Isolation And Identification Of Fungal Causing Pitaya Canker Disease And Physiological And Molecular Basis Studies On Pitaya Canker-resistance

Canker disease,caused by fungus Neoscytalidium dimidiatum,is the most severe disease affecting the pitaya(Hylocereus spp.)industry because of its rapid and widely spread.The occurrence of canker disease not only influences yields but also affects the quality of pitaya in most commercial pitaya growing areas around the world.We isolated,identified and characterized this fungal pathogen based on its morphology,molecular characteristics,and pathogenicity in this study.Also,metabolomics,transcriptomics,bioinformatics and molecular biology were used to elaborate the living life of fungus and screen the fungicides and resistant genes.Further bioinformatics analysis and genes expression profiles were made of HpLRR and HpPR family genes.The jobs about HpPR1 cloning,subcellular location and interacted proteins screening also were done.All the results were described as follows:1.Neoscytalidium dimidiatum was the fungus to cause canker disease of pitaya.Inoculated on PDA culture plates,the fungal hyphae were radial and cycloidal.In the initial stage of culture,the fungal hyphae were white.Then,from day three onward,the color of the hyphae turned gray,and after that,hyphae color changed to black with pigment.Under the microscope,the stained hyphae appeared branched,the conidia were rod-shaped,arthrospore and chlamydospore were chain-like.However,individual arthrospore was rodshaped,individual chlamydospore was oval-shaped,and the cell wall of chlamydospore was thicker than conidia and arthrospore.The diseased stems were small,circular,sunken,orange spots or reddish brown that developed into canker.Tender plants are more susceptible to this disease than old ones.The canker disease occurs in all growth periods of pitaya,especially in the hot and rainy period from May to September and after typhoon in Hainan.2.Biological fungicides can inhibit the growth of pathogens,while high temperature and some fungicides can kill the pathogens.Extraction solution of three kinds of biological residues which included in oil tea residues,rapeseed residues and peanut residues and the 12 selected fungicides in this study inhibited the growth of the N.dimidiatum fungal.Extraction solution of oil tea residues was all of the best.The order of inhibit efficacy of three biocides was oil tea residues>rapeseed residues>peanut residues.All of the 12 fungicides,iprodione,prochloraz-manganese chloride complex,propiconazole,and flusilazole were the four most effective fungicides in inhibiting the growth of N.dimidiatum strain.No hyphae grew on PDA media containing iprodione,prochlorazmanganese chloride complex,propiconazole,or flusilazole even 30 days after inoculation.Pitaya canker strain N.dimidiatum have tolerance of high temperature.Treated with 70?more than 4 h or 80? more than 1 h could kill the N.dimidiatum strain.3.Wax layer of pitaya was an important barrier against external pathogens.Inoculated experiments showed that it was not susceptible to the N.dimidiatum when the older stems of pitaya infected with wax layer.However,it would be diseased when infected with N.dimidiatum after tearing the wax layer.Results of transmission electron microscopy(TEM)suggested the wax layer of tender stems were uneven,cells array loosely and more folds in surface which was beneficial to the attachment and colonization of fungal.On the contrary,fungal was hard to grow on the older stems with tight cell arrangement and a smooth surface.From the section structure,the wax layer of tender stems was arranged loosely and more gaps between the cells.While,the wax layer of older stems was arranged closely,the cells were filled with plant wax and evenly distributed.Compared with redfleshed pitaya(Hylocereus polyrhizus),the cells of white-fleshed pitaya(Hylocereus undatus)were arranged more closely in tender stems and filled with more wax in older stems.Results of metabolomics showed that,pitaya wax layer was comprised of lipids and lipid-like molecules,organooxygen compounds,phenylpropanoids and polyketides,nucleosides,nucleotides,and analogues and benzenoids.The identified different ion mainly participated in metabolic pathways,biosynthesis of secondary metabolites,terpenoid biosynthesis,alkaloid biosynthesis,unsaturated fatty acid,phenolic compounds,aromatic heterocyclic compounds containing oxygen,quinone and ABC transporters pathways.The types and contents of lipid and lipid-like molecules determined the differences between tender stems and older stems,also the differences between tender stems and older stems red-fleshed and white-fleshed pitaya.The lipid types of pitaya mainly included in fatty acyls,glycerolipids,glycerophospholipids,polyketides,prenol lipids,saccharolipids,sphingolipids,sterol lipids.There have great differences between tender and older stems in types,components and contents of wax,while little differences between different pitaya varieties.Results of TEM and metabolomics explained the reasons for why tender stems were susceptible with dimidiatum but old stem was not and white-fleshed pitaya was more resistant but not obvious than red-fleshed pitaya.4.Comparative studies on transcriptome of pitaya resistant to canker disease.There were 151.88 Mb raw reads and 144.01 Mb clean reads were obtained in the study of comparative transcriptome analysis of normal and diseased pitaya stems.There were 33584 unigenes monitored.The N50 of Unigene were distributed in 610 bp-1081 bp.The guaninecytosine(GC)content was 40%-50%.The above data showed that the overall sequencing quality and assembly quality were good.The results of the qRT-PCR assay showed that qRTPCR conformed to the RNA-Seq data,with an accuracy of 87%.These results indicated that the RNA-Seq findings were both stable and reliable.These unigenes shared a similarity of with Vitis vinifera,Theobroma cacao,Nelumbo nucifera,and Jatropha curcas.There were 832 unigene encoding transcript factor which mainly included MYB,MYB-related,AP2-EREBP,C3H,WRKY,bHLH,NAC,GRAS.In this study,1073 different expression genes(DEGs)were identified,of which 1032 were up-regulated and 41 down-regulated.Mostly DEGs were associated with catalytic activity,cellular processes,metabolic processes,membrane and binding processes.Pathway analysis showed that mostly DEGs were significant enriched in plant-pathogen interaction,RNA transport,amino sugar and nucleotide sugar metabolism pathways.Thirteen categories and twenty-four DEGs were associated with plant-pathogen interaction pathways.These DEGs mainly were annotated to calcium ion-dependent protein kinase(CDPK),respiratory burst oxidase homologs(Rboh),cyclic nucleotide-gated channel(CNGC),calmodulin and calmodulin-like(CaM/CML),flagellin-sensing(FLS)and WRKY.5.HpLRR and HpPR were two important resistance genes of Pitaya.HpLRR and HpPR family genes were identified using transcriptome method.Bioinformatics analysis indicated that HpLRR family genes mainly included in LRR-STK,FBXL,NBS-LRR,LRRRLK,PIRL subfamilies with LxxLxLxxNxL conserved domain.The HpPR family genes mainly included in pathogenesis-related protein,pathogenesis-related genes transcriptional activator and pathogenesis-related protein Bet v I family without typical domain.Genes expression of HpLRR and HpPR family genes in special tissues,different infected stages and SA,MeJA,ABA treatment showed that the expression of these genes was different in different tissues,varieties,expression periods and treatment of disease-related hormones.Bioinformatics analysis combined with expression analysis indicated that HpLRR and HpPR family genes were two important resistance genes of pitaya.6.HpPR1 is a crucial gene involved in resistance of canker disease.HpPRl,which encodes 166 amino acids,is a small cysteine rich secretory protein with a leading signal peptide coding by 28 amino acids.It is classified to CAP super family protein.Subcellular experiments indicated that it was located in cell membrane.Yeast two hybrid(Y2H)experiments showed that the bait expression vector HpPRl-pGBKT7 has no self-activating activity and was non-toxic to yeast cell Y2H Gold.Five kinds of interacted proteins which included in suppressor-like protein,Cactus virus X isolate,mannitol dehydrogenase,chloroplast and cysteine proteinase were screened by Y2H.