title: Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines.
abstract: Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.
authors.affiliation: National Centre for Advanced Bio-Protection Technologies, Lincoln University, Canterbury, New Zealand. ridgwh@lincoln.ac.nz
authors.forename: Hayley J
authors.initials: HJ
authors.lastname: Ridgway
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Steyaert
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Pottinger
authors.forename: Margaret
authors.initials: M
authors.lastname: Carpenter
authors.forename: David
authors.initials: D
authors.lastname: Nicol
authors.forename: Alison
authors.initials: A
authors.lastname: Stewart
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000235:genetics* / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005656:Fungal Proteins / Q000235:genetics; D005819:Genetic Markers; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013170:Spores / Q000302:isolation & purification; D027843:Vitis / Q000382:microbiology*
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D005819:Genetic Markers; C414954:moxY protein, Aspergillus parasiticus; D010105:Oxygenases; C056434:endodeoxyribonuclease BsrI; D015252:Deoxyribonucleases, Type II Site-Specific