| Potentilla anserina L.is a perennial herb of Rosaceae family,which is a typical rhizome special cash crop.The distribution of P.anserina in China is most prominent in Qinghai Province.At present,P.anserina has typical root rot symptoms in the field(such as obvious black spots and corruption),which bring great threats to its yield and quality safety.Therefore,the timely and effective detection of the root rot pathogen of P.anserina and the study of its pathogenic related mechanism will provide a solid foundation to grasp the root rot of P.anserina and carry out subsequent disease prevention and control.In this paper,we conducted a field survey of P.anserina root rot in Qinghai Province,the isolation and identification of pathogenic fungus,the morphological and histological observation of disease samples,and carried out related research work using electron microscopy and transcriptomics techniques.The main research results are as follows:1.Investigation of P.anserina root rotWe carried out investigation and research on root rot in P.anserina fields with different planting years in different areas of Qinghai Province.In the early stage of the investigation,the typical symptoms of P.anserina root rot were clarified,namely:black-brown spots appeared at the beginning of the disease,and the spots were obvious in the middle of the disease,and as the disease worsened,the root of the P.anserina became browned seriously,and even softened,decayed with white mycelium phenomenon.The severity of the disease was determined by establishing a grading scale for the disease,which classified P.anserina root rot into 0 to 5 grades according to the degree of earthnut lesion.The disease of P.anserina root rot in different planting years and regions is different.The incidence and index of the disease were positively correlated with the number of years of planting,with no significant disease in fields within 2 years,and a common disease occurring in the fourth year,with a7-year incidence of more than 40%.Root rot disease in different areas showed some variability due to the combined effect of various factors.2.Isolation and identification of pathogenic fungi of P.anserina root rotFrom the isolation and purification of 15 parts of P.anserina root disease samples,62 fungal strains(belonging to 23 species in 12 genera)were isolated and purified,with the highest separation frequency(43.55%)of Fusarium strains.The pathogenic fungi identified as P.anserina root rot were: F.acuminatum,F.tricinctum,F.incarnatum,F.avenaceum,F.equiseti and F.torulosum.In the study,it was found that strain DLZ-4(identified as F.avenaceum)was the strain with the highest isolation frequency and the most severe epilepsy.So it was selected as a representative strain for follow-up experiments.3.Morphological and histiocytic studies of P.anserina in response to fungal infestation caused by root rotMorphological and anatomical observations were made on the roots of healthy P.anserina and disease-susceptible P.anserina.It was found that the root of the field P.anserina has a natural cracking phenomenon,and the root was rich in starch granules and parenchymal cells,which facilitated the invasion and multiplication of P.anserina root rot pathogens.The pathogenic fungus of P.anserina root rot existed in a variety of forms such as hyphal rings,fragmented hyphal segments,mycelium clustering and spores,and invaded through the natural pores and mechanical wounds of the root,with strong vitality and selective infection.Obvious lesions such as dark spots,decay,and tissue softening appeared in the diseased roots;apparent cell gaps were evident,even sunken and fractured,exposing the internal structure;invading fungi were visible on the periderm,bast and xylem;the internal cell structure was destroyed,with the characteristics of plasma wall separation,cell wall lignification and thickening,cytoplasmic concentration,cell deformation,and chaotic digestion of inclusions.The fungal infestation process was from shallow to deep,and the response of the root to it could be divided into three stages: fungus invaded but no obvious lesions in the root at the initial stage(1-2 d);fungus invaded the phloem in large numbers,some parenchymal cells turned necrotic,and the root lesions were obvious in the later stage(4-8 d);the fungus was dense,most of the phloem parenchymal cell structure was severely damaged,the starch granules were reduced or even disappear,and the root lesions were serious in the late stage(12 d).4.Transcriptomic study of P.anserina in response to pathogenic fungal infestation of root rotThrough transcriptomic analysis of P.anserina root rot,a total of 107434 Unigene after de-redundancy was obtained by sequencing assembly,and 14737 differential genes of P.anserina response to pathogenic infection of Fusarium rot were obtained,of which 12547 were upregulated and 2190 were down-regulated.The functional enrichment analysis of differential genes showed obvious differences between P.anserina-inducing disease strains and healthy strains.The GO enrichment results showed that the upregulated genes were mainly enriched in DNA template transcription,hydrolase activity and DASH synthesis,and the downregulated genes were mainly enriched in hydrolase activity and exosomes based on vascular motion and hydrolyzed O-glycosyl compounds.KEGG enrichment results showed that all differential genes were mainly concentrated in metabolism and genetic information processing.Screening of key genes for differential expression showed that there were differential expressions and expression patterns of key genes of important functionally related types such as cell wall reinforcement and degradation,antioxidant stress,disease resistance signal transduction,toxin production,and plant hormone signal transduction.10 genes were screened for fluorescence quantitative verification,and the expression trend was basically consistent with the results of the transcriptome,which verified the reliability of the transcriptome sequencing results. |
