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Extraction of allelochemicals from germinated wheat seeds and their inhibitory effects on cucumber Jingwen Bai , Xuedong Bai, Yu Yang *, Bo Tao *o College of Arts and Sciences, Northeast Agricultural University, Harbin 150030, China' College of Agronomy, Northeast Agricultural University, Harbin 150030, China * Corresponding author. Tel.: +86 451 551911442; fax: +86 451 55190243 E-mail address: yangyu_002@163.com(Y. Yang). * Corresponding author. Tel.: +86 451 551910990; fax: +86 451 55190243 E-mail address: Botaol@163.com (B. Tao). Abstract Microwave-assisted extraction (MAE) ofallelochemicals from germinated wheat seeds was optimized by using response surface methodology. Under the optimal conditions70 °C, power 283W, liquid-to-solid ratio 17 mL/g and time 20.0 min), the inhibitory rate ofallelochemicals on cucumber seeds germination was 64.21%±1.99%. Three mainallelochemicals including p-Hydroxybenzoic acid, 3-methoxy-4-hydroxycinnamic acid and 3,5-dimethoxy-4-hydroxybenzoic acid were identified by LC-MS/MS analysis. These This article has been accepted for publication and undergone full peer review but has not been throughthe copyediting, typesetting, pagination and proofreading process, which may lead to differencesbetween this version and the Version of Record. Please cite this article as doi: 10.1002/agj2.20703. This article is protected by copyright. All rights reserved. allelochemicals significantly inhibited the growth of cucumber seeding and root. oo malondialdehyde content increased and root vitality and amylase activity decreased after thetreatment of allelochemicals. Glutathione content, glutathione peroxidase and glutathionetransferase activities rose first and then fell with the allelochemicals concentration increasedThese results suggested that the extract from germinated wheat seeds could inhibit the growthof cucumber by affecting the physiological and biochemical processes in cucumber. Keywords: Germinated wheat seeds; Allelochemicals; Microwave-assisted extraction;indicators; Physiological activities 1. Introduction Blind and indiscriminate use of chemical herbicides has caused crop loss and weedresistance in most arable lands worldwide. Furthermore, herbicides also lead to soil, waterpollution, and herbicide residues in food, which was a big threat to food safety and humanhealth (Farhoudi and Lee 2013). Allelopathy, as a common biological phenomenon in whichan organism produces biochemicals that influence the growth, survival, development andreproduction of other organisms, may be a possible alternative weed management strategy(Macias et al. 2014). Recently, allelochemicals have drawn much attention because of their.potential applications, such as growth regulators, herbicides, insecticides, and antimicrobialcrop protection products (Cheng and Cheng 2015; Lam et al. 2012; Macias et al. 2014). ao Wheat (Triticum aestivum L.) is the second most produced food among the cereal crops inthe world, and has been extensively studied for its allelopathic potential for the managementweeds (Marta et al. 2006; Tischler et al. 2018). Previous studies revealed that the allelopathic activity of wheat was due to the release of a broad range of allelochemicals, such as phenolicacids, hydroxamic acids and short-chain fatty acids (Albuquerque et al. 2011; Wu et al.Many researchers have confirmed that the diluted extracts of wheat seedlings, straws, roots,shoot tissues, leaves, stems, and residues displayed allelopathic effects on the germinationgrowth of agricultural weeds (Wu et al. 2000; Wu et al. 2001). However, limited reports onallelochemicals from germinated wheat seeds were available. Marta et al. (2006) found thattype of metabolites in the germinated wheat seeds depended strongly on the working upprocedure of the plant material, as well as the plant growth stage. In addition, allelochemicalscan affect the physiological and biochemical processes of plants when they are absorbed intothe plant circulatory system (Weir et al. 2004). Zhang et al.(2011) revealed thatmalondialdehyde (MDA) content and glutathione (GSH) level in M. aeruginosa cellswith the increase of allelochemicals concentration and the prolongation of exposure time. Tannic acid can reduce the synthesis of amylase and acid-phosphatase in the endosperm (Cheng and Cheng 2015). Glutathione peroxidase (GSH-Px) and glutathione transferase increased remarkably under allelochemical stress, suggesting that they might serve as an effective defense tool in avoidance of oxidative damage (Erica Marusa Pergo et al. 2011). Nowadays, the allelopathic effect of allelochemicals from germinated wheat seeds on thegrowth of other plants has not been studied, and the mechanism is unclear. In general, allelochemicals extraction from different plants is the first step for the study ofao allelopathy mechanism. During the past decades, solvent extraction with distilled water,ethanol, and acetone at ambient temperature is the most common method (Tesio et al. 2011;Wu et al. 2001). However, this method is time consuming, solvent wasting, and low yield. Atpresent, microwave-assisted extraction (MAE) is increasingly widespread for different activecompounds extraction (Farhadpour et al. 2016; Moreira et al. 2017; Yang et al. 2020; Zhao etal.2021).MAE performance depends on its operational mode of heating, and allows theextraction of bioactive components without distillation and evaporation, and thereby isfor extracting thermal-labile compounds (Felkai-Haddache et al. 2016; Zhao et al. 2021).Generally, response surface methodology (RSM) model can be established to simultaneouslydesign and optimize the variables, and moreover can predict the operating condition for theresponses (Felkai-Haddache et al. 2016; Yang et al. 2020). To our knowledge, no detailedinvestigation has been conducted to predict and optimize the allelochemicals extraction fromgerminated wheat seeds with MAE using RSM. In the present study, MAE conditions (temperature, liquid-to-solid ratio, power, andfor allelochemicals from germinated wheat seeds were optimized based on the inhibitory rateusing RSM. Then, the inhibitory effect of allelochemicals on the growth of cucumber was analyzed. Finally, root vitality, GSH-Px and GST activities, MDA, GSH and amylasewere determined. 2. Materials and methodsao 2.1.Materials Nineteen wheat seed varieties were obtained from Northeast Agricultural University inHarbin, which were coded as DN-1 to DN-19. The wheat seeds including Longfu series(from Longfu-1 to Longfu-16) and the other five varieties were provided by HeilongjiangInstitute of Agricultural Science (Harbin, China). Seventeen indicating crop seeds werebought from Xinhua seed company (Harbin). All other chemicals were of analytical grade. 2.2. Germination ofwheat seeds Wheat seeds were surface-sterilized for 15 min with 2.5% (v/v) sodium hypochloritesolution, and then rinsed with sterilized distilled water. Two pieces of filter paper (9 cmdiameter) soaked with 5.0 mL sterilized aqueous were placed in each Petri dish, and then 20wheat seeds were placed. The dishes were incubated in darkness in a growth chamber at 22C and 80% humidity. After 36 h of incubation, the seeds with embryo root breaking throughseed coat 2 mm were collected. 2.3. Screening ofindicating crop Seventeen crops,including cucumber, chicory, valerian, spinach, beans, lettuce, carrot,tomato, loofah, big radish, mustard, ramie, xanthium, radish, shallot, Chinese cabbage, and 5 This article is protected by copyright. All rights reserved. ooo rape were treated with the same concentration of extract from germinated wheat seeds. Then,the suitable indicating crop was selected according to response index (RI) of germinationrate, root length, seeding height, and seeding fresh weight. RI was determined according tothe previous reference (Williamson and Richardson 1988): if T> C, then RI=1-(C/T), ifT
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