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.Species of the genus Pythium (Oomycota) are cosmopolitan. Several species are facultative parasites of plants and of animals such as fish or crustaceans. Some species are parasites of other fungi, and others are primarily saprophytes. One species, Pythium insidiosum, is the etiological agent of pythiosis in animals and humans, with symptoms such as arteritis (49), keratitis (4), and cutaneous or subcutaneous infections (45). Several species of Pythium are ubiquitous in soils and cause severe declines in crop yield either as sole pathogens or in complexes with Fusarium spp. and Rhizoctonia spp. The wide distribution and host range of this genus demonstrate its ecological importance and its impact on many human activities.

Pythium cederbergensesp. nov. and related taxa fromPythiumclade G associated with the South African indigenous plantAspalathus linearis(rooibos)

“…Therefore, 55 C was selected for all membrane hybridizations. This temperature is close to that (54 C) used by Tambong et al (2006) in their Pythium macro-array. The two Pythium RB I/P.…”

“…In addition to the four Pythium RB I/P. cederbergense and RB II oligonucleotides, 54 oligonucleotides from Tambong et al (2006) also were spotted onto the membrane and included the (i) clade G oligonucleotides iwa51 (iwayamai group), iwa52 (P. iwayamai), and vioIwa (P. violae, P. iwayamai), (ii) 12 oligos that detect other Pythium species that have been identified from rooibos (Bahramisharif 2012) and (iii) nine oligos that detect pathogenic Pythium species that frequently are found in South Africa (e.g. P. aphanidermatum [Edson]) Fitzp., P. ultimum, P. vexans de Bary).…”

“…Identification of Pythium species from rooibos seedlings and nursery soils through isolation studies has proven to be time consuming and currently cannot be done on a routine basis for making management decisions. On the other hand, the use of DNA-based technologies, such as the Pythium DNA-macro-array developed by Tambong et al (2006), would allow rapid, simultaneous detection and identification of pathogenic Pythium species within a sample. The identification of non-pathogenic species is also important in that these species hold promise for biological control of pathogenic species on rooibos.…”

Automatic Analysis of Dot Blot Images

“…Currently, macroarrays, i.e., hybridization using as support a membrane of nitrocellulose or nylon, allows for a higher cost-benefit in routine analysis than the much more expensive and elaborate microarray platform. In this context, an effort has been made in the last decade to develop macroarrays for detection/identification of various microorganisms, as demonstrated for a large variety of targets including bacteria pathogenic on potato (Fessehaie, De Boer, & Lévesque, 2003), phytopathogenic Pseudomonas (Vieira et al, 2007), Lactobacillus species (Poltronieri, D'urso, Blaiotta, & Morea, 2008), Pythium species (Tambong, De Cock, Tinker, & Lévesque, 2006) and Aeromonas spp (Khushiramani, Girisha, Karunasagar, & Karunasagar, 2009), among others.…”

Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

“…With automated molecular genotyping techniques, appropriate DNA databases [11] and a better knowledge of ITS variability within fungal species [12], identification of fungal taxa in environmental samples can now be expanded from the aforementioned methods to high-throughput molecular diagnostic tools, such as phylochips [13]. So far, DNA arrays have been mainly used for genome-wide transcription profiling [14,15], but also for the identification of bacterial species from complex environmental samples [16] or for the identification of a few genera of pathogenic fungi and Oomycetes [17,18]. Phylochips may comprise up to several thousand probes that target phylogenetic marker genes, such as 16S rRNA in bacteria or the internal transcribed spacer (ITS) region in fungi [19]; indeed, the latter is one of the most widely used barcoding regions for fungi [20].…”