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.
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ABSTRACT: The suitability of random amplified polymorphic DNA for identification of Pythium aphanidermatum was investigated. Three oligoprimers were selected after testing three isolates of P. aphanidermatum and one isolate of P. ultimum with a total of 40 primers. The selected 10-mer primers were used with 20 isolates of P. aphanidermatum, four isolates of P. deliense, two isolates of P. ultimum, two isolates of P. irregulars and one isolate of P. paroecandrum. Most of the P. aphanidermatum isolates (13 of 20), were obtained from samples of Cucumis sativus, or water from cucumber greenhouses. The three selected primers gave identical fingerprints for 18 of the 20 P. aphanidermatum isolates, including all the isolates from cucumbers. Two of the primers gave fingerprints that could be used to differentiate between isolates of the Pythium species studied. The banding pattern of P. aphanidermatum given by the third primer could not easily be distinguished from the fingerprint of P. deliense. However, when used in conjunction with the other two primers, the third primer can be used to verify the identity of P. aphanidermatum.Mycological Research. 01/1998;
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ABSTRACT: Physical maps of mitochondrial DNA from eight Oomycete species were constructed to determine genome size and to detect inverted repeats (IRs).Aplanopsis terrestris (mtDNA 45.0 kb) andLeptolegnia caudata (53.0 kb) had IRs of about 10 kb arranged very much like those inAchlya species investigated earlier (M. E. S. Hudspeth, D. S. Shumard, C. J. R. Bradford, and L. I. Grossman, 1983.Proc. Natl. Acad. Sci. USA80: 142–146;D. A. Boyd, T. C. Hobman, S. A. Gruenke, and G. R. Klassen, 1984.Canad. J. Biochem. Cell Biol.62: 571–576).Pythium paddicum (61.3 kb) andPythium irregulare (60.0 kb) had large IRs (about 20 kb) similar to those in otherPythium species (S. A. McNabb, D. A. Boyd, A. Belkhiri, M. W. Dick, and G. R. Klassen, 1987.Curr. Genet.12: 205–208), andSapromyces elongatus (53.0 kb) had an IR of intermediate size (about 15 kb).Phytophthora cryptogea (40.7 kb),Apodachlya pyrifera (40.0 kb), andApodachlya brachynema (36.4 kb) had no IRs. Approximate mtDNA complexity (genome size minus one arm of the IR) in these species and in others investigated earlier is very uniform, ranging from 36.2 to 45.3 kb with an average value of 39.9 kb. This uniformity is in contrast to the nonuniformity observed in other fungal groups and is reminiscent of that observed in angiosperm chloroplast genomes, where the presence of the IR is the rule. The detection of the IR in Oomycetes may be important for phylogenetic studies as the regularities observed are consistent with accepted taxonomic groupings. The regularities suggest that the IR is an ancestral character for Oomycetes and that the absence of the IR is a derived character useful for phylogenetic analysis within the group.Experimental Mycology. 01/1988;
- Physiological and Molecular Plant Pathology - PHYSIOL MOLEC PLANT PATHOL. 01/1992; 41(4):265-281.