Molecular and Behavioural Characterisation of Dothistroma septosporum in Great Britain and New Zealand

Abstract

Dothistroma septosporum is a foliar pathogen that causes Dothistroma needle blight (DNB), affecting over 80 species of tree. The pathogen is of serious concern; it has spread globally and is of particular significance in pines used in the timber industry leading to quarantine status in several countries. This study examined isolates of D. septosporum originating in Great Britain and New Zealand with molecular and behavioural characterisation and identification based on phenotype and genotype. Subcultures grown from 19 archived isolates from five distinct genetic population clusters of D. septosporum from Great Britain and New Zealand were used to create inocula for in vitro and in planta experiments. An in vitro biomass experiment investigated the dependency of fungal growth on temperature, the isolates and their country of origin, population cluster and latitude of the original sample. A second experiment was carried out in planta, inoculating freshly collected Scots pine (Pinus sylvestris Linnaeus), lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Watson) and Corsican pine (Pinus nigra subsp. laricio (Poir.) Palib. ex Maire) needles with D. septosporum conidial suspensions from the chosen isolates. Both experiments were conducted over the temperature range 15°C to 40°C. The fungal DNA concentration recovered from lesions on the needles was estimated using qPCR, and the pathogen species and genotypes for each sample were determined from β-tubulin 2 marker gene sequences and BLAST comparisons. The results for both experiments were statistically analysed using linear mixed-effects modelling to determine any relationship between temperature, the isolates and their country of origin, population cluster and latitude of the original sample, and species of inoculated pine needle. In the case of the in planta results, the mean concentration of DNA recovered from lesions on the three species of inoculated pine needles was also compared as a proxy measure for the fungal burden of the infection in the needles. This study has shown the temperature-dependent growth and fungal burden of D. septosporum for Great Britain and New Zealand population clusters peaks between 20°C and 25°C, and the growth falls away significantly at temperatures on either side of this small range. There appears to be some variation in temperature for peak growth and tolerance of temperature above and below this peak, depending on the population cluster. Differences in the profiles of fungal mass produced in the biomass experiment and the DNA concentrations estimated from lesions in the needle bioassay provided additional information on the variation between population clusters and isolates, both as a function of temperature, and the pine host. A significant difference in growth and fungal burden between isolates from Great Britain and New Zealand was also found at 30°C, and additionally in the pattern of results between in vitro and in planta experiments at this temperature. It is suggested that these differences might be due to gene expression and the role of effector proteins and dothistromin in determining the growth and fungal burden of the pathogen, based on evidence from other studies. A description of the Dothistroma septosporum developmental stages was proposed for in vitro and in planta, which accounts for growth and pathogenicity phenotype behaviour with temperature. It was established that isolates of D. septosporum do not achieve higher levels of infection on species of pine needles that correspond with the original host from which the isolate was sampled, compared to the other two species of pine. The results of this study could help in the management of DNB around the world as the climate changes. Based on the behaviour of the pathogen in response to temperature, consideration needs to be given to the susceptibility of host species and the pathogenicity of different population clusters of D. septosporum. This study has shown that the lesions on needles on some species of pine had much higher fungal burdens than others at lower temperatures and this could assist forest management in planting less susceptible species appropriate to expected summer temperatures.