杧果露水斑病病原菌枝状枝孢霉的巢氏PCR检测方法的建立

Development of a nested PCR detection method for Cladosporium cladosporioides causing mango sooty blotch disease

【目的】杧果露水斑病是当前生产上影响杧果产业的重要病害之一,因潜伏期长常在发病初期不易被察觉而错过防治适期,建立其主要致病菌Cladosporium cladosporioides的快速准确检测方法,对于该病的早期诊断和及时防控尤为重要。【方法】将该病菌的ITS序列与NCBI中的数据大量比对后,在差异位点大的区域设计了8对引物,通过筛选获得两对特异性良好的引物及其扩增条件。【结果】两对特异性引物分别是ML-SF9/ML-SR5和ML-SF10/ML-SR10,扩增条件:94℃4 min;94℃45 s,65℃45 s,72℃1 min,36周;72℃10 min,特异条带大小分别为408 bp和424 bp。为了提高检测的灵敏度,又与真菌通用引物ITS1/ITS4结合后,建立了杧果露水斑病病原菌的两套巢式PCR快速检测体系,可检测病菌DNA的含量最低为3.55×10-9ng·μL-1,比常规PCR灵敏度提高了1万倍,且能实现对潜伏期果实的特异性检测。【结论】此技术操作简单、特异性强、灵敏度高,为杧果露水斑病的早期诊断提供了新方法。

[Objective]Mango sooty blotch disease,a new disease on mango,has occurred in Hainan,Sichuan,Guangxi and Fujian provinces that are major planting areas of mango in China.Mango varieties like Guifei and Jinhuang were easy to be infected in mango orchards.Generally,the young fruit can be infected,but a lot of spots appeared during the harvest time for its slow expansion and long incubation period.Water-soaked and irregular dark lesion with dark green mould appeared on those infected mango peels,which seriously affected the appearance of mango and caused great loss of commodity value.The incidence of some poorly managed orchards was as high as 100%.Sofar mango sooty blotch disease has been one of the important diseases affecting mango industry.It is often difficult to be detected at the early stage for the long incubation period and cause to miss optimum period for control.To timely prevent this disease,it is important to establish the rapid and accurate method for Cladosporium cladosporioides detection,which is the main causal agent of mango sooty blotch disease.[Methods]The Genomic DNA of pathogens was extracted from mycelium using Fungal DNA Kit(OMEGA).Field samples of genomic DNA were extracted from mangoes by the CTAB method.Based on nucleotide differences in the internal transcribed spacer(ITS)sequences of Cl.cladosporioides and ITS of other pathogens from the data in NC-BI,eight sets of primer pairs were designed and screened for use in PCR assay in the present work.Eleven Cl.cladosporioides strains from leaf,branch and fruit samples were collected from diseased symptomatic mango plants from orchards in the mango growing areas in Hainan and Sichuan regions.Eleven pathogenic strains of other mango diseases such as Botryodiplodia theobromae,Colletotrichuma cutatum,C.gloeoporioides,B.theobromae,Xanthomonas campestris pv.mangiferae,Sphaceloma mangiferae,Trichothecium roseum,Pestalotiopsis mangiferae,Fusarium mangiferae,F.proliferatum and F.decemcellμlare were used to determine the specificity of the primers.Each PCR reaction mixture contained 12.5 μL 2×Taq PCR Master Mixture(TaKaRa),1 μL of genomic DNA,1 μL of 10 μmol· L^-1 primers 1/2,and ddH2 O 9.5 μL in a total volume of 25 μL.The PCR thermal cyclingreaction was started by denaturation at 94 ℃ for 4 min,followed by 36 cycles of 94 ℃ for 45 s,Tm℃ for 45 s,and 72 ℃ for 1 min,and then a final extension at72 ℃ for 10 min,and annealing temperature Tm included 51 ℃,54 ℃,57 ℃,60 ℃,63 ℃,65 ℃ and67 ℃ for screening the optimizing PCR amplifying program.To increase the sensitivity,two nested-PCR protocols were further established.The first round PCR amplification was performed by using the universal fungal primers ITS1(5’-TCCGTAGGTGAACCTGCGG-3’)and ITS4(5’-TCCTCCGCTTATTGATATGC-3’)with the similar PCR reaction mixture.PCR was performed using the following parameters:one cycle at 94 ℃ for 3 min,30 cycles at 94 ℃ for 45 s,56 ℃ for 45 s,and 72 ℃ for 1 min,followed by one cycle at 72 ℃ for 10 min.Then,1 μL PCR product was used as templates for the second round of PCR amplification with the primers ML-SF9/ML-SR5 and ML-SF10/ML-SR10 using the same conditions previously screened.To determine the sensitivity of the PCR and nested-PCR protocols,the genomic DNA of Cl.cladosporioides was diluted from 0 to 10^10 with ddH2 O by preparing 10-fold serial dilutions,respectively.To test the feasibility of early detection from the Cl.cladosporioides,the mango peel after8 d inoculation with Cl.cladosporioides and six young mango fruits showing the typical,atypical and no visible disease symptoms of sooty blotch disease from Tainong and Jidan mangoes were collected from the field,and total DNA samples were extracted for PCR and nested-PCR amplifications.The amplified PCR products were separated on 1.5% agarose containing goldview,visualized and documented in a Biorad UV Trans illuminator.[Results]A total of 21 fungal and one bacterial DNA samples were subjected to PCR amplification with 8 pairs of primers.The results showed that only two pairs of primers ML-SF9/ML-SR5 and ML-SF10/ML-SR10(ML-SF9:5’-TAGCCTCCCGAGCACCCTT-3’/ML-SR5:5’-GTTCATAACCCTTTGTTGTCC-3’and ML-SF10:5’-TAGCCTCCCGAGCACCCTT-3’/ML-SR10:5’-GTTTACCACCGGGATGTTCATAAC-3’)were highly specific for Cl.cladosporioides when the annealing temperature was 65 ℃.A 408 bp and 424 bp unique bands were respectively obtained from each of the 11 Cl.cladosporioides strains collected from leaf,branch and fruit samples in various areas in China,but not from other fungal and bacterial species.The results showed that the minimum amount of the fungal DNA could be detected by the PCR using the primer combination ML-SF9/ML-SR5 and MLSF10/ML-SR10 of about 3.55×10-4 ng· μL^-1and 3.55×10-5 ng· μL^-1respectively,while nested-PCR could detect as low as 3.55×10^-9 ng· μL^-1of the fungal genomic DNA,which indicated at least 10 000-fold higher sensitivity than the conventional PCR method.Using PCR,the fungal DNA molecules were not detected from the mango peel after 8 d inoculation with Cl.cladosporioides,while nested-PCR could amplify target band,which indicated nested-PCR could sensitively detect target pathogen.The nested-PCR could amplify the PCR band from six young mango fruits showing the typical,atypical disease symptoms of sooty blotch disease from Tainong and Jidan mangoes,but could not amplify the band from no visible disease symptoms of fruits,which did not appeared symptoms of sooty blotch disease all the time.[Conclusion]This study has developed two conventional PCR methods and two nested-PCR method for the detection of Cl.cladosporioides with higher sensitivity and specificity.The findings would be a good tool for various applications,such as mango quarantine,early diagnostic applications and surveillance of mango sooty blotch disease,which would be useful for timely preventing this disease.