Absence of induced resistance in Agaricus bisporus against Lecanicillium fungicola.

Lecanicillium fungicola causes dry bubble disease and is an important problem in the cultivation of Agaricus bisporus. Little is known about the defense of mushrooms against pathogens in general and L. fungicola in particular. In plants and animals, a first attack by a pathogen often induces a systemic response that results in an acquired resistance to subsequent attacks by the same pathogen. The development of functionally similar responses in these two eukaryotic kingdoms indicates that they are important to all multi-cellular organisms. We investigated if such responses also occur in the interaction between the white button mushroom and L. fungicola. A first infection of mushrooms of the commercial A. bisporus strain Sylvan A15 by L. fungicola did not induce systemic resistance against a subsequent infection. Similar results were obtained with the A. bisporus strain MES01497, which was demonstrated to be more resistant to dry bubble disease. Apparently, fruiting bodies of A. bisporus do not express induced resistance against L. fungicola.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen.

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [ gdh], and the gene encoding the extracellular protein factor [ epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.

Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays.

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.

A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic kinkage map of Mycosphaerella graminicola, the septoria tritici leaf blotch pathogen of wheat.

An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.

Multiplex detection of plant pathogens through the Luminex MagPlex bead system.

Here we describe a versatile multiplex method for both the serological and molecular detection of plant pathogens. The Luminex MagPlex bead system uses small paramagnetic microspheres ("beads"), either coated with specific antibodies or oligonucleotides, which capture respectively viruses and/or bacteria or PCR products obtained from their genetic material. The Luminex MagPlex bead system allows true multiplex detection of up to 500 targets in a single sample on a routine basis. The liquid suspension nature of the method significantly improves (1) assay speed, (2) detection limits and (3) dynamic range. It can also considerably reduce labor and consumables costs.

Development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens.

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.

Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets.

Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.