Specific detection of Pythium aphanidermatum from hydroponic nutrient solution by booster PCR with DNA primers developed from mitochondrial DNA

Phytoparasitica (Impact Factor: 0.72). 01/2002; 30(5):473-485. DOI: 10.1007/BF02979752

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 genus Pythium, with slightly over 280 described species, has been classified traditionally with other filamentous, coenocytic, sporangia-producing fungi as “Phycomyetes”. However, with recent advances in chemical, ultrastructural and molecular studies, Pythium spp. are now considered as “fungus-like organisms” or “pseudo-fungi” and are placed in the Kingdom Chromista or Kingdom Straminopila, distinct from the true fungi of the Kingdom Fungi or Kingdom Mycota. They are widely distributed throughout the world as soil saprophytes or plant pathogens. Because of the warm moist maritime climate, Taiwan, China, is especially rich in Pythium species. To date, 48 species of Pythium have been reported from Taiwan, China, with the dominant species being Py. vexans, Py. spinosum, Py. splendens, Py. aphanidermatum, Py. dissotocum and Py. acanthicum. There is no definite geographical distribution of Pythium spp. in Taiwan, China. Twenty nine species of Pythium have proven to be plant pathogens attacking a wide variety of woody and herbaceous plants primarily causing pre- and post-emergence seedling damping-off, root rot, stem rot and rotting of fruits, tubers and ginger rhizomes, resulting in serious economic losses. The most important plant pathogenic species include Py. aphanidermatum and Py. Myriotylum, which are most active during the hot and wet summer months; whereas Py. splendens, Py. spinosum, Py. ultimum and Py. irregulare cause the greatest damage in the cool winter. Most Pythium spp. are non-specific pathogens, infecting mainly juvenile or succulent tissues. This review attempts to assess the taxonomic position of the genus Pythium and provide details of the historical development of the study of Pythium as pathogens in Taiwan, China, causing diseases of sugarcane, trees, vegetables, fruits, specialty crops and flowering plants, as well as measures to control these diseases. Of special note is the introduction of the S-H mixture which, when used as soil amendment, effectively controls many soil-borne Pythium diseases during the early stages of plant growth. The diversity of Pythium species in Taiwan, China, is discussed in comparison with the situation in the mainland of China and suggestions are made to fully utilize Pythium spp. as agents for biological control, in industry and medicine.
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    ABSTRACT: The objective of this study was to develop a multiplex PCR detection method for the high-temperature-growing pathogens Pythium aphanidermatum, P. helicoides and P. myriotylum. Species-specific primer pairs were designed that targeted the rDNA ITS regions. The multiplex PCR was constructed with a universal primer pair for eukaryotes directed at the 18S rDNA as a positive control, in addition to the three species-specific primer pairs. When the multiplex PCR was applied to naturally infested soils, the expected species were reliably identified, suggesting that the method is suitable for the detection of the three Pythium pathogens in environmental samples.
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