This level uses results from Tree level and literature to gain applicable models and data for management and will test management options. Applied results are expected during time of project.

Long-term Change of Forest Structure

Forest composition change to increase resilience

Czech authorities are now mandating change of tree species and varieties and forest composition to polycultures. Will these changes give the increased storm resilience and/or will they decrease insect herbivore damage as predicted by the Semiochemical Diversity Hypothesis (SDH) (Zhang & Schlyter 2003)?

Landsat data from Sweden indicate less attack on standing spruce in landscapes with larger fraction of birch stands present (Kärvemo et al. 2014). Using the natural stands and managed stands, which are attacked in Czechia, we could quantify different stand structural types and its resistance and resilience against the bark beetle attack.

iLand, one of models to be used.

We will use a novel simulation approach, combined with the field based data collection, to determine the stand species composition and structure resilient towards disturbance effects in Norway spruce forest landscape. The approach consists of field data on individual processes that affect disturbance, and then linking these data through a mechanistic simulation model. This model will then serve as a predictor of dynamics of susceptibility to disturbance over a forest stand development, spatial distribution of susceptible forests, likelihood of its potential triggers (windstorm events), and contingent processes such as bark-beetle dynamics, dynamics of dead wood mass, and tree regeneration.

We will be able to

test the SDH hypothesis and further therefore provide guidelines for the future forest management under climatic change.
better define disturbance regimes and characterize the range of natural forest variability.
In particular, we suppose that we will be able to answer important questions concerning forest dynamics and disturbance regimes of the mountain mixed and pure Norway spruce forests in European temperate zone and provide fundamental answers for forest management.

Spruce protection by birch corridors “Potemkin style”

We propose wind, sun, and semiochemical protection by non-host tree corridors “Potemkin style” to be established along conifer forest edges at risk. The idea is to establish rapid-growing trees of different non-host species (Betula, Populus, and Salix) and sizes in single/double rows to be compared along stands at risk. Additional biodiversity effects on ecosystem service pollination could come from Salix due to its early flowering.

Data from Dulaurent et al. (2012) on reduced Pine Processionary Moth (PPM) infestation on stand edges behind broad-leafs like oak, alder, or birch, France. (Figure courtesy of H. Jactel, INRA)

Short-term Semiochemical and Sensory Management

Tree protection against beetles by non-host volatiles application

We aim to improve of the effect in lowering beetle attacks by “artificial semiochemical diversity” based on the concept of the Semiochemical Diversity Hypothesis (SDH) (Zhang & Schlyter 2003) by direct application of synthetic Non-Host Volatiles (NHV) in dispensers, including “push pull” with pheromone traps (Schiebe et al. 2011).

A meta-analysis of Ips typographus and Mountain Pine Beetle in large scale field-test and management experiments have shown an effect size of ca 1.0 standard deviation (SD) unit of lower tree attacks in the NHV & verbenone treated areas compared to untreated areas (Raffa et al. 2016).

Norway spruce with 2 NHV-dispensers (close-up in grey box) in Tatry Mts, part of a successful experiment (Schiebe et al. 2011).

Our hypothesis is that new active semiochemicals, from literature and those newly identified here by highly developed analytical techniques at G and T levels, as well as their combinations in newer dispenser types and in new spatial arrangements will increase the protective effect of NHV blends considerably.

New blend combinations will be tested for efficacy at stand section scale and found superior, together with new components.
Overall effect size of treatment vs untreated is expected to double in magnitude.

Manipulation of bark to interfere with beetle landing behaviours

We aim to manipulate landing mainly by modulating visual stimuli. Combining the use of two quite different sensory modalities we could expect very strong effects by both visual and chemical non-host stimuli inputs.

We follow basic designs of earlier authors by simple colour changes of lower tree bole. Depending on paint variety, there might as well be effects of paint cover on bark roughness, increasing slipperiness (Ferrenberg &Mitton 2014). The change to lower bark temperatures due to white paint application is quantified by the competence and instrumentation built up under T level study.

In a final, more management oriented experiment; we will test for possible synergistic effects of combining the best chemical NHV stimuli with the best visual deterrent.

Bark texture of trees varies within species: (c) a limber pine with a high proportion of smooth-textured bark and (d) a limber pine with MPB attacks present (Orange Arrows) on rough/crenulated bark, but absent on nearby smooth bark (Ferrenberg &Mitton 2014).

Genetic Understanding and Long-term Improvement of Norway spruce

Genetic dissection of complex resilience traits in Norway spruce

Most traits in forest tree species are associated with low heritability and complex genetic architecture, with very many quantitative trait loci (QTL) of marginal effects confounded by polygenic background. Most efficient approaches of genetic dissection by marker-based analysis are not suitable to outbred forest tree species, resulting in significantly decreased power of QTL detection (Lynch &Walsh 1998). The very large genome size (Nystedt et al. 2013) and additional population attributes require existence of high number of markers in linkage disequilibrium with respective genes influencing target resilience traits (e.g., phenolics, terpenoids, and alkaloids) and additional correlated characteristics that might influence resistance on a tree-level (White et al. 2007).

DNA / SNP sampling in Czech Republic (Photo M Lstiburek).

We aim to investigate the genetics of resilience traits using two approaches:

Association study to quantify potential QTL’s contributing to trait’s variability as a first precondition to functional analyses,
Genomic-based genetic evaluation utilizing optimally both phenotypic, like bark secondary chemistry, and genotypic data to rank individuals based on their genetic predisposition as a basis to artificial selection and most suitable management practices in both forestry and natural conservation.

Genotyping by Sequencing (GBS) approach (Syvänen 2005) will be used in selected natural stands covering an altitude gradient of Norway spruce in Central Europe. The GBS is a fast and highly efficient method to genotyping, lately used in conifers as it enables to score huge number of single nucleotide polymorphisms SNP loci (>50 000 and more) without much information needed on that particular species. We propose it for whole genome sequencing to discover large numbers of SNP for exploring within-species diversity.

Faculty of Forestry and Wood Sciences has experience with population and quantitative genetics in forest tree species. Currently, a large-scale project is conducted applying Genomic Selection in Norway spruce in collaboration with the Genomic Diversity Facility at the Cornell University.

Genotyping by Sequencing (GBS) workflow with contributing institutes (Graphics M Lstiburek).

We will sample 1 500 trees in three geographical areas, all believed to be closed to the natural composition and locally adapted to altitudinal gradient, 500 trees per location.

Volatiles will be analysed by GCxGC-MSTOF method (see T level page) that allows not only precise evaluation of highly complex host odour, but in combination with chiral column is also an excellent tool for full resolution of all enantiomers of all compounds. Non-volatiles as phenolics and alkaloids will be analysed by HPLC.

Modification of terpene precursor enantiomers of insect pheromone production

We will establish a selection program focusing on superior parental trees based on their genetic values, followed by general recurrent selection strategy capturing general combining ability. Focus will first be on enantiomers of ?-pinene, a precursor of cis-verbenol in Ips, for a proof-of-concept. The role of epigenetics needs to be assessed as a basis for functional selection strategy. In addition, we propose a scientifically elaborate approach utilizing direct genetic modification, which is primarily dependent on genetic segregation model in studied traits (from research item above).

Field data on insect pheromone-components ratios strongly effected by natural variation between attacked trees (Lindström et al. 1989). Note: no trees attacked below 30 % cis-isomer of insect pheromone, corresponding to 40% (-)-(S)-enantiomer of ?-pinene in tree bark.

In a first step, we propose a more traditional breeding program based on genomic breeding values of parental trees to establish progeny trials.

Following the dissection of observable traits (above), we plan to investigate the stability of the respective trait expression using repeated measurements (different stress levels of host organisms). This equals functional changes in the genome that do not involve a change in the primary DNA structure “Epigenetics”. Adaptive traits in trees are influenced by epigenetic changes, where also the magnitude of the epigenetic memory in different families in Norway spruce appears to be a heritable genetic trait.

We aim to investigate 500 selected trees (same locations as for above) periodically (4 years measurement) in order to reveal and describe epigenetic modification (i.e. silencing and altering of gene expression).

Modifications by tree breeding will be realised on the level of selected populations. Parental plus trees will possess an implied relationship, which enables the recommended Genomic Best Linear Unbiased Prediction (GBLUP) method from previous Research Item. In a first step, we propose a more traditional breeding program based on genomic breeding values of parental trees to establish progeny trials.

In second step, “plus trees” will be selected based on their GBLUP ranking or QTL scores and consequent progeny tests will be established in multiple environments. Both genomic selection and genetic engineering are considerable means of modification in conifers. Some of the outputs of these strategies are beyond this project duration, but generative copies of superior phenotypes (plus trees) will be captured.

In addition, the genomic information now at hand for Norway spruce will be mined for genes directly included in terpene synthesis (Nystedt et al. 2013; Boutanaev et al. 2015).

Chemical analyses are done as above and at T level. Proof of principle is sought by bioassays of beetle pheromone production and feeding progress with bark from small trees selected and unselected for high (+)-?-pinene and other potential defence compounds making tree tissue less suitable for insect reproduction.

Marker-based tree breeding modification of terpene pathways for precursor enantiomers could disrupt beetle pheromone production – a novel approach to beetle management.

Boutanaev AM, Moses T, Zi J, Nelson DR, Mugford ST, Peters RJ, Osbourn A. 2015. Investigation of terpene diversification across multiple sequenced plant genomes. Proceedings of the National Academy of Sciences of the United States of America 112: E81-E88

Dulaurent AM, Porté AJ, van Halder I, Vetillard F, Menassieu P, Jactel H. 2012. Hide and seek in forests: colonization by the pine processionary moth is impeded by the presence of nonhost trees. Agricultural and Forest Entomology 14: 19-27

Ferrenberg S, Mitton JB. 2014. Smooth bark surfaces can defend trees against insect attack: resurrecting a ‘slippery’ hypothesis. Functional Ecology 28: 837-845

Kärvemo S, Rogell B, Schroeder M. 2014. Dynamics of spruce bark beetle infestation spots: Importance of local population size and landscape characteristics after a storm disturbance. Forest Ecology and Management 334: 232-240

Lindström M, Norin T, Birgersson G, Schlyter F. 1989. Variation of enantiomeric composition of a-pinene in Norway spruce, Picea abies, and its influence on production of verbenol isomers by Ips typographus in the field. Journal of Chemical Ecology 15: 541-548

Lynch M, Walsh B 1998 Genetics and analysis of quantitative traits. Vol. 1. Sinauer Sunderland, MA.

Nystedt B, Street NR, Wetterbom A, Zuccolo A, Lin Y-C, Scofield DG, . . . Jansson S. 2013. The Norway spruce genome sequence and conifer genome evolution. Nature 497: 579-584

Raffa KF, Andersson MN, Schlyter F. 2016. Host selection by bark beetles: Playing the odds in a high-stakes game. In: Blomquist G, Tittinger C (eds), Advances in Insect Physiology. Vol. 50, Elsevier Ltd., pp 1–74. 10.1016/bs.aiip.2016.02.001

Schiebe C, Blazenec M, Jakus R, Unelius CR, Schlyter F. 2011. Semiochemical diversity diverts bark beetle attacks from Norway spruce edges. Journal of Applied Entomology 135: 726–737

Syvänen A-C. 2005. Toward genome-wide SNP genotyping. Nature Genetics 37: S5-S10

White TL, Adams WT, Neale DB 2007 Forest genetics. CABI Publishing, Oxfordshire. 1845932854.

Zhang Q-H, Schlyter F. 2003. Redundancy, synergism, and active inhibitory range of non-host volatiles in reducing pheromone attraction in European spruce bark beetle Ips typographus. Oikos 101: 299-310