The Allelopathy Effects Of The Extracts Of Decomposed Myosoton Aquaticum On Barnyardgrass And Its Weeding Mechanism

Barnyardgrass（Echinochloa）is one of the main malignant weeds in paddy fields,and chemical herbicides are still the most important means to control barnyardgrass.However,the excessive use of chemical herbicides has lead to the rapid development of weed resistance,and which brings new challenges to weed control.Botanical compounds with herbicidal active have the characteristics of safety,low toxicity and not easy to cause herbicide resistance.They have a wide perspective application in green agricultural production and the development of organic agriculture.In this study,we tested the allelopathic effect of 27 decomposed weed solution on barnyardgrass.We found that the solution of decomposed Myosoton aquaticum showed the best inhibitory effects on barnyardgrass,and then isolated and identified the predominant inhibitory substance in the decomposed M.aquaticum solution,and found dihydrocoumarin was the most active allelochemical.We investigated the effects of dihydrocoumarin on the growth of rice,on the growth,physiology and biochemistry of barnyardgrass seedlings,and preliminarily explored the mechanism of dihydrocoumarin for barnyardgrass.The results were as follows:（1）It was screened that the solution of decomposed M.aquaticum intestined had high inhibitory activity on the growth of barnyardgrass by the bioassay method.Through two separate tests,we found that the solution of decomposed M.aquaticum completely suppressed the germination of barnyardgrass seeds at a concentration of 20 g/L,and showed the best inhibitory effects in all 27 decomposed weed solution.The pot experiments showed that more than 20 g/kg fresh M.aquaticum decomposition inhibited the germination of barnyardgrass seeds and the growth of seedlings.（2）Dihydrocoumarin was isolated and identified as the main allelopathic substance in the solution of decomposed M.aquaticum.The decomposed M.aquaticum solution was extracted and separated by fractional extraction and silica gel column chromatography,and the M2 and M3 fractions was found to be the most active in all 31 different fractions.M2 and M3 fractions were identified by high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）,and 32 main compounds were selected according to the reliability and relative content of common compounds.Dihydrocoumarin was found to be the most active of eleven candidate allelochemical compounds using a bioassay-guided method,and it significantly inhibited the shoot and root growh of barnyardgrass at 0.01 g/L.（3）Dihydrocoumarin had strong inhibitory activity on the growth of barnyardgrass root,and it significantly inhibited the shoot growh of barnyardgrass at 10 mg/L.Dihydrocoumarin caused oxidative stress to barnyardgrass,and affected the physiological and biochemical processes such as the development of barnyardgrass root hair,but had no significant inhibitory effect on the growth of rice seedlings at 3-4 leaf stage.Compared the effects of dihydrocoumarin and pretilachlor,dihydrocoumarin and coumarin on the growth of barnyardgrass,the results showed that the inhibitory effect of dihydrocoumarin was greater on the root growth of barnyardgrass than that of pretilachlor and coumarin at low concentration（20 mg/L）.It showed that dihydrocoumarin inhibited the root and shoot growth of rice seed by Petri dish method,but had no significant inhibitory effect on rice seedlings at 3-4 leaf stage by pot method.The physiological and biochemical results of dihydrocoumarin on barnyardgrass showed as follow:the root hair was inhibited when the concentration was higher than 5 mg/L,and promoted between 5 to 1 mg/L,and there was no effect on root hair below1mg/L.Dihydrocoumarin caused a large amount of O^2-in the root tip of barnyardgrass when the concentration was more than 50 mg/L,and the cell membrane of barnyardgrass was damaged by dihydrocoumarin.Dihydrocoumarin increased the permeability of cell membrane of barnyardgrass root significantly when the concentration was more than 5 mg/L.With the increase of treatment time,the activities of SOD,POD and CAT were maximally inhibited 24hours after treatment,and then increased.（4）Transcriptome sequencing showed that dihydrocoumarin may inhibit the normal growth of barnyardgrass by affecting the signal transduction of various plant hormones and inhibiting the expression of key enzymes such as phenylalanine ammonia lyase（PAL）and 4-coumarate coenzyme A ligase（4CL）in phenylpropane biosynthesis pathway.We studied the mechanism of dihydrocoumarin on the growth of barnyardgrass seedlings by application of RNA-seq,qRT-PCR and detection the activities of PAL and 4CL.The results showed that dihydrocoumarin caused significant differential expression of auxin,cytokinin,ABA,SA and other endogenous hormone signal transduction genes in barnyardgrass,and the transduction of plant hormone was influenced by the abnormal transcriptional expression of many key proteins,such as Aux/IAA,CRE1,TGA.Dihydrocoumarin interfered with the expression of many genes of phenylpropanoid biosynthesis in barnyardgrass,and it significantly inhibited the expression of PAL,4CL and other genes after 48 hours which changed the activities of PAL and 4CL and affected the production of a variety of important metabolites.In conclusion,the decomposed M.aquaticum solution showed higher inhibitory effects on barnyardgrass,and dihydrocoumarin was the main allelochemical.Dihydrocoumarin caused oxidative stress to barnyardgrass,and affected the growth of barnyardgrass by regulating plant hormone signal transduction and inhibiting phenylpropanoid biosynthesis.