Tuula Piri

Population structure of a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato

We describe a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato. This virus, designated as Heterobasidion RNA virus 6 (HetRV6), is taxonomically distant from all previously known viruses of Heterobasidion species, but somewhat related to the Curvularia thermal tolerance virus and the Fusarium graminearum virus 4. Based on a population analysis including 35 virus strains from Heterobasidion abietinum, Heterobasidion parviporum, Heterobasidion annosum sensu stricto and Heterobasidion occidentale, HetRV6 showed a considerable degree of geographical and host-related differentiation. The North American and Eurasian virus populations were clearly separated. In Eurasia, we observed cases of discrepancy between virus and host taxonomy, suggesting interspecies virus transfer. HetRV6 was also successfully transmitted between the three European species H. abietinum, H. annosum and H. parviporum. Based on growth rate tests on agar plates and spruce stem pieces, HetRV6 seemed to be cryptic or slightly mutualistic to its host.

Virus Community Dynamics in the Conifer Pathogenic Fungus Heterobasidion parviporum Following an Artificial Introduction of a Partitivirus

Viruses infecting the conifer pathogenic fungus Heterobasidion annosum sensu lato are intracellular and spread via anastomosis contacts. In the laboratory, these viruses transmit readily even between somatically incompatible isolates, but their dispersal capacity in natural conditions has not been previously studied. We introduced a mycovirus to a heavily diseased forest site by inoculating Norway spruce stumps with heartrot decay using a mycelial suspension of Heterobasidion parviporum strain RT3.49C hosting the partitivirus strain HetRV4-pa1. The Heterobasidion population at the sample plot was screened for mycoviruses prior to and after the inoculation. Based on sequence analysis, the resident H. parviporum strains harbored six different strains of the virus species Heterobasidion RNA virus 6 (HetRV6) and one strain of HetRV4 prior to the inoculation. After three growth seasons, the inoculated H. parviporum host strain was not detected, but the introduced virus had infected two resident H. parviporum genets. The presence of a preexisting HetRV6 infection did not hinder spread of the introduced partitivirus but resulted in coinfections instead. The resident HetRV6 virus population seemed to be highly stable during the incubation period, while the single indigenous HetRV4 infection was not detected after the inoculation. In laboratory infection experiments, the introduced virus could be transmitted successfully into all of the resident H. parviporum genets. This study shows for the first time transmission of a Heterobasidion virus between somatically incompatible hosts in natural conditions.

Viruses accumulate in aging infection centers of a fungal forest pathogen

Fungal viruses (mycoviruses) with RNA genomes are believed to lack extracellular infective particles. These viruses are transmitted laterally among fungal strains through mycelial anastomoses or vertically via their infected spores, but little is known regarding their prevalence and patterns of dispersal under natural conditions. Here, we examined, in detail, the spatial and temporal changes in a mycovirus community and its host fungus Heterobasidion parviporum, the most devastating fungal pathogen of conifers in the Boreal forest region. During the 7-year sampling period, viruses accumulated in clonal host individuals as a result of indigenous viruses spreading within and between clones as well as novel strains arriving via airborne spores. Viral community changes produced pockets of heterogeneity within large H. parviporum clones. The appearance of novel viral infections in aging clones indicated that transient cell-to-cell contacts between Heterobasidion strains are likely to occur more frequently than what was inferred from genotypic analyses. Intraspecific variation was low among the three partitivirus species at the study site, whereas the unassigned viral species HetRV6 was highly polymorphic. The accumulation of point mutations during persistent infections resulted in viral diversification, that is, the presence of nearly identical viral sequence variants within single clones. Our results also suggest that co-infections by distantly related viral species are more stable than those between conspecific strains, and mutual exclusion may play a role in determining mycoviral communities.

Effects of Nutrient Availability on Pest Resistance of Trees

The effects of nutrient availability on the pest resistance of trees has been studied by surveying damage occurrence and pest populations in fertilised field experiments (population-level studies), and by rearing herbivores and pathogens on fertilised trees or cuttings in the laboratory (individual-level studies). These experiments have clearly shown that fertilisers can have marked effects on herbivores (Stark 1965, Waring and Cobb 1992, Kyto et al. 1996b). However, the effects of nutrient availability on individual insects can differ from those at the population level, because the indirect effects on the population can be so strong that they override the effects at the individual level. Nitrogen fertilisation improves the nutritional quality of the foliage for insects by increasing the concentration of free amino acids and by decreasing the concentrations of defence compounds. In spite of this, fertilisation has had only a negligible effect at the insect population level. One explanation is that predators and parasitoids, as well as folivores, also benefit from fertilisation. Lush understorey vegetation and increased herbivore populations support larger predator and parasitoid populations, which restricts any increase in the folivore populations (Fig. 1). The response of folivores to a change in food quality depends on their degree of specialisation. Herbivores that are host-specific, but nitrogen generalists, respond less strongly to changes in food quality than host generalists that depend on specific forms of nitrogen (Prestidge and McNeill 1983).

Heterobasidion Partitivirus 13 Mediates Severe Growth Debilitation and Major Alterations in the Gene Expression of a Fungal Forest Pathogen

ABSTRACT The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the boreal forest region. In this study, we showed that the alphapartitivirus Heterobasidion partitivirus 13 from Heterobasidion annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, of which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and Heterobasidion parviporum. The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing medium, while only two of six H. annosum isolates tested showed significant debilitation. Based on reverse transcription-quantitative PCR (RT-qPCR) analysis, the response toward HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum. We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates. IMPORTANCE A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry, as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of €800 million in Europe. We used virus curing and reintroduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a low growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.

Ectomycorrhizal fungi increase the vitality of Norway spruce seedlings under the pressure of Heterobasidion root rot in vitro but may increase susceptibility to foliar necrotrophs

We tested if root colonisation by ectomycorrhizal fungi (EMF) could alter the susceptibility of Norway spruce (Picea abies) seedlings to root rot infection or necrotic foliar pathogens. Firstly, spruce seedlings were inoculated by various EMF and challenged with Heterobasidion isolates in triaxenix tubes. The ascomycete EMF Meliniomyces bicolor, that had showed strong antagonistic properties towards root rot causing Heterobasidion in vitro, protected spruce seedlings effectively against root rot. Secondly, spruce seedlings, inoculated with M. bicolor or the forest humus, were subjected to necrotrophic foliar pathogens in conventional forest nursery conditions on peat substrates. Botrytis cinerea infection after winter was mild and the level of needle damage was independent of substrate and EMF colonisation. Needle damage severity caused by Gremminiella abietina was high in seedlings grown in substrates with high nutrient availability as well as in seedlings with well-established EMF communities. These results show that albeit M. bicolor is able to protect spruce seedlings against Heterobasidion root rot in axenic cultures it fails to induce systemic protection against foliar pathogens. We also point out that unsterile inoculum sources, such as the forest humus, should not be considered for use in greenhouse conditions as they might predispose seedlings to unintended needle damages.