Characteristics Of Leifsonia Xyli Subsp.xyli And Influences Of Its Inoculation On Physiology And Gene Expressions In Sugarcane

Ratoon stunt widely spread in sugarcane planting regions,which is an important restrictive factor of cane yield.The disease has a great influence on growth of ratoon crop of sugarcane and resulted in economic loss in sugarcane production.The incidence of high susceptible variety is up to 93%,resulting to the cane yield loss is up to 27%.So far,there are no effective chemicals to control ratoon stunt.Planting resistant varieties is considered the most effective method to control the disease.Unfortunately,there is no resistant variety up to now.Ratoon stunt is caused by Leifsonia xyli subsp.xyli(Lxx),a gram-positive bacterium.Lxx is very difficult to isolate in vitro and it requires rich nutrients to prepare specific medium,so the research about mechanism of interactions between Lxx and host is limited.In this study,Lxx was successfully isolated from sugarcane variety Badila(high susceptible).The morphology of Lxx was observed under scanning electron microscope(SEM)and transmission electron microscope(TEM),and the genome comparisons between Lxx in this study and Lxx CTCB07 from Brazil were conducted by high-throughput sequencing.The anatomic structure of +1 leaf and chloroplast in Lxx-infected sugarcane were observed under paraffin section and TEM,respectively.Moreover,Lxx was inoculated into disease-free cane setts.The photosynthesis,defense enzymes activities,endogenous hormones and gene differential expressions were analyzed.The results are as follows.1.Lxx was successfully isolated from sugarcane variety Badila(high susceptible)and identified by PCR using specific primers designed according to the 16S-23S intergenic sequences.The PCR amplified products were sequenced by Sanger platform and Blast in NCBI and the results confirmed the isolate was Lxx,also the amplified sequence was submitted to NCBI and got the accession number KR132244.The isolate was named as LxxGXBZ01.Lxx colonized and many small particles adhered in pits and walls of vascular vessel.The anatomic structures of+1 leaf was observed using paraffin section and showed that motor cell of Lxx-infected plant was plump with narrow upper and wide bottom,which was bigger than the healthy one.The results of chloroplasts observed under TEM showed that chloroplasts were smaller and turned into short oval from long oval,the number of osmium particles increased and starch grains decreased,chloroplast membrane was destroyed,but grana lamella and thylakoid had no obvious changes.2.LxxGXBZ01 was collected,and genomic DNA was extracted and re-sequenced by Illumina Hiseq2000.The results showed that 262 Mb data was produced with 500 bp insert size,the genome size was 2,446,908 bp,the GC content was 67.94%,the number of scaffold was 82,and the number of contig was 2,724.A contig N50 size of 1,088 bp and a scaffold N50 size of 53,480 bp were obtained.The re-sequencing result has been registered in GenBank with the accession number LFYU00000000.The results of comparative genomics analysis showed the coverage was 99.99%and the average depth was 130.There were differences between LxxCTCB07 and LxxGXBZ01 in 13 SNPs including 3 synonymous mutations,2 non-synonymous mutations and 8 indels consisting of 2 insertions and 6 deletions.Two indels were found in the coding sequence which might cause frame shifting.The ANI(average nucleotide identity)between these two strains was 93.61%calculated with EzBioclound software,which indicated that there was a high degree of similarity between LxxCTCB07(NCBI GenBank accession number is AE016822)and LxxGXBZ01.3.Pure culture of Lxx was inoculated into disease-free cane sett of three varieties(GT11,Badia and GT29)and the results indicated that Lxx infection reduced the net photosynthetic rate,stomatal conductance,intercellular carbon dioxide and transpiration rate in GT11 and Badila,also reduced the PEPC activity in GT29 with different infection time,The O2·-content,superoxide dismutase and peroxidase activity had different changes depending on different sugarcane varieties and time after inoculation of Lxx pathogen.About the Brix of cane juice,there was no significant difference between Lxx-infected plant and the heathy one,which indicated that Lxx infection did not affect sugarcane quality.4.The contents of gibberellins(GA3),abscisic acid(ABA)and indoleacetic acid(IAA)in sugarcane were determined at processing maturity stage after Lxx inoculation in plant and first ratoon crops.For the stalk in variety GT11,Lxx infection increased the contents of GA3 and ABA in plant crop,but decreased the contents of GA3 and ABA and increased the content of IAA in ratoon crop.For the leaf +1 in variety GT11,Lxx infection decreased the contents of GA3 and IAA in plant crop,but increased the content of GA3 in ratoon crop.For the stalk in variety Badila,Lxx infection decreased the contents of ABA and IAA in plant crop,decreased ABA content and increased IAA content in ratoon crop.For the leaf+1 in variety Badila,Lxx infection decreased the contents of GA3 and IAA in plant crop,but showed no significant effects on the contents of GA3,ABA and IAA in ratoon crop.These results indicated that the influence of Lxx infection on the endogenous hormones in sugarcane depending on varieties,tissues and crop types;meanwhile,the response of sugarcane to Lxx infection involved in various hormones interaction.5.Pure culture of Lxx was inoculated into disease-free cane sett of variety Badila.Basal stalks were sampled on 60th and 90th days after Lxx inoculation,then RNA in stalks was extracted and RNA libraries was constructed and subsequently sequenced using Illimina Hiseq platform to analyze gene differential expressions under Lxx infection.299,199 transcripts and 156,713 unigenes were obtained after RNA-seq.The N50 size of transcripts and unigenes were 1,875 bp and 1,348 bp,and the N90 size of transcripts and unigenes were 442 bp and 279 bp,respectively.Compared to healthy plants,there were 37 differentially expressed genes in Lxx-infected plants after 60 days' inoculation;therein 9 were up-regulated and 28 down-regulated.There were 371 differentially expressed genes after 90 days' inoculation;therein 267 were up-regulated and 104 down-regulated.These differentially expression genes involved in many metabolic pathways,and the metabolic pathways related to responses to stress were phenylpropanoid metabolism,terpenoid biosynthesis,signal transduction,glutathione metabolism,ABC transport protein,phenylalanine,tryptophan,tyrosine biosynthesis,cysteine,methionine metabolism,and so on.These results indicated that the responses of sugarcane to Lxx infection involved in multiple metabolic pathways,which were very complicated.