Specific detection of Pythium aphanidermatum from hydroponic nutrient solution by booster PCR with DNA primers developed from mitochondrial DNA
ABSTRACT Pythium aphanidermatum causes damping-off and root rot of vegetable crops in hydroponic systems. A DNA probe was isolated and modified from a library
ofHindIII-digested mitochondrial DNA ofP. aphanidermatum that strongly hybridized to DNA ofP. aphanidermatum and weakly hybridized to DNA ofPythium deliense. Cross-hybridizing sequences were absent from DNA of plants and other related fungi. The probe detected as little as 5 ng
ofP. aphanidermatum DNA and 250 ng ofP. deliense DNA in slot-blot assays.P. aphanidermatum was detected by a hybridization assay of total DNA extracted directly from infected roots. A pair of oligonucleotide primers
P1 and RP2, which allowed amplification of a specific 0.65 kb DNA fragment ofP. aphanidermatum using polymerase chain reaction (PCR), was designed from a specific DNA probe. Specific amplification of this fragment fromP. aphanidermatum was highly sensitive, detecting template DNA as low as 0.1 pg total DNA by booster PCR. Specific booster PCR amplification
using P1 and RP2 was successful in detectingP. aphanidermatum in naturally infected nutrient solution and roots of vegetables in a field hydroponic system.
- SourceAvailable from: usda.gov[Show abstract] [Hide abstract]
ABSTRACT: ABSTRACT A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10(-5) dilution of extracted tissue of artificially infected rhododendron 'Cunningham's White', and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.Phytopathology 05/2006; 96(4):336-45. · 2.97 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Twelve isolates of Pythium species (P. aphanidermatum, P. deliense, P. ultimum var. ultimum and P. ultimum var. sporangiiferum) from different hosts were compared from morphological, pathological and molecular viewpoints. Minimum, optimum and maximum temperatures of P. aphanidermatum and P. deliense were similar while those of P. ultimum var. ultimum and P. ultimum var. sporangiiferum were also similar. All tested isolates were highly virulent against cucumber seedlings with 100% damping-off. RAPD data using three different primers revealed that strains of P. ultimum var. ultimum and P. ultimum var. sporangiiferum are distinct from each other. This data can be used to separate those species from P. aphanidermatum and P. deliense. In contrast, RAPD data cannot be used to separate P. aphanidermatum and P. deliense. Sequence analysis of the ribosomal DNA internal transcribed spacers (ITS) was used to establish phylogenetic relationships among the tested isolates.Archives of Phytopathology and Plant Protection 08/2005; 38(3):193-208.
- [Show abstract] [Hide abstract]
ABSTRACT: The polymerase chain reaction (PCR) technology has been used to rapidly detect, characterize, and identify a variety of organisms. In a PCR diagnosis study, the development of PCR primers is one of the most important steps. This paper reviews several major approaches that have been used successfully for designing PCR primers specific to various phytopathogenic fungi. These approaches include using species-specific genes or DNA regions, or anonymous unique DNA regions to design PCR primers. Since the problem with PCR inhibitors is prevalent in PCR diagnosis of plant diseases, we also review various techniques that have been used to circumvent PCR inhibitors derived from plant tissues, soil, air, and water samples.Crop Protection. 01/2007;