Mechanism Of Kaolin Particle Film Regulating Grape Leaves Against Downy Mildew

Downy mildew(Downy mildew)is one of the major diseases affecting viticulture worldwide,and it often brings serious losses to grape production.At present,the most common way to control downy mildew is still spraying chemical fungicides,but frequent and largescale spraying of chemical fungicides often causes problems such as toxic residues,environmental pollution,and pathogen resistance,which seriously threatens environmental protection and healthy of human beings.Kaolin particle film(KPF)is an inert mineral with kaolin as the main component,which has been widely used to control crop pests,relieve summer stress,and improve crop yield and quality.KPF can evenly attach to the surface of crops,so it can be used as a barrier to prevent direct contact between plant surface and the external environment,including pathogens.And because the composition does not contain chemically active substances,it will not cause environmental pollution and pathogen resistance.There is great application potential for the prevention of grapevine downy mildew.In this study,the leaves of Vitis vinifera cv.Cabernet Sauvignon were used as experimental materials to investigate the effect of KPF pretreatment on the control effect of downy mildew on detach leaves and under field conditions in two consecutive years(2020 and 2021)and its effect on the temporal dynamics of downy mildew in the field.This study aims to provide a theoretical basis for studying the role and mechanism of KPF in grape disease-resistant cultivation.The primary conclusions were as follows:(1)The control effect and degree of oxidative stress of different concentrations of KPF on leaf downy mildew were investigated under detach leaves inoculation and field natural disease conditions,respectively.The results showed that in infected detach leaves,1%,2%and 3% KPF treatments significantly reduced the leaf disc disease index,and the control effects were 31.99,44.33 and 65.74%,respectively.The growth of intercellular mycelium was observed by fluorescence microscope,and the results showed that the development of mycelium between mesophyll cells in the KPF treatment group was retarded,and the formation of mycelium was reduced.Under the field natural disease conditions,the disease index conformed to the S-shaped growth curve.Due to insufficient rain tolerance,compared with the control group,3% KPF treatment had no significant effect on disease index,but 6%KPF treatment significantly reduced the field disease index in both 2020 and 2021,and the control effect exceeded 30% in both years.Compared with the control group,the field onset date of the 6% KPF treatment group was delayed by 1-2 weeks,and the exponential growth period and the logistic growth period were shortened by about 20 days.KPF treatment significantly reduced the contents of hydrogen peroxide,superoxide anion and malondialdehyde in susceptible leaves under both in vitro and field conditions.Among the non-inoculated treatments,KPF treatment alone did not affect the hydrogen peroxide,superoxide anion and malondialdehyde content of healthy leaf discs.(2)The surface condition and stomatal structure of the leaves treated with KPF were observed by scanning electron microscope.The results showed that the stomatal opening of the leaves in the control group was well,and the sporangium could be seen in the stomata.With the increase of KPF concentration,the coverage of kaolin particles on the leaf surface increased,and the degree of stomatal blockage increased.(3)Mixtures of different concentrations of KPF and pathogen sporangia were prepared,the effect of low concentration KPF treatment on the release of sporangia zoospores was observed by microscope,and the effect of KPF on pathogen spore formation and attack index was explored through in vitro inoculation.The results showed that 0.10%,0.25% and 0.50%KPF treatments significantly reduced the rate of zoospore release.Compared with the control group,treatment with 1%,2% and 3% KPF did not affect the incubation period and incidence of pathogens,but significantly reduced pathogen sporulation efficiency(SPO),daily cumulative proportion(DCP)and attack index(IA),thereby reducing the disease index.(4)KPF treatment significantly increased the activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)in infected leaf discs and natural disease conditions in the field.The content of glutathione(GSH)and ascorbic acid(ASA)was increased,and the content of proline was decreased by KPF treatment.In addition,KPF treatment also significantly increased salicylic acid(SA),gibberellin3(GA3),brassinolide(BR),abscisic acid(ABA)and methyl jasmonate(JA-me)in susceptible leaves.However,in the noninoculation treatments,only KPF treatment had almost no significant effect on the above physiological indicators.(5)KPF treatment significantly increased phenylalanine ammonia lyase(PAL),β-1,3-glucanase(PR2)and chitinase(PR4)in infected leaves and field natural disease conditions.KPF treatment rapidly up-regulated Vv PR1,Vv PR2,Vv PR4,Vv PAL,Vv EDS1,Vv PAD4,Vv TGA1 and Vv NPR1 genes in leaf discs at the early stage of infection compared with the inoculated control group.However,with the progress,the above genes in the inoculated control group showed a slow activation trend,and the expression of the above genes in the KPF treatment group was greatly reduced,which was significantly lower than that in the inoculated control group at the later stage of infection.However,in the non-inoculated leaves,only KPF treatment had almost no significant effect on the indicators above healthy leaf discs.The results showed that KPF treatment could promote the activation of defense responses in infected leaves at the early stage of infection,but KPF itself did not affect leaf metabolism and defense responses.In conclusion,KPF can effectively control the leaf downy mildew of Cabernet Sauvignon under both detach leaves and field conditions,and comprehensively regulate leaves against downy mildew by hindering pathogen infection,reducing pathogenicity and improving early leaf defense response.