Fungicidal Activities Of Cuminic Acid And Its Lethal Mechanisms Against Fusarium Oxysporum F. Sp. Niveum

Cuminic acid（p-isopropyl benzoic acid）,isolated from the seed of Cuminum cyminum,could severely inhibit several plant pathogens,whereas the specific fungicidal mechanisms of cuminic acid are still unknown.In this research,five studies including:spectrum determination of cuminic acid;activity and biochemical characteristics of cuminic acid against Sclerotinia sclerotiorum,Valsa mail and Fusarium oxysporum f.sp.niveum（FON）;functional identification of genes vel1 and lae1 from velvet complex and bik2,bik3 parted in biosynthesis of bikaverin in FON by gene knockout as a possible target of cuminic acid and molecular docking of cuminic acid with potential target proteins were processed,then following results were obtained:1.Fungicidal activities of 12 chemical compounds of botanical aromatic acid against plant pathogens were systemically studied.Results indicated that among the 12 acids,cuminic acid was confirmed with the most significant effect and the broadest spectrum.The EC₅₀ values of cuminic acid against S.sclerotiorum,V.mail and FON were 4.17,18.76 and22.87μg/ml,respectively.Meanwhile,bioactiveties of cuminic acid were based on its reactive group,rather than for acidic environment by cuminic aicd.2.From in vivo and in vitro experiments,cuminic acid showed significant control effects against three pathogens above,and protective effects were more significant than therapeutic effects,control effects of cuminic acid at 1000μg/ml against these three pathogens were all more than 50%.After treatment with cuminic acid,these three pathogens were found with similar biochemical alterations,including mycelia cluster and twine intensified,surface atrophy and collapse;increase of membrane permeability and glycerol content of mycelia;decrease of main pathogenic factors such as oxalic acid,pectase and fusarium acid toxins.Besides,self-defensive enzymes of host plants,including SOD,POD,CAT and PAL were enhenced by cuminic acid.These results indicate that cuminic acid not only has high fungicidal activity,but also can improve the host plant system defense.3.Four gene deletion mutants of FON,including velvet complex genes vel1 and lae1（?fovel1,?folae1）,and bikaverin biosynthetic genes bik2 and bik3（?fobik2,?fobik3）,as well as their corresponding complementation mutants（?fovel1-C,?folae1-C,?fobik2-C and?fobik3-C）were constructed and positive mutations were confirmed.4.Comparison of physiological phenotypes between wild-type strains,knockout strains,and replacement strains was processed with following results:（1）for?fovel1,?folae1,mycelium color disappears,cell membrane integrity is impaired,mycelial growth rate and conidia volume are reduced;（2）forΔfobik2 andΔfobik3 mutants,pigments were disappeared completely under dark conditions,sensitivities to oxidative pressure were increased,and POD activities were decrease;（3）All changes in the knockout mutants are restored in their corresponding replacement strains.All these results indicated that biocide synthesis and bacterial resistance to host system defense were regulated by bik2 and bik3genes,and cell membrane structure,mycelial growth,conidial production,bikacin and fusarium toxin synthesis and pathogenicity in FON were regulated by vel1 and lae1 genes.5.Effects of physiological and biochemical changes of each strain by cuminic acid showed that compared with EC₅₀ values of the acid against the wild strains,that values againstΔfovel1,Δfolae1,Δfobik2,andΔfobik3 mutants were increased by 3.41,2.23,1.16,and 1.57 times,respectively.The effects on the physiological effects toΔfovel1 andΔfolae1are weaker than those to the wild strains,indicating that velvet family proteins are one of the targets of fulvic acid.6.Homology models of fovel1,folae1,fobik2 and fobik3 were constructed with the online server SWISS-MODEL.Molecular docking of cuminic acid with fovel1,folae1,fobik2 and fobik3 were performed using Autodock software.Docking results with the minimized reaction energy were selected and binding sites were analyzed with PyMOL software.The results indicated that ARG206,ASN208 and Glu273 at lae1protein are binding sites for cuminic acid with 5 hydrogen bonds,and bond distances were 1.9,2.3（ARG206）,2.2（ASN208）and 2.1,2.4（GLU273）,respectively,with a binding energy of-4.64 kcal/mol;ARG104 and ARG163 at vel1 protein were the binding sites for the cuminic acid with 4 hydrogen bonds,and the bond distances were2.2,1.9 and 2.3（ARG104）,and 1.7（ARG163）,respectively,with a binding energy of-5.34 kcal/mol.In conclusion,this study confirmed the potential of cuminic acid as a natural alternative to commercial fungicides or a leading compound to develop new fungicides for the control of plant diseases.The fungicidal mechanism of cuminic acid against FON was investigated as following:cuminic acid directly acts on folae1 and fovel1 from the velvet complex to reduce the production of bikaverin and fusaric acid,which can significantly reduce the tolerance of FON to host defense and decrease its virulence on watermelon plants.