Seminar on Forest and Plant Health, November 11th, 2022

1. Seminar on Forest and Plant Health,
November 11th
, 2022
Natural Resources Institute Finland, Viikki, Helsinki
Photo: Symptoms of blackleg and soft rot caused by Dickeya and Pectobacterium
species in potatoes (picture provided by Yeshitila Degefu)

2. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
EVO restoration experiment: twenty-year dynamics of dead wood and
epixylic vegetation
Ekaterina Shorohova, Ilkka Vanha-Majamaa
Natural Resources Institute Finland (Luke), Helsinki, Finland
Email: ekaterina.shorokhova@luke.fi
In managed Fennoscandian forests, prescribed burning, creation of dead wood, and leaving retention
trees are used as restoration treatments. However, their long-term effects on biodiversity are not yet
well understood. The EVO restoration experiment was established in 2001 in mature Norway spruce
forests in Southern Finland with a general goal to analyse the short- and long-term effects of restoration
treatments on the biodiversity and ecosystem functioning (Vanha-Majamaa et al. 2007). The experiment
includes partial cuttings with standing retention of 50 m3
ha-1
with or without prescribed burning and
three levels of downed retention: 5, 30 and 60 m3
ha-1
, as well as controls in both upland and paludified
biotopes, with three replicates each.
In the first sub-study, we inventoried dead wood by size, tree species, decay class and position before,
one and 16 years after the treatments. Main results of this sub-study show that both volume and diversity
of dead wood 1) increased with the level of downed retention, 2) were higher in burnt than in unburnt
forest stands, and 3) were higher in upland than in paludified biotopes. In the second sub-study, we
analyzed decomposition rate of bark and wood; carbon (C), nitrogen (N) and other element dynamics;
as well as cellulose and lignin in the Norway spruce and birch logs 20 years after restoration treatments:
tree and downed wood retention and burning. The methodology and working hypotheses are discussed
in the presentation. The third sub-study was designed to study the twenty-year dynamics of epixylic
communities in a Norway spruce forest after standing and downed retention and restoration burning.
Our main objectives are to: 1) characterize the composition and diversity of epixylic communities
before, and 1, 2, 3, 4, and 20 years after restoration treatments; 2) estimate the influence of substrate
attributes (size and tree species of logs, height above ground, decomposition stage, litter cover, bark
cover, charring) and ecosystem attributes (biotope, total volume and diversity of CWD, tree stand
characteristics etc.) on the log- and community-level epixylic diversity; 3) reveal the substrate
specialization of common and threatened epixylic plants and lichens; 4) discuss implications of the
results for forest management and ecological restoration in boreal spruce forests. Currently, more than
5000 samples of bryophytes and lichens are being identified. Some threatened species Cladonia
norvegica, Nowellia curvifolia, Riccardia palmata, Crossocalyx hellerianum, Lophozia guttulate were
found so far.
Even preliminary findings from Evo experiment highlight the long-term importance of prescribed
burning, standing and downed retention as nature-based forest management methods for maintaining
dead wood diversity and the diversity of wood-inhabiting communities.
Further information
Vanha-Majamaa, I., Lilja, S., Ryömä, R., Kotiaho, J.S., Laaka-Lindberg, S., Lindberg, H., Puttonen, P., Tamminen, P.,
Toivanen, T. and Kuuluvainen, T., 2007. Rehabilitating boreal forest structure and species composition in Finland through
logging, dead wood creation and fire: The EVO experiment. Forest Ecology and Management, 250(1-2), pp.77-88.

3. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Soil factors and genetic variation regulate intraspecific growth in Norway
spruce (Picea abies)
Matti J. Salmela, Sannakajsa Velmala, Katri Himanen, Tiina Ylioja, Taina Pennanen
Natural Resources Institute Finland (Luke), Latokartanonkaari 9, FI-00790 Helsinki
Email: mattijsalmela@gmail.com
Maintenance of biodiversity is based on trade-offs. Intraspecific diversity is maintained among
genotypes when they vary in the expression of functional traits such that their performance ranking
changes from one environment to another.
We examined genotypic trade-offs in Norway spruce (Picea abies) in Finland using field data from
breeding trials and a subsequent progeny experiment with detailed seedling-wide phenotyping. In the
field, genotypes exhibited significant genotype × environment interactions in growth across a former
agricultural field and a native forest site that arose partly from genotypic rank order shifts. To determine
whether soil type could be a driving force behind trade-offs, we established a progeny experiment with
a subsample of the field genotypes and grew seedlings in three soil treatments in a growth chamber. We
found that variation in 14 shoot and root functional traits could be summarised by four distinct trait
dimensions and that all traits exhibited phenotypic plasticity in response to treatment. Seed origin had
a minor effect on growth and phenology, with seedlings from the more southern former agricultural
field growing slightly larger and exhibiting delayed phenology. Despite genetic variation in most
functional traits, no genotype × environment interactions emerged in seedlings, and genetic differences
in seedling functional traits were not related to sapling or adult growth in the field. This may be due to
the soil treatments not capturing the definite aspects of soil variability that induce interactions in natural
settings, or to a genotypic sample that was not representative of the complete range of genetic diversity
in the field trials.
Collectively, the results indicate that soil variation and genetic factors have major effects on
intraspecific functional trait variation in forest trees, but further research is required to identify
environmental agents that have potential to cause environment-specific patterns of genotypic growth in
economically valuable tree species.

4. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
What constrains the expansion of the Spruce Bark Beetle in northern
Europe? The making of a new genomic data set to study adaptation and
population dynamics of a forest pest in Finland
Chedly Kastally
Department of Forest Sciences, University of Helsinki, Finland
Email: chedly.kastally@helsinki.fi
In this project, I will investigate the factors that constrain the expansion of insect populations to northern
Europe. Complex population dynamics in insects can result in genomic consequences that influence
migration capacity and adaptation. The adaptation to growing season itself has effects on the number
of generations in a given year. To improve our understanding of the interplay of these processes, at the
heart of the current expansion of many pest insects in northern Europe, I will study the European Spruce
Bark Beetle, Ips typographus, an ecologically and economically important forest insect, characterized
by a complex population dynamic. Outbreaks of this insect in Europe have been well documented
during the last century making it an excellent model to explore the effects of population expansion and
contraction on genetic variation. Moreover, I. typographus is widely distributed and has evolved
flexible mechanisms of photoperiodic diapause, making it an interesting model to study the current and
rapid adaptation of pest insects in northern Europe.
The project is organized in three main parts. First, I will identify the past and current migration routes
of I. typographus by analysing genome sequences from across Europe. I will evaluate the effects on the
genome of the population expansion at the continental scale. Second, I will collect genomic data in sites
of outbreaks of the species in southern Finland to identify admixture and demographic events at a higher
spatial and temporal resolution. This will allow me to investigate the relative importance of local
population growth and migration in the on-going expansion of the pest in northern Europe. Third, to
examine the genetic basis of the photoperiodic diapause, I will combine a gene-environment association
study using samples from across the latitude with a bulk-segregant analysis based on a crossing
experiment of individuals from northern and southern populations. This will allow to identify and map
the genes underlying the photoperiodic control of diapause in this species.
Together, these analyses will help understand how key genomic areas for adaptation to seasonal
variations have evolved during the expansion of I. typographus in northern Europe. These results will
also have the potential to provide a new understanding of the future population dynamics of this pest
species in the context of climate change.

5. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Interactions of microbiome present in
Heterobasidion fruiting body and infected wood tissues
Wenzi Ren1
, Reijo Penttilä2
, Risto Kasanen1
, Fred O. Asiegbu1*
1
Department of Forest Sciences, University of Helsinki, Helsinki, Finland,
2
Natural Resources Institute of Finland (Luke), Helsinki, Finland
Email: wenzi.ren@helsinki.fi
Microbiome community in Heterobasidion infected materials have been extensively studied for
understanding the pathogenesis, but not much is known on the interactions between bacteria and fungi
component of the microbiota.
In this study, we aim to 1) Unravel the contribution and development of bacteria community and other
fungi during Heterobasidion induced wood decay; 2) Uncover how the environmental factors and the
conifer pathogen drive the microbiome community structure and functional changes; 3) Identify the
core microbiome during the Heterobasidion mediated decay process.
ITS and 16S amplicon sequencing data from Heterobasidion fruiting body and its adhering woody
tissue were used in this analysis. Wood samples collected from managed and unmanaged forest were
classified into four decay classes. Our result showed that bacteria and fungi have different abundance
and activeness in the study materials. The Heterobasidion ecological correlation network was found to
be conservative especially in the fruiting body. The core microbiome defined in our analysis highlighted
their potential in the microbiome community functioning. A particular Heterobasidion OTU was found
to have the most important impact on the community and disease development. The study further
revealed that forest management had the biggest impact on the microbiome community, and the fungal
community showed to be more sensitive to environment changes than bacteria.

6. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Potato mycobiome and disease biocontrol in Estonia
Kaire Loit
Estonian University of life Sciences, Institute of Agricultural and Environmental Sciences,
Fr.R. Kreutzwaldi 5, 51006 Tartu, Estonia
Email: kaire.loit@emu.ee
The roots of agricultural plants are hosts for various microorganisms. These include many harmful,
often soilborne, pathogens and beneficial organisms such as plant growth- and health‐promoting
arbuscular mycorrhizal (AM) fungi.
In this study, diversity, abundance, and community composition of putative pathogenic fungi and
symbiotic AM fungi in potato (Solanum tuberosum L.) fields in Estonia was studied. We found that
abundance of AM fungi in potato roots is significantly higher at the end of the growing season compared
to the beginning. Cultivar influenced root-pathogenic fungal richness and community composition more
than sampling time or sampling time × cultivar interaction. Additionally, cultivar had a significant effect
on root fungal pathogen relative abundance. In soil, the community composition of putative pathogenic
fungi changed significantly over time. Our potato, wheat and barley mycobiome analysis revealed that
crop species is the main driver in shaping root fungal diversity and composition under the same
environmental conditions. Of the ten most abundant OTUs in different crop species, at least half were
pathogens. Under the increasing threat of plant disease outbreaks, the speed of detection of plant disease
agents is essential for avoiding large-scale crop losses.
Of the third-generation sequencing methods, the phone-sized DNA sequencing device MinION has
received much attention, allowing immediate data streaming for rapid results. We aimed to test the
suitability of MinION for ultrarapid plant pathogen diagnostics. Our work demonstrated that the third-
generation sequencing device MinION is well suited for rapid, PCR-free diagnosis of fungal pathogens.
A 2-year field study was conducted to test commercially available AM inoculum in natural conditions.
The results of this study bring attention to the importance of the legislation and quality control of
products marketed as biofertilizers. Key findings were that either granulated or liquid formulations did
not contain almost any sequences belonging to AM fungi. Secondly, inoculums were contaminated and
contained plant pathogenic fungi and plant parasites.

7. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Latency and pathogenesis of the blackleg and soft rot Pectobacteriaceae in
potatoes
Yeshitila Degefu
Natural Resources Institute Finland (Luke), Plant Health. Latokartanonkaari 9, 00790 Helsinki.
Email: yeshitila.degefu@luke.fi.
Five species of bacteria which belong to the family Pectobacteriaceae and the genera Dickeya and
Pectobacterium, namely Dickeya solani, Pectobacterium atrosepticum, Pectobacterium brasiliense,
Pectobacterium carotovorum and Pectobacterium parmentieri are known to cause a destructive
diseases commonly referred blackleg and soft rot on potatoes. The bacteria are seed borne and latent
bacteria present in the tuber are the main source of infection. The latent nature of the pathogens pose
diagnostic and disease management challenges since the bacteria are deep seated in the tuber hidden
from visual inspection and not accessible to tuber surface chemotherapeutic and thermotherapeutic
treatments. In addition, the exceptionally long period of latent existence of the bacteria in storage and
transit help the pathogen to travel with the host with the possibility of reaching new geographical area.
Aided by globalization, international trade and climate change, there is frequent northward (poleward)
expansion and establishment new species of the bacteria particularly in north Europe including Finland.
To date rapid, specific, and sensitive molecular and PCR based diagnostic methods are widely used for
the detection of latent infections at the port of entry and symptomless stored seed tubers.

8. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Figure. Pathogenesis and latent development of blackleg and soft rot caused by Dickeya and
Pectobacterium species in potatoes. Bacteria from infected mother tuber moves upward and infect the
stems where it causes blackening and rotting of the stem. It then moves down through the stolon and
enters the daughter tubers through the stolon ends. Depending on the prevailing weather condition,
the daughter tubers may rot or remain symptomless, and the bacteria remain latent until the next
season. (Degefu, Luke)
The bacteria remain latent until favorable conditions prevail for initiation of infection. The main driver
of the shift from latency to active infection and disease development is the presence of free water on
tubers which triggers a cascade of events leading to the onset of rotting. These include anaerobiosis,
swelling of cortical cells, breaching of the phelloderm layer in the lenticels which tends to open.
As confirmed by many years of research, there is general positive correlation between laboratory PCR
detection of the blackleg Pectobacteriacae to field infection. Therefore, targeting the latent bacteria and
ensuring bacteria free seed is fundamental to the management of blackleg in potatoes.
Further information
Yeshitila Degefu, Elina Virtanen and Tiina Varyrynen. 2009. Pre-PCR Processes in the Molecular Detection of Blackleg and
Soft Rot Erwiniae in Seed Potatoes. Journal of Phytopathol 157:370–378
Pérombelon, MCM 2002. Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathology 51:1-12

9. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Heterobasidion viruses in the Czech Republic
László Benedek Dálya1
, Leticia Botella2,3
, Anna Poimala4
, Eeva Vainio4
, Jarkko Hantula4
1
Department of Forest Protection and Wildlife Management, Faculty of Forestry and Wood
Technology, Mendel University in Brno, Zemědělská 3, 613 00 Brno, Czechia
2
Phytophthora Research Centre, Department of Forest Protection and Wildlife Management, Faculty
of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 3, 613 00 Brno, Czechia
3
Department of Genetics and Agrobiotechnology, Faculty of Agriculture and Technology, University
of South Bohemia in České Budějovice, Na Sádkách 1780, 370 05 České Budějovice, Czechia
4
Natural Resources Institute Finland (Luke), Latokartanonkaari 9, FI-00790 Helsinki, Finland
Email: laszlo.dalya@mendelu.cz
The basidiomycete species complex Heterobasidion annosum s.l. comprises some of the most
devastating pathogens of conifers. In this study, we examined the presence of viruses in a collection of
H. annosum s.l. specimens originating from different regions of Czechia, with the long-term goal of
finding a virus conferring hypovirulence to its host. The presence of dsRNA elements was examined in
101 fungal isolates. RNA-Seq was done using pooled libraries constructed from 16, 16 and 13 strains
of Heterobasidion annosum s.s. (twice) and Heterobasidion parviporum, respectively. RNA-Seq was
repeated for a single H. annosum strain infected by multiple viruses. The bioinformatics pipelines for
in silico data mining for viruses included de novo assembly of raw reads and host-unmapped reads.
Contigs were examined with Blastx and Blastn searches against viral and host protein and nucleotide
databases. The identity of virus-hosting Heterobasidion strains was revealed by RT-PCR. The 5’ or 3’
ends of incomplete coding regions were amplified by RACE and cloned. Amplicons were Sanger
sequenced. Our analysis did not reveal any clear dsRNA particles. However, 19 distinct ssRNA viruses
were discovered in the RNA-Seq datasets. Eleven ambi-like viruses, two ourmia-like viruses, one
mitovirus, one fusarivirus, one tobamo-like virus, one bisegmented narna-like virus, the RNA1 segment
of another one and one cogu-like virus were found. The presence of each virus was verified in up to
three fungal genotypes. Coinfections of up to nine viruses were recorded in six isolates, while five
isolates harbored a single virus. In H. parviporum, only one strain of the recently described
Heterobasidion ambi-like virus 3 was detected. Intraspecies pairwise nucleotide identities of ORFs of
virus strains ranged between 88.4–99.4%. In an attempt to eliminate viruses, ten monohyphal cultures
of three selected isolates were created by hyphal tipping. RT-PCR screening has shown that the viruses
were retained in the monohyphal cultures to varying degrees (0–100%); low-titer viruses were generally
less stable. Four cultures with different combinations of viruses and the original isolates were thermally
treated to compare the effects of the two curing methods. Overall, these findings indicate that the virome
of Heterobasidion populations in Central Europe is highly diverse, and seems to differ from that in the
boreal region. Notably, the most frequently occurring virus species of the pathogen, Heterobasidion
orthocurvulavirus was not found among 50 Heterobasidion strains from Czechia. Likewise, the family
Partitiviridae, with 21 members described to date from Heterobasidion, was not represented in our
datasets.

10. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Effect of co-inoculation of pine seedlings with Tricholoma sp. and/or
Streptomyces on Heterobasidion pathogenesis and host growth
Hafiz Umair Masood Awan1,2
, Kai Wang1,3
, Fred Asiegbu1
1
Department of Forest Sciences, University of Helsinki, PO Box 27, Helsinki FIN-00014, Finland.
2
Helclean Consultancy Services, Asiakkaankatu 6B 29, 00930 Helsinki, Finland
3
College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Email: hafiz.awan@helsinki.fi
Forest trees frequently interact with a diverse range of microorganisms including ectomycorrhiza,
bacteria, and fungal pathogens. Plant defense responses to individual pathogen have been widely
studied, but very little is known on the effect of co-inoculation on host defenses. To study the impact
of co-inoculation or tripartite interaction on plant growth and host defenses, Scots Pine (Pinus
sylvestris) seedlings were inoculated with either Tricholoma sp or Streptomyces sp or both together with
a root pathogen Heterobasidion annosum for three months. The inoculation with Streptomyces or
Heterobasidion alone had negative effect on plant growth whereas co-inoculation of Tricholoma and
Streptomyces sp in presence of the pathogen seems to promote plant growth (root length, number of
lateral roots, seedling weight) of Scots pine over time. Based on the phenotypic examination, it was
concluded that the ectomychorrhizal Tricholoma sp and Actinobacterial Streptomyces sp counteracts
negative effect of H. annosum on plant growth. RNA-seq analysis of seedlings inoculated with
Tricholoma sp, Streptomyces sp, infected with H. annosum will be analyzed for for identification of
differentially expressed genes (DEGs). The potential of pre-inoculation of seedlings to protect seedling
roots before out-planting deserves to be further explored
Keywords: actinobacteria, ectomycorrhiza, microbe, root pathogen, Scots pine

11. Seminar on Forest and Plant Health, November 11th
2022
Natural Resources Institute Finland (Luke), Helsinki
Diplodia tip blight in Finland - Current situation and future prospects
Eeva Terhonen
Natural Resources Institute Finland (Luke), Natural resources / Forest health and biodiversity,
Latokartanonkaari 9, FI-00790 Helsinki, FINLAND
Email: eeva.terhonen@luke.fi
Diplodia sapinea (Fr.) Fuckel is an ascomycete that cause shoot blight on Scots pine (Pinus sylvestris
L.) and other conifers. This fungus has spread unnoticed because it undergoes a symptomless
endophytic life stage during the disease cycle. Disease symptoms come visible when trees have been
weakened by abiotic stress, usually related to temperature and drought. It has been expected to appear
in Finland, since it was found in 2009 in Estonia (Hanso & Drenkhan 2009) and 2013 in Sweden (Oliva
et al. 2013). In 2015 D. sapinea was discovered in Finland as saprophyte on Scots pine cones (Müller
et al. 2019). The endophytic mode of this fungus was discovered in 2019 in healthy Scots pine twigs
(Terhonen et al. 2021). Finally, the disease, Diplodia tip blight, was observed in 2021 (Terhonen 2022).
Nowadays this disease is observed regularly in Scots pine (P. sylvestris) sites in Europe, increasing
dramatically in the last decades. Changes in climatic conditions will gradually increase the damage of
this pathogen because it is favoured by elevated temperature and the host trees will be more susceptible
due to climate-change related environmental stress. In a project, funded by Alfred Kordelin foundation
and Luke, we have collected D. sapinea strains from areas where the typical disease has been observed
by citizens of Finland. Based on the samples collected during the year 2022, D. sapinea is more common
than previously thought. The current knowledge over the distribution is that the diseased trees are in the
coastal areas. Luke continues to collect and receive Scots pine twig samples to monitor the distribution
of D. sapinea.
Further information
Hanso M, Drenkhan R (2009) Diplodia pinea is a new pathogen on Austrian pine (Pinus nigra) in Estonia. Plant Pathol 58:
797–797.
Müller MM, Hantula J, Wingfield M, Drenkhan R (2019) Diplodia sapinea found on Scots pine in Finland. For Pathol 49:
e12483.
Oliva J, Boberg J, Stenlid J (2013) First report of Sphaeropsis sapinea on Scots pine (Pinus sylvestris) and Austrian pine (P.
nigra) in Sweden. New Dis Rep 27: 23–23.
Terhonen E. 2022. Havuparikkaan aiheuttamaa tautia, etelänversosurmaa, tavattiin Suomessa ensimmäisen kerran 2021.
[Diplodia tip blight observed first time in Finland in 2021]. In: Melin, M. (edit.), Terhonen, E (edit.), et al. 2022. Metsätuhot
vuonna 2021. Luonnonvara- ja biotalouden tutkimus 38/2022. Luonnonvarakeskus. Helsinki. pp. 21–23.
Terhonen E, Babalola J, Kasanen R, Jalkanen R, Blumenstein K (2021). Sphaeropsis sapinea found as symptomless
endophyte in Finland. Silva Fenn 55: 13.