Written by John Daudu (far right) at the University of Hertfordshire. This is the report from a BSPP Junior Fellowship. Click here to read more/apply for one yourself.
I consider myself fortunate to have been awarded a junior fellowship by the BSPP to support my studies on a mycovirus from Dothistroma septosporum, the causative agent of pine needle blight. This fellowship has helped me immensely in supporting collaborative work with Dr Katherine Tubby and her colleagues at Forest Research UK.
Dothistroma needle blight (DNB), caused by Dothistroma septosporum, has emerged in the British Isles as a significant threat to Corsican pine, lodgepole pine and Scots pine over the past few decades. DNB is endemic on indigenous pine populations in the Northern hemisphere and is a significant world-wide tree health issue. This is particularly true for the UK, where DNB affects the commercial pine tree industry, causing premature defoliation in forest tree nurseries and in some cases tree mortality. It has been reported that over 63 countries and over 82 pine species have been affected by DNB. The economic significance is challenging to evaluate; however, in the UK, an estimated £8.6 million is lost per annum, mainly due to DNB.
Recent studies confirm that mycoviruses (fungal viruses) can reduce the growth and pathogenicity of fungal plant pathogens of their hosts (hypovirulence) and therefore have mostly unexplored potential as biological control agents. One of the most successful mycovirus-mediated biocontrol applications utilised mycovirus infected isolates of Cryphonectria parasitica, the causative agent of chestnut blight in chestnut trees (Castanea spp.). Cryphonectria parasitica hypovirus 1, a member of the family Hypoviridae significantly reduced the growth rate of secondary infections by the host fungus, protecting the chestnut trees.
My PhD project focuses on one D. septosporum isolate infected with a tetrapartite mycovirus. My aim was to characterise this novel mycovirus by sequencing its double-stranded RNA genome using genome walking and recombinant DNA technology; to eradicate the mycovirus and assess the effects of infection on fungal phenotype and growth utilising virus-free and virus-infected isogenic isolates and to investigate the pathogenicity of virus-free and virus-infected isogenic isolates on pine tree saplings and pine needles. This last section specifically concerned my BSPP fellowship.
The four viral genomic segments of the mycovirus were cloned and sequenced; each one contained a single open reading frame (ORF) flanked by 5’ and 3’ untranslated regions. The ORFs encode respectively an RNA-dependant RNA polymerase (RdRP) to replicate the virus, the capsid protein, a protein of unknown function and a putative protease. All protein sequences were closely related to members of the family Chrysoviridae particularly to Isaria javanica chrysovirus. Phylogenetic analysis of the RdRP sequence showed that the D. septosporum mycovirus belongs to the genus Alphachrysovirus, family Chrysoviridae and was nominated DsCV-1 following the discovery of conserved functional motifs in the RdRP sequence. Further data analysis revealed the presence of conserved Eukaryotic Linear Motifs (ELMs) in the DsCV-1 RdRP sequence. ELMs are small compact protein interaction sites of specific amino acid sequence that play crucial roles in cell regulatory processes and can often be used to manipulate host cellular machinery. Alphachrysoviruses were predicted to affect fungal growth and development together with gene expression based on their conserved ELMs. Subsequently, DsCV-1 infected and DsCV-1 free isogenic lines were generated using ribavirin to eliminate the virus and their phenotype, growth rate and virulence were compared in terms of radial growth on solid pine minimal media with glucose (PMMG), biomass in liquid PMMG, production of the mycotoxin dothistromin and pathogenicity in pine tree nurseries.
To conduct the pathogenicity assays in pine tree nurseries I have visited the laboratory of Dr Katherine Tubby at Forest Research UK on over 10 occasions, where excellent facilities are available and outstanding work is done on tree and plant pathology. Forest Research is located at Alice Holt Lodge, Wrecclesham, Farnham, Surrey. I took advantage of my visits there to learn new technology, such as growing and maintaining pine trees, inoculation with fungi and sampling, together with quantitative polymerase chain reaction (qPCR) amplification. Additionally, I was granted access to the state-of-the-art growth chambers which mimicked the required conditions for D. septosporum pathogenicity assays and allowed the fungus to grow under optimal conditions on pine tree saplings. The DsCV-1 infected and DsCV-1 free isogenic lines and six other isolates of D. septosporum are being investigated in two duplicate trials over a period of six months. These experiments are currently ongoing as pine infection of D. septosporum takes eight to twelve weeks to be visually confirmed. Following sampling the levels of D. septosporum will be quantified using a qPCR protocol devised in the Forest Research laboratories. Any effects of DsCV-1 infection on the saplings will be monitored by PCR comparing virus concentration, fungal growth, fungal toxin levels and pine tree growth.
I wish to acknowledge Drs Ioly Kotta-Loizou (Imperial College) and Avice Hall (University of Hertfordshire) for their support and Dr Robert Coutts (University of Hertfordshire) for his supervisory assistance with the project and for introducing me to Dr Katherine Tubby and James Snowden at Forest Research. Finally, I wish to thank the BSPP for funding this endeavour.
John Daudu (far right)
University of Hertfordshire.