Based On Omics Technology Decipher Genetic Evolution And Winter Pathogenicity Of Pathogens Causing Sugarcane Pokkah Boeng Disease

Sugarcane pokkah boeng disease(PBD)is a significant constraint to sugarcane production and re-emerging as a prevalent foliar disease in most sugarcane producing areas,including Brazil,India,and China.This air bore PBD disease is caused by Fusarium species complex(FSC),the most critical phytopathogenic fungi.We aim to decipher the evolution,diversity,and pathogenicity of FSC by high-throughput omics and pathological analysis.Two whole-genomes of F.sacchari CNO-1 and F.proliferatum YN41 were sequenced,assembled,annotated at the chromosome level.The comparative genome of six representative Fusarium genomes was carried out to reveal the collinearity of the genome,gene families,and evolutionary relationships.To parse why PBD occurred in winter,the multiple-omics analysis was performed to clarify its adaptability and pathogenic link in low temperature,including transcriptome,proteome,and metabolome.It will provide insight into the scientific references for control of multi-line sugarcane varieties and to breed new varieties of resistance to PBD.A survey was conducted in guangxi province.Pokkah boeng occurrence was investgated accroding to the 90 sugarcane varieties(lines).Guitang 02-619,RB83-5486,Shuidian 25 belonged to high susceptible(HS)grade;Guitang 09-03,Guitang 05-3846,CP96-1252,Guitang05-378,Guitang 00-122,ROC22,Guitang 02-761,Gannan 99-591,Yuetang 97-976,Yunzhe 89-159,Yuetang 94-128 belonged to high resistance(HR)grade;Other 76 varieties(lines)belonged to medium resistance.There were distinct different sugarcane varieties(lines)regarding the susceptibility and resistance against pokkah boeng disease.A total of 123 Fusarium strains were collected,isolated,and identified from Ecological Zone Division of China(Guangxi,Yunnan,Guangdong,Zhejiang,Hainan,and Fujian)during 2012～2019.To reveal the genomic diversity of these Fusarium stains,the specific genomic regions were amplified using 30 primer pairs selected from previously available references.A total of 81 representative ones from 123 strains were selected for re-sequencing to identify the species-specific SNP.F.oxysporum BS2-6 and F.commune GD-46 were additionally sequenced as an outgroup species.Evolutionary relationships on the basis of SNP loci showed the genetic conservation in 14 strains of F.proliferatum,all of which were collected from the colder regions in winter.In addition,31 strains belong to F.sacchari,35 strains belong to F.andiyazi,and only one strain belongs to F.verticillidides.Among them,two of the leading pathogens are F.sacchari and F.proliferatum.Yunnan regions have a rich genetic diversity.The difference in genetic diversity in different regions and the multiple pathogenic Fusarium species can explain the complex causes of sugarcane pokkah boeng disease in China.The whole genomes of F.sacchari CNO-1 and F.proliferatum YN41 were sequenced by a combination of the single-molecule real-time(SMRT)sequencing with the Pac Bio Sequel platform and the paired-end sequencing with the Illumina Hi Seq platform.The genomes were assembled in the chromosome level of 13 scaffolds,including twelve chromosomes and one mitochondrial genome.The genome size for CNO-1 was 44.59 Mb,with N50 of 4.3 Mb and GC content of 47.81 %,whereas for YN41,it was 44.05 Mb with 4.4 Mb of N50 and 48.29 % of GC content.The assembly quality was assessed using Benchmarking Universal Single-Copy Orthologs(BUSCO)and Core Eukaryotic Genes Mapping Approach(CEGMA).Collectively,our results suggested the high quality of chromosome-level genomes of two Fusarium species with over 99% of the complete and single-copy BUSCOs.Combined with the evidence of Ab initio,homolog,and transcriptome-assistance strategies,a total of 14,670(CNO-1)and 14,796(YN41)genes were annotated.We performed a comparative analysis for the assembled genomes of CNO-1,and YN41 coupled with the whole genome of four related Fusarium species(F.fujikuroi IMI58289,F.verticillioides 7600,F.oxysporum 4287 and F.circinatum FSP34).The lineage-specific regions include six chromosomes(chromosomes 5,7,9,11,and 12).We calibrated the evolutionary rate of CNO-1 and YN41,through estimation of divergence time.The result shows that YN41 and CNO-1 belonged to the most conservative Asian clade.Additionally,we compared the gene families related to core secondary metabolites enzymes,transporter proteins,cytochrome P450,transcription factors,and other secreted proteins,and revealed the difference in gene numbers among the Fusarium gene families.Noticeably,genome sequence analysis revealed the presence of Gibberellins gene cluster in both CNO-1 and YN41 species.However,despite the presence of the Gibberellins cluster in YN41,we identified that core gene P450-4 missed in YN41.Contrary to Gibberellins cluster,the entire gene cluster for Fumonisin was detected in the ninth chromosome of YN41,while CNO-1 showed the presence of 38 kb inserts instead of Fumonisin cluster.F.sacchari is a dominant pathogen in summer or the warmer area,while F.proliferatum occurs in winter or the colder area.Interestingly,F.proliferatum isolates remarkably showed better adaptation to low temperature as compared to F.sacchari.We compared the transcriptomic,proteomic and metabolomic profiling of CNO-1 and YN41 exposed to16 ℃ and 32 ℃ to gain deep insight into the pathogenic factor,to understand the effect of temperature on pathogenicity behavior,and to examine F.proliferatum regulation in response to low temperature.Transcriptomic data indicated that 403 and 545 differentially expressed genes(DEGs)were up-regulated and 611 and 643 down-regulated in CNO-1 and YN41,respectively.Noticeably,low temperature enhanced expression level of four pathways associated with RNA processing,energy production and conversion,lipid transport and translation,ribosomal structure and biogenesis in YN41.From the proteomic analysis,we have identified 204 and 610 up-regulated,and 182 and 416down-regulated differentially expressed proteins(DEPs)at low temperature in YN41 and CNO-1,respectively.We also marked the fold change in YN41 low temperature associated DEGs and DEPs.We observed that 12 genes were markedly up-regulated at both transcriptional and post-translational levels in YN41 exposed to 16 ℃.Among them,the up-regulation of pathogenesis-related proteins,alcohol dehydrogenase,and lysophospholipid acyltransferase is worth to further study.A comparison of metabolic profiles resulted in a total of 90 and 56 significantly altered metabolites in YN41 or CNO-1,respectively.We integrated the differentially expressed metabolites(DEMs)with the aforementioned DEGs and DEPs data and mapped 16 common pathways regulated at low temperatures.Our findings indicated that YN41 adapted to low temperature,mainly due to the difference in carbohydrate,amino-acid,and lipid metabolic pathways.Multi-Omics results showed that YN41 kept active for growth and pathogenicity kept at low temperatures.Some genes of ALDH,ALDH2,TGL4,LPT1,which are related to energy,were up-regulated in YN41.Arginine and proline metabolic pathways and glycine,serine,and threonine metabolic pathways also could provide energy.Similarly,the up-regulation of glycerol DEMs remarkably render YN41,a cryotolerant species.YN41 also up-regulated the virulence and pathogenicity related DEGs and DEPs,such as ALDH2,mal Z,and P5 CDH at low temperature.It is vital to reveal why sugarcane pokkah boeng disease caused by F.proliferatum is occurring in winter now.