Study On The Allelopathy Mechanism Of Coumarin Inhibiting Lolium Multiflorum

In the face of the destruction of natural ecosystems caused by chemical herbicides,China proposes zero growth of fertilizers and pesticides by 2020.At present,the development of a new type of green bioherbicide based on allelochemicals has become a research hotspot.The coumarin has the advantages of small molecular weight,high bioavailability and simple synthesis,and can be fully exploited as a natural herbicide.Therefore,this study used coumarin as a donor and Lolium multiflorum as the recipient plants,determining the content of hormone and storage substance during seed germination under different coumarin concentration and different treatment times,observing the changes of seed internal structure under scanning electron microscope and transmission electron microscope,testing the changes of physiological and biochemical indicators,gene expression,and coumarin residues in seedlings.Provide theoretical basis for the development of new biological source herbicides of coumarin.Experiment 1:Effect of seed germination of Lolium multiflorum under coumarinLaboratory bioassay was used to test the initial stages of seed germination determining the changes of storage substances content and hormones.The results showed that different concentrations of coumarin inhibited the germination of seeds significantly(P<0.05),and the inhibitory effect increased with increasing concentration;In the process of germination,the occurrence of germination delay and unevenness was observed;There are lots of stems breaking through the seed coat firstly in the treatment group,while roots breaking through seed coat firstly in the control group.Starch content in the treatment group was higher than control group significantly(P<0.05),and the higher treatment concentration,the higher starch content;The content of soluble sugar in the treatment group was lower than that in the control group.Coumarin inhibits GA and IAA hormone secretion and promotes ABA hormone secretion.The observation of scanning electron microscope showed that the starch in the control group was polygonal and evenly distributed,and the starch granule volume became larger,appearing amyloid composed of multiple starch granules after treatment,and the number increases with increasing concentrations;The original ellipsoid becomes more concave and flat,showing adhesion phenomenon under 200 mg/L treatment;Transmission electron microscopy showed that the treated cell wall became thicker or even deformed;The organelles were degraded and large vacuoles were decomposed into small vacuoles.Experiment 2:Effect of physiological and biochemical Indexes and gene expression of Lolium multiflorum under coumarinThe experiment was conducted in pots in the greenhouse.Selection of experimental concentrations was according to the fatality rate of the plants in the preliminary experiments.The important physiological and biochemical indicators and the expression of antioxidant enzyme genes during the growth of the seedlings were determined.The results showed that plant height,root length,stem fresh weight and dry weight,chlorophyll a,chlorophyll b,and total chlorophyll of Lolium multiflorum were decreasing with increasing coumarin concentration.Relative conductivity increases significantly with increasing treatment concentration(P<0.05);The content of MDA in the treatment group was higher than that in the control group.The activity of CAT and POD in the treatment group was higher than control group,SOD activity was higher than the control group in the 3,7,and 14 d,and lower than the control group in 21 d;The gene expression levels of CytCu/ZnSOD,MnSOD,POD and CAT antioxidant enzymes were significant upregulation(P<0.05).Experiment 3:Effect of ultrastructure and coumarin content of Lolium multiflorum under coumarinThe experiment was conducted in pots in the greenhouse.The leaf cell structure was observed and analyzed from the microscopic level and the coumarin content in Lolium multiflorum was quantitatively analyzed by LC-MS.The results showed that the chloroplasts changed from oval,fusiform to spherical and osmiophilic granules increased;The lamellae appeared scattered,fractured,discontinuous phenomenon,and some mitochondria were deformed under the treatment with coumarin;The central large vacuoles were lysed,the chloroplasts no longer closely aligned with the cell wall and disintegrated,the cell structure was almost destroyed under the 500 mg/kg concentration.After treatment for 21 days,ryegrass was back to normal by self-regulation under 50 mg/kg concentration,and starch granules increased in resistantance to stress under 200 mg/kg concentration,and organelles were almost degraded under 500 mg/kg concentration.The content of coumarin in Lolium multiflorum increased with the increase of concentration on the same day;The coumarin content reached a maximum at 1 d,followed by a decreasing trend with the increase of treatment time under same concentration treatment.