| Previous studied of the research group showed that Amaranthus tricolor extract has inhibitory effect on a variety of plant pathogenic bacteria,such as: Acidovorax avenae subsp.citrulli、Xanthomonas oryzae pv.oryzicola、Xanthomonas axonopodis pv.citri,etc.which substances are playing a role,and what are their components.Aiming at this scientific problem,this study used Acidovorax avenae subsp citrulli as the target bacteria,carried out the separation and purification of amaranth extract,and analyzed the active components by TLC,column chromatography,NMR and other technologies,in order to provide theoretical basis for the next development of antibacterial lead compounds.The main results are as follows:(1)Determination and identification of pathogenicity of target bacteria: the activity of crude extract of amaranth to A.avenae subsp.citrulli was evaluated by the method of indoor plate bacteriostatic circle.The results showed that the leaves of watermelon seedlings infected by target bacteria showed water spots after 3 days of acupuncture treatment,and the spots gradually extended to the base of cotyledons into strip or irregular dark green water spots,and then turned into brown necrosis spots,accompanied by yellow halos.The results showed that the target bacteria had pathogenicity to watermelon,met the test requirements,and had the conditions to be the target bacteria.After activation of target bacteria,DNA,PCR amplification,gel electrophoresis and phylogenetic analysis revealed that the homology between the target strain(strain LQ-2)and the model strain ATCC(NR＿102856.1)was 99%,so it was identified as A.avenae subsp.citrulli.(2)Selection of the best varieties of amaranth: the activity of crude extracts of different amaranth varieties to A.avenae subsp.citrulli was evaluated by the method of bacteriostatic circle.The results showed that HYY had the best bacteriostatic effect,the diameter of bacteriostatic circle was 1.86 cm,the next was GYH,and the others were in the order of the diameter of bacteriostatic circle from big to small: YHY,HLY,XBY,XHL,GYH,QYJ and YSX.HYY(Amaranthus tricolor)is the best materials for antibacterial test.(3)Preliminary study on chemical constituents of Amaranthus tricolor leaves: the components of Amaranthus tricolor leaves were separated and identified by HS–SPME–GC–MS.42 substances were identified,including alkanes,ketones,esters,alcohols and organic acids,with the relative peak areas of 36.43%,28.84%,11.35%,10.03%and 8.51% respectively.The results showed that there were organic acids,phenols,alkaloids,flavonoids,coumarins,lactones,steroids,sterols,saponins,anthraquinones,glycosides and cardiotonides in the extracts.The results showed that there were flavonoids,lactones and organic acids in the extracts.(4)Isolation,purification and structure identification of Amaranthus tricolor leaf ester extract: the antimicrobial components of Amaranthus tricolor leaf ester extract were separated,purified and identified by various chromatography and NMR techniques.Six pure compounds were obtained by separation and purification.Compounds 1,2,3,4,5 and 6were identified as P-hydroxybenzoic acid,Anchoic acid,4-hydroxy-5-methoxy-benzoic acid,Salicylic acid,Benzofuran-2-carboxaldehyde and P-coumaric acid.Among them,compound2,compound 3 and compound 5 were isolated from this genus for the first time.(5)Indoor toxicity test of pure compounds: the inhibitory activity of six compounds on A.avenae subsp.citrulli was determined by the method of indoor plate bacteriostatic circle.The results showed that compound 1 and compound 2 had a good inhibitory effect on A.avenae subsp.citrulli,among which compound 1 had the best effect,the diameter of bacteriostatic circle was 3.23 cm,and the diameter of bacteriostatic circle was 1.36 times that of compound 2.The mic and MBC of compounds 1 and 2 were further determined by plate double dilution method.The results showed that mic value of compound 1 to A.avenae subsp.citrulli was 125 μg/mL,MBC was 500 μg/mL,MIC of compound 2 to A.avenae subsp.citrulli was 250 μg/mL,MBC value was 1000 μg/mL.It is suggested that compounds1 and 2 have the potential to further evaluate the bacteriostatic ability.(6)Determination of bacteriostatic spectrum of pure compounds: the inhibition effect of compounds 1 and 2 on four plant pathogenic bacteria was evaluated by the method of indoor plate bacteriostatic circle.The results showed that both compound 1 and compound 2 had inhibitory effect on all the tested plant pathogenic bacteria,and the inhibitory effect was higher than that of the antibiotic in the positive control.The antimicrobial activities of the two compounds against the tested bacteria were in the order of the diameter of the inhibition circle from large to small,and they were all against Xanthomonas oryzae pv.oryzae,Xanthomonas axonopodis pv.citri,Ralstonia solanacearum and Xanthomonas oryzae pv.oryzicola.The growth rate method was used to determine the inhibitory effect of compound1 and compound 2 on 7 plant pathogenic fungi.Compound 1 had the highest inhibitory effect on Phytophthora capsici(52.98%),followed by Colletotrichum capsici,Verticillium dahliae.And Pyricularia oryzae.Compound 2 had the strongest inhibition on Phytophthora capsici,with the inhibition rate reaching 100%.The others were Botrytis cinerea,Sclerotinia sclerotiorum,Rhizoctonia solani,Verticillium dahliae and Colletotrichum capsici in the order of the inhibition rate from large to small,which had no inhibition on the rice blast.The results show that compound 1 and compound 2 have broad spectrum of antibacterial activity. |
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