Studies On Fungicide Resistance And Screening Of Biocontrol Strains Of Apple Ring Rot Fungus Botryosphaeria Dothidea

China is the top apple producer,with output and planting area accounting for more than 50%in the world.In apple orchards,apple ring rot is common and serious,which results in the decline of apple yield and quality,thus causing great economical losses.In this study,we collected apple ring rot samples from different orchards in 9provinces of China during 2018 and 2019.Pathogens were isolated and identified,and a reliable detection method was developed for this pathogen.Then,sensitivities of B.dothidea to 4 fungicides were assayed,and corresponding resistance mechanism was demonstrated.In addition,a LAMP method was developed to rapidly and specifically identify resistant isolates.Finally,we investigated the efficiency and possible mechanism of the biocontrol agent,Bacillus velezensis P2-1 against B.dothidea.This study provides the technical and theoretical bases for the diagnosis and integrated management of apple ring rot.1)Identification and detection of causal agent of apple ring rot.During 2018 and2019,apple ring rot samples were collected from 9 different provinces or municipality including Shandong,Shaanxi,Henan,Shanxi,Jiangsu,Beijing,Hebei,Liaoning and Tianjin.From them,440 isolates were obtained by tissue separation method,and representative isolates were selected for morphological and molecular identification.All of the isolates were identified as B.dothidea.In order to specifically differentiate B.dothidea from other fungal species,the conventional PCR,nested PCR,and LAMP assays were developed based on internal transcribed spacer（ITS）sequences.Results showed that the LAMP assay was as sensitive as 10^-6ng/μL of fungal DNA,and was10000-fold higher than the conventional PCR and 10-fold higher than nested PCR.We successfully utilized these nested PCR and LAMP assays to detect diseased apple fruit and branch tissue samples in the field.Therefore,the LAMP assay represents the most sensitive,simple and highly specific approach to detect apple ring rot disease caused by B.dothidea.2)Resistance assays of B.dothidea to four fungicides.The sensitivities of 80representative B.dothidea isolates to carbendazim,tebuconazole,boscalid and pyraclostrobin were determined based on mycelium growth inhibition method.Then,fungicide resistance of all the 440 B.dothidea isolates were evaluated using discrimination doses assay.Altogether 19 carbendazim-resistance isolates were detected from Jiangsu,Henan,and Hebei provinces with the resistance frequencies at33.33%,9.67%and 5.97%respectively for each location,and 4.32%for all locations.The resistance stability and fitness of carbendazim-resistant B.dothidea isolates were also evaluated.Results revealed that the resistance was stable and the fitness parameters of carbendazim-resistant isolates were similar with carbendazim-sensitive isolates in terms of pathogenicity,sporulation,and mycelial growth.Finally,the corresponding resistance mechanism was also investigated.Results showed that theβ-tubulin gene in carbendazim-resistant isolates existed a mutation at codon 198（GAG to GCG）resulting in the substitution of glutamic acid with alanine（E198A）.All isolates were sensitive to tebuconazole,pyraclostrobin and boscalid.These results indicated that carbendazim resistance has emerged in B.dothidea isolates from apple in China.Moreover,the resistant isolates could compete with sensitive ones,showing a high risk of spreading.3)A Loop-mediated isothermal amplification（LAMP）technique was established for rapidly and specifically identifying carbendazim-resistant B.dothidea isolates on apple.The E198A point mutation sequence was used to design LAMP primers.The DNA bearing E198A mutation could be specifically identified by the LAMP technique.After optimization of reaction system and conditions,the results showed that the established LAMP technique had excellent specificity,sensitivity and reproducibility.The LAMP method can be used for the resistance monitoring of B.dothidea to carbendazim on apple crops widely,contributing to the management of such resistance.Moreover,DNA extraction was simplified and crude DNAs of apple fruits were extracted quickly for the LAMP technique,and the B.dothidea isolates bearing the E198A mutation in diseased apple fruits could also be correctly detected.4)Isolation and characterization of B.velezensis strain P2-1 for biocontrol of apple ring rot caused by B.dothidea.In this study,biocontrol bacteria were isolated from healthy apple branches.The results of physiological and biochemical assays and molecular identification showed that the biocontrol strain P2-1 was B.velezensis.It was also shown that the strain P2-1 exhibited strong inhibitory effects against B.dothidea isolates no matter with or without E198A mutation.The cell-free fermentation supernatant of strain P2-1 exhibited antifungal activity against B.dothidea.At the same time,the strain P2-1 could colonize and survive in apple fruits.B.velezensis P2-1 treatment significantly reduced the ring rot caused by B.dothidea,and had no significant effect on apple fruit quality（measured in fruit firmness,titratable acid ascorbic acid,and soluble sugar）.In addition,the results of biocontrol mechanism showed P2-1 strain could lead to mycelial abnormality of B.dothidea.PCR assay revealed that the strain P2-1 harbored the gene clusters（itu D,itu A,srf AD,bae A,mln A,bae A/B,bmy A and dfn A）required for the biosynthesis of antifungal lipopeptides and polyketides.q RT-PCR assay revealed that strain P2-1 treatment significantly enhanced the expression levels of pathogenesis-related genes（Md PR1and Md PR5）in apple fruits.These results suggest that the B.velezensis P2-1 might be a useful biocontrol agent against B.dothidea.