Analysis On The Resistance Effects Induced By Exogenous Betaine And Chitosan During The Pathogen Infection Of Glomerella Leaf Spot

In recent years,Glomerella leaf spot（GLS）,as a major fungal disease affecting the healthy development of apple industry,has occurred widely in the main production areas of apple in China,which has seriously reduced the yield and quality of apple and caused huge economic losses to fruit farmers.It is urgent to control this desease effectively.Chemical fungicides have traditionally been used to treat fungal diseases.However,with the concept of environmental protection and green health gradually in-depth,so the method of using green pollution-free plant immune attractants to control plant diseases came into being.At present,many studies have reported that plant immune attractants could improve the resistance to GLS,but there were few studies on the resistance of apple to this disease.Therefore,in this study,‘Gala-3’（‘GL-3’）rooting seedlings were pretreated with exogenous betaine（GB）and chitosan（CHT）,and in vitro apple leaves were inoculated with Glomerella cingulata.Firstly,suitable concentrations of betaine and chitosan were screened,and then the changes of phenotype,antioxidant enzymes activities,hormones levels,amino acids contents,phenolic substances contents,expression levels of disease-resistance related proteins and key genes in plants treated with or without exogenous treatment were compared.The aim was to reveal the effection of induced resistance to GLS by exogenous GB and CHT,which would provide some theoretical and technical basis for preventing and controlling GLS in apple producing areas to promote the healthy development of apple industry.The main results were as follows:1.On the 6^th day after inoculation with the G.cingulata,apple leaves in vitro produced disease spots,but the application of exogenous GB could reduce the area of disease spots to a certain extent.The lesion area,relative electrolytic leakage and relative chlorophyll content of isolated leaves changed to different degrees when GB was sprayed with different concentrations.GB could inhibit the infection of apple leaves after they were sprayed with exogenous GB,and 2 mM GB was the more appropriate concentration for exogenous application.With the time of inoculation going,the activities of catalase（CAT）,peroxidase（POD）and superoxide dismutase（SOD）increased by 353.3%,151.0%and 168.7%,respectively,compared with GLS group,after pretreatment with 2 mM GB.The contents of endogenous hormones,salicylic acid（SA）and jasmonic acid（JA）were increased rapidly in the early stage of inoculation,while the contents of abscisic acid（ABA）,1-aminocyclopropanocarboxylic acid（ACC）and indoleacetic acid（IAA）were decreased by37.9%,34.0%and 55.0%respectively in the late stage of inoculation.Meanwhile,the contents of proline（Pro）,glutamate（Glu）,valine（Val）,tyrosine（Tyr）and histidine（His）decreased at different periods,while the contents of chlorogenic acid,gentianic acid,caffeic acid and ferulic acid decreased at the early stage of inoculation.The expression of PR1,PR4,PR8,PAL,chitinase,β-1,3-glucanase was up-regulated,but the expression of ERF3 was down-regulated.All showed that G.cingulata could induce leaf resistance response,and exogenous 2 mM GB pretreatment could make the resistance response occur more quickly,and result in the more accumulation of resistant substances and the high express of disease resistance related genes more quickly.2.On the 6^th day after inoculation,G.cingulata caused lesion spots on isolated apple leaves,but applying exogenous CHT played an important role in reducing the area of the spots.After spraying CHT with different concentrations,it was observed that transparent film was formed on the leaf surface,and the area of lesion spot,relative electrolytic leakage and relative chlorophyll content of leaves were changed to different degrees compared with 0 g·L^-1 CHT.The spraying with exogenous CHT could resist the infection of G.cingulata to a certain extent.The 0.5 g·L^-1 CHT was the optimum concentration for external application.Compared with GLS group,pretreatment with 0.5 g·L^-1 CHT increased the activities of CAT and SOD by 2.0and 1.5 times,but had no effect on POD activity.The contents of endogenous SA and JA increased rapidly in the early stage of inoculation,while the contents of ABA,ACC and IAA decreased by 12.0%,34.0%and 27.5%in the late stage of inoculation.The contents of Pro,Glu,threonine（Thr）,alanine（Ala）and His decreased by 25.3%,47.4%,37.4%,7.0%and 34.9%respectively at the later stage of inoculation,and the contents of catechin,chlorogenic acid,coumaric acid,rutin,hyperin and phloside increased at the early stage of inoculation and then decreased at the later stage.Meanwhile,the pathogenesis related genes PR1,PR2,PR4,PR8,PAL,chitinase,β-1,3-glucanase were highly expressed,while the aging gene ERF3 was down-expressed.All showed that G.cingulata triggered the disease resistance response of leaves.Exogenous 0.5 g·L^-1 CHT could stimulate a series of defense responses of leaves more quickly,leading to the rapid synthesis of disease-resistant substances and the rapid expression of disease-resistant genes.In conclusion,the spraying with exogenous 2 mM GB and 0.5 g·L^-1 CHT respectively could induce the resistance effect forapple leaves,inhibit the infection of G.cingulata.At the same time,it could enhance the activity of antioxidant enzyme system,regulate the levels of endogenous hormones and the contents of amino acids and phenolics,and strengthen the expression levels of disease related proteins and key genes of disease resistance.It resulted in the enhanced disease resistance of apple leaves.