| Peanut scab(Elsino? arachidis)is an important disease in peanut industry in China.The disease is widespread and serious,becoming an urgent problem of plant protection in the healthy development of peanut industry.At present,researchers concerning the etiology,occurrence investigation,transmission characteristics and chemical control have been reported.Little is known about pathogenic mechanism,toxin biosynthesis pathway and regulatory network due to the lack of genome information and molecular research platform.Mutant analysis is an effective tool for studying the interaction between pathogen and host,pathogenic mechanism and genetic evolution.In this paper,mutant library of E.arachidis was constructed,and several pathogenicity mutant strains were screened out.The biological characteristics,toxicity secondary metabolism,infection process and re-sequencing were systematically studied.The results provide materials for the biosynthesis pathway and regulatory network of toxin produced by E.arachidis,and lay a theoretical foundation for the innovation of pathogenic mechanism,disease resistance breeding and control strategy innovation.The main results are as follows:1.Construction of mutant library of E.arachidis and characterization of its mutants The mutant library of E.arachidis was established.The characteristics,toxin accumulation and pathogenicity of typical and stable mutants were studied.A total of 1194 mutants were obtained,of which 360 were phenotypically different from wild-type strain(WT).The results showed that the colony color of E.arachidis was mostly red or orange,and the growth rate was 0.65 mm/d.The color of tested 22 mutants was rich and varied,ranging from yellow,orange,red,brown to black,and the growth rate was 0.23-0.71 mm/d.The accumulation of mutant toxins was significantly different.Two mutants produced 2 and 1.5 folds of WT toxin level,respectively.The toxin accumulation of eight mutants decreased in varying degrees.Toxin was not detected in nine mutants.The pathogenicity of mutants has also undergone abundant mutations,of which 8 mutants have no lesions.Molecular characteristics showed that the mutants had abundant intraspecific genetic diversity,and could be divided into six genetic lineages when the genetic similarity coefficient was 0.83.The construction and characterization of mutant library of E.arachidis provide abundant research materials for the follow-up study of pathogenic mechanism,toxin biosynthesis pathway and regulatory network of E.arachidis.2.The biological characteristics and secondary metabolism of pathogenic mutant strains The biological characteristics of pathogenic mutants Sa Sp-4 and Sa Uv-62 were systematically studied.The results showed that the optimum temperature and nitrogen source for the mycelial growth of three strains was 25 ℃ and yeast extract powder,respectively.The colony color of Sa Uv-62 was black both in light and darkness,of which optimum p H was 4,and optimum carbon source was galactose.The colony grew slowly and the growth rate was only 0.50mm/d.The colony color of Sa Sp-4 was black in darkness,while the color was black first and then pink,of which optimum p H was 6,and optimum carbon source was glucose.The colony growth rate of mutant Sa Sp-4 was 0.75 mm/d faster than that of WT(0.65 mm/d).Sa Uv-62 was detected intracellular melanin,but no extracellular melanin and ESC toxin.Sa Sp-4 was detected intracellular and extracellular melanin,while WT was only detected ESC toxin,no intracellular and extracellular melanin.3.Infection process and extracellular matrix analysis of pathogenic mutant strains The morphology,incubation period and infection probability of conidia of Sa Sp-4,Sa Uv-62 and WT strains were studied by optical microscopy and scanning electron microscopy.The results showed that conidia produced germ tubes,and the apex produced appressorium-like structure,which infected the leaves through stomata or epidermis.There was no significant difference in conidia morphology and color between the tested strains.They were all oval cells,transparent and colorless,but the conidia of Sa Sp-4 and Sa Uv-62 turned darker and thicker than those of WT over time.In the course of scanning electron microscopy observation,it was found that both Sa Sp-4 and Sa Uv-62 conidia had filamentous structure on the contact surface with leaves during germination and infection.Through the analysis of specific staining agents of extracellular matrix components,it was clear that conidia of E.arachidis could secrete mucous substances to help the pathogen adhere and fix during infection.The main components of the substances were starch carbohydrates and hydrophobic proteins.4.Genome rearrangement analysis of the weak pathogenic mutant Sa Uv-62 The whole genome rearrangement analysis of the weak pathogenic mutant Sa Uv-62 was carried out by Pac Bio RSII sequencing technology,and the differential genes were detected and annotated.The results showed that 4,267,158 reliable reads were obtained from Sa Uv-62.The coverage of Sa Uv-62 with reference genome of Peanut Scab pathogen was 97.37%.By comparing with the reference genome,5660 single nucleotide polymorphisms(SNPs)of Sa Uv-62 were detected,5605 in the non-coding region and 55 in the coding region,of which 16 were synonymous and 39 were non-synonymous,resulting in 37 gene mutations.There were 84 small fragment insertions and deletions(In Del)in Sa Uv-62,77 in non-coding region and 7 in coding region,including 4 code-shifting mutations,2 codon insertions and 1 codon deletion,which resulted in 7 gene mutations.Referring to GO,COG and KEGG databases,44 mutant genes were annotated.Mutant genes participated in primary metabolism(5),biosynthesis and catabolism of secondary metabolites(1),signal transduction(4)and genetic information processing(2),respectively.Rearrangement analysis of Sa Uv-62 provides molecular information and theoretical basis for screening and identifying genes related to pathogenicity,toxin biosynthesis and metabolism of E.arachidis. |
PDF Full Text Request