Characterization and origin of infection of Rhizoctonia solani associated with Brassica oleracea crops in the UK

An extensive study was conducted to determine where in the production chain Rhizoctonia solani became associated with UK module-raised Brassica oleracea plants. In total, 2600 plants from 52 crops were sampled directly from propagators and repeat sampled from the field. Additional soil, compost and water samples were collected from propagation nurseries and screened using conventional agar isolation methods. No isolates of R. solani were recovered from any samples collected from propagation nurseries. Furthermore, nucleic acid preparations from samples of soil and compost from propagation nurseries gave negative results when tested for R. solani using real-time PCR. Conversely, R. solani was recovered from 116 of 1300 stem bases collected from field crops. All the data collected suggested R. solani became associated with B. oleracea in the field rather than during propagation. Parsimony and Bayesian phylogenetic studies of ribosomal DNA suggested the majority of further classified isolates belonged to anastomosis groups 2-1 (48/57) and AG-4HGII (8/57), groups known to be pathogenic on Brassica spp. in other countries. Many R. solani isolates were recovered from symptomless plant material and the possibilities for such an association are discussed.

Molecular tools to investigate Rhizoctonia solani distribution in soil

Real-time PCR protocols were developed to detect and discriminate 11 anastomosis groups (AGs) of Rhizoctonia solani using ribosomal internal transcribed spacer (ITS) regions (AG-1-IA, AG-1-IC, AG-2-1, AG-2-2, AG-4HGI+II, AG-4HGIII, AG-8) or beta-tubulin (AG-3, AG-4HGII, AG-5 and AG-9) sequences. All real-time assays were target group specific, except AG-2-2, which showed a weak cross-reaction with AG-2tabac. In addition, methods were developed for the high throughput extraction of DNA from soil and compost samples. The DNA extraction method was used with the AG-2-1 assay and shown to be quantitative with a detection threshold of 10-7 g of R. solani per g of soil. A similar DNA extraction efficiency was observed for samples from three contrasting soil types. The developed methods were then used to investigate the spatial distribution of R. solani AG-2-1 in field soils. Soil from shallow depths of a field planted with Brassica oleracea tested positive for R. solani AG-2-1 more frequently than soil collected from greater depths. Quantification of R. solani inoculum in field samples proved challenging due to low levels of inoculum in naturally occurring soils. The potential uses of real-time PCR and DNA extraction protocols to investigate the epidemiology of R. solani are discussed.

The N-terminal SH2 domain of Syk is required for (hem)ITAM, but not integrin, signaling in mouse platelets

We have used a novel knockin mouse to investigate the effect of disruption of phosphotyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2, and integrin αIIbβ3. The Syk(R41Afl/fl) mouse was crossed to a PF4-Cre(+) mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. Syk(R41Afl/fl;PF4-Cre) mice are born at approximately 50% of the expected frequency and have a similar phenotype to Syk(fl/fl;PF4-Cre) mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the 2 vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in Syk(R41Afl/fl;PF4-Cre) platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2-stimulated platelets, suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signaling by integrin αIIbβ3 is not altered, but it is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that αIIbβ3 regulates Syk through an ITAM-independent pathway in mice and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2.