Oligonucleotide array for identification and detection of pythium species

Abstract

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.

FIG.1.

Summary of hybridization patterns of digoxigenin-labeled PCR amplicons of Pythium species to an array of species- and group-specific oligonucleotides on nylon membranes. Chemiluminograms were scanned on a 16-bit gray scale with Fotolook Ps 2.08 software on an ARCUS II scanner, and gray scale values of each dark spot, computed with GenePix Pro 3.0.6 (AXON Instruments, Inc., Calif.), are indicated by the following symbols: □, <500 (not detected); ▪, 501 to 1,000; ⊡, 1,001 to 10,000; , 10,001 to 20,000; , 20,001 to 30,000; ▪, 30,001 to 65,000 (maximum reaction signal). The locations of the oligonucleotides given at the top correspond to their locations on the membranes in the array. Oligonucleotides are presented in Table 1. Symbols: §, phylogenetic clusters as described by Lévesque and de Cock (25); *, oligonucleotides that exhibited cross-hybridization with nontarget Pythium species; +, oligonucleotides that did not produce detectable signals; #, oligonucleotides from potential new species that were spotted but not hybridized with corresponding digoxigenin-labeled DNA.

FIG.1.

Summary of hybridization patterns of digoxigenin-labeled PCR amplicons of Pythium species to an array of species- and group-specific oligonucleotides on nylon membranes. Chemiluminograms were scanned on a 16-bit gray scale with Fotolook Ps 2.08 software on an ARCUS II scanner, and gray scale values of each dark spot, computed with GenePix Pro 3.0.6 (AXON Instruments, Inc., Calif.), are indicated by the following symbols: □, <500 (not detected); ▪, 501 to 1,000; ⊡, 1,001 to 10,000; , 10,001 to 20,000; , 20,001 to 30,000; ▪, 30,001 to 65,000 (maximum reaction signal). The locations of the oligonucleotides given at the top correspond to their locations on the membranes in the array. Oligonucleotides are presented in Table 1. Symbols: §, phylogenetic clusters as described by Lévesque and de Cock (25); *, oligonucleotides that exhibited cross-hybridization with nontarget Pythium species; +, oligonucleotides that did not produce detectable signals; #, oligonucleotides from potential new species that were spotted but not hybridized with corresponding digoxigenin-labeled DNA.

FIG. 2.

Chemiluminograms showing hybridization patterns of digoxigenin-labeled amplicons obtained after PCR amplification of DNAs of pure cultures of P. aristosporium (A) and P. grandisporangium (B). Amino-linked oligonucleotides were spotted in duplicate, hybridized overnight, exposed on Kodak X-ray film for 45 min, and developed as described in Materials and Methods. Pairs of diagonally arranged dark spots indicate positive hybridization signals. Spots A1 to A5 are universal, family- or genus-specific oligonucleotides as described in Table 1, while A20 is a validating standard for digoxigenin efficiency. Chemiluminograms were repeated at least once, and similar patterns were obtained.

FIG. 3.

Hybridization patterns of digoxigenin-labeled PCR amplicons obtained by direct processing of soil. One representative sample out of four replicates that had very similar hybridization patterns and showed the same species complex. Spots A1 to A5 are universal, family- or genus-specific oligonucleotides as described in Table 1. Chemiluminograms were repeated at least twice, and similar patterns were obtained. The species detected in all soil samples were P. acanthicum (G12, H12), P. arrhenomanes (B16, C16, A16), P. volutum (G18, I18), P. torulosum (A18, C18, D18), P. vanterpolii (H19), P. monospernum (B13, D13), P. acrogynum (C7), P. minus (F7), P. attrantheridium (F9), P. sylvaticum (C10, D10, E10), P. heterothallicum (D5), P. nunn (G6, H6), and P. rostratum (I20).

FIG. 4.

Trees obtained by the unweighted-pair group method using average linkages and based on ITS sequences including the 5.8S rRNA gene subunit from 65 strains obtained by soil dilution plating (A) or baiting (B) compared to ex-type or representative strains (species name followed by culture collection number) which show the species detected on the oligonucleotide array. Reference Pythium species not clustering with any other isolates were not detected or isolated by traditional methods. Taxa (in bold) not clustering with a reference Pythium species were not detected on the oligonucleotide array. Sequences of taxa with asterisks were obtained by subcloning the PCR amplicons.

FIG. 5.

Sequence match between oligonucleotides and ITS sequences of new species from GenBank as assessed through BLAST analyses. Only the oligonucleotides that perfectly matched any of the sequences are shown. The only exception is P. cystogenes, which did not match two bases at the 3′ end of the oligonucleotide. Asterisks indicate species with ITS sequences identical or nearly identical to those of known species. ▪, 100% match; ⊡, 90% match.