Pathogenicity Analysis And Whole Genome Sequencing Of Banana Sheath Rot Pathogen Isolate XJ12

Banana is an important fruit crop in tropical and subtropical regions of China.However,banana Sheath Rot,a bacterial disease,was found in recent years and has caused great loss to banana production in China.Because the disease was not identified as Dickeya dadantii until 2014,little research has been done on it.In this paper,a series of strains were isolated from different banana varieties and different symptomatic types of banana.Moreover,On the basis of the identification,the pathogenicity tests of different strains was carried out.In addition,a real-time fluorescent PCR method was established,and the whole genome sequence of a strain XJ12 was determined.The main results are as follows:1.Through the tissue dilution method,15 strains were isolated and purified from different kinds of banana plants,and the DNA of 15 strains of strain was amplified by Dickeya specific primers ADE1 and ADE2,and it was confirmed to be Dickeya genus.The16 S rDNA sequence could not distinguish the species,so we then used the nucleotide sequence analysis and phylogenetic tree of 6 housekeeping genes(FusA,Gyr A,RecA,GryB,DnaJ,DnaX)to further clarify that these isolates belonged to D.dadantii.2.Pathogenicity of 15 isolates was determined by in vitro needle inoculation and living plant needling inoculation.The results showed that the isolates could cause decay in the isolated stems,which could cause the decay of the leaf sheath and the pseudostem brown rot,and the banana leaves were broken,but the pathogenicity showed different degrees of variation.The strains with strong pathogenicity were XJ2-1-2,XJ3,XJ4,XJ7 and XJ11,while the strains with stronger pathogenicity were XJ1,XJ8,XJ12,and the strains with moderate pathogenicity were XJ5,XJ6,XJ9,XJ10,it is 33.3%,20% and33.3%,respectively.3.A conventional PCR and a real-time fluorescence quantitative PCR(qPCR)methodfor Banana Sheath Rot pathogen were respectively established by primer design and optimization of amplification conditions.The best system of the conventional PCR is that,primer concentration 0.1mol/L,reaction conditions: 95? predenaturation 5min;94 ?denatured 40s;53? annealing 30s;72? extension 1min;35 cycles after 72? extension7 min,PCR products at 4? preservation;and the real time fluorescent quantitative PCR primer concentration was 0.2 mol/L,the annealing temperature is 58 ?.The sensitivity comparison between the two PCR methods showed that the real-time fluorescence PCR is100 times higher than that of the conventional PCR.Moreover,the real-time fluorescent quantitative PCR can be amplified in an independent closed system when the sample is completed,which reduces the human pollution and does not need gel electrophoresis.To a certain extent,in addition to more accurate knowledge of the pathogen expression,it saves time,and has achieved rapid,sensitive and accurate.4.In order to understand the pathogenicity of Banana Sheath Rot at the molecular level,a strain XJ12 was chosen and its whole genome sequencing was first carried out.The results showed that the nucleotide size of the strain was 4972118 bp,the GC content was56.30%,and the coencoding gene was 4696,accounting for the whole genome of the genome.85.18% of the length.There are 75 RNA(tRNA)and 22 ribosome RNA(rRNA),which contain eight of 5S rRNA,seven of 16 S rRNA and 23 S R RNA each,and reveal the possible roles of many predicted structural proteins.In the co-linear analysis of nucleic acids,the ratio of the bacteria to the other reference strains of the same genus and the similarity between XJ12 and 3937 was the highest,which sharing the similarity at 96.54%in the nucleotide.The sequencing of the strain will lay a solid foundation for further research on the pathogenesis and interaction between pathogen and banana.