Research On Root Exudate Analysis Based On Microextraction And Allelopathic Effect

Root exudate is one of the important releasing ways of allelochemicals, most of allelochemicals are organic compounds which are insoluble in water with low concentration. The research of collection, isolation, identification of root exudate and allelopathic effect has been the frontier and difficulty in the field due to the complex chemical properties and many influencing factors. Therefore, it is important to develop a simple, friendly and in situ extraction method for restoring the realcomponentsand studying the allelopathic effect. Root exudate was studied based on hot pepper as a model plant. Three analytical methods were used to extract different components from root exudate, respectively. The analytical performance was estimated comparing with solid phase extraction (SPE) and liquid-liquid extraction (LLE). The higher sensitivity and lower limit of detection were obtained when the developed methods were applied for the real sample under the optimized condition. Seed germination and seedling growth were comprehensively used to study allelopathic potential of different components from root exudate. The most appropriate donor plant and potential allelochemicals were determined with different concentrations according to biometric indicator. The exogenous potential allelochemicals were applied solely and synergistically in the donor plant to study allelopathic effect.Headspace solid phase microextraction (HS-SPME) with gas chromatography was developed to extract root exudative dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DIBP), dibutyl phthalate (DBP) and dioctyl phthalate (DOP) of Capsicum annuum in hydroponic solution. Parameters affecting extraction and desorption efficiency of5PAEs were optimized. The result showed that the optimal condition is extraction fiber of65μm PDMS/DVB, extraction temperature of80℃, extraction time of50min, stirring rate of1000r·min-1, sodium chlorideof18%, sample volume of10mL. The highest extraction efficiency of5PAEs was obtained, and5PAEs were completely desorbed at the250℃for10min. Analytical performance of HS-SPME and LLE were compared from relative standard deviation (RSD), and recovery. Recovery ranging from83.4%to104.7%, and the RSDs below8.74%were obtained when HS-SPME can be applied for the determination of5PAES in real sample under the optimum condition.PDMS tube microextraction after derivatization was applied for the determination of2,3-dimethyl phenol (2,3-DMP),2,6-di-tert butyl phenol (2,6-DTBP) and4,4’-(1-methylethyliden) bisphenol (4,4’-(1-M)BP) in aqueous solution using gas chromatography-mass spectrometry (GC-MS). Different derivatization reagents and their volume were studied.The phenolic compounds were fully derivatized using200μL pentafluoropyridine as derivative reagent. PDMS microtube was applied to extract derivative product at the extraction time of30min and extraction temperature of60℃.1.5mL isooctane can completely elute three derivative products from PDMS microtube. The high sensitivity, good reproducibility, low detection limit and satisfactory recovery were obtained for target analytes by PDMS tube microextraction after derivatization under optimal conditions comparing with SPE method when PDMS tube microextraction after derivatization was used to determine2,3-DMP,2,6-DTBP and4,4’-(1-M)BP in root culture solution of hot pepper.Hollow fiber liquid phase microextraction (HF-LPME) with three derivatization methods was developed to extract1-naphthylamine (1-NA), diphenylamine (DPA) and N-phenyl-2-naphthaleneamine (N-P-2-NA). HF-LPME with in situ derivatization was the appropriate extraction method. The effect of different derivatization reagents on extraction efficiency was studied. When15μL toluene and aceticanhydride (v/v=80:20,v/v%) were used in the hollow fiber as extraction and derivatization reagent, the optimum extraction efficiency of1-NA, DPA and N-P-2-NA was obtained using the following parameters: extraction time of30min, sample solution pH of12; extraction temperature of50℃; stirring rate of800r·min-1. Analytical performances of HF-LPME with in situ derivatization for1-NA, DPA and N-P-2-NA from RSD, LOD and recovery was better that of LLE The recovery rate ranging from90.2%to96.1%and the RSDs below9.1%were obtained when HF-LPME with in situ derivatization was applied to determine root exudate1-NA, DPA, and N-P-2-NA. This proposed method is simple, inexpensive, and environmentally friendly.Therefore, HF-LPME with in situ derivatization can be applied for the determination of the for phenylamine compound in aqueous samples.Three developed analytical methods provided new separation technique for the allelochemicals analysis based on root exudates of hot pepper. Six donor plants including Lycopersicon esculentum, Cucumis sativus, Lactuca sativa, Cucurbita moschata, Raphanus sativu and Capsicum annuum were used to study the allopathic potential of root exudates in hot pepper by bioassay. Lactuca sativa was the appropriate acceptor plant using the combination of subordinate function values and allelopathic response index.28compounds (similarity≥80%) including alkanes, phthalateester, phenolsanilinesand carboxylic acid were isolated and identified from aqueous root exudate by the combination of GC-FID and GC-MS. Six concentrations of exogenoussuspected substances (0,2,4,6,8,10μg·mL-1) were separately applied for lettuce seed for the bioassay according to the measured concentration range in sample solution. N-P-2-NA and phthalicacid (PA) were considered as root exudative potential allelochemicals by the research of germination rate, germinationpotential, root length and shoot length. Six different concentration of N-P-2-NA and PA including0,5,10,15,20,25μg·mL-1were solely and synergistically applied for hydroponic lettuce seedling for7days,Tthe effect of two potential allelochemicals on photosynthesis, respiration, plant hormone content, carbon and nitrogen metabolism, ion absorption and antioxidant ability of lettuce seedling was studied. The result showed that potential allelochemicals of N-P-2-NA and PA inhibited seed germination and seedlings growth in the concentration range from0to10μg·mL-1, and the allelopathic effect of N-P-2-NA is greater than that of PA. However, the allelopathic effect of PA is stronger that of N-P-2-NA between15and25μg·mL-1. The allelopathic interaction of N-P-2-NA and PA showed inhibitory synergistic effect.