Screening Effective Biocontrol Agents Against Litchi Downy Blight Caused By Peronophythora Litchii And Mechanism Of Disease Suppression

The yield and quality of litchi fruit are severely affected by Litchi downy blight,caused by the oomycete Peronophythora litchii,occurring in almost all the litchi production regions of China.Therefore,there is urgent need for a riskless and sustainable biological control strategy.This research show that the Biocontrol Agents(BCAs)and bacterial volatile organic compounds(VOCs)supressed the disease LDB.We determined the efficacy of BCAs on different application methods,their colonization ability and micro-ecological diversity on the litchi pericarp,and the indexes related to induced resistance responses.We further conducted time course transcriptome sequencing on infected litchi fruit with or without BCA pretreated and predicted key resistance genes with potential roles in plant defense responses.The prospective research findings not only will provide a theoretical basis,but also may give an outlook on probable processes as well as the development of other fruit.The main findings are as follows:1.188 isolates of bacteria were obtained from different habitats(soils,leaves,fruit)of litchi.82 isolates were selected for sequencing and cluster analysis for assignment evaluation,which were 12 clusters and 21 species.The efficacy of 20 isolates were assessed to“Guiwei”branch and“Baitangying”fruit under the LT(laboratory trial)mode,which with biocontrol efficacy of 0-35.42%,30.16-100%,and fresh-keeping of 2.22-72.22%,respectively.In 2016,disease index of 7 BCAs on cvs.“Huaizhi”,“Feizixiao”and“Nuomici”was analyzed under the LT and FLT(field plus laboratory trial)modes.The biocontrol efficacy was found to be different between the two modes.PP19 and SI17showed stable control efficacy(40%-90%)and provided the highest at 48-60 hpi.In 2017,the 5 BCAs were tested on cvs.“Huaizhi”and“Feizixiao”under the LT mode again,SI17and PP19 showed 40-85%efficiency(36-60 hpi),while with 30%-50%fresh-keeping effect(168-180 hpi).Disease index was measured on cv.“Feizixiao”(65%ripening degree)pretreated by BCAs for 7 d in the field with two modes(1DS/2DS).Under the 1DS mode,PP19,SI17,LI24 and PI26 showed 90%control efficacy at 48 hpi,along the control efficacy decreased;particularly,the fungicide decreased to 3.95%and PP19 to 24.69%at60 hpi.Under the 2DS mode,PP19 showed an efficiency of 23.58%at 60 hpi,while the chemical fungicide still kept on 90%.Field experiments showed that the efficacy of SI17and SP6 was 40-57%and 21-52%(90-168 hpi),respectively,while the fungicide was approximately 75%-90%.The effects of PP19 and SI17 on the enzyme activity and the relevant compound content were analyzed.Without the P.litchii inoculation,the BCAs first increased the enzyme activities for a period of time,however,there was no significant difference between them.With the P.litchii inoculation,BCAs reduced the effect due to the pathogen on enzyme activity.They activated the related enzyme activity 12-24 h earlier than the control.Furthermore,it found that no significant difference between BCAs and the control in fruit quality,indicating that BCAs were safe and reliable for litchi fruit.BCAs may provide control efficacy by inducing resistance to litchi downy blight of litchi fruit.Moreover,they most likely work by priming.2.Colonization ability of five BCAs(PP19/SI17/PI26/LI24/HS10)were detected in FLT condition,the curve of 2DS was higher than that of 2RS;the same one BCA appeared similar trend in different cvs.“Feizixiao”and“Huaizhi”;interestingly,PP19 is much more lovely to colonize“Huaizhi”while SI17 is fond of“Feizixiao”.The activity of 11 enzymes or content associated with resistance and fresh preservation were tested with P.litchii inoculation.The activity of CAT,SOD,?-1,3-Glucanase,PAL,PPO and the content of anthocyanin of the fruit was increased by BCAs compared to the control,while reduced anthocyaninase activity.SI17 was more effective in improving the chitinase activity,and PP19 appeared a better impact on improving much more enzyme activity.Further high-throughput sequencing analysis of PP19 found that there is an improvement of the micro-ecology diversity,affecting the distribution of some certain group of bacteria in the fruit pericarp,such as Gluconobacter.3.The biocontrol efficacy of volatile organic compounds(VOCs)from 5 BCAs were tested.The highest control efficacy was 56.55%with SP and 49.15%with FP.Five bacterial VOCs produced of 24,36,48,60,and 72 h were analyzed by SPME-GC-MS,which are ketones,alcohols,acids,hydrocarbons,benzene,or alkene.Eleven of them and four other components reported in the literature were tested for antagonism against P.litchii in dish plate assays.Eight of them(3-Aminobutanoic acid?Bicyclo[4.2.0]octa-1,3,5-triene?Pentadecane?2-Nonanone??-Farnesene?1-Tridecene?2-Heptanone?6-methyl-,2,6-Di-tert-butyl-p-cresol)showed no inhibitory activity,while the other 7 components with inhibitory activity,and EA?BTH?SA had a EC₅₀of 170.29,146.39,and 175.97 mg/L,respectively.EA,BTH,AF,BABA,SA,Me JA were tested at three concentration of 1000,500,200,and 100 mg/L,respectively,it observed a higher control at 100 mg/L of them.Moreover,tendency of eight enzymes activities with treated bacterial VOCs of PP19 and SI17 at the condition of the P.litchii inducted were detected.It showed an“increase-decrease”tendency across 0 to 24 h,and a“decrease increase decrease”trend24-72 hpi.Totally,anthocyanase,?-1,3-glucansae,chitinase,and total phenols presented better effects.It can speculate that bacterial VOCs probably induced resistance in litchi fruit by affecting activities of certain plant defense resistant enzymes;some components,such as BTH or AF could be promising for the control of LDB on harvested litchi fruit.4.Analysis the relative contents of P.lichii treated with Bc PP19 revealed a significantly lower content at 36 hpi and 48 hpi,despite no significant difference at the time point of 12 and 24 hpi compared with the control.Further transcriptomic analysis showed that a number of genes were significantly expressed in the 36 hours post inoculation(hpi),suggesting that the pretreatment with Bc PP19 substantially altered the plant response to P.litchii at transcriptional level.We clustered the genes with absolute fold changes greater than 1.5 based on their time course expression patterns and found four clear types.Further gene ontology enrichment analyses showed that functions related to fructose metabolism,regulation of cell plasma membrane,ethanol biosynthesis,and regulation of defense response were over-represented in the four clusters,respectively;and the most significant was associated with the genes revelant plant defense resistance.In summary,Bc PP19 can indeed induce the resistance of litchi fruit to P.litchii,and might regulate some genes related to disease resistance and preservation to avoid plant browning and pathogen infection.