Physiological And Biochemical Responses Of Stylosanthes Leaves To Colletotrichum Gloeosporioides Infection And The Identification Of Antagonistic Metabolites

Stylo(Stylosanthes spp.)is a leguminous forage and green manure crop which is widely planted in tropical and subtropical regions.It has high economic and ecological value with good grass quality,drought tolerance,acid tolerance,and ability to repair and improve soil.Anthracnose is the main disease that endangers the production of stylo.At present,the prevention and control measures of stylo anthracnose in production mainly focus on planting disease-resistant varieties and chemical control,but the lack of new disease-resistant varieties in practical applications and chemical control often cause problems such as environmental pollution.Therefore,studies on the metabolic response of stylo in response to infection and identification of the antifungal metabolites of stylo can lay the foundation for further analysis of the resistance mechanism of stylo and the development of green biocontrol drugs.In this study,Stylosanthes guianensis cv.Reyan No.2 was used as the material to inoculate Colletotrichum gloeosporioides,and the changes in the physiological and biochemical indexes and metabolites of Stylosanthes guianensis cv.Reyan No.2 post inoculation were detected.At the same time,metabolites wtih the antifungal activity were screened through in vitro inhibition assay.The main findings are as follows:(1)The concentration of soluble phosphorus,potassium,calcium,iron,sodium,total phenols,flavonoids,proanthocyanidins,glucose,soluble sugars and malondialdehyde(MDA)in the leaves of stylo was differentially increased,whereas concentration of soluble magnesium was significantly reduced,compared with the control group at 96 hours after inoculation of Colletotrichum gloeosporioides in stylo.In addition,the Cu,Zn-superoxide dismutase(Cu Zn-SOD)activity,total superoxide dismutase(T-SOD)activity,peroxidase(POD)activity,phenylpropane ammonia-lyase(PAL)activity,β-N-acetylglucosaminidase(NAG)activity and total antioxidant capacity(T-AOC)were also significantly increased.The results suggested that the stylo leaves increased the total antioxidant capacity,including the enzymatic and non-enzymatic antioxidant system,in response to the oxidative damage caused by anthracnose infection.(2)Five types of metabolites,including free amino acids,fatty acids,organic acids,alkaloids,coumarin compounds were detected by mass spectrometry in response to Colletotrichum infection.A total of 39 free amino acids,14 fatty acids,29 organic acids,6coumarins and 5 alkaloids were detected.Among them,the concentration of 13 amino acids,11 fatty acids,10 organic acids,2 coumarins and 1 alkaloids was significantly increased post inoculation,indicating these metabolites may participate in the defense responses.(3)In vitro growth inhibition assay was performed with 15 metabolites significantly induced by inoculation,including carnosine,alanine,β-alanine,aspartic acid,azelaic acid,dodecanedioic acid,maleic acid,creatine,gamma-aminobutyric acid,3-hydroxybutyric acid,syringic acid,4-guanidinobutyric acid,dihydrocoumarin,6-methylcoumarin and betaine.Five metabolites have shown the inhibitory effect,including 6-methylcoumarin,dihydrocoumarin,alanine,β-alanine and azelaic acid with inhibitory rates of 42.02%,31.30%,27.90%,12.44% and 11.19% respectively.Among them,6-methylcoumarin has shown the best antifungal effect with a half-inhibitory concentration(IC50)of 138.70 mg/L on the growth of Colletotrichum gloeosporioides.In summary,this study analyzed the response of stylo leaf during Colletotrichum gloeosporioides infection at the physiological,biochemical and metabolic level,and initially identified 6-methylcoumarin as a metabolite with a good antifungal effect on Colletotrichum gloeosporioides.The research results will provide a theoretical basis for the green biocontrol of stylo anthracnose.