Identification Of Main Pathogenic Fungus Species Isolated From Fritillaria Thunbergu Miq. And Their Sensitivity To Fungicides

Objective Fritillaria thunbergii Miq.,as one of "Zhejiang Bawei""is an important economic crop in Zhejiang Province.Due to vegetative propagation for a long time and continuous cropping barrier of F.thunbergii,the fungal diseases are increasingly prominent.As a result of the irrational use of fungicides,the problem of pesticide remnant,environmental pollution,resistance of the pathogenic fungi is more and more serious,which has seriously restricted the sustainable development of F.thunbergii.Therefore,we isolated and identified the main pathogenic fungi from F.thunbergii.Monoclonal antibody(McAb)against the main pathogenic fungi isolated from F.thunbergii were prepared,which establishes the groundwork for subsequent development of colloidal gold immune chromatography test paper.The sensitivity of pathogenic fungi to different fungicides was determinated to provide scientific basis and technical support for the rational selection of fungicides to control disease,and improve the yield and quality of F.thunbergii.Methods(1)The pathogen fungi isolated from F.thunbergii collected from Panan,Zhejiang Province,were preliminary identified through observation of morphological features and culture characters.The pathogenicity of fungi was determined by inoculation test.The Internal Transcribed Spacer(ITS)and Translation Elongation Factor 1 Alpha(TEF-la)sequences were used as the DNA barcodes to further clarify the species of pathogenic fungi.(2)Bal b/c mice were immunized with the mixture of mycelium and spores.Using hybridoma technology,the McAbs against the pathogen fungi isolated were prepared.The characters of McAbs including titer,specificity,sensitivity,subclasses,target proteins,and so on were studied by indirect ELISA and Western blot.(3)Through inhibition zone method and mycelium growth rate method,the sensitivity of the pathogen fungi to three novel fungicides(Prochloraz,Difenoconazole,Tebuconazole)were studied in laboratory.Results(1)Four pathogenic fungi were isolated and purified from disease plants of F.thunbergii.Isolate 1 was identificated as the pathogen of black spot,isolate 2 and 3 were identificated as the pathogen of dry rot,and isolate 4 was identificated as the pathogen of leaf spot.The felt colony of isolate 1 was gray white,green brown to black brown later;conidia was clavate in shape with cross and longitudinal walls,dark brown,borne in single or chains,breaks without forks.The sequence of ITS and TEF-la of isolate 1 was found to match 100%with the sequence of A.alternata in GenBank.The colony of isolate 2 was regularly round in shape with white front and purple back,the macroconidium are falciform in shape with 1～4 septum,microconidium usually abundant,elliptic and botuliform in shape with 0～1 septum,accumulating in slimy heads.The sequence of ITS and TEF-la of isolate 2 was found to match 100%with the sequence of F.oxysporum in GenBank.The colony of isolate 3 was primarily white,gradually camel,macroconidium usually abundant,falciform in shape with 3～6 septum,chlamydospore grow on middle of mycelium,brown,spherical,the sporulation pattern were monophialides and polyhialide.The sequence of ITS and TEF-la of isolate 3 was found to match 100%with the sequence of F.incarnatum in GenBank.The colony of isolate 4 was round cultured on PDA,the colony’s center was carnation with slightly protuberance,the outer ring was gray white,and matrix showed reddish-brown later;the colony cultured on OA,showed primarily white,gradually red and light green color on the medium.The sequence of ITS of isolate 4 was found to match 100%with the sequence of Phoma sp.Anhui-Rfsb04(accession number:KX219595)in GenBank,and the sequence of TEF-1α of the isolates was found to match 86%with the sequence of P.fungicola strain 7F90(accession number:KC357259)in GenBank.(2)Obtained two hybridoma cell strains named 14A9 and 5B6,respectively,which could secrete monoclonal antibody against Fusarium sp..14A9 belonged to IgM,and 5B6 belonged to IgG1.As demonst rated by indirect ELISA,the titers of the purified ascetic fluids of 14A9 and 5B6 were 1.311×107 and 4.096×105,respectively.The sensitivity of 14A9 to F.incarnatum was 0.125 μg/mL;the sensitivity of 5B6 to F.oxysporum was 0.0625 μg/mL.The specificity identification results show that six strains of Fusarium sp.include F.proliferatum,F.lateritium,F.sacchari,F.equiseti,F.incarnatum,and F.oxysporum have cross reaction with 14A9,and no cross reaction with A.alternata or Phoma sp.,while only F.incarnatum and F.oxysporum,which isolated from F.thunbergii,have cross reaction with 5B6.It was demonstrated by western blot that 14A9 combinated with 51 kDa and 37 kDa protein of F.incarnatum,and 5B11 combinated with 37 kDa and 39 kDa protein of F.oxysporum.(3)The results of the inhibition zone method showed that Prochloraz had the highest inhibition effect on F.oxysporum,F.incarnatum and A.alternate.The results of mycelium growth rate method showed that Prochloraz had the highest inhibition effect on F.oxysporum,F.incarnatum and Phoma sp.,the EC50 was estimated to be 0.001 mg/L,0.042 mg/L and 0.124 mg/L,respectiely.The order of toxicity to A.alternate was Difenoconazole>Prochloraz>Tebuconazole,the EC50 of Difenoconazole to A.alternate was 0.016 mg/L.Conclusion(1)Four pathogenic fungi were isolated from Fritillaria thunbergii.The results showed that A.alternate is the pathogen to cause black spot disease,F.oxysporum and F.incarnatum could result in dry rot disease and Phoma sp.could cause leaf spot disease in F.thunbergii.F.incarnatum and Phoma sp.were isolated from F.thunbergii for the first time(2)Two hybridoma cell strains,secreting McAbs against Fusarium sp.,were obtained.5B6 has a great specificity.(3)Prochloraz can restrained the spore germination of F.oxysporum and F.incarnatum,inhibit the mycelial growth of F.oxysporum,F.incarnatum and Phoma sp..A.alternate has a high sensitivity to Prochloraz,while the inhibition of Difenoconazole to mycelial growth was stronger than other two fungicides.