Isolation And Identification Of Pantoea Stewartii Subsp. Stewartii Causing Leaf Wilt On Sugarcane

Four important bacterial diseases of sugarcane include ratoon stunting disease,leaf scald,red stripe,and gumming,which are important diseases that threaten the safe production of sugarcane.Recently,we found the symptoms of bleaching on both sides of the leaf margins,followed by leaf blight and necrosis,and then plant death in some sugarcane cultivars in Zhanjiang,Guangdong province of China.In this study,we carried out molecular detection and performed the pathogenic bacteria isolation and identification from these putative diseased leaf samples.First,these leaf samples were detected by nested-PCR using primers PS1/PS4(outer primers)and Ps2r/Ps3r(inner primers)specific to Pantoea stewartii subsp.stewartii(Pss),a causal agent of bacterial wilt of maize.The PCR products were cloned and sequenced,and the results showed that the leaf samples were infected by Pss.Subsequently,a bacterial strain PSCN1 was successfully isolated from leaves with bleaching and blight from YZ08-1095 cultivar.Sequence analysis showed that PSCN1 had the highest homology with Pss based on 16 S r DNA sequences.In additional,cell morphology and physiological and biochemical indexes of PSCN1 strain were similar to Pss.Moreover,According to Koch’s Postulates,the PSCN1 strain was confirmed to be pathogenic to sugarcane host,causing similar symptoms that were observed in the field.Meanwhile,PSCN1 isolates were re-isolated from inoculated leaves and were further verified by the nested PCR assay.Lastly,the complete genome sequence of PSCN1 strain was obtained using the Nanopore sequencing platform.Phylogenetic analysis revealed that PSCN1 strain had the closest phylogeny with the Pss strain DC283 based on whole genome sequences of PSCN1 along with 12 reference strains of P.stewartii.PSCN1 and DC283 strain of Pss were clustered into a same clade.Pairwise sequence identity analysis indicated that PSCN1 were 97.51-98.85% identity with other tested P.stewartii strains.Taken together,in this study we reported that Pss is a pathogenic bacterium in sugarcane based on the biological characteristics,pathogenicity test,and genomic sequence analysis,and then we proposed this disease name as bacterial wilt of sugarcane.Our findings provide important basis for spread and epidemics,inspection and quarantine,as well as prevention and control in this disease in sugarcane.