pot (in triplicate)

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followed by growth in a greenhouse at 20 ± 2°C, a relative humidity of 70 ± 5%, and



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an L:D ratio of 14 h:10 h. Then each pot received 20 mL of a Rhizoctonia cerealis

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suspension (OD420=0.56). Disease incidences and indices were recorded at 7, 14, and

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21 days after emergence. At the booting stage, sharp eyespot severity was rated on a

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scale from 0 to 7 as follows: 0, no lesions; 1, lesions on the sheath but not on the stem;

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3, lesions covering 50% of the

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stem circumference, accompanied by plant wilt or head blight; and 7, lodging and

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dying. Disease severity was calculated as follows26:

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Disease severity = [∑(number of diseased plants with a certain index value × index value)/(total number of plants investigated × highest disease index)]× 100%. Control = [(disease severity in the control group - disease severity in the treated group)/disease severity in the control group]× 100%. Chlorophyll measurement. Chlorophyll content of Jimai 22 was extracted into 80%

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(v/v) acetone and the levels spectrophotometrically determined as described by

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Lichtenthaler and Wellburn.27

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Extraction and assay of antioxidant enzymes. Leaf samples of Jimai 22 (0.1 g)

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were ground in a chilled mortar with 1% (w/v) polyvinylpolypyrrolidone and then

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homogenized in 1.2 mL 50 mM potassium phosphate buffer (pH 7.8) containing 1

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mM Na2EDTA and 0.3% (v/v) Triton X-100. The homogenates were centrifuged at

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13,000 g for 20 min at 4°C and the supernatants assayed. Peroxidase (POD) activity

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was calculated by measuring OD470 with extinction coefficient 26.8mM-1·cm-1.28

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Superoxide dismutase (SOD) was assayed with the method of Beauchamp and

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Fridovich.29 The reaction mixture contained 1.17 × 10–6 M riboflavin, 0.1 M


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methionine, 2 × 10–5 M potassium cyanide (KCN), and 5.6 × 10–5 M NBT dissolved

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in 3 mL 0.05 M sodium phosphate buffer (pH 7.8). Each reaction mixture was added

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to 1 mL enzyme solution and the mixtures (in glass tubes) illuminated by two 40 W

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Philips fluorescent tubes at 30°C for 1 h. Identical solutions kept in the dark served as

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blanks. Absorbances were read at 560 nm and SOD activity expressed in U mg–1

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protein. One unit was defined as the amount of enzyme that changed the absorbance

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by 0.1 h–1 mg protein–1.

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Extraction and assay of disease-related enzymes. PAL activity was

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spectrophotometrically assayed according to the method of Khan and Vaidyanathan.30

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Leaf samples of Jimai22 (0.1 g) were homogenized in a chilled mortar in 2 mL

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amounts of 5% (w/v) polyvinylpolypyrrolidone in 100 mM Tris-HCl buffer (pH 7.5)

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containing 14 mM β-mercaptoethanol, 5 mM dithiothreitol, 10% (v/v) Triton X-100,

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and 1% (w/v) Albumin from bovine serum(BSA). After centrifugation, 1 mL amounts

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of the supernatants were applied to a PD10 column equilibrated with 100 mM

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Tris-HCl (pH 7.5); proteins were eluted in 2 mL elution buffer and subjected to

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immediate assay. We determined PAL activity spectrophotometrically by measuring

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the OD290 with extinction coefficient 17.4mM–1·cm–1. A mixture lacking

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L-phenylalanine served as the control.

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Malondialdehyde (MDA) assay. Plant material was homogenizedin 3 mL 0.5%

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(w/v) thiobarbituric acid in 20% (w/v) trichloroacetic acid. Each homogenate was

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incubated at 100°C for 30 min and then transferred to ice or cool water to stop the

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reaction. All samples were centrifuged at 10,000 × g for 10 min and the absorbances



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of the supernatants recorded at 450, 532, and 600 nm. The MDA concentration was

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determined as follows: CMDA (µmol L–1) = 6.45(A532 – A600) – 0.56A450. This yielded

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absolute MDA concentrations (µmol g–1FW).31

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Determination of root vigor. Root vigor was explored with triphenyltetrazolium

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chloride (TTC) method.32 5 mL of 0, 0.005%, 0.01%, 0.02%, 0.03%, 0.04% TTC

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solution were taken into the test tubes, and 5 mL ethyl acetate and a small amount of

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Na2S2O4 (about 2 mg of the same amount of each tube) were added. The mixtures

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were shaken sufficiently to produce red TTF, and the ethyl acetate layer was

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transferred. Add another 5 mL ethyl acetate for TTF extract, and then take the ethyl

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acetate solution to determine OD485. Finally, standard curve was drawn with the TTC

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concentration as the abscissa and the OD485 as ordinate.

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Root samples of Jimai 22 (0.5 g) were immersed in a mixture of 10 mL

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Na-phosphate buffer (pH 7.0) and 10 mL 0.4% (w/v) TTC for 1 h at 37°C in the dark,

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and then 2 mL of 1 M H2SO4 was added to stop the reaction. The root samples were

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removed and ground with silica sand suspended in ethyl acetate to extract 1, 3,

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5-triphenylformazan (TTF), followed by measurement of absorbance at 485 nm to

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calculate the loss of TTC. Root vigor was calculated as in eq. (1):

TTC loss (mg) × 1,000 (1) root weigh t (g) × time (h)

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Root vigor =

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RNA extraction and real-time quantitative RT-PCR. Leaf tissue of wheat grown

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from seeds exposed to 4 and 8 g LNT/100 kg was collected 7 days after seedling

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emergence and stored at –80°C. Total RNA was extracted using a Plant Total RNA Kit

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(Omega, USA) according to the manufacturer’s instructions. RNA integrity was


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confirmed via 1% (w/v) agarose gel electrophoresis, and OD260 and OD280 values

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were measured using a micro-spectrophotometer. First-strand cDNA was synthesized

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using a RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, USA).

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RT-PCR reactions were performed in triplicate on each of three replicates. The

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relative level of transcripts coding for wheat was determined using Actin (Forward-CP

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5′-CACTGGAATGGTCAAGGCTG-3′and

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5′-CTCCATGTCATCCCAGTTG-3′) as internal control. The values were averaged

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and normalized using the ∆∆CT method, and the control level was subtracted from the

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mean test levelto determine the fold change afforded by treatment. The relative

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transcript levels were expressed as 2–∆∆CT values.33 Wheat resistance related genes34-37

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(Xgwm526, Xwmc364, csGS etc.), salicylic acid pathway related genes(NbPR1a,

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NbrbohB)38 and chitinase related alleles(IR5, IR2 etc.)39,40 were selected for RT-PCR

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test. Specific primers were designed using the Primer Express software (Sandon

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Biotech, Shanghai, China)(Table 1).

Reverse-CP

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Statistical analyses. The effects of data processing were examined using

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analysis of variance (ANOVA) and when the F-test was significant (P < 0.05), means

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values were compared using the Student-Newmane-Keuls test (SPSS, version 18.0 for

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Windows).

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RESULTS AND DISCUSSION

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The results indicated that fludioxonil, thifluzamide, validamycin and carbendazim

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had higher toxicity on the hypha growth of Rhizoctonia cerealis, with EC50 all lower

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than 10 mg·L-1. Meanwhile, LNT, a biological resistance induce agent, showed poor



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direct fungicidal effect on this pathogen, and EC50 was 178.94 mg·L-1 (Table 2).

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Previous studies demonstrated that, biological elicitors could control plant diseases

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via stimulation of plants disease resistance41, even they have no obvious fungicidal

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effect.

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Effects of LNT seed dressing on wheat germination and growth. When the

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control group germination rate was 50%, the LNT treated germination rates were

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relatively higher. With a dressing rate of 4 g LNT/100 kg seeds, the germination rates

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of Jimai 22, Shannong 23, and Luyuan 502 were 53.50%, 52.75%, and 51.50%

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respectively, which were higher than 50.25%, 50.50%, and 50.50% of the control. The

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complete germination rates of the LNT treated groups were similar with those of the

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controls. Thus, LNT could slightly accelerate the germination rate of wheat (Table 3).

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The plant heights in biological elicitor treated groups were significantly higher than

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those in control, and the LNT effect was dose dependent. For example, 7 days after

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emergence, the heights of Jimai 22, Shannong 23, and Luyuan 502 seed treated with 8

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g LNT/100 kg were 9.52, 8.52, and 10.51 cm, respectively, the figures for groups of 4

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g LNT/100 kg seed were 8.85, 7.51, and 8.50 cm, respectively, and the control groups

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were 7.35, 6.28, and 7.01 cm. However, 21 days after complete emergence, plant

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heights did not differ significantly between the two LNT groups, although both groups

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were significantly higher than the control group (Table 4).

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Effects of LNT seed dressing on sharp eyespot of wheat. As an efficient

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chemical fungicide, fludioxonil is widely used for wheat sharp eyespot control in the

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field.42 From table 5, we can see that with concentration increasing of fludioxonil, the


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prevention and control effect on sharp eyespot of wheat were obviously increased. At

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the 4 g/100 kg dose, 7 days after wheat complete germination, their control effects

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were all higher than 80%. However, control effects of seed treated LNT on sharp

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eyespot of wheat was much lower than those of fludioxonil. The higher LNT dose

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reduced sharp eyespot development only by 30% 7 days after complete germination.

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Interestingly, seed dressing of LNT could exert longer duration for the control of

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sharp eyespot of wheat. With the 8 g/100 kg dose of LNT, sharp eyespot indexes for

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Jimai 22, Shannong 23, and Luyuan 502 cultivars were reduced by 30.8%, 26.5%, and

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31.4% 21 days after complete germination. And resistance induction effect of LNT to

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Rhizoctonia cereal is in wheat maybe the mechanism.

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Effects of LNT on defense enzyme levels and MDA content. In plants, resistance

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induced by plant activators would provide defense effect against pathogens, and

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resistance could manifest various defensive responses, such as oxidative burst,

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cell-wall reinforcement, and phytoalexin synthesis.43 Figure 2 shows that LNT

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dressing affected PAL, POD, and SOD activity and MDA content differently.

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Phenylpropanoids increase plant resistance to pathogens. PAL catalyzes the first,

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essential rate-limiting step in phenylpropanoid synthesis.44 And it is considered a

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defense enzyme because both the intermediate products (phenols) and final products

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(lignin, flavones, and isoflavones) of the PAL pathway mediate resistance to plant

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pests.45 After seed dressing with 4 and 8 g LNT/100 kg, the PAL activity after 3, 7,

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and 21 days were higher than those of the control, being most obvious 7 days after



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emergence. The effect of high-dose LNT was slightly greater than that of low-dose

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LNT.

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Plants have evolved both enzymic and nonenzymic mechanisms that efficiently

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scavenge excess reactive oxygen species (ROS),46 SOD and POD are important in this

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context. SOD protects against oxidant stress by disrupting the superoxide anion to

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oxygen and H2O2. In addition, H2O2 may be involved in phenol oxidation during plant

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defense, such reactions are catalyzed principally by POD (induced by various

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stressors to biosynthesize phenolic compounds).47 We found that POD activity tended

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to increase early and then decrease. POD activity after LNT treatment was higher than

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that of the control, and the activity after high-dose LNT treatment was slightly higher

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than after low-dose treatment. POD activity peaked 7 days after emergence. SOD

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activity did not vary greatly among the control and test samples at 3, 7, 14, or 21 days

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after emergence.

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MDA, which mediates membrane lipid peroxidation,48 severely damages various

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enzymes and membranes. Compared to the control, LNT at 4 and 8 g/100 kg seed

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significantly reduced the MDA level, but the two LNT doses did not differ in this

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regard. Overall, LNT increased defense enzyme activity and decreased MDA content,

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most notably on day 7 after emergence(Fig. 2).

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Effects of LNT seed treatment on chlorophyll content. Chlorophylls a and b are

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essential for converting light energy to stored chemical energy. Leaf chlorophyll

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content is closely related to the level of plant stress and the stage of senescence.49

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Figure 3 shows that the chlorophyll a and b contents of plants changed similarly over


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time, first increasing rapidly and then gradually stabilizing. Both LNT doses increased

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the levels of leaf chlorophyll a and b to different extents, peaking at 7 days after

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emergence. The effect of LNT at 8 g/100 kg seeds was slightly higher than that of the

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lower dose, but the difference was not significant.

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Effects of LNT seed treatment on root vigor. Root vigor is important for

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terrestrial plant growth, reflecting efficient nutrient absorption and disease

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resistance.50Figure 4 shows that the root activity of wheat treated with 4 g and 8 g

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LNT/100 kg seed was significantly higher than that of the control. The root vigor of

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wheat whose seeds were treated with 8 g LNT/100 kg was 0.19, 0.22, 0.21, and 0.18

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mg·g–1·h–1 at 3, 7, 14, and 21 days after emergence, thus 1.73-, 1.57-, 1.62-, and

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1.29-fold that of the control, respectively. LNT seed dressing was particularly

294

effective during early development.

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Effects of LNT seed treatment on transcription of disease resistance–related

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genes. The effects of LNT seed treatment on transcript levels of resistance related

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genes differed among the various cultivars. In Jimai 22, LNT increased the relative

298

expression levels of both disease resistance–related genes and the chitinase gene. The

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relative expression levels of the AOX- and RS33-encoding genes increased 6.964- and

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7.499-fold, respectively, when seeds were treated with 8 g LNT/100 kg; the figures

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for the lower LNT dose were 1.76- and 1.37-fold, respectively. In addition, at the

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lower and higher LNT doses, the relative expression levels of the β-1,3-glucanase

303

(GLU)-encoding gene were 6.93 and 6.48, and those of the salicylic acid signal

304

pathway-related gene NbPR1a were 4.64 and 3.83. For Shandong 23, the relative



305

expression levels of AOX--encoding gene and NbPR1a were 3.14- and 1.38-fold

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higher in plants grown from seeds treated with the higher rather than lower LNT dose.

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In addition, the relative expression levels (compared to controls) of the

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GLU-encoding gene were 6.41 and 6.28 in plants grown from seeds treated with the

309

higher and lower doses of LNT, respectively; the figures for the broad-spectrum

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resistance–related gene encoding Lr46/Yr29 were 5.91 and 5.06. For Luyuan 502, the

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relative expression levels of the AOX- and Lr46/Yr29-encoding genes in plants grown

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from seed treated with 8 g LNT/100 kg were 3.32- and 3.69-fold those of plants

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grown from seed treated with 4 g LNT/100 kg. In addition, in plants grown from seed

314

treated with 4 and 8 g LNT/100 kg, the relative expression levels of the

315

GLU-encoding gene were 3.32 and 3.69 and those of the NbPR1a3.51 and 3.26

316

compared to controls. Thus, LNT treatment significantly improved expression of the

317

AOX, GLU, NbPR1a, and RS33 genes. AOX expression was dependent on the dose of

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LNT in all three wheat varieties, and we thus speculate that increased AOX expression

319

may mediate the observed increase in resistance to sharp eyespot of wheat induced by

320

LNT(Fig. 5).

321

In summary, LNT improved the wheat germination rate, promoted early growth,

322

and countered sharp eyespot of wheat. LNT seed treatment somewhat increased PAL,

323

POD, and SOD activity; reduced the MDA content; increased chlorophyll a and b

324

levels; and enhanced root vigor. All of these effects peaked 7 days after germination.

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LNT treatment enhanced transcription of AOX, GLU, NbPR1a, and RS33. The


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increase in AOX transcription was dependent on the dose of LNT; we thus speculate

327

that AOX is the principal LNT-targeted gene.

328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348



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REFERENCES (1)

FAOSTATS.

[Online].

Food

and

Agriculture

Organization,

United

351

Nations(2017).Available:http://faostat.fao.org/site/339/default.aspx,data accessed on

352

10th July 2017.

353

(2) Hamada, M. S.; Yin, Y. N.; Chen, H. G.; Ma, Z. H. The escalating threat of

354

Rhizoctonia cerealis, the causal agent of sharp eyespot in wheat. Pest Manag Sci.2011,

355

67, 1411-1419.

356 357 358 359 360 361

(3)Jung, H. W.; Tschaplinski, T. J.; Wang, L.; Glazebrook, J.; Greenberg, J. T. Priming in systemic plant immunity. Science. 2009, 324, 89-91. (4) Jones, J. D. G.; Dangl, J. L. The plant immune system. Nature. 2006, 444, 323−329. (5) Shibuya, N.; Minami, E. Oligosaccharide signalling for defence responses in plant. Physiol Mol Plant P. 2001, 59, 223-233.

362

(6) Klarzynski, O.; Descamps, V.; Plesse, B.; Yvin, J. C.; Kloareg, B.; Fritig, B.

363

Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and

364

systemic resistance against tobacco mosaic virus. Mol Plant Microbe In. 2003, 16,

365

115-122.

366

(7) Klarzynski, O.; Plesse, B.; Joubert, J. M.; Yvin, J. C.; Kopp, M.; Kloareg, B.;

367

Firtig, B. Linear β-1,3glucans are elicitors of defense responses in tobacco. Plant

368

Physiol. 2000, 124, 1027-1038.


Page 19 of 37


369

(8) Mercier, L. C.; Lafitte, G.; Borderies, X.; Briand, M. T. Esquerré-Tugayé;

370

Fournier, J. The algal polysaccharide carrageenans can act as an elicitor of plant

371

defence. New Phytol. 2001, 149, 43–51.

372

(9) Ménard, R.; de Ruffray, P.; Fritig, B.; Yvin, J. C.; Kauffmann, S. Defense and

373

resistance-inducing activities in tobacco of the sulfated β-1,3 glucan PS3 and its

374

synergistic activities with the unsulfated molecule. Plant Cell Physiol. 2005, 46,

375

1964-1972.

376

(10) Kopp, M.; Rouster, J.; Fritig, B.; Darvill, A.; Albersheim, P. Host-pathogen

377

interactions: XXXII. A fungal glucan preparation protects Nicotianae against infection

378

byviruses. Plant Physiol. 1989, 90,208–216.

379 380

(11) Reglinski, T.; Lyon, G. D.; Newton, A. C. Induction of resistance mechanisms in barley by yeast-derived elicitors. Ann Appl Biol. 1994, 124, 509–517.

381

(12) Ménard, R.; Alban, S.; de Ruffay, P.; Jamois, F.; Franz, G.; Fritig, B.; Yvin, J.;

382

Kaufmann, S. β-1,3 Glucan sulfate, but not β-1,3 glucan, induces the salicylic acid

383

signaling pathway in tobacco and Arabidopsis. Plant Cell. 2004, 16, 3020-3032.

384

(13) Trouvelot, S.; Varnier, A. L.; Allegre, M.; Mercier, L.; Baillieul, F.; Arnould, C.;

385

Daire, X. et al. A β-1,3glucansulfate induces resistance in grapevine against

386

Plasmopara viticola through priming of defense responses, including HR like cell

387

death. Mol Plant Microbe In. 2008, 21, 232–243.

388 389

(14)

Inui,

H.;

Yamaguchi,

Y.;

Hirano,

S.

Elicitor

actions

of

N-acetylchitooligosaccharides and laminarioligosaccharides for chitinase and



390

L-phenylalanine ammonia-lyase induction in rice suspension culture. Biosci Biotech

391

Biochem. 1997, 61, 975–978.

392 393

(15) Anusuya S, Sathiyabama M. Foliar application of β-D-glucan nanoparticles to control rhizome rot disease of turmeric. Int J Biol Macromol, 2015, 72, 1205-1212.

394

(16) Saito, H.; Ohki, T.; Sasaki, T. A 13C-nuclear magnetic resonance study of

395

polysaccharide gels. Molecular architecture in the gels consisting of fungal, branched

396

(1→3)-β-D-glucans (lentinan and schizophyllan) as manifested by conformational

397

changes induced by sodium hydroxide. Carbohydr. 1979, 74, 227–40.

398

(17) Konno, N.; Nakade, K.; Nishitani, Y.; Mizuno, M.; Sakamoto, Y. Lentinan

399

degradation in the lentinula edodes fruiting body during postharvest preservation is

400

reduced by down regulation of the exo-β-1, 3-glucanase EXG2. J Agr Food Chem.

401

2014, 62, 8153-8157.

402

(18) Rincao, V. P.; Yamamoto, K. A.; Ricardo, N. M. P. S.; Soares, S. A.; Meirelles,

403

L. D. P.; Nozawa, C.; Linhares, R. E. C. Polysaccharide and extracts from Lentinula

404

edodes: structural features and antiviral activity. Virol J. 2012, 9 ,1–6.

405

(19) Wang, J.; Wang, H. Y.; Xia, X. M.; Li, P. P.; Wang, K. Y. Synergistic effect of

406

Lentinula edodes and Pichia membranefaciens on inhibition of Penicillium expansum

407

infections. Postharvest Biol Technol. 2013, 81, 7-12.

408

(20) Wang, J.; Yu, G.; Li, Y.; Shen, L.; Qian, Y.; Yang, J.; Wang, F. Inhibitory

409

effects of sulfated lentinan with different degree of sulfation against tobacco mosaic

410

virus (TMV) in tobacco seedlings. Pestic Biochem Phys, 2015, 122, 38-43.


Page 20 of 37

Page 21 of 37


411

(21) Liu, Y.; Liu, Z.; Hamada, M. S.; Yin, Y.N.; Ma, Z.H. Characterization of

412

laboratory pyrimethanil-resistant mutants of Aspergillus flavus from groundnut in

413

China. Crop Prot. 2014, 60, 5-8.

414

(22) Brandt, U.; Schägger, H.; Von, J.G. Characterisation of binding of the

415

methoxyacrylate inhibitors to mitochondrial cytochrome c reductase. Eur J Biochem.

416

1988, 173, 499–506.

417

(23) El-Sayed, E. S. A.; El-Didamony, G.; El-Sayed, E. F. Effects of mycorrhizae

418

and chitin-hydrolysing microbes on Viciafaba. World J Microbiol. Biotechnol. 2002,

419

18, 505–515.

420

(24) Qayyum, M. F.; Abid, M.; Danish, S.; Saeed, M. K.; Ali, M. A.Effects of

421

various biochars on seed germination and carbon mineralization in an alkaline soil.

422

Pakistan J Agric Sci. 2015, 51, 977-982.

423

(25)Liu, T.; Zhu, L.; Xie, H.; Wang, J.; Wang, J.; Sun, F.; Wang, F. Effects of the

424

ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate on the growth of

425

wheat seedlings. Environ Sci Pollut R, 2014, 21, 3936-3945.

426

(26)Peng, D.; Li, S.; Chen, C.; Zhou, M. Combined application of Bacillus subtilis

427

NJ-18 with fungicides for control of sharp eyespot of wheat. Biol Control, 2014, 70,

428

28-34.

429

(27) Lichtenthaler, H. K.; Wellburn, A. R. Determinations of total carotenoids and

430

chlorophylls b of leaf extracts in different solvents. Biochem Soc Trans. 1983, 4,

431

142–196.



432

(28) Rao, M. V.; Paliyath, G.; Ormrod, D. P. Ultraviolet-B- and ozone-induced

433

biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol.

434

1996, 110, 125–136.

435 436 437 438

(29) Beauchamp, C.; Fridovich, I. Superoxide dismutase: improve assays and an assay applicable to acrylamide gels. Ann. Biochem. 1971, 44, 276–287. (30) Khan, N.; Vaidyanathan, C. A new simple spectrophotometric assay of phenylalanine ammonia-lyase. Curr Sci. 1986, 55, 391–393.

439

(31) Qiu, Z. B.; Guo, J. L.; Zhu, A. J.; Zhang, L.; Zhang, M. Exogenous jasmonic

440

acid can enhance tolerance of wheat seedlings to salt stress. Ecotox Environ Safe.

441

2014, 104, 202-208.

442

(32) Kong, X. S.; Yi, X. F. Measurement of plant root vigor. In: Experimental

443

technical of plant physiology. Chinese Agriculture Press, Beijing, China. 2008, 67-71.

444

(33) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using

445

real-time quantitative PCR and the 2− ∆∆CT method. Methods. 2001, 25, 402–408.

446

(34) Mohler, V.; Zeller, F. J.; Wenzel, G.; Hsam, S. L. Chromosomal location of

447

genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em

448

Thell.). 9. Gene MlZec1 from the Triticum dicoccoides-derived wheat line Zecoi-1.

449

Euphytica. 2005, 142, 161-167.

450

(35)Xiao, M.; Song, F.; Jiao, J.; Wang, X.; Xu, H.; Li, H. Identification of the gene

451

Pm47 on chromosome 7BS conferring resistance to powdery mildew in the Chinese

452

wheat landrace Hongyanglazi. Theor Appl Genet. 2013, 126, 1397-1403.


Page 22 of 37

Page 23 of 37


453

(36)Herrera-Foessel, S. A.; Singh, R. P.; Huerta-Espino, J.; Rosewarne, G. M.;

454

Periyannan, S. K.; Viccars, L.; Lagudah, E. S.Lr68: a new gene conferring slow

455

rusting resistance to leaf rust in wheat. Theor Appl Genet. 2012, 124, 1475-1486.

456

(37)Zheng, S.; Li, Y.; Lu, L.; Liu, Z.; Zhang, C.; Ao, D.; Wu, Y. Evaluating the

457

contribution of Yr genes to stripe rust resistance breeding through marker-assisted

458

detection in wheat. Euphytica. 2017, 213, 50.

459

(38) Yang, W.; Xu, X.; Li, Y.; Wang, Y.; Li, M., Wang, Y.; Chu, Z. Rutin-Mediated

460

Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA

461

Signal Pathway. PloS one. 2016, 11, e0146910.

462

(39) Nishijima, R.; Yoshida, K.; Motoi, Y.; Sato, K.; Takumi, S. Genome-wide

463

identification of novel genetic markers from RNA sequencing assembly of diverse

464

Aegilops tauschii. Mol genet genomics. 2016, 291, 1681-1694.

465

(40) Wang, J.; Xu, H.; Liu, H.; Li, M.; Kang, Z. Expression Analysis of Three

466

Wheat Resistance-related Genes Induced by Blumeria Graminis. Chinese Agricultural

467

Science Bulletin.2011, 27, 48-51.

468 469

(41)Walters, D. R.; Ratsep, J.; Havis, N. D. Controlling crop diseases using induced resistance: challenges for the future. J Exp Bo. 2013, 64, 1263-1280.

470

(42)Hamada, M. S.; Yin, Y.; Chen, H.; Ma, Z. The escalating threat of Rhizoctonia

471

cerealis, the causal agent of sharp eyespot in wheat. Pest Manag Sci. 2011, 67,

472

1411-1419.



473

(43) Gan, X., Hu, D., Wang, Y., Yu, L., Song, B. Novel trans-Ferulic Acid

474

Derivatives Containing a Chalcone Moiety as Potential Activator for Plant Resistance

475

Induction. J Agr Food Chem, 2017.65,4367-4377

476

(44) Mauch-Mani, B.; Slusarenko, A. J. Production of salicylic acid precursors is a

477

major function of phenylalanine ammonia-lyase in the resistance of Arobidopsis to

478

Peronospora parasitica. Plant Cell. 1996, 8, 203–212.

479

(45) Zhang, L.; Chang, J. H.; Luo, Y. W. Activity changes of POD, PPO, PAL of the

480

different Sorghum Genotypes invaded by Aphis sacchari. Zehnter. Chinese

481

Agricultural Science Bulletin. 2005, 21 40–42. (in Chinese with English abstract)

482 483

(46) Inze, D.; Montagu, M.V. Oxidative stress in plants. Cur.Opin.Biotechnol. 1995, 6, 153–158.

484

(47) Hashmi, M. S.; East, A. R.; Palmer, J. S.; Heyes, J. A. Hypobaric treatment

485

stimulates defence-related enzymes in strawberry. Postharvest Biol Tec. 2013, 85,

486

77-82.

487

(48) Bailly, C.; Benamar, A.; Corbineau, F.; Come, D. Changes in malondialdehyde

488

content and in superoxide dismutase, catalase and glutathione reductase activities in

489

sunflower seed as related to deterioration during accelerated aging. Plant Physiol.

490

1996, 97, 104–110.

491

(49) Merzlyak, M. N.; Gitelson, A. A.; Chivkunova, O. B.; Rakitin, V. Y.

492

Nondestructive optical detection of leaf senescence and fruit ripening. Physiol Plant.

493

1999, 106, 135–141.


Page 24 of 37

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494

(50) Kajikawa, M.; Morikawa, K.; Abe, Y. Yokota, A.; Akashi, K.1 Establishment

495

of a transgenic hairy root system in wild and domesticated watermelon (Citrullus

496

lanatus) for studying root vigor under drought. Plant Cell Rep. 2010, 29, 771-778.

497



498

FIGURE CAPTIONS

499

Fig. 1 The chemical structural formula of Lentinan (LNT)

500

Fig. 2 Effect of seed treated lentinan on defense enzyme activities and MDA content of wheat

501

(Jimai22)

502

Fig. 3 Effect of seed treated lentinan on chlorophyll content of wheat(Jimai22)

503

Fig. 4 Effect of seed treated lentinan on root vigor of wheat(Jimai22)

504

Fig. 5 Effect of seed treated lentinan on transcript quantity of wheat disease resistance-related

505

genes

506 507 508 509 510 511 512 513 514 515 516 517 518 519


Page 26 of 37

Page 27 of 37


520 521 522

Table 1 Groups of real-time quantitative PCR (RT-qPCR) primers used to amplify genes pecific regions

523 Primer IR5 IR2 DQ-GLU IR6 WMC44 csGS WMS533 NbPR1a NbrbohB Xgwm526 Xwmc364 GFP-RS33 Actin

Sequence 5’- GCCGTTCGCATAGTCAATC-3’ 5’- CGCACCATTATTCGCTTGT-3’ 5’- GACATCCATTTCCAGGGGC-3’ 5’- GCGGTCTGGGCATTCATC-3’ 5’- GCTGGAAAGGATGTTGCT-3’ 5’- TGCCCGTTACACTTGGAT-3’ 5’- CACTGGGTCGTGACACTTCT-3’ 5’- CCTCCTCTTCCTTGTATGCTG-3’ 5’- GGTCTTCTGGGCTTTGATCCTG-3’ 5’- TGTTGCTAGGGACCCGTAGTGG-3’ 5’- AAGATTGTTCACAGATCCATGTCA-3’ 5’- GAGTATTCCGGCTCAAAAAGG-3’ 5’- AAGGCGAATCAAACGGAATA-3’ 5’- GTTGCTTTAGGGGAAAAGCC-3’ 5’- CGTTGAGATGTGGGTCAATG-3’ 5’- CCTAGCACATCCAACACGAA-3’ 5’- GTGATGCTCGTTCTGCTCTT-3’ 5’- CTTTAGCCTCAGGGTGGTTG-3’ 5’- CAATAGTTCTGTGAGAGCTGCG-3’ 5’- CCAACCCAAATACACATTCTCA-3’ 5’- ATCACAATGCTGGCCCTAAAAC-3’ 5’- CAGTGCCAAAATGTCGAAAGTC-3’ 5’-TGGAGAGGACAGCCCATGGAGTTGGTAGTAGGTGC-3’ 5’-GCCCTTGCTCACCATGCTGCTGATAACATGATCCA-3’ 5’-CACTGGAATGGTCAAGGCTG-3’ 5’-CTCCATGTCATCCCAGTTG-3’

524 525



526

Page 28 of 37

Table 2 Indoor toxicity test of LNT and fungicides to Rhizoctonia cerealis Fungicide

Regression equation

R2

EC50 value (mg·L-1)

95% Confidence interval

Fludioxonil Thifluzamide Validamycin Carbendazim

Y=0.76+1.47X Y=1.09+1.05X Y=1.57+2.22X Y=-2.52+3.05X

0.985 0.986 0.952 0.967

0.312 4.695 6.532 8.222

0.081~0.574 3.863~5.399 4.741~8.323 2.249~12.319

Lentinan

Y=-3.11+1.40X

0.982

178.94

121.3~234.6

527 528


Page 29 of 37


529

Table 3 Results of LNT seed dressing germination test of wheat

530 Germination rate (%)

Dosage Wheat variety

Jimai 22

Shannong 23 Luyuan 502

(g a.

Control

i./100kg

germination

seed)

50%

CK

-

50

95

97

Lentinan

4

52

97

97

Lentinan

8

54

97

98

CK

-

49

96

97

Lentinan

4

51

96

97

Lentinan

8

52

98

98

CK

-

48

95

97

Lentinan

4

50

96

98

Lentinan

8

51

97

98

Agentia

Control total germination

531 532


3 days after control total germination


533

Page 30 of 37

Table 4 Effect of LNT seed dressing on the growth of wheat seedlings

534 Height (cm) Wheat

Dosage (g a. i./100kg seed)

7d

14 d

21 d

CK

-

7.35±0.57c

18.50 ±0.91b

27.50±1.36b

Lentinan

4

8.85±0.43 b

19.70 ±1.12ab

29.37± 1.61a

Lentinan

8

9.52±0.26 a

20.97±0.98 a

30.98± 1.83a

CK

-

6.28±0.37c

15.50 ±0.84b

26.50±1.65b

Lentinan

4

7.51 ±0.53b

15.96±0.69 b

26.97± 1.53b

Lentinan

8

8.52 ±0.41a

17.98± 1.08a

28.58± 1.34a

CK

-

7.01±0.45c

17.50 ±0.74b

26.50± 1.58b

Lentinan

4

8.50 ±0.62b

19.96 ±1.13a

27.98± 1.82a

Lentinan

8

10.51± 0.73a

20.27±1.06 a

28.02± 1.97a

Agentia variety

Jimai 22

Shannong 23

Luyuan 502 535

Note: Numbers with the same letter in the same column are not significantly different at P = 0.05 according to

536

Duncan's multiple range tests, vice versa, and the same as follows.

537


Page 31 of 37


538

Table 5 Control effect of seed treatment on wheat sharp eyespot Wheat

Agentia

Dosage (g a. i./100 kg

Control effect（%）

seed)

7d

14 d

21 d

Fludioxonil

2

60.6±6.3c

58.5±7.4c

49.7±3.6c

Fludioxonil

4

82.5±9.1d

78.4±6.2d

66.1±4.1d

Lentinan

4

25.8±1.9a

23.2±1.5a

22.8±1.6a

Lentinan

8

33.4±3.7b

31.7±2.9b

30.8±1.8b

Fludioxonil

2

58.6±3.6c

56.3±2.8c

51.9±6.2c

Shannong

Fludioxonil

4

82.3±6.5d

76.3±5.3d

71.5±2.5d

23

Lentinan

4

25.6±2.0a

23.3±1.8a

22.5±1.9a

Lentinan

8

30.3±1.7b

28.6±1.4b

26.5±3.5b

Fludioxonil

2

50.2±3.7c

44.8±3.5c

41.2±2.6c

Luyuan

Fludioxonil

4

81.6±2.5d

76.3±4.1d

71.2±5.2d

502

Lentinan

4

35.7±3.6a

32.1±1.9a

29.4±2.3a

Lentinan

8

37.9±2.7b

34.5±2.1b

31.4±1.6b

variety

Jimai 22

539 540



541

542 543 544 545

Fig. 1 The chemical structural formula of Lentinan(LNT)


Page 32 of 37

PAL

40 35 30 25 20 15 10 0

5 CK

15

20

25

POD 140 120 100 80 60 40 20 0 0

LNT 8

5

10

SOD

3 2.5 2 1.5 1 0.5 0

15

Time (d) LNT 4

CK

20

25

LNT 8

MDA 2.5 MDA (µmol·g-1)

SOD Activity (U·g-1)

546

547 548 549 550 551

10

Time (d) LNT 4

POD Activity (U·min-1·g-1)


PAL Activity (U·min-1·g-1)

Page 33 of 37

0

5 CK

10 15 Time (d) LNT 4

20

25

LNT 8

2 1.5 1 0.5 0 0

5 CK


20

25

LNT 8

Fig. 2 Effect of seed treated lentinan on defense enzyme activities and MDA content of wheat (Jimai22)

552



1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

553 554

Chlorohpyll b 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Content (mg·g-1)

Content (mg·g-1)

Chlorophyll a

5 CK


Page 34 of 37

20 LNT 8

25

-5 CK

5 15 Time (d) LNT 4

25 LNT 8

Fig. 3 Effect of seed treated lentinan on chlorophyll content of wheat(Jimai22)

555


Page 35 of 37


Root vigor

0.3

Vigor (mg·g-1·h-1)

0.25

556 557

0.2 0.15 0.1 0.05 0 0

5 CK

10 Time (d) LNT 4

15

20

25

LNT 8

Fig. 4 Effect of seed treated lentinan on root vigor of wheat(Jimai22)

558 559



Jimai 22 Relative transcript level

9 8 7 6 5 4 3 2 1 0

Gene CK

560

LNT 4

LNT 8

Relative transcript level

Shannong 23 8 7 6 5 4 3 2 1 0

Gene CK

LNT 4

LNT 8

561 Luyuan 502

Relative transcript level

6 5 4 3 2 1 0

CK

562 563 564

Gene LNT 4

LNT 8

Fig. 5 Effect of seed treated lentinan on transcript quantity of wheat disease resistance-related gene

565


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566 567

TOC

Graphic

568 569

570 571 572 573


Use of Lentinan To Control Sharp Eyespot of ... - ACS Publications

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