Rebecca McDougal

The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism

Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.

Dothistroma needle blight.

What does Dothistroma needle blight look like? Dothistroma needle blight first appears as dark green, water-soaked spots on the needles. The spots become tan, yellow, or reddish-brown, and may encircle the needles to form bands. The tip of the needle beyond the band eventually dies leaving the base of the needle alive and green. Young trees are more likely to suffer damage than older trees. Seedlings (< 1 yr. old) can be killed within a year after infection.

A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum.

Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.

Evolution of polyketide synthesis in a Dothideomycete forest pathogen

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.

Development of a high throughput optical density assay to determine fungicide sensitivity of oomycetes

A high-throughput assay was developed to screen Phytophthora species for fungicide sensitivity using optical density measurements for unbiased, automated measurement of mycelial growth. The efficacy of the optical density assay (OD) to measure phosphite sensitivity in Phytophthora species was compared to two widely used methods, radial growth (RG) and dry weight (DW) assays. Three isolates of each of Phytophthora cinnamomi, P. multivora and P. pluvialis, with known phosphite exposure and three isolates of each species with no prior phosphite exposure, were screened for phosphite sensitivity using the three assays. Mycelial growth measurements were taken after culturing for 6, 14 and 15 days for the OD, DW and RG assays respectively. Mycelial growth inhibition at 15, 80, 200 and 500 μg/mL phosphite relative to growth on control media was used to determine effective concentration values for 50% growth reduction (EC50). The species varied in their tolerance to phosphite with P. cinnamomi being the least sensitive followed by P. multivora and P. pluvialis. No significant differences in tolerance were found between isolates within the same species using any method. The OD assay produced comparable EC50 values to the RG and DW assays. The growth of the three species was more sensitive to phosphite in the DW than the RG and OD assays, however limited sample throughput and greater variation in measuring small amounts of mycelia in the dry weight assessment increase variability and limits throughput. The OD assay offers a fast method to enable an inventory of chemical resistance and is particularly advantageous for slow growing species as it requires less time and offers greater throughput than existing RG and DW methods.

Molecular tools for differentiating Cyclaneusma minus morphotypes and assessing their distribution in Pinus radiata forests in New Zealand

BackgroundCyclaneusma needle cast (CNC) is a pine disease caused by the ascomycetous fungus Cyclaneusma minus (Butin) DiCosmo, Peredo and Minter. The pathogen occurs worldwide but is of particular significance in New Zealand where it infects Pinus radiata D. Don plantations. There are two morphological types of C. minus, termed C. minus ‘simile’ and C. minus ‘verum’, recently shown by multigene phylogenetic analysis to belong to distinct clades and therefore proposed to be two separate species. It is currently unknown whether one or both of these molecular operational taxonomic units (MOTUs) are responsible for CNC.MethodsIn this study, DNA analysis of the rDNA internal transcribed spacer (ITS) region was carried out on 120 isolates of C. minus collected in New Zealand since 1969 to distinguish C. minus simile from C. minus verum. Specific primers for C. minus simile and C. minus verum were developed for molecular differentiation from pure cultures and direct amplification from infected needles. Using these specific primers, the distribution of C. minus simile and verum was determined from isolates collected throughout New Zealand.ResultsThe C. minus simile and C. minus verum primers developed were specific to these morphotypes only and were successfully used to identify simile and verum MOTUs from culture. Morphological typing of cultures was consistent with specific primer results in only 56% of isolates tested, demonstrating the high level of skill and experience required for accurate morphotyping from culture alone. C. minus simile was more common throughout New Zealand including regions where CNC is known to be more prevalent. From a collection of 120 isolates, 89% of North Island isolates and 59% of the South Island isolates were C. minus simile. The C. minus simile and verum specific primers were used to detect Cyclaneusma DNA in symptomatic P. radiata needles. C. minus simile was detected in 39% of the needle samples and C. minus verum was only detected in 11%. However, Cyclaneusma DNA was not detected by PCR in 50% of the sampled needles.ConclusionsThe molecular methods developed for differentiation of C. minus simile and C. minus verum can be used to rapidly identify these morphotypes from cultures or from infected needles, which reduces the time otherwise required for morphological identification. Analysis using these tools for characterisation of C. minus sensu lato, isolated from P. radiata plantations in New Zealand since 1969, revealed that C. minus simile was more common than C. minus verum.