Isolation And Identification Of The Pathogens Of Leaf Spot On Poplars And Preliminary Research On Plant Immune

Poplar is an important tree species which is widely used in industrial production and ecological protection,and which is widely cultivated in China.Most of the poplars planted in China are clonal breeding with a single forest structure,which can lead to the outbreak of poplar pests and diseases easily.Poplar leave spot is one of the main diseases on poplars,which is a major threat to the forestry economy and ecosystem.Therefore,it has practical values on the healthy development of poplar industry to carry out the investigation of poplar leaf spots,isolation and identification of pathogens,exeplation of phylogenetic relationships between different pathogens and pathogenicity test.In addition,the interaction between pathogens and plants is thorough studied in recent years.Several plant immune pattern recognition receptors were successfully identified.We hope to create model plants with immune deficiency by knockout of the genes of pattern recognition receptors.In this way,we make some preparations for the future study on the pathogenic mechanism of poplar pathogens by using model plants as vectors.Based on the above content,this paper mainly carried out the following research:1.Isolation and identification of pathogens of poplar leaf spots: Three kinds of pathogens were isolated from poplar disease leaves by tissue separation.The pathogens were identified by morphology and ITS、LSU、 RBP2、TEF1-α characteristic sequences.The results showed that the pathogen causing the leaf spot disease on P.tomentosa is the first reported new species.We named it Elsino?.tomentosae;The pathogen of the leaf spot disease on P.deltoides is E.australis;The pathogen of the leaf spot disease on P.deltoides is Venturia phaeosepta.2.Pathogenicity test: The pathogen inoculation test showed that the pathogen E.tomentosae can successfully infect P.tomentosa and P.davidiana×P.bolleana.E.australis can infect P.deltoids×P.euramericana and cause cell death on P.tremula×P.tremuloides because of hypersensitive response.Venturia phaeosepta is able to infect P.deltoides×P.euramericana and P.tremula×P.tremuloides.3.Non-host resistance of Nicotiana benthamiana: All strains of the pathogens E.tomentosae,E.australis and Venturia phaeosepta were used to inoculate Nicotiana benthamiana.None of them can cause pathogenicity to Nicotiana benthamiana.The expression levels of immune reporter genes Nb Pti5 and Nb Acre31 of Nicotiana benthamiana were improved after inoculating with pathogens,which caused non-host resistance of Nicotiana benthamiana.4.Nb FLS2 genes knockout: In order to knock out the pattern recognition receptor Nb FLS2,we construct the CRISPR/Cas9 vectors which can knockout Nb FLS2-1、Nb FLS2-2 and both of them.After genetic transformation of Nicotiana benthamiana by agrobacterium,the mutant plants with immune deficiency of Nb FLS2-1、Nb FLS2-2 and both of them were obtained.