Study On The Function Of Succinate Dehydrogenase In Mango Colletotrichum Gloeosporioides

Mango(Mangifera indica L.)is the world’s second largest tropical fruit,known as the‘tropical fruit king’ reputation,widely distributed in the world in more than 100 countries and regions,China is the world’s third largest mango producer,planting area of more than3.3×105acres.Colletotrichum gloeosporioides is one of the most common and harmful fungal diseases in mango production.Chemical control is often used as the main integrated control measures.In recent years,microbicides targeted at succinate dehydrogenase have developed most rapidly and attracted the attention of researchers.Succinate dehydrogenase(SDH),is widely distributed in eukaryotes and combined with inner mitochondrial membrane,it is an important constituent of the fungal chain of attraction,and it is the only polysubunit enzyme integrated in membrane in the TCA cycle,its inhibitor is a common fungicide,called succinate dehydrogenase inhibitor(SDHIs),which belongs to amide fungicide.This study is an interdisciplinary field of pesticide and plant pathology,through field efficacy test,resistance evaluation,bioinformatics analysis,gene editing,q RT-PCR,and other techniques,a functional study of the succinate dehydrogenase gene of Colletotrichum gloeosporioides was carried out,which broadened the understanding of the functions of 4subunits in the succinate dehydrogenase of Colletotrichum gloeosporioides.It is of great production significance and academic value to explore the bactericidal mechanism of succinate dehydrogenase inhibitors and the pathogenesis-related mechanism of succinate dehydrogenase.The main results are as follows: The results of field test showed that SDHIs had certain control effect on anthracnose in field,taking 55 strains of Colletotrichum gloeosporioides in the main producing area of mango and inhibitors of thifuramide,pyridamide,flupyroxamide with concentration of 1000 mg/L as the research object,it was found that the growth of mycelium and the formation of Conidia of Colletotrichum gloeosporioides were significantly inhibited by the three inhibitors,there were obvious differences in sensitivity and sporulation among the tested strains.The inhibition effect of flupyroxamide on mycelium growth and conidia was the most significant,followed by that of thifuramide and pyridamide,the results indicated that there were significant differences in the bacteriostatic efficacy of the succinate dehydrogenase inhibitors among the tested Colletotrichum gloeosporioides populations,which also implied the risk of drug resistance.Genome-wide data and bioinformatics analysis revealed that Colletotrichum gloeosporioides succinate dehydrogenase consists of 4 subunits: Flavin(SDHA),Ferritin(SDHB)and two membrane anchored proteins(SDHC and SDHD).q RT-PCR analysis showed that 4 subunits were involved in the infection process of the pathogen to some extent,among which CgSDHB,CgSDHC,CgSDHD and other subunits were stressed by succinate dehydrogenase inhibitors.By constructing gene editing CRISPR/Cas9 vectors of CgSDHB,CgSDHC and CgSDHD,the protoplasts of Colletotrichum gloeosporioides were transformed by PEG mediated protoplast transformation,the gene editing mutants of CgSDHB,CgSDHC and CgSDHD subunits,named B14,C61 and D70,were obtained by resistance screening,resistance gene amplification and Sanger sequencing,the CRISPR/Cas9 system was successfully established in Colletotrichum Gloeosporioides,which laid a foundation for studying the function of succinate dehydrogenase subunits.Compared with wild-type strain 171-1,the 3 CRISPR/Cas9 mutants of succinate dehydrogenase had significantly slower mycelium growth,less virulence,and stronger inhibition of ATP energy formation,and reduced activities of cell wall degrading enzymes and pectinase associated with pathogenicity.When the pathogen was treated with SDHIs such as thifuramide,pyridamide and flupyroxamide,the same phenotype was obtained as that of the mutant deletion.A comprehensive analysis would speculated that the function of Colletotrichum gloeosporioides succinate dehydrogenase was to maintain the dynamic equilibrium between succinic acid and fumaric acid,and to keep the ATP energy produced continuously by the TCA cycle for mycelial growth and normal life activities.When the succinate dehydrogenase subunits of Colletotrichum gloeosporioides were mutated or treated with inhibitors,the inhibition of the assembly of succinate dehydrogenase protein complex,the decrease of enzyme activity,the change of the balance between succinic acid and fumaric acid,the decrease of ATP production,the decrease of mycelium activity and the loss of pathogenicity,the suitable concentration of inhibitors can cause mycelial death and protect the host from the disease.