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Live plants are often traded in winter or early spring when the number of pests is potentially low, in part because deciduous trees lack foliage. However, the pests that may be associated with traded dormant plant material are likely to be largely unknown. Pest risk assessments can only be performed for known pest species and it is therefore important to identify potential pests before they are moved to a new area. In addition, it is difficult to predict whether introduced pests, even if already well known, may shift host after their arrival. Several studies attempted to estimate the likelihood of host shifts using literature about known pest-host associations, but the number of pests in these studies was often limited and the data were unlikely collected in the same place and at the same time. To overcome these problems, we performed a global study which aimed at detecting potential insect pests and fungal pathogens on congeneric native and exotic woody plant species that belong to six studied tree genera. Sampling was done simultaneously in 33 countries around the world following a common sampling protocol. At each location, twenty 50 cm long, dormant twigs were collected from each tree species and kept in containers with water at room temperature. Emerged insects were collected and identified based on morphology and DNA barcoding of the mDNA COI region. Fungal DNA was extracted from pooled buds, twig parts and needles for species identification using a metabarcoding approach based on the rDNA ITS region. Here, we present preliminary results of this study and discuss them in the context of pest risk assessment.

Common scab, caused by species from the bacterial genus S treptomyces, is an important disease of potato (S olanum tuberosum) crops worldwide. Early tuberization is a critical period for pathogen infection; hence, studies of host gene expression responses during this developmental stage can be important to expand our understanding of the infection process and to identify putative resistance genes. In an infection experiment with the highly susceptible potato cultivar Saturna and the relatively resistant cultivar Beate, transcription profiles were obtained by RNA sequencing at two developmental stages: the early hook stage and the early tuber formation stage. Our results indicate that ‘Beate’ mounts an early and sustained response to infection by S . turgidiscabies, whereas the defence response by ‘Saturna’ ceases before the early tuber formation stage. Most pronounced were the putative candidate defence‐associated genes uniquely expressed in ‘Beate’. We observed an increase in alternative splicing on pathogen infection at the early hook stage for both cultivars. A significant down‐regulation of genes involved in the highly energy‐demanding process of ribosome biogenesis was observed for the infected ‘Beate’ plants at the early hook stage, which may indicate an allocation of resources that favours the expression of defence‐related genes.

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are caused by ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’, respectively. These are the main phytoplasma diseases on pome and stone fruit that cause severe diseases on commercial crops. The main goal of the APOPHYT project is to improve the understanding of the multitrophic interactions between plants – vectors – phytoplasmas in order to develop disease management strategies and finally to reduce the impact of phytoplasma diseases in Europe. The different work packages aim to deeper analyse parts of the multitrophic interactions: 1. Phytoplasma Is it possible to improve diagnostics and to develop more sensitive detection methods (WP5) ? Are phytoplasma present as latent infections in nuclear plantings and nurseries (WP2) ? How are phytoplasma distributed in Europe (WP2) ? What are the population dynamics and the diversity of AP strains within apple trees (WP3) ? Is it possible to evidence virulence differences of AP strains (WP3) ? 2. Host plant and plant – phytoplasma interactions What is influencing the disease development at different locations in Europe (WP4) ? How different climatic conditions influence symptom expressions (WP4) ? Can symptom expressions be linked with phytoplasma titre (WP4) ? 3. Vector and vector – host plant interactions Can all vectors be found at all location in Europe (WP6) ? What is the percentage of vectors infested with phytoplasma (WP6) ? What is the influence of host plants on vector behaviour (WP6) ?, Scientific report of the Euphresco funded project 'Evaluation of factors determining distribution, impact, detection and characterization of fruit tree phytoplasmoses' (APOPHYT)

Fruit tree canker caused by Neonectria ditissima is a major bark canker disease on apple especially in temperate regions with high humidity and precipitation. The fungus can infect plant material through natural or artificial wounds. It can lead to loss of yield with possible high impact on economy for the apple producer. It has been reported that there is more fruit tree canker attack on apple in recent years. To evaluate susceptibility of genotypes to fruit tree canker, one year old potted tree and detached shoots from top of main stem of one year old trees were tested in the greenhouse and growth chamber. All experiments were inoculated with macroconidia suspension after artificial wounding. The inoculum used in these trials was kept in deep freezer and activated on rootstock before inoculation on wounded areas. In general, infection percent and area under the disease progress curve (AUDPC) are commonly calculated to examine resistance of apple genotypes to fruit tree canker. This study showed that infection percent or AUDPC values alone were not enough to conclude about resistance level to fruit tree canker in apple genotypes. It may be beneficial to determine the resistance level by using several parameters, as evaluation of several parameters together seem to ensure more reliable results. In this study, a range of parameters were applied for recording symptom development. External lesion size was measured at fixed points during experiment period, also giving number of days until the first symptoms were visible. Several extra parameters were recorded at the last observation date, such as internal lesion size, wilting, girdling, swelling, flakes, bubbles, ring pattern, color and sporulation. In general, the developing of lesion surface size increased rapidly 4 weeks after first symptoms was visible in potted tree trial and in detached shoot trial symptoms increased rapidly already one week after first symptoms were visible. We investigated susceptibility of apple cultivars/genotypes by analysis of datasets including or not including inoculation points with no infection symptoms. It was a clear pattern that when including only inoculation points with infection in data analysis, susceptibility of cultivars with low infection percentage can be overestimated. The number of days until the first symptoms were visible after inoculation seem to be a good parameter to investigate susceptibility to fruit tree canker, together with other parameters such as AUDPC level and lesion size at the end of experiment. Wilting and girdling are important parameters to include when examining resistance of apple genotypes, both parameters presented a good association with other parameters such as AUDPC level and external lesion size at last iii recording date. Swelling is a parameter that may discriminate significant difference among apple cultivars. ‘Aroma’ is known to have low to medium susceptibility to N. ditissima, and on swelling character ‘Aroma Fagravoll’ differed from the susceptible cultivars ‘Elise’ and ‘Discovery’. PCA plot showed a relationship between the characters swelling and bubbles. These two characters might be related to resistance, while flakes seem to be related to susceptibility. The tested apple genotypes differ in level of susceptibility to fruit tree canker. MA042 10041 and ‘Bramley’s Seedling’ showed stronger resistance against the canker than ‘Aroma’ in this study. The study shows that recording of several symptoms can give a clearer picture of susceptibility to canker in apple genotypes. Phenotyping with several characters is necessary to get reliable results on resistance in breeding material for use as parents and for introduction of new resistant cultivars. Such phenotyping is also necessary when searching for resistance genes or markers for resistance genes. Frukttrekreft forårsaket av soppen Neonectria ditissima er en viktig plantesykdom på eple både på treet og fruktene, spesielt i omåder med mildt og fuktig klima. Soppen kan infisere plantemateriale gjennom naturlige sår som for eksempel frostskade, sårdannelse etter bladfall (bladarr) eller beskjæring. Angrep av frukttrekreft kan medføre økonomisk tap for epleprodusenter. Det er rapportert at det er mer frukttrekreft nå enn det har vært før. For å evaluere motstandsevne mot angrep av frukttrekreft hos nummersorter og sorter ble det gjennomført forsøk på ettårige tre (pisker) og avskårne toppskudd av ettårige tre i veksthus og vekstrom. Plantemateriale i alle forsøk ble inokulert med makrokonidia etter såring, der inokulum ble lagret i fryseboks og aktivert på grunnstammer før det ble brukt i forsøkene. Vanligvis blir infeksjonsprosent og «area under the disease progress curve» (AUDPC) beregnet for å kartlegge motstandsevne mot angrep av fruktrekreft hos eplesorter. I denne studien fant vi at infeksjonsprosent og AUDPC-verdier alene ikke var nok til å konkludere om motstandsevne. Evaluering av flere parameter sammen ser ut til å sikre mer pålitelige resultater. I denne studien ble et vidt spekter av parametre brukt til å registrere ulike symptomer i forsøkene. Lengde av ytre skade ble registrert med faste intervall undervegs i løpet av forsøksperioden, og dette ga samtidig antall dager til første symptomer ble synlig etter inokulering. Sluttregistrering omfattet registrering av både ytre symptomer og indre symptomer, som for eksempel visning, ringing (sår vokser rundt tre), svelling, flassing, bobling, ringmønster, farge og sporulering. M-PV

Silver leaf disease, caused by the basidiomycete Chondrostereum purpureum, is a growing problem in Norwegian plum production. It affects the overall health and lifespan of the fruit trees. The inoculation experiments in this study set out to attain more knowledge regarding disease development of the fungus under Norwegian conditions. They were performed at NIBIO Ullensvang and Ås between fall 2019 and fall 2020. Different inoculation methods were used on the main plum cultivars used in Norwegian production (Edda, Jubileum, Mallard, Opal, and Reeves) utilizing five C. purpureum isolates of different origin. The results of the inoculations, using map-pins with mycelium placed in newly developed side shoots and 1-year old side shoots in an established field, were unreliable and the method was found ill suited for field inoculations. The map-pin method did work well in more controlled conditions with whips in pots. Map-pin inoculation of whips developed leaf symptoms after 45 days outdoors and 35 days in growth chamber experiments. Fruiting bodies developed in August on one root stump that was left in the field. In addition, two trees in the outdoor experiment had silver leaf infections that had come prior to inoculations. Young graftings, inoculated via agar pieces with mycelium, showed leaf symptoms 77 days after inoculation no matter if the inoculation point being in the rootstock or scion. Almost all graftings displayed leaf symptoms at this time. The fact that the graftings were very small appeared to be the reason behind the many severe infections in this experiment. Five dead graftings, inoculated in the rootstock, developed fruiting bodies in the field in August before final assessments and were identified as C. purpureum. This is a good example of how infected trees can become inoculum sources in the field if not removed. Inoculations with mycelium, performed in fall and winter, developed leaf symptoms already at leaf emergence in May and did not seem to be inhibited by the colder conditions during inoculation and wintertime. Overall, the methods using agar pieces with mycelium caused the most infections and were the most severe in younger trees. For future studies susceptibility of the different cultivars should be investigated to provide a preference for Norwegian fruit farmers. Studies investigating disease development spanning over multiple seasons should be done on well-established fields to see long term effects of the fungus. Such an experiment could also provide better insight to which cultivars are more resilient than others and would be of helpful for future farm practices in Norway. M-PV

The octoploid strawberry (Fragaria × ananassa) is highly valued horticultural crop. The fruit crop is often challenged by several phytopathogens including a hemibiotrophic oomycete, Phytophthora cactorum. The pathogen has been known to cause two distinct diseases in strawberry: crown rot and leather rot. Both diseases are reported to cause significant economic losses in strawberry production globally. The high heterozygosity, absence of qualitative/natural resistance and progressive phaseout of chemical means of disease control has made it obvious for identification and characterization of putative resistance genes from their wild accessions. In this study, two Fragaria vesca genes, WAK (Wall Associated Kinases) and FvCDIP1 (Fragaria vesca Cell Death Inducing Protein 1), were selected for their functional characterization. Agrobacterium tumefaciens mediated transient expression of WAK gene in Nicotiana benthamiana was performed followed by an inoculation of P. cactorum. Diseased area in terms of percentage was calculated to analyse the effect of WAK gene expression on defence. A reduced disease severity in comparison to the mock controls was observed for the WAK gene expressed plants. M-PV

One of the greatest challenges faced by humanity today is to feed the estimated population of 10 billion people by 2050 in the face of climate change. Plant pathogens are still a major challenge for agriculture and one of the most promising ways for efficient and sustainable management of plant pathogens is the use of gene editing tools to generate new improved crop cultivars. The plant pathogen Sclerotinia Sclerotiorum causes a disease commonly known as white mold on a wide range of host plants, including Lactuca Sativa (lettuce). This brings significant losses on lettuce yields (reportedly up to 30 %) and results in agricultural and economic challenges for the farmers. The aim of this study is to conduct functional analysis of genes possibly involved in the S. sclerotorium pathogenesis in lettuce, by developing CRISPR-Cas9 knockout-constructs for targeted mutagenesis on candidate genes. Ultimately gene edited L. sativa lines with enhanced resistance against the plant pathogen S. Sclerotiorum would be generated. Bioinformatical analysis, literature search and RT-qPCR gene expression analysis of infected and uninfected lettuce were used to identify target genes. CRISPR-constructs with sgRNA specifically designed for three target genes were constructed to perform the study. The cloning system used in this thesis consisted of Escherichia Coli, for transformation and propagation of the CRISPR expression vectors, and Agrobacterium Tumefaciens binary vector system, which delivered the CRISPR-constructs into the L. Sativa plants. The CRISPR-constructs were tested in both stable and transient A. tumefaciens-mediated plant transformation. Unfortunately, the plant transformation experiments were not completed due to the COVID-19 outbreak which caused the Norwegian government to inflict lock-down on most public institutions, including Nibio. En av de største utfordringene menneskeheten står ovenfor i dag er å produsere mat til den forventede befolkningen på 10 milliarder innen 2050. Plantepatogener er fremdeles en stor utfordring for landbruket, og en av de mest lovende metodene for en effektiv og bærekraftig forvaltning av plantepatogener er bruken av genredigeringsverktøy for å generere nye og forbedrede kultivarer. Plantepatogenet Sclerotinia Sclerotiorum forårsaker råtesopp på en rekke vertsplanter, inkludert Lactuca Sativa (salat). Råtesoppen forårsaker betydelige tap for salatavlingene (opptil 30%) og resulterer i landbruks- og økonomiske utfordringer for bøndene. Målet med dette studiet er å gjennomføre en funksjonell analyse av gener som muligens er involvert i S. sclerotorium patogenese i salat ved å utvikle CRISPR-Cas9 knockout konstrukter for målrettet mutagenese på kandidatgener. Hensikten er å genere genredigerte L. sativa-linjer med forbedret resistens mot plantepatogenet S. sclerotiorum. Bioinformatisk analyse, litteraturstudier og RT-qPCR genekspresjonsanalyse av infisert og frisk salat ble brukt til å identifisere målgener. CRISPR-konstrukter med sgRNA, spesifikt designet for tre målgener, ble konstruert for å gjennomføre studiet. Kloningssystemet som ble brukt i denne oppgaven besto av Escherichia Coli for transformasjon av CRISPR ekspresjonsvektorer og Agrobacterium Tumefaciens binær vektorsystem, som overførte CRISPR-konstruktene til L. Sativaplantene. CRISPR-konstruktene ble testet med to typer A. tumefaciens-mediert plantetransformasjon. Dessverre ble ikke transformasjonseksperimentene fullført på grunn av COVID-19 utbruddet som førte til at den norske regjeringen innførte unntakstilstand som berørte de fleste offentlige institusjoner, inkludert NIBIO. M-BIOTEK

Botrytis cinerea is a necrotrophic pathogen that is reported to cause disease in more than 1400 plant species. Each year the fungi cause yield losses of great economic importance and is the most important pathogen in Norwegian strawberry production. Priming has been shown to enhance defense in plants, and epigenetic gene regulations are hypothesized to be part of the underlying mechanisms of the priming effect. Epigenetic gene regulation is mediated by specific enzymes that alter DNA or the associated histones without changing the underlying DNA sequence. In the first part of this thesis I have investigated the effect of defense priming. Plants of F. x ananassa were primed for defense by soil drenching with the chemical agent β-aminobutyric acid (BABA) and leaves were detached and drop-infected with B. cinerea. The disease development was scored phenotypically to determine if primed plants were more or less resistant to the pathogen compared to non-primed plants. After phenotypic scoring, candidate defense genes were analyzed by RT-qPCR. An enhanced susceptibility to B. cinerea was observed in the phenotypic scoring of the primed leaves compared to the non-primed leaves, and the increase in gene expression of defense related genes were mainly found to be in response to B. cinerea and not due to the priming effect of BABA. In the second part of the thesis, new constructs for CRISPR/Cas9 gene editing was made to target putative epigenetic modifiers homologous to important epigenetic modifiers in Arabidopsis thaliana to identify their role in defense against B. cinerea in F. vesca. To this end, CRISPR-vectors with different U6-promoters of F. vesca were designed and tested in a transient expression system. The vector with the highest U6-promoter-driven sgRNA-expression was then selected to knock out target genes involved in regulation of defense responses. Norges forskningsråd ; NIBIO M-PV

Common scab disease of potato is distributed worldwide and not easily controlled. It is caused by Streptomyces species. The virulence mechanism of Streptomyces species is the production of thaxtomin A. Although the disease cycle of common scab of potato is well understood, the molecular processes underlying disease development and disease resistance are little known. The work performed in this thesis is a part of an on-going project on common scab resistance in potato, studying gene expression in response to S. turgidiscabies infection. Transcription profiles established by RNA-sequencing indicate a number of putative defense-associated genes which uniquely expressed in resistant cultivar Beate at the initial stage of tuber development. Seven candidate genes of which were selected to investigate the correlation between their expression and common scab resistance in potato, using real-time PCR method. Apart from these seven genes, two candidate genes putatively encoding TXR1 proteins involving in thaxtomin A transport, two putative candidate auxin signalingassociated genes and a gene putatively encoding aminotransferase ALD1 were also investigated. Comparisons of candidate gene expressions in resistant cultivars and susceptible cultivars indicate two selected candidate genes performed correlations between their expressions and common scab resistance, which are gene putatively encoding auxin binding protein 1 and gene putatively encoding aminotransferase ALD1. These findings putatively conceived of an idea that auxin signaling might promote common scab resistance in potato. On the other hand, the results are not in the agreement with the hypothesis that putative defenseassociated genes which uniquely expressed in resistant cultivar Beate at the initial stage of tuber development might enhance common scab resistance. M-PV