Study On Functions Of Diaporthe Citri Effectors And Development Of Effective Management Strategies For Citrus Melanose

Citrus melanose,caused by Diaporthe spp.,is the most common fungal disease in citrus production worldwide.The pathogens not only cause citrus melanose and gummosis,but also cause stem rot during fruit storage.The Diaporthe genus shows high species diversity.Diaporthe citri is the major pathogenic species in citrus melanose fungi.Based on next-generation sequencing and third-generation sequencing,the entire genome of D.citri was sequenced and assembled.The candidate effectors were predicted and functions of some effectors were analyzed.The functions of DcNLPs in D.citri were explored in this study.The efficient fungicides for inhibiting conidia germination were screened and selected.By mixing with kresoxim-methyl and SMARTFILM,the usage of mancozeb was reduced in citrus melanose control.The main results were as follows:(1)Genome of D.citri strain NFHF-8-4 was sequenced,assembled and annotated,which laid the foundation for revealing the pathogenic mechanisms.The genome size of D.citri was 63.68 Mb,coverage was 271×.It contained 34 scaffolds,and the longest sequence length was 12 Mb,N50 was 5.47 Mb.The quality of genome data was good,and the assembly results were complete.Repeat sequences accounted for 0.6 %,lower than that found in normal pathogenic fungi.The total gene length of D.citri was 26.00 Mb,and 15,921 genes were predicted.CAZymes analysis showed glycoside hydrolase family genes were the most in D.citri genome,and they were up to 2.82%.GO term analysis annotated 7,880 genes(49.9%)in the D.citri genome,and most genes were associated with cell components.In biological process branch,genes associated with cellular process(4,824)and metabolic process(4,490)accounted for main part,while genes belonging to the molecular function were the least,mostly enriched in catalytic activity(5,068)and binding(4,310).KOG analysis annotated 7,400 genes(up to 46.48%),mostly enriched in secondary metabolite biosynthesis,transport and catabolism.KEGG analysis showed that 4,024 genes(up to 25.27%)were annotated in 44 pathways.(2)Study on effectors in D.citri is helpful in revealing the mechanisms of interaction between pathogens and hosts.From 15,921 predicted proteins,4,528 proteins were considered as candidate effectors,of which 512 were apoplast effectors and 3,514 were cytoplasmic effectors.Fourteen effectors were randomly selected to make the further analysis and the secretory function was verified with the Agrobacterium-mediated transient expression system.INS49＿006387 and INS49＿007175 inhibited XEG1-induced plant cell death in Nicotiana benthamiana,while INS49＿012443 triggered cell death in N.benthamiana leaves.INS49＿012443 was found to contain the NPP1 domain.INS49＿007175 could promote Phytophthora capsica infection on N.benthamiana leaves.(3)DcNLPs function analysis in D.citri.SMART analysis showed that six candidate effectors contained the NPP1 domain in D.citri genome.The signal peptides of six candidate effectors had secretory functions which were verified by the yeast secretion system.During the D.citri infection process,the expression level of DcNLP2 was low,while the expression levels of DcNLP4 and DcNLP5 were high.DcNLP5 caused cell death in N.benthamiana leaves,DcNLP2 and DcNLP4 inhibited DcNLP5-induced cell death and ROS burst in N.benthamiana leaves.DcNLP5 could not induce cell death in Nb RLP23-silenced N.benthamiana,but expression of homologous gene CcRLP23 complemented the function of Nb RLP23,leading to DcNLP5 induced cell death.The results indicated that both Nb RLP23 and CcRLP23 participated in recognition of DcNLP5 in plants,stimulating plant immune defense responses.Through yeast two-hybrid system,luciferase complementation and pull-down assays,CcRLP23 was confirmed to be interacted with DcNLP2,DcNLP4 and DcNLP5,respectively.We hypothesized that DcNLP2 and DcNLP4 inhibited plant recognition of DcNLP5 by competitively binding to CcRLP23.To further identify the function of DcNLPs in D.citri infection,the knockout and overexpression transformants were obtained through protoplast transformation.The mycelial growth rates of OEDcNLP2 and OEDcNLP4 were slower than the wild type.ΔDcNLP2 was more resistant to ROS stress.Pathogenicity assay indicated that the virulence of OEDcNLP5 was increased,but was reduced in OEDcNLP2.(4)In combination with kresoxin-methyl and SMARTFILM,the application of mancozeb was reduced in the field.Mancozeb is a common fungicide in citrus melanose control,and the recommended dose is 1.34 g/L.Because of non-standard orchard management,the dose of mancozeb was used up to 2.66 g/L in some orchards.The conidial germination inhibitions of 11 fungicides were screened.The inhibition rate of mancozeb was 95.96% at 1 μg/m L,the inhibition rate of kresoxim-methyl and fludioxonil was 100% at 0.1 μg/m L.Evaluation of fungicide efficacy on detached citrus fruits showed 1 g/L mancozeb mixed with 0.1 g/L kresoxim-methyl and 2 g/L SMARTFILM were better than 1 g/L mancozeb.Through investigating the prevalence of citrus melanose in Nanfeng,Jiangxi Province,we found that the key management period for citrus melanose was from May to July.Control efficacy of different fungicide combinations in fields showed that 2.66 g/L mancozeb had the best control effect in a series of mancozeb treatments;treatment with 1 g/L mancozeb mixed with 2 g/L SMARTFILM and 0.1 g/L kresoxim-methyl were equivalent to that of 2.66 g/L mancozeb alone,leading to 62.41%reduction of mancozeb usage.