An Oligonucleotide Array for the Identification and Differentiation of Bacteria Pathogenic on Potato

Abstract

Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.

Oligonucleotide Array for Identification and Detection of Pythium Species

“…The oligonucleotide arrays were prepared and spotted as reported earlier by Fessehaie et al (15). The oligonucleotides listed in Table 1 were synthesized with a C6 5Ј amino group, which acts as a covalent linker to the membrane.…”

“…Universal primers for the ITS of eukaryotes, UN-up18S42 (forward, 5Ј-CGTAACAAGGTTTCCGTAGGTGAAC-3Ј) and UN-lo28S22 (reverse, 5Ј-GTTTCTTTTCCTCCGCTTATTGATATC-3Ј), were used to amplify DNA fragments. Simultaneous PCR amplification and digoxigenin labeling were performed with Titanium/Ultratherm Taq DNA polymerase (9:1; BD Biosciences Clontech, Inc., Palo Alto, CA, and Tetra Link International Inc., Buffalo, NY, respectively), 10 to 15 ng of template DNA, digoxigenin-labeled dUTP, and other standard reaction substrates as reported by Fessehaie et al (15). PCR was carried out in a Genius thermal cycler (Techne Ltd., Cambridge, United Kingdom) with initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 45 s, 68°C for 45 s, and 72°C for 90 s and a final extension at 72°C for 8 min.…”

“…In plant pathology, array technology targeting the rRNA gene cluster was successfully applied to identify some oomycete fungi (26) and to differentiate bacterial pathogens (Clavibacter spp., Ralstonia solanacearum, and Erwinia spp.) of potato (15). It was also applied to differentiate and detect Verticillum species (29) and species of the nematode genus Pratylenchus (50).…”

DNA Sequencing and Crop Protection

“…Also real-time PCR allows the simultaneous detection of different targets by using probes with different fluorescent reporter dye (Weller et al 2000. Microarray technology is an emerging tool in crop protection for unlimited multiplexing analyses (Fessehaie et al, 2003;Lèvesque et al, 1998;Lievens et al, 2003;Uehara et al, 1999;Mumford et al 2006;Boonham et al 2007), but nowadays existing microarray methods are still complex and relatively insensitive, and a widely accepted diagnostic format has yet to be adopted.…”

DNA microarray-based detection and identification of bacterial and viral pathogens of maize

“…The DNA microarrays were used for detection of potato viruses (Bystřická et al 2003), cucurbit-infecting tobamoviruses (Lee et al 2003), plum pox virus (Pasquini et al 2008), grapevine viruses (Nicolaisen 2011) and for detection of a number of plant viruses in a multiplex assay (Engel et al 2010). Other microarray studies involve both complex detection of a wide range pathogens (Zhang et al 2013) and targeted identification of pathogens in a particular plant host, e.g., tomato (Tiberini et al 2010) or potato (Fessehaie et al 2003). Nevertheless, there is no specific DNA microarray available for detection of microbial pathogens of maize (Zea mays).…”