Analysis of Transcriptome and Terpene Constituents of Scots Pine Genotypes Inherently Resistant or Susceptible to Heterobasidion annosum.

Root and stem rot caused by Heterobasidion annosum is a severe problem in boreal Scots pine. Dissecting the features of disease resistance is generally an essential step in resistance breeding in plants and forest trees. In this study, we explored inherent resistance factors of Scots pine against H. annosum. A total of 236 families consisting of 85 full-sib (FS), 35 half-sib population mix (HSpm), and 116 half-sib (HS) families of Scots pine seedlings were inoculated with a H. annosum isolate. We sampled needle tissues before inoculation for terpene measurements and RNA sequencing. Based on the lesion area, the extremes of 12 resistant and 12 susceptible families were selected for further analyses. Necrotic lesions resulting from fungal infection were in a weak to moderate relationship with the plant height. Monoterpenes were the principal terpene compounds observed in Scots pine seedlings. Concentrations of 3-carene were significantly higher in pine genotypes inherently resistant compared with susceptible seedlings. By contrast, susceptible genotypes had significantly higher proportions of α-pinene. Gene ontology analysis of differential expressed transcripts (DETs) revealed that response to biotic factors was enriched in resistant seedlings. Functional characterization of individual DETs revealed that higher expression of transcripts involved in response to abiotic stress was common in susceptible genotypes. This observation was supported by the annotation of hub genes in a key module that was significantly correlated with the lesion trait through weighted gene co-expression network analysis (WGCNA) of 16 HS and HSpm samples. These findings contribute to our understanding of constitutive resistance factors of Scots pine against Heterobasidion root and stem rot diseases.

Molecular and Chemical Screening for Inherent Disease Resistance Factors of Norway Spruce (Picea abies) Clones Against Conifer Stem Rot Pathogen Heterobasidion parviporum.

Root and stem rot of conifer trees caused by Heterobasidion annosum species complex leads to huge economic losses in Europe, yet not much is known about the molecular and chemical basis for host resistance. To identify inherent chemical or molecular markers in clones found to be either resistant or susceptible, we sampled needle tissues of all the clones before pathogen inoculation. We conducted a short-term resistance screening by using the pathogen H. parviporum to inoculate 70 Norway spruce clones. Based on lesion size, subsets of highly susceptible and resistant clones were further analyzed. Terpene detection and RNA sequencing were performed to explore inherent variations in genotypes differing in resistance to pathogenic challenge at chemical and transcriptional levels. A negative correlation emerged between resistance and growth. Terpene profiles of resistant clones showed higher content of monoterpenes and sesquiterpenes, with concomitant increased transcript abundance of genes involved in the terpenoid pathway. A set of upregulated genes relevant to flavonoid biosynthesis was observed in resistant genotypes, whereas higher transcripts of lignin biosynthetic genes were prevalent in susceptible clones. Genes involved in flavonoid and lignin biosynthesis as well as terpene content may have a role in facilitating resistance of Norway spruce against H. parviporum. Our results provide strong support on the feasibility of sampling needle tissues before pathogen inoculation, and the approach could be of value for large-scale screening of novel biomarkers for durable resistance. The additional insights could form a basis for further research on resistance screening in this pathosystem.

Evaluation of potential genetic and chemical markers for Scots pine tolerance against Heterobasidion annosum infection.

MAIN CONCLUSION: Two terpene compounds and four genes were identified as potential biomarkers for further evaluation for Scots pine susceptibility or tolerance against Heterobasidion annosum. Scots pine (Pinus sylvestris) is one of the main sources of timber in the boreal zone of Eurasia. Commercial pine plantations are vulnerable to root and butt rot disease caused by the fungus Heterobasidion annosum. The pathogen affects host growth rate, causes higher mortality and decreases in timber quality, resulting in considerable economic losses to forest owners. Genetic and biochemical factors contributing to Scots pine tolerance against H. annosum infection are not well understood. We assessed the predictive values of a set of potential genetic and chemical markers in a field experiment. We determined the expression levels of 25 genes and the concentrations of 36 terpenoid compounds in needles of 16 Scots pine trees randomly selected from a natural population prior to artificial infection. Stems of the same trees were artificially inoculated with H. annosum, and the length of necrotic lesions was documented 5 months post inoculation. Higher expression level of four genes included in our analysis and encoding predicted α-pinene synthase (two genes), geranyl diphosphate synthase (GPPS), and metacaspase 5 (MC5), could be associated with trees exhibiting increased levels of necrotic lesion formation in response to fungal inoculation. In contrast, concentrations of two terpenoid compounds, ß-caryophyllene and α-humulene, showed significant negative correlations with the lesion size. Further studies with larger sample size will help to elucidate new biomarkers or clarify the potential of the evaluated markers for use in Scots pine disease resistance breeding programs.

Dual RNA-seq analysis provides new insights into interactions between Norway spruce and necrotrophic pathogen Heterobasidion annosum s.l.

BACKGROUND: Root and butt rot of conifer trees caused by fungi belonging to the Heterobasidion annosum species complex is one of the most economically important fungal diseases in commercial conifer plantations throughout the Northern hemisphere. We investigated the interactions between Heterobasidion fungi and their host by conducting dual RNA-seq and chemical analysis on Norway spruce trees naturally infected by Heterobasidion spp. We analyzed host and pathogen transcriptome and phenolic and terpenoid contents of the spruce trees. RESULTS: Presented results emphasize the role of the phenylpropanoid and flavonoid pathways in the chemical defense of Norway spruce trees. Accumulation of lignans was observed in trees displaying symptoms of wood decay. A number of candidate genes with a predicted role in the higher level regulation of spruce defense responses were identified. Our data indicate a possible role of abscisic acid (ABA) signaling in the spruce defense against Heterobasidion infection. Fungal transcripts corresponding to genes encoding carbohydrate- and lignin-degrading enzymes, secondary metabolism genes and effector-like genes were expressed during the host colonization. CONCLUSIONS: Our results provide additional insight into defense strategies employed by Norway spruce trees against Heterobasidion infection. The potential applications of the identified candidate genes as markers for higher resistance against root and butt rot deserve further evaluation.

Tissue Microbiome of Norway Spruce Affected by Heterobasidion-Induced Wood Decay.

Plants live in close association with microbial symbionts, which may affect the host fitness, productivity, and tolerance against biotic and abiotic stressors. The composition of plant microbial communities is influenced by many biotic and abiotic factors, but little is known about the effect of plant pathogens on the structure of these communities. In this study, we investigated the structure of bacterial communities associated with different tissues of asymptomatic and symptomatic (Heterobasidion-rotten) Norway spruce (Picea abies (L.) Karst.) trees. Our results demonstrated that each of the investigated anatomic tissues (root, bark, down stem, upper stem, and needles) harbored a unique bacterial assemblage. However, the health status of the host trees had little effect on the structure of bacterial communities, as the only significant differences among asymptomatic and symptomatic trees were found in the composition of the bacterial communities of needles. Proteobacteria was predominant in all anatomic regions with the highest abundance in needles (86.7%), whereas Actinobacteria showed an opposite trend, being more abundant in the woody tissues than in needles. Additionally, we performed profiling of terpenoid compounds present in spruce xylem and phloem. Total concentrations of monoterpenes and sesquiterpenes were considerably higher in asymptomatic trees. However, we found no significant correlations between terpenoid profiles of spruce trees and the composition of their bacterial communities. Our results provide an insight into the diversity of bacteria associated with Norway spruce tree tissues. At the same time, the health status and terpenoid content of host trees had a limited effect on the composition of bacterial communities in our survey.

Climate Change Effects on Secondary Compounds of Forest Trees in the Northern Hemisphere.

Plant secondary compounds (PSCs), also called secondary metabolites, have high chemical and structural diversity and appear as non-volatile or volatile compounds. These compounds may have evolved to have specific physiological and ecological functions in the adaptation of plants to their growth environment. PSCs are produced by several metabolic pathways and many PSCs are specific for a few plant genera or families. In forest ecosystems, full-grown trees constitute the majority of plant biomass and are thus capable of producing significant amounts of PSCs. We summarize older literature and review recent progress in understanding the effects of abiotic and biotic factors on PSC production of forest trees and PSC behavior in forest ecosystems. The roles of different PSCs under stress and their important role in protecting plants against abiotic and biotic factors are also discussed. There was strong evidence that major climate change factors, CO2 and warming, have contradictory effects on the main PSC groups. CO2 increases phenolic compounds in foliage, but limits terpenoids in foliage and emissions. Warming decreases phenolic compounds in foliage but increases terpenoids in foliage and emissions. Other abiotic stresses have more variable effects. PSCs may help trees to adapt to a changing climate and to pressure from current and invasive pests and pathogens. Indirect adaptation comes via the effects of PSCs on soil chemistry and nutrient cycling, the formation of cloud condensation nuclei from tree volatiles and by CO2 sequestration into PSCs in the wood of living and dead forest trees.

Understorey Rhododendron tomentosum and Leaf Trichome Density Affect Mountain Birch VOC Emissions in the Subarctic.

Subarctic vegetation is composed of mountain birch [Betula pubescens ssp. czerepanovii (MB)] forests with shrubs and other species growing in the understorey. The effects of the presence and density of one understorey shrub, Rhododendron tomentosum (RT), on the volatile emissions of MB, were investigated in a Finnish subarctic forest site in early and late growing season. Only MB trees with an RT-understorey emitted the RT-specific sesquiterpenoids, palustrol, ledol and aromadendrene. Myrcene, which is the most abundant RT-monoterpene was also emitted in higher quantities by MB trees with an RT-understorey. The effect of RT understorey density on the recovery of RT compounds from MB branches was evident only during the late season when sampling temperature, as well as RT emissions, were higher. MB sesquiterpene and total emission rates decreased from early season to late season, while monoterpene emission rate increased. Both RT and MB terpenoid emission rates were linked to density of foliar glandular trichomes, which deteriorated over the season on MB leaves and emerged with new leaves in the late season in RT. We show that sesquiterpene and monoterpene compounds emitted by understorey vegetation are adsorbed and re-released by MB, strongly affecting the MB volatile emission profile.

Needle removal by pine sawfly larvae increases branch-level VOC emissions and reduces below-ground emissions of Scots pine.

Climate warming is expected to increase the frequency of insect outbreaks in Boreal conifer forests. We evaluated how needle removal by the larvae of two diprionid sawfly species affects the composition and quantity of VOC emissions from Pinus sylvestris L. saplings. Feeding damage significantly increased the rate of localized VOC emissions from the damaged branch. The emissions of total monoterpenes (MTs) were dominating (96-98% of total VOCs) and increased by14-fold in Neodiprion sertifer-damaged branches and by 16-fold in Diprion pini-damaged branches compared to intact branches. Emissions of δ-3-carene, α-pinene, sabinene, and ß-phellandrene were most responsive. Feeding damage by N. sertifer larvae increased the emission rates of total sesquiterpenes by 7-fold (4% of total VOCs) and total green leaf volatiles by 13-fold (<1% of total VOCs). The VOC emissions from N. sertifer larvae constituted nearly 25% of the total branch emissions. N. sertifer feeding in the lower branches induced 4-fold increase in MT emissions in the top crown. Defoliation of Scots pine by D. pini significantly reduced the below-ground emissions of total MTs by approximately 80%. We conclude that defoliators could significantly increase total VOC emissions from the Scots pine canopy including MT emissions from resin storing sawfly larvae.