This document summarizes the presentations from a seminar on forest pathology held on November 2nd, 2017 at the Natural Resources Institute Finland in Viikki, Helsinki. There were several presentations on various topics relating to forest pathogens: 1) Interactions, omics, and novel applications from wood-decaying fungi. This presentation discussed genomics research on white-rot and brown-rot fungi. 2) Climate change and a forestry pathosystem: fungal strain-specific variation in disease severity. This presentation discussed research on the effects of climate change on the interaction between Endoconidiophora polonica and Norway spruce. 3) Lecanosticta acicola on

1. Seminar on Forest Pathology,
November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Photo: Ips typographus outbreak in Iitti, Finland (photo provided by Riikka Linnakoski)

2. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Interactions, omics, and novel applications from
wood-decaying fungi
Taina Lundell, Mari Mäkinen, Firoz Shah, Hans Mattila & Tuulia Mali
Fungco group, Department of Microbiology, Faculty of Agriculture and Forestry, Viikki
Campus, University of Helsinki, Finland
Email: taina.lundell@helsinki.fi
Basidiomycota Agaricomycetes order Polyporales fungi are able to depolymerize wood-
lignocellulose components: white-rot species decompose all biopolymers including lignin
while brown-rot species are efficient in decomposing wood polysaccharides, mainly cellulose
[1]. Accumulating fungal genomic data is prominently improving our understanding on
fungal biology and ecology, but exact details of wood degradation processes and the genetic,
biochemical and proteomic factors involved are not yet fully understood. Our aim is to
combine fungal genomics to functional transcriptomics and proteomics including testing on
various lignocellulose and plant biomass substrates for growth and bioconversions, in order
to identify the key genes, proteins and metabolites necessary for decomposition of plant
biomasses in ecologically important fungi, also presenting applicability in industrial and
environmental biotechnology processes. Screening of wood-decaying CAZyme and
oxidoreductase enzyme activities in wood-supplemented cultures have demonstrated the
proficiency of white-rot Polyporales phlebioid clade species [2], which were further
investigated for bioethanol production from recyclable waste lignocelluloses [3]. Genome
sequencing of the best, model species Phlebia radiata aided in description of the species’
functional transcriptome and proteome on spruce wood [4], and more recently, under
fermentative, ethanol producing conditions. Differentially expressed key genes have been
identified to elucidate the principal metabolic pathways and putative transcription factors
operating under fermentative versus respirative conditions, also to promote systems biology
and metabolic engineering approaches in near future. Novel bioproducts and bioactive
metabolites are actively searched in wood-decay fungi. Our additional concern is to elucidate
brown-rot decay of wood conducted by ecologically significant Polyporales fungi,
concentrating on Fomitopsis pinicola, and its interactions with white-rot fungi [5]. In nature,
fungal communities are under dynamic changes, and co-habitation of several species is
common upon wood decay. Hyphal contacts and interactomes upon decomposition of
lignocelluloses and plant biomasses are challenging to investigate, but may offer us new tools
for fungal bioproductions and bioconversions supporting more sustainable bioeconomy.
[1] Lundell T (2014) Adv Bot Res 70:329-370; [2] Kuuskeri J (2015) BMC Microbiol
15:217; [3] Mattila H (2017) Bioresour Technol 225:254-261; [4] Kuuskeri J (2016)
Biotechnol Biofuels 9:192; [5] Mali T (2017) PLoS ONE 12(9):e0185171

3. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Climate change and a forestry pathosystem: fungal strain-
specific variation in disease severity
Riikka Linnakoski1,2
, Junko Sugano1
, Samuli Junttila1
, Michael J. Wingfield3
, Pertti
Pulkkinen2
, Kristian M. Forbes4
& Fred O. Asiegbu1
1
Department of Forest Sciences, University of Helsinki, Latokartanonkaari 7, 00790,
Helsinki, Finland
2
Natural Resources Institute Finland, Helsinki, Finland
3
Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria,
Pretoria, South Africa
4
Department of Virology, University of Helsinki, Finland
Email:riikka.linnakoski@luke.fi
Forest pests and pathogens represent some of the greatest threats to global forest health under
climate change. However, little is known as how disease severity in forestry pathosystems
will be impacted by expected anthropogenic climate change during the next decades. The
aims of our experiments have been to test in vivo the effects of a changed growing
environment on Endoconidiophora polonica infected Norway spruce seedlings. The first
experiment was conducted under the ambient summer temperature and CO2 levels, and under
the predictions for the years 2030 and 2100 in Finland. The seedlings were inoculated with
different E. polonica strains or mock-inoculated. In the second experiment, the seedlings
were randomized to high or low water availability treatment groups, and inoculated with E.
polonica strains. Seedling mortalities were monitored throughout the annual growing season,
and total seedling growth and lesion length indices were measured at the experiment
conclusions. The results indicate that increased temperatures have a negative impact on P.
abies susceptibility to E. polonica, with the higher temperature increases likely to be the most
detrimental to tree health. Disease severity was not universally greater for water-restricted
seedlings, and the effects varied among fungal strains. Our results suggest that predicted
climate changes have the potential to alter the damage caused to Norway spruce by E.
polonica, but also demonstrate that such effects can vary markedly among fungal strains.
Therefore, we emphasize the need for a strain-specific level of understanding of the disease
agents.

4. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Lecanosticta acicola on Pinus sylvestris and the pathogen
population structure in Estonia
Marili Laas, Kalev Adamson & Rein Drenkhan
Estonian University of Life Sciences, Institute of Forestry and Rural Engineering, Fr.R.
Kreutzwaldi, 5, 51 014 Tartu, Estonia
Email: rein.drenkhan@emu.ee
Lecanosticta acicola is a quarantine pathogen that infects different species of pine (Pinus
spp.) and causes them needle disease Brown Spot Needle Blight (BSNB). The pathogen has
been present in Estonia since 2008, but so far only exotic pine species were found to be
infected. In October 2016 several young Pinus sylvestris trees in a mixed stand of P. mugo
and P. sylvestris were noticed to have symptoms of BSNB. Needle samples were collected
for laboratory analyses from symptomatic trees. The pathogen was isolated from both host
species and identified using molecular methods. The ITS sequences of the pathogen from
both host species were deposited in GenBank. This is the first time L. acicola infecting native
Scots pine is molecularly confirmed in Estonia and northern Europe.
Since there were no data on the population genetics of the pathogen in northern Europe, a
population study was conducted, that would allow to evaluate the population viability. In the
study 104 L. acicola isolates from Estonia were analysed using microsatellite and mating type
markers. Results of the population study indicate that there have been several separate
introductions of the pathogen to Estonia and anthropogenic activity has affected the spread of
L. acicola in the country. Both mating types of the pathogen are present in Estonia, still
mating type MAT1-1 is more common. This means that sexual reproduction of the pathogen
is probable and genetic diversity and virulence of the population may increase.

5. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Pine weevil, major risk for planted seedlings in forest
regeneration and factors affecting to damage risk
Heli Viiri1
& Katri Himanen2
1
Natural Resources Institute Finland (Luke), Yliopistokatu 6, 80100 Joensuu, Finland
2
Natural Resources Institute Finland (Luke), Juntintie 154, 77600 Suonenjoki, Finland
Email: heli.viiri@luke.fi
Pine weevil (Hylobius abietis) is the most harmful pest in forest regeneration in Eurasia.
Adult weevils gnaw bark and phloem from seedlings. Even 80% seedlings can die in first
year after planting. Forest regeneration and seedling production in Finland was presented as
background information. Several factors in seedling quality and forest regeneration affect the
pine weevil damage risk. Conifer seedlings are typically planted to depths of 3–5 cm in
boreal forests. Given that machine planting has become more common, there is a need to
demonstrate that conifer seedlings can be planted deeper using a machine without reducing
field performance. To investigate the effects of deep planting on Norway spruce (Picea abies)
container seedlings, we have established field experiments in Central Finland. On a site with
medium coarse soil, planting depths were 0, 2, 5 and 8 cm and on fine-textured soil, planting
depths were 3, 6 and 10 cm. Deeper planted seedlings grew better during the first 4 years
after planting and at the end of the fourth season, initial differences in shoot length among
planting depths had disappeared. Incidence of insect damage was relatively low, and no
differences among planting depths were found in two field experiments. Risk for pine weevil
insect damage was also tested in a cage experiment and the number of deep gnawing scars on
stems was significantly lower in deeper planted seedlings. Thus, deep planting improves
growth of Norway spruce container-grown seedlings and is an advantage especially in dry
conditions.

6. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Expression analysis of genes encoding effector-like proteins of
Norway spruce pathogen (Heterobasidion parviporum)
Zilan Wen, Tommaso Raffaello, Zhen Zeng & Fred O. Asiegbu
Department of Forest Sciences, University of Helsinki, Latokartanonkaari 7,
00790 Helsinki, Finland
Email: zilan.wen@helsinki.fi
Heterobasidion annosum sensu lato (s.l.), as a species complex containing H. annosum sensu
stricto (s.s.), H. parviporum and H. abietinum, is necrotrophic pathogen responsible for root
and butt rot in pine, spruce and fir respectively in Europe. In Heterobasidion-conifer
interaction, the fungus is believed to deploy a repertoire of small secreted proteins (SSP),
including known effector proteins, to promote the infection of the host and sustain the disease
development. Recent analysis of the H. parviporum genome sequence has revealed the
presence of 268 SSP-encoding genes. Since H. parviporum and its host are not yet amenable
to functional genetic manipulation, heterologous delivery-expression system such as
Agrobacterium–Nicotiana benthamiana have been used currently to gain insight into effector
functional genomics and assess putative interaction partners. Two candidate genes encoding
HpSSPs in the transient expression reveals their ability to induce cell death in N.
benthamiana. In particular, HpSSP35 could induce a rapid, strong and consistent cell death at
2 day post-infiltration. Gene expression profiling of HpSSPs is monitored by using qPCR
during infection between H. parviporum and Norway spruce seedling roots, revealing that
HpSSP35 encoding gene showed a significant induction at 2 dpi. Thus HpSSP35.8 is
regarded as a prime candidate for future functional studies.

7. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Metatranscriptomic profiling of asymptomatic and symptomatic
Norway spruce trees naturally infected by Heterobasidion spp.
Mengxia Liu, Zhen Zeng, Mukrimin Mukrimin, Tommaso Raffaello, Andriy Kovalchuk &
Fred O. Asiegbu
Department of forest sciences, University of Helsinki, Latokartanonkaari 7,
00790 Helsinki, Finland
Email: mengxia.liu@helsinki.fi
Through the previous work of my colleagues, fungal abundance in different parts of Norway
spruce has been analyzed. They found Heterobasidion existed in both healthy and naturally
infected trees. Thus, terms healthy/infected have been replaced by
asymptomatic/symptomatic. After attacked by pathogens, conifers can trigger a series of
defense responses, including induction of defense-related genes. In this study, expressed level
of genes of pathogenesis-related (PR) proteins and enzymes involved in lignin synthesis and
terpenoid pathway were discussed based on RNA sequencing data analyzed by R. In
differential expressed PR protein genes, there were 8 down- and 33 up-regulated such as
chitinase, dirigent-like protein and antimicrobial peptide. For each gene, different expressed
level between asymptomatic and symptomatic was presented by heatmaps. In most genes, no
obvious differences were found in the comparison except for terpene synthase. Interestingly,
within on gene family, some genes were highly induced but others were not. In conclusion,
several defense-related genes were highly induced in symptomatic trees compared to
asymptomatic trees; Genes involved in terpene synthase were strongly induced in
symptomatic trees suggesting they might have a role in the resistance of Norway spruce
against pathogens; Different expressed level of genes in the same family complicated the
interpretation of the precise role of individual gene in host defense. Besides, RNA-seq reads
were mapped to Heterobasidion genome. 14 up- and 3 down-regulated genes were listed in
differential expressed genes. The function of up-regulated genes might relate to protein
synthesis, but what kind of protein it is will be studied in the future.

8. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Metabolome and expression analysis of genes involved in terpene
biosynthesis in Scots pine trees infected with
Heterobasidion annosum
Mukrimin Mukrimin1, 2
, Andriy Kovalchuk1
, Hui Sun1,3
, Jarmo Holopainen4
&
Fred O. Asiegbu1
1
Department of Forest Sciences, University of Helsinki, Latokartanonkaari 7, P.O. Box 27,
00790 Helsinki, Finland
2
Department of Forestry, Hasanuddin University, Jalan Perintis Kemerdekaan Km. 10,
90245 Makassar, Indonesia
3
Collaborative Innovation Center of Sustainable Forestry in Southern China, College of
Forestry, Nanjing Forestry University, Nanjing, China
4
Department of Environmental and Biological Sciences, University of Eastern Finland,
Kuopio, Finland
Email: mukrimin.mukrimin@helsinki.fi
In the northern hemisphere, the forest area mostly covered by conifer trees faced a serious
problem of root-rot disease. This disease is caused by Heterobasidion annosum s.l. species
complex which affects the growth rate and mortality of the host as well as reduces the
economic value. Scots pine as the main host for H. annosum has both constitutive and
induced chemical defenses producing resin and terpene as response against fungi and insect
attack. Hence, the chemical and transcript profiles have the potential to serve as markers for
pathogen resistance.
In this project, fourty (15-year-old) Scots pine trees at Research Forest, Lapinjärvi-Finland
were tested to determine their response to H. annosum infection under field conditions. After
five months, the necrotic lesion sizes were documented. The expression patterns of genes
related to terpene synthesis and chemical compounds of terpenes were also evaluated.
The results showed that Scots pine had different levels of resistance to H. annosum infection.
The most resistant of Scots pine had 2 mm necrotic lesion size in xylem tissue, while the
most susceptible of Scots pine had 90 mm necrotic lesion in xylem tissue. Additionally,
Genes (-)-alpha pinene synthase and geranyl diphosphate synthase positively correlated with
vertical necrosis in xylem tissue, while gene 3-carene 2 synthase positively correlated with
the Scots pine’s diameter. This study found that the monoterpene and sesquiterpene
compounds varied in needle tissue of Scots pine, although none had significantly statistical
correlation with both vertical and horizontal lesion length.

9. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
The quantification of genome segments' (RNA) transcripts in
viral coinfections of Heterobasidion annosum
Muhammad Kashif, Jaana Jurvansuu, Eeva Vainio & Jarkko Hantula
Natural Resources Institute Finland (LUKE), PO Box 2 (Latokartanonkaari 7),
FI-00791 Helsinki, Finland
Email: muhammad.kashif@luke.fi
The frequency of partitiviruses among forest pathogenic fungi of the genus Heterobasidion is
relatively low, i.e. 15-17 %. These mycoviruses are transmitted horizontally among strains
via mycelial anastomoses. Fungal dual cultures were used to find whether pre-existing virus
infections affect virus transmission rates among strains of H. annosum. The presence of
HetPV15-pa1 was found to be detrimental to its host as in single infection and this virus
strain significantly enhanced the transmissibility of congeneric alphapartitiviruses, namely
HetPV13-an1 (nt similarity ~70% based on RdRp segment), when present in the recipient.
On contrary, the pre-existing virus infections of species HetPV1 diminished subsequent
infections to their close conspecific relatives (98% nt sequence similarity based on RdRp).
The results suggest that co-infections developed by distantly related virus strains would be
more stable than those of closely related strains, and therefore mutual exclusion may play a
vital role determining the stability of coinfections. The transmission of these coinfections was
followed by quantification of mRNA based viral transcripts by RT-qPCR. The quantification
of CP and RdRp mRNA showed distinctive expression of transcripts for each virus strain.
Overall, the expression of HetPV15-pa1 RdRp was found to be 90-139 times higher than CP
in both single and in co-infections whereas the expression of HetPV13-an1 RdRP was only 3-
5 times higher than that of its CP. Interestingly the amounts of transcripts of three viruses out
of four was significantly reduced in coinfections which shows that the transmission of
partitiviruses is influenced by other viruses existing in the recipient Heterobasidion host.

10. Seminar on Forest Pathology, November 2nd
, 2017
Natural Resources Institute Finland, Viikki, Helsinki
Species communication and Open Data
Urmas Kõljalg
Institute of Ecology and Earth Sciences, University of Tartu, Estonia
Email: urmas.koljalg@ut.ee
The Unified system for the DNA based fungal species linked to the classification is available
at: https://unite.ut.ee