title: Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit.
abstract: Pilidiella granati, a causal agent of twig blight and crown rot of pomegranate, is an emerging threat that may cause severe risk to the pomegranate industry in the future. Development of a rapid assay for the timely and accurate detection of P. granati will be helpful in the active surveillance and management of the disease caused by this pathogen. In this study, a nested PCR method was established for the detection of P. granati. Comparative analysis of genetic diversity within 5.8S rDNA internal transcribed spacer (ITS) sequences of P. granati and 21 other selected fungal species was performed to design species-specific primers (S1 and S2). This primer pair successfully amplified a 450 bp product exclusively from the genomic DNA of P. granati. The developed method can detect 10 pg genomic DNA of the pathogen in about 6 h. This technique was successfully applied to detect the natural infection of P. granati in the pomegranate fruit. The designed protocol is rapid and precise with a high degree of sensitivity.
authors.affiliation: Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China.|Scientific Observing and Experimental Station of Crop Pests in Hefei, Ministry of Agriculture, China.|Laboratory of Quality and Safety Risk Assessment for Agro-Products (Hefei), Ministry of Agriculture, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Yang
authors.affiliation: Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China.|Institute of Industrial Biotechnology, Government College University, Lahore 54000, Pakistan.
authors.forename: Uzma
authors.initials: U
authors.lastname: Hameed
authors.affiliation: Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China.|Scientific Observing and Experimental Station of Crop Pests in Hefei, Ministry of Agriculture, China.|Laboratory of Quality and Safety Risk Assessment for Agro-Products (Hefei), Ministry of Agriculture, China.
authors.forename: Ai-Fang
authors.initials: AF
authors.lastname: Zhang
authors.affiliation: Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China.|Scientific Observing and Experimental Station of Crop Pests in Hefei, Ministry of Agriculture, China.|Laboratory of Quality and Safety Risk Assessment for Agro-Products (Hefei), Ministry of Agriculture, China.
authors.forename: Hao-Yu
authors.initials: HY
authors.lastname: Zang
authors.affiliation: Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China.|Scientific Observing and Experimental Station of Crop Pests in Hefei, Ministry of Agriculture, China.|Laboratory of Quality and Safety Risk Assessment for Agro-Products (Hefei), Ministry of Agriculture, China.
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Gu
authors.affiliation: Institute of Plant Protection and Agro-products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China.|Scientific Observing and Experimental Station of Crop Pests in Hefei, Ministry of Agriculture, China.|Laboratory of Quality and Safety Risk Assessment for Agro-Products (Hefei), Ministry of Agriculture, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Anhui Academy of Agricultural Sciences, Hefei 230031, China.|Key Laboratory of Genetic Improvement and Ecophysiology of Horticultural Crop, Anhui Province, China.
authors.forename: Yi-Liu
authors.initials: YL
authors.lastname: Xu
mesh_terms: D001203:Ascomycota / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005638:Fruit / Q000382:microbiology; D029561:Lythraceae / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012340:RNA, Ribosomal, 5.8S