| Morus alba L.,a kind of perennial woody plant,has multiple economic applications.Mulberry diseases affect the quality and yield of mulberry leaves.Especially the mulberry mosaic dwarf disease(MMD),lacking the effective detection and prevention of its potential pathogens,is becoming more and more serious.In order to explore the etiology and occurrence of MMD,our study collected 590 leaf samples associated MMD adapted from 11 provinces in China from 2017 to 2019.Molecular detection and genomics analysis were conducted on MMDaV,MVBaV and Ca.P.asteris MD,and establishing efficient molecular detection technology and products respectively.In order to explore the pathogenic molecular mechanism,our study compare the cellular and molecular levels based combining transcriptome and electron microscopy etc.,technologies.The results are as follows:(1)Coexistence of one or more pathogens among MMDaV、MVBaV and Ca.P.asteris MD associated MMD reference to result of PCR assay for detection of 590 samples.The detection rate of MMDaV,MVBaV,Ca.P.asteris MD,Ca.P.asteris MD+MMDaV,Ca.P.asteris MD+MVBaV,MMDaV+MVBaV and Ca.P.asteris MD+MMDaV+MVBaV is53.72%,23.90%,48.14%,28.98%,6.27%,11.02%,1.02% respectively.Among them,the detection rate of MMDaV and Ca.P.asteris MD is the highest in summer,while the detection rate of MVBaV is 0,which show the masked symptom。(2)The genome sequence of Ca.P.asteris MD-GZ-1 was assembled by Next Generation Sequencing(NGS)methods.Whole genome length of Ca.P.asteris MD-GZ-1is 620028 bp,and contain a plasmid named p MDGz-1 with a full length of 4502 bp.(3)Phylogenetic analysis showed that MMDaV was closely related to citrus chlorotic dwarf virus(CCDaV)(JQ920490)with 75.65%-78.26% sequence similarity,and MVBaV was closely related to pepper chlorotic virus(Ca CV)(DQ256124,DQ256125,DQ256123)with 75.13%-76.54% sequence similarity,Ca.P.asteris MD-GZ-1 was closely related to Ca.P.asteris OY-M(AP006628)with 99% sequence similarity based on 90% sequence coverage.(4)Based on the whole-genome analysis,the target genes Rep A,N and ltr A of MMDaV,MVBaV and Ca.P.asteris MD were screened respectively for establishing molecular detection technology system.MMDaV-LAMP detection kit was developed in combination with Rep A(Chinese patent application number: 201810599352.9),MVBaV-LAMP detection kit was developed in combination with N(Chinese patent application number: 201810998181.X),Ca.P.asteris MD-special PCR detection kit was developed in combination with ltr A(Chinese patent application number: 201910927209.5).(5)Based on transcriptomics analysis,showed that the DEGs of mulberry responded to MMDaV and Ca.P.asteris MD infection were 835 and 3664 respectively.Shared high catalytic activity and membrane function in cells etc.,GO items and starch and sugar metabolism,plant hormone signal transduction,and interaction of plant pathogens etc.,KEGG pathways in both cases.The q PCR assay of the mulberry Kunitz type protease inhibitor gene 1(Mn KPI-1)in which DEGs analysis was significantly differentially expressed showed that Mn KPI-1 was up-regulated after infection by the two pathogens and may be involved in the mulberry immunity to related pathogens.(6)Electron microscopy results showed that MMDaV virions and Ca.P.asteris MD cell can be clearly observed in the diseased mulberry tissue cells,and the organelles and endometrial system of the diseased mulberry leaves have changed significantly: The cells of the diseased mulberry leaf were obviously shrunken,deformed,plasma membrane separated from the cell wall,the cell wall became thicker,the starch granules became larger,the chloroplast swelled and cracked,the number of lipid-like droplets increased and the volume became larger,the multivesicular body(MVB)increased,the exosome increased,xylem autolysis and other phenomena occurred.(7)Successfully isolated mulberry leaf exosomes,which are about 100 nm in diameter and contain regular shaped crystalline particles.In conclusion,this study was firstly reported the genome of Ca.P.asteris MD,and innovatively developed of three pathogens’ molecular detection techniques and products of MMDaV,MVBaV and Ca.P.asteris MD,respectively.It was found that the mulberry tree induced the up-regulation of its own Mn KPI-1 gene expression,and more Multivesicular bodies or exosomes appeared in the disease-like plant tissue cells.This study will provide a theoretical reference for the green prevention and control of mulberry virus and phytoplasma disease. |
