Dimeric oligonucleotide probes enhance diagnostic macroarray performance.

Disease management can be improved with rapid and accurate pathogen detection and identification techniques. Here we describe the development of a macroarray diagnostic technique with enhanced detection sensitivity and only small reduction in specificity. With probes designed based on the internal transcribed spacer sequences of the rRNA genes of fungal and oomycete strains, we produced a macroarray, which included five types of oligonucleotide probes: monomers (20-24nt), dimers (40-48nt), dimers with a poly-A spacer of 10 bases between the two repeats (50-58nt), monomers with a poly-A tail of 10 (30-34nt) and 20 (40-44nt) bases. The use of repeat sequence probes (dimers) greatly improved the sensitivity of the macroarray. The dimeric probes could reliably detect 0.01fg target genomic DNA, which is lower than the detection limits of most currently available molecular diagnostic methods, such as the conventional PCR and real-time PCR. Dimer probes also had lower signal variability, thereby increasing the macroarray signal uniformity. However, in a few cases, specificity was reduced in the dimer probes. Cross-hybridization occurred in highly similar sequences where the mismatched base was located near the end or in a chain of the same base, but this should be prevented in future array probe design.

Stem and Crown Rot of Chickpea in California Caused by Sclerotinia trifoliorum.

The identities of Sclerotinia isolates obtained from chickpea plants showing stem and crown rot were determined using morphological characteristics, variations in group I introns, and internal transcribed spacer (ITS) sequences. Isolates could be separated into two groups based on growth rates at 22°C, fast growing (about 40 mm per day) versus slow growing (about 20 mm per day). All fast-growing isolates induced stronger color change of a pH-indicating medium than did slow-growing isolates at 22°C. The slow-growing isolates contained at least one group I intron in the nuclear small subunit rDNA, whereas all fast-growing isolates lacked group I introns in the same DNA region. ITS sequences of the slow-growing isolates were identical to sequences of Sclerotinia trifoliorum. Those of the fast-growing isolates were identical to sequences of S. sclerotiorum. Finally, the slow-growing isolates showed ascospore dimorphism, a definitive character of S. trifoliorum, whereas the fast-growing isolates showed no ascospore dimorphism. Isolates of both species were pathogenic on chickpea and caused symptoms similar to those observed in the field. This study not only associated the differences between S. sclerotiorum and S. trifoliorum in growth rates, group I introns, ITS sequences, and ascospore morphology, but also represented the first report that S. trifoliorum causes stem and crown rot of chickpea in North America.