Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses

Abstract

A rapid identification assay for Waitea circinata (anamorph: Rhizoctonia spp.) varieties zeae and circinata causing patch diseases on turfgrasses was developed based on the universally primed PCR (UP-PCR) products cross-blot hybridization. Tester isolates belonging to the two varieties of W. circinata were amplified with a single UP primer L21, which generated multiple DNA fragments for each variety. Probes were prepared with UP-PCR products of each tester isolate by labeling with digoxigenin. Fieldcollected W. circinata isolates and representative isolates of different R. solani anastomosis groups (AG) and AG subgroups were amplified with L21, immobilized on nylon membrane and cross hybridized with the two probes. Isolates within a W. circinata variety cross-hybridized strongly, while non-homologous isolates did not cross-hybridize or did so weakly. Closely related W. circinata varieties zeae and circinata were clearly distinguished with this assay. Sequencecharacterized amplified region (SCAR) markers also were developed from UP-PCR products to identify isolates of Thanatephorus cucumeris (anamorph: R. solani) AG 1-IB and AG 2-2IIIB. These two AGs are commonly isolated from diseased, cool-season turfgrasses. The specific SCAR markers that were developed could differentiate isolates of AG 1-IB or AG 2-2IIIB groups. These SCAR markers did not amplify a product from genomic DNA of nontarget isolates of Rhizoctonia. The specificities and sensitivities of the SCAR primers were tested on total DNA extracted from several field-grown, cool-season turf species having severe brown-patch symptoms. First, the leaf samples from diseased turf species were tested for the anastomosis groups of the causal pathogen, and thereafter the total DNA was amplified with the specific primers. The specific primers were sensitive and unique enough to produce a band from total DNA of diseased turfgrasses infected with either AG 1-IB or AG 2-2IIIB.

The genusBipolaris

“…The monotypic genus Porocercospora is a recently described genus introduced by Amaradasa et al. (2014) , placed between the genera Bipolaris and Curvularia in their phylogenetic analysis.…”

“…Porocercospora is morphologically distinct from Bipolaris in having densely aggregated conidiophores arising from brown stroma with characteristic conidial morphology. The cylindrical or subcylindrical conidia generally have a sub-obtuse apex and obconically truncate base with a distinct thickened and brown hilum ( Amaradasa et al. 2014 ).…”

Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris

“…Thus, although the ITS (ITS-5.8S-ITS2) primary sequences of all the three isolates of Rtul formed a well-supported clade in the phylogenetic tree (Figure 6), molecular morphometric analyses clearly differentiated them as three potentially distinct sexual species within R. tuliparum, sensu lato, per Müller et al [45] and Wolf et al [31] (Figure 8; Table S4), and call for further investigation. Moreover, from disease epidemiology and management viewpoints, our findings call for accurate identification of pathogen genotypes as well as determination of sensitivities of genetically diverse isolates of Rhizoctonia pathogens to commonly used fungicides [13,43,[47][48][49][50].…”

SCAR marker: A potential tool for authentication of agriculturally important microorganisms

“…morsprunorum (races-1 and 2) [27] Puccinia striiformis f. sp. tritici [28,29] Ralstonia solanacearum race 2 [30] Rhizoctonia solani AG 1-IB [31] AG 1-IB and AG 2-2IIIB [32] Plasmodiophora brassicae P1 [33] Sclerospora graminicola Pathotype 1 [34] Synchytrium endobioticum Pathotype D1 [20] Xanthomonas axonopodis pv. dieffenbachiae [35,36] Xanthomonas campestris pv.…”

“…Requires additional cost and effort for probe designing and fluorescent labeling [32,80] Monoclonal antibiotics with immunocapture PCR Sensitive detection and feasible at the genus level Time taking and tedious to develop antibodies for each microbial strain. [35] Abbreviations: PCR, polymerase chain reaction; SCAR, sequence characterized amplified region.…”