An improved artificial pathogenicity assay for Dothistroma needle blight on Pinus radiata

Dothistroma septosporum causes Dothistroma needle blight (DNB) on pines throughout the world. An increase in incidence and severity of DNB, associated with climatic factors, raises an urgent need to screen for resistance in plant hosts and to detect changes in virulence of the pathogen. The availability of the genome sequence for this fungal pathogen also opens up possibilities for biochemical and molecular studies leading to new methods of disease control. All these activities require a reliable and reproducible DNB pathogenicity assay, but this has been notoriously difficult to achieve in controlled glasshouse conditions. We therefore aimed to improve DNB assays by investigating the effects of various pathogen, environment and host factors, using Pinus radiata. The production of spores in sufficient quantity and quality for infection was optimised by using pine needle minimal medium with glucose (PMMG). The critical environmental parameter for achieving high levels of DNB (>80 % of needles with disease symptoms) was leaf wetness; use of individual covers for 4–7 days post inoculation, followed by incubation in covered chambers containing water foggers for the remainder of the infection cycle, enabled reliably high rates of disease incidence. Using this assay, it was possible to distinguish between trees from families with different levels of field resistance, using clones taken as cuttings from young trees (<1 year). This method will facilitate screening for quantitative resistance traits as well as enabling functional studies of pathogen effectors and virulence factors.