Study On Diversity And Resistance Mechanism Of Naked Barley Crown Rot Disease Caused By Fusarium Avenaceum

Naked barley?Hordeum vulgare L.var.Nudum Hook.F.?was a kind of cereal crops of the genus Barley,which was mainly distributed in the Qinghai-Tibet Plateau and the high cold regions.It was the main fodder and food crop in these regions,and was widely used in forage,food and brewing.On the basis of identifying the symptoms and pathogens of crown rot disease,the dominant pathogen,Fusarium avenaceum was used as the research object,and NQK-01-03 and Ganqing?were used as resistant and susceptible varieties.Diversity and resistance mechanism of naked barley crown rot disease caused by F.avenaceum were studied,and the following results were obtained:?1?91 F.avenaceum strains were used to identify the genetic diversity and toxin chemical types of six populations of F.avenaceum in Gansu and Qinghai provinces along Qinghai-Tibet plateau.The genetic diversity of population and the related toxin chemical types were analyzed by SSR molecular marker method.The results showed that 14 alleles and 13 polymorphic loci were detected in 91 strains by SSR primers,and the percentage of polymorphic bands was 92.86.The number of allele in 6 populations was 1.8215,the effective number of allele was 1.5530,the diversity index of Nei's gene was 0.3156 and Shannon information index was 0.4644,the number of polymorphic sites was 11.5 and the percentage of polymorphic loci was 82.15.The Nei's genetic similarity and genetic distance of 6 populations were 0.8325⁰.9869 and 0.0132⁰.8705,respectively.There was no significant correlation between genetic distance and geographic distance,genetic similarity and altitude difference among populations.The group of F.avenaceum was divided into three groups,group?was composed of Lintan County,Hezuo Municipality and Zhuoni County,Gansu Province,and Group?was composed of Tuk Autonomous County and Gangcha County,Qinghai Province,and Group?was composed of Haiyan County,Qinghai Province.The genetic variation of F.avenaceum species mainly came from within the population,accounting for 93.63 percent of the total.The toxin chemical types of F.avenaceum were divided into three groups,NIV,DON and 3-AcDON,and there was not15-AcDON.DON was distributed in all of six different geography.The results provided theoretical basis for the identification of genetic diversity of F.avenaceum population and geographical distribution of toxin chemical type.?2?A loop-mediated isothermal amplification?LAMP?was first developed in F.avenaceum with SYBR Green I as a fluorescence indicator in this study.Based on the internal transcribed spacer?ITS?sequence of DNA genes in F.avenaceum,a set of four LAMP primers was designed using software.The reaction systems and conditions were optimized.The specificity and sensitivity of LAMP were assayed by electrophoresis on 2.0%agarose gel and SYBR Green I stain.Diseased naked barley tissue and F.avenaceum spores in soil were evaluated.The LAMP assay efficiently amplified the target DNA in 1 h at 65?and 20 min at 80?with the optimum reaction temperature at 65?.The results of color changes could be directly determined using naked eye by adding SYBR Green I before the amplification.A positive yellow green color was only observed in the presence of F.avenaceum,whereas all other isolates showed orange color as negative control.The detection limit of the LAMP assay for F.avenaceum was 10 pg/?L of target DNA or 10spores in 0.25 g soil in a high sensitivity.The diagnostic validation was performed to detect F.avenaceum in 13 out of 20 suspect diseased naked barley samples collected from Lintan and Zhuoni county in Gannan state,Gansu province.This study provided a new approach for the detection of F.avenaceum.?3?The structure of cell membrane and chloroplast of naked barley leaves,the response of light and CO₂ and the content of each component were tested by inoculation.Microscope observation showed that the normal leaves in colour were uniform and dark green,while diseaed naked barley leaves showed green fading,yellow green or yellow-white alternating symptoms,and light transmittance increased.Transmission electron microscopy showed that the cell membrane and chloroplast of the diseased leaves were seriously damaged and broken,and the mesophyll cells were crumpled and deformed.Net photosynthetic rate,stomatal conductance and transpiration rate of Ganqing?and NQK-01-03 were decreased,while the concentration of intercellular CO₂ increased.Light saturation point,maximum net photosynthetic rate and dark respiration rate decreased,while light compensation point increased.CO₂ saturation point decreased,while CO₂ compensation point and photorespiration rate increased.Water content decreased,while crude fiber content increased.The contents of total nitrogen and total phosphorus in naked barley plants were decreased.These results provided theoretical basis for elucidating the pathological mechanism of naked barley crown rot disease.?4?Naked barley seedlings leaves and roots physiological characteristic and biochemical activity,Pro,MDA,soluble sugar,soluble protein,POD,SOD and CAT content and their change of plant height,root length,plant fresh weight and root fresh weight were tested in pot culture condition with an inoculation method.The results showed that the content of Pro,MDA and POD in diseased leaves of susceptible GanQing?were higher than those in healthy ones,while soluble sugar,soluble protein,SOD and CAT in diseased leaves were lower than healthy ones.And the content of Pro,MDA,POD and SOD in diseased roots of susceptible GanQing?were higher than those in healthy ones,while soluble sugar,soluble protein and CAT in diseased roots were lower than healthy ones.Meanwhile,the content of Pro and CAT in diseased leaves of resistant NQK-01-03 were higher than those in healthy ones,while soluble sugar,soluble protein,MDA,POD and SOD in diseased leaves were lower than healthy ones.And the content of Pro,soluble protein,MDA and POD in diseased roots of resistant NQK-01-03 were higher than those in healthy ones,while soluble sugar,SOD and CAT in diseased roots were lower than healthy ones.At the same time,the content of Pro,POD and CAT in diseased leaves of susceptible GanQing?were higher than those in healthy ones of resistant NQK-01-03,while soluble sugar,soluble protein,MDA and SOD in diseased leaves of susceptible GanQing?were lower than healthy ones of resistant NQK-01-03.And the content of Pro,MDA,POD and CAT in diseased roots of susceptible GanQing?were higher than those in healthy ones of resistant NQK-01-03,while soluble sugar,soluble protein and SOD in diseased roots of susceptible GanQing?were lower than healthy ones of resistant NQK-01-03.The plant height,root length,plant fresh weight and root fresh weight of both susceptible naked barley GanQing?and resistant NQK-01-03 were decreased after F.avenaceum infection.There was a negative correlation between the content of Pro,POD,CAT and naked barley crown rot disease,and a positive correlation between soluble protein and SOD.This study provided theoretical basis on physiological characteristic and biochemical activity mechanism of F.avenaceum infection naked barley leaves and roots.?5?In order to clarify the interaction mechanism between naked barley and F.avenaceum causing crown rot disease,the two varieties,NQK-01-03 and Ganqing?were used as materials,and Illumina HiSeq Xten was used as the platform to sequence the transcriptome.The results showed that the plant height,root length,plant fresh weight and root fresh weight of susceptible variety decreased after infection by F.avenaceum,and the plant height and root length,plant fresh weight and root fresh weight of susceptible variety Ganqing?were lower than that of resistant variety NQK-01-03.Through the annotation of gene expression,a total of 29676 DEGs were obtained in the crown tissues of diseased naked barley,of which 16274 were up-regulated and 13592 were down-regulated.The annotated information was obtained by comparing the BLAST software with the database NCBI-NR,Pfam,Swiss-Prot and the eggNOG database.The most annotated Unigene in the NCBI-NR database was 28799,accounting for 99.76%of the total annotated genes.Comparing the DEGs database with KEGG database,the significant enrichment metabolic pathways in DEGs were found.A total of 28869 DEGs were enriched in 240 metabolic pathways,among which there were 21 metabolic pathways with Q-value?0.05 threshold.There were 5 types of physiological and signal transduction pathways involved in differential expression gene KEGG,which were involved in metabolism,synthesis,photosynthesis,processing and interaction.The KEGG pathway of differentially expressed genes mainly concentrated on phenylpropyl biosynthesis,phenylalanine metabolism,plant-pathogen interaction,isoquinoline alkaloids biosynthesis,tyrosine metabolism,diterpene biosynthesis and glutathione metabolism.The KEGG pathway of differentially expressed genes mainly included phenylpropyl biosynthesis,phenylalanine metabolism,starch and sucrose metabolism,tyrosine metabolism,cysteine and methionine metabolism,and aminosaccharide and nucleotides metabolism.The molecular interaction mechanism between naked barley and F.avenaceum was preliminarily revealed at the transcriptional level.