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Abstract

The oomycete pathogen Phytophthora cactorum causes crown rot, a major disease of cultivated strawberry. We report the draft genome of P. cactorum isolate 10300, isolated from symptomatic Fragaria x ananassa tissue. Our analysis revealed that there are a large number of genes encoding putative secreted effectors in the genome, including nearly 200 RxLR domain containing effectors, 77 Crinklers (CRN) grouped into 38 families, and numerous apoplastic effectors, such as phytotoxins (PcF proteins) and necrosis inducing proteins. As in other Phytophthora species, the genomic environment of many RxLR and CRN genes differed from core eukaryotic genes, a hallmark of the two-speed genome. We found genes homologous to known Phytophthora infestans avirulence genes including Avr1, Avr3b, Avr4, Avrblb1 and AvrSmira2 indicating effector sequence conservation between Phytophthora species of clade 1a and clade 1c. The reported P. cactorum genome sequence and associated annotations represent a comprehensive resource for avirulence gene discovery in other Phytophthora species from clade 1 and, will facilitate effector informed breeding strategies in other crops.

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Abstract

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr-vnt1 effector, recognized by the potato Rpi-phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi-Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C-terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large-scale cultivation of plants containing the Rpi-Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi-phu1 gene were found, the corresponding Avr-vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.

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Abstract

Phytophthora cryptogea, P. gonapodyides, P. lacustris, P. megasperma, P. plurivora, P. taxon paludosa and an unknown Phytophthora species were isolated from waterways and soil samples in Christmas tree fields in southern Sweden. In addition, P. megasperma was isolated from a diseased Norway spruce (Picea abies) plant from one of the fields in Svalöv. Inoculation tests were sequentially carried out with one isolate from each of the three species P. cryptogea, P. megasperma, and P. plurivora, all known pathogens on conifers. The same three isolates were used to study a few morphological features to confirm the identification, and temperature-growth relationships were carried out to see how well the organisms fit into Swedish climatic conditions. Seedlings of Norway spruce and Nordmann fir (Abies nordmanniana) were inoculated in the roots and the stems. None of the isolates caused extensive root rot under the experimental conditions, but all three species could be re-isolated from both Norway spruce and Nordmann fir. Phytophthora root rot is currently of minor concern for Christmas tree growers in Sweden. However, the Phytophthora isolations from soil and water indicate the presence of this damaging agent, which may lead to future problems.

Abstract

Plants are exposed to various pathogens in their environment and have developed immune systems with multiple layers of defence to fight-back. However, often pathogens overcome the resistance barriers, infect the plants to cause the disease. Pathogens that cause diseases on economically important crop plants like strawberry incur huge losses to the agriculture industry. For example, The 2016 outbreak of strawberry grey mould (Botrytis cinerea) in Norway caused up to 95% crop losses. Outbreaks like this underline the importance of developing novel and sustainable tools to combat plant diseases, for example by increasing the plants’ natural disease resistance. Priming plant defences using chemical elicitors may be effective in providing the enhanced resistance against multiple pathogens. We have used β-aminobutyric acid (BABA) as a chemical priming agent to induce resistance in Fragaria vesca against Botrytis cinerea. Effects of BABA on disease progression and defence responses of Fragaria are being characterized using molecular tools like RNAseq, RT-PCR and ChIP. As priming chemicals may induce an epigenetic memory in treated plants, we also plan to study the histone methylation patterns in primed plants and the genes that are regulated. Our long-term aim is to understand the duration of the epigenetic memory and its cross-generational transmission to the progeny in Fragaria. Our results will help guide various crop protection strategies in addition to providing new insights to develop novel tools for plant disease management.

Abstract

Pathogenic soft rot Enterobacteriaceae (SRE) belonging to the genera Pectobacterium and Dickeya cause diseases in potato and numerous other crops. Seed potatoes are the most important source of infection, but how pathogen-free tubers initially become infected remains an enigma. Since the 1920s, insects have been hypothesized to contribute to SRE transmission. To validate this hypothesis and to map the insect species potentially involved in SRE dispersal, we have analyzed the occurrence of SRE in insects recovered from potato fields over a period of 2 years. Twenty-eight yellow sticky traps were set up in 10 potato fields throughout Norway to attract and trap insects. Total DNA recovered from over 2,000 randomly chosen trapped insects was tested for SRE, using a specific quantitative PCR (qPCR) TaqMan assay, and insects that tested positive were identified by DNA barcoding. Although the occurrence of SRE-carrying insects varied, they were found in all the tested fields. While Delia species were dominant among the insects that carried the largest amount of SRE, more than 80 other SRE-carrying insect species were identified, and they had different levels of abundance. Additionally, the occurrence of SRE in three laboratory-reared insect species was analyzed, and this suggested that SRE are natural members of some insect microbiomes, with herbivorous Delia floralis carrying more SRE than the cabbage moth (Plutella xylostella) and carnivorous green lacewing larvae (Chrysoperla carnea). In summary, the high proportion, variety, and ubiquity of insects that carried SRE show the need to address this source of the pathogens to reduce the initial infection of seed material.

Abstract

The plant pathogenic fungus Fusarium langsethiae produces the highly potent mycotoxins HT-2 and T-2. Since these toxins are frequently detected at high levels in oat grain lots, they pose a considerable risk for food and feed safety in Norway, as well as in other north European countries. To reduce the risk of HT-2/T- 2-contaminated grain lots to enter the food and feed chain, it is important to identify factors that influence F. langsethiae infection and mycotoxin development in oats. However, the epidemiology of F. langsethiae is unclear. A three-year survey was performed to reveal more of the life cycle of F. langsethiae and its interactions with oats, other Fusarium species, as well as insects, mites and weeds. We searched for inoculum sources by quantifying the amount of F. langsethiae DNA in crop residues, weeds, and soil sampled from a selection of oat-fields. To be able to define the onset of infection, we analysed the amount of F. langsethiae DNA in oat plant material sampled at selected growth stages (between booting and maturation), as well as the amount of F. langsethiae DNA and HT-2 and T-2 toxins in the mature grain. We also studied the presence of possible insect- and mite vectors sampled at the selected growth stages using Berlese funnel traps. The different types of materials were also analysed for the presence F. graminearum DNA, the most important deoxynivalenol producer observed in Norwegian cereals, and which presence has shown a striking lack of correlation with the presence of F. langsethiae in oat. Results show that F. langsethiae DNA may occur in the oat plant already before heading and flowering. Some F. langsethiae DNA was observed in crop residues and weeds, though at relatively low levels. No Fusarium DNA was detected in soil samples. Of the arthropods that were associated with the collected oat plants, aphids and thrips species were dominating. Further details will be given at the meeting.

Abstract

Sclerotinia stem rot (SSR) is the most important disease of oilseed Brassica crops in Norway. Fungicide applications should be aligned with the actual need for control, but the SSR prediction models used lack accuracy. We have studied the importance of precipitation, and the role of petal and leaf infection for SSR incidence by using data from Norwegian field and trap plant trials over several years. In the trials, SSR incidence ranged from 0 to 65%. Given an infection threshold of 25% SSR, regression and Receiver Operating Characteristics (ROC) analysis were used to evaluate different precipitation thresholds. The sum of precipitation two weeks before and during flowering appeared to be a poor predictor for SSR infection in our field and trap plant trials (P = 0.24, P = 0.11, respectively). Leaves from three levels (leaf one, three, five), and petals were collected at three to four different times during flowering from nine field sites over two years and tested for SSR infection with real-time PCR. Percentage total leaf and petal infection explained 57 and 45% of variation in SSR incidence, respectively. Examining the different leaves and petals separately, infection of leaf three sampled at full flowering showed the highest explanation of variation in later SSR incidence (R2 = 65%, P < 0.001). ROC analysis showed that given an infection threshold of 45%, both petal and leaf infection recommended spraying when spraying was actually needed. Combining information on petal and leaf infection during flowering with relevant microclimate factors in the canopy, instead of the sum of precipitation might improve prediction accuracy for SSR.

Abstract

Helminthosporium solani causes silver scurf, which affects the quality of potato. The biocontrol agent Clonostachys rosea greatly limited the severity of silver scurf symptoms and amount of H. solani genomic DNA in laboratory experiments. Transcriptomic analysis during interaction showed that H. solani gene expression was highly reduced when coinoculated with the biocontrol agent C. rosea, whereas gene expression of C. rosea was clearly boosted as a response to the pathogen. The most notable upregulated C. rosea genes were those encoding proteins involved in cellular response to oxidative stress, proteases, G-protein signaling, and the methyltransferase LaeA. The most notable potato response to both fungi was downregulation of defense-related genes and mitogen-activated protein kinase kinase kinases. At a later stage, this shifted, and most potato defense genes were turned on, especially those involved in terpenoid biosynthesis when H. solani was present. Some biocontrol-activated defense-related genes in potato were upregulated during early interaction with C. rosea alone that were not triggered by H. solani alone. Our results indicate that the reductions of silver scurf using C. rosea are probably due to a combination of mechanisms, including mycoparasitism, biocontrol-activated stimulation of plant defense mechanisms, microbial competition for nutrients, space, and antibiosis.

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Abstract

Soft rot and blackleg of potato caused by pectinolytic bacteria lead to severe economic losses in potato production worldwide. To investigate the species composition of bacteria causing soft rot and black leg of potato in Norway and Poland, bacteria were isolated from potato tubers and stems. Forty-one Norwegian strains and 42 Polish strains that formed cavities on pectate medium were selected for potato tuber maceration assays and sequencing of three housekeeping genes (dnaX, icdA and mdh) for species identification and phylogenetic analysis. The distribution of the species causing soft rot and blackleg in Norway and Poland differed: we have demonstrated that mainly P. atrosepticum and P. c. subsp. carotovorum are the causal agents of soft rot and blackleg of potatoes in Norway, while P. wasabiae was identified as one of the most important soft rot pathogens in Poland. In contrast to the other European countries, D. solani seem not to be a major pathogen of potato in Norway and Poland. The Norwegian and Polish P. c. subsp. carotovorum and P. wasabiae strains did not cluster with type strains of the respective species in the phylogenetic analysis, which underlines the taxonomic complexity of the genus Pectobacterium. No correlation between the country of origin and clustering of the strains was observed. All strains tested in this study were able to macerate potato tissue. The ability to macerate potato tissue was significantly greater for the P. c. subsp. carotovorum and Dickeya spp., compared to P. atrosepticum and P. wasabiae.

Abstract

The genus Microbacterium contains bacteria that are ubiquitously distributed in various environments and includes plant-associated bacteria that are able to colonize tissue of agricultural crop plants. Here, we report the 3,508,491 bp complete genome sequence of Microbacterium sp. strain BH-3-3-3, isolated from conventionally grown lettuce (Lactuca sativa) from a field in Vestfold, Norway. The nucleotide sequence of this genome was deposited into NCBI GenBank under the accession CP017674.

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Abstract

Dollar spot is a destructive and widespread disease affecting most turfgrass species, but until recently it has been absent from the Scandinavian countries of northern Europe. In the fall of 2014, disease symptoms consistent with dollar spot were observed on a golf course fairway in Sweden. A fungus was isolated from symptomatic turf and identified as Sclerotinia homoeocarpa on the basis of ribosomal deoxyribonucleic acid (rDNA) internal transcribed spacer (ITS) sequences, morphology, and culture characteristics. The ITS sequence was identical to isolates of S. homoeocarpa from the eastern and midwestern United States. Koch’s postulates were fulfilled, confirming the S. homoeocarpa isolate as the causal agent. This is the first report of turfgrass dollar spot in Sweden and only the second report of the disease from Scandinavia. Because pesticides are rarely used in the cultivation of Scandinavian turfgrass, dollar spot disease may prove difficult to control through conventional means and potentially represents a major threat to the industry.

Abstract

Knowledge about the reproduction strategies of invasive species is fundamental for effective control. The invasive Fallopia taxa (Japanese knotweed s.l.) reproduce mainly clonally in Europe, and preventing spread of vegetative fragments is the most important control measure. However, high levels of genetic variation within the hybrid F. × bohemica indicate that hybridization and seed dispersal could be important. In Norway in northern Europe, it is assumed that these taxa do not reproduce sexually due to low temperatures in the autumn when the plants are flowering. The main objective of this study was to examine the genetic variation of invasive Fallopia taxa in selected areas in Norway in order to evaluate whether the taxa may reproduce by seeds in their most northerly distribution range in Europe. Fallopia stands from different localities in Norway were analyzed with respect to prevalence of taxa, and genetic variation within and between taxa was studied using amplified fragment length polymorphism (AFLP). Taxonomic identification based on morphology corresponded with identification based on simple sequence repeats (SSR) and DNA ploidy levels (8× F. japonica, 6× F. × bohemica and 4× F. sachalinensis). No genetic variation within F. japonica was detected. All F. × bohemica samples belonged to a single AFLP genotype, but one sample had a different SSR genotype. Two SSR genotypes of F. sachalinensis were also detected. Extremely low genetic variation within the invasive Fallopia taxa indicates that these taxa do not reproduce sexually in the region, suggesting that control efforts can be focused on preventing clonal spread. Climate warming may increase sexual reproduction of invasive Fallopia taxa in northern regions. The hermaphrodite F. × bohemica is a potential pollen source for the male-sterile parental species. Targeted eradication of the hybrid can therefore reduce the risk of increased sexual reproduction under future warmer climate.

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Abstract

The natural occurrence of fungi, mycotoxins and fungal metabolites was investigated in 100 samples of maize grains collected from south and southwestern Ethiopia in 2015. The maize samples were contaminated by Fusarium, Aspergillus and Penicillium species. Using liquid chromatography tandem mass spectrometry 127 secondary metabolites were analysed. Zearalenone was the most prevalent mycotoxin, occurring in about 96% of the samples. Zearalenone sulfate was the second most prevalent, present in 81% of the samples. Fumonisin B1 was detected in 70% of the samples with a mean level of 606 μg kg−1 in positive samples, while FB2, FB3 and FB4 were detected in 62%, 51% and 60% of the maize samples with mean levels of 202, 136 and 85 μg kg−1, respectively. Up to 8% of the samples were contaminated with aflatoxins, with a maximum level of aflatoxin B1 of 513 μg kg−1. Results were higher than earlier reports for maize from Ethiopia.

Abstract

The genus Pectobacterium, which belongs to the bacterial family Enterobacteriaceae, contains numerous species that cause soft rot diseases in a wide range of plants. The species Pectobacterium carotovorum is highly heterogeneous, indicating a need for re-evaluation and a better classification of the species. PacBio was used for sequencing of two soft-rot-causing bacterial strains (NIBIO1006T and NIBIO1392), initially identified as P. carotovorum strains by fatty acid analysis and sequencing of three housekeeping genes (dnaX, icdA and mdh). Their taxonomic relationship to other Pectobacterium species was determined and the distance from any described species within the genus Pectobacterium was less than 94% average nucleotide identity (ANI). Based on ANI, phylogenetic data and genome-to-genome distance, strains NIBIO1006T, NIBIO1392 and NCPPB3395 are suggested to represent a novel species of the genus Pectobacterium, for which the name Pectobacterium polaris sp. nov. is proposed. The type strain is NIBIO1006T (=DSM 105255T=NCPPB 4611T).

Abstract

Important losses in strawberry production are often caused by the oomycete Phytophthora cactorum, the causal agent of crown rot. However, very limited studies at molecular levels exist of the mechanisms related to strawberry resistance against this pathogen. To begin to rectify this situation, a PCR-based approach (NBS profiling) was used to isolate strawberry resistance gene analogs (RGAs) with altered expression in response to P. cactorum during a time course (2, 4, 6, 24, 48, 96 and 192 h post-infection). Twenty-three distinct RGA fragments of the NB-LRR type were identified from a resistance genotype (Bukammen) of the wild species Fragaria vesca. The gene transcriptional profiles after infection showed that the response of most RGAs was quicker and stronger in the resistance genotype (Bukammen) than in the susceptible one (FDP821) during the early infection stage. The transcriptional patterns of one RGA (RGA109) were further monitored and compared during the P. cactorum infection of two pairs of resistant and susceptible genotype combinations (Bukammen/FDP821 and FDR1218/1603). The 5′ end sequence was cloned, and its putative protein was characteristic of NBS-LRR R protein. Our results yielded a first insight into the strawberry RGAs responding to P. cactorum infection at molecular level.

Abstract

The prevalence of Fusarium dry rot in potatoes produced in Norway was investigated in a survey for three consecutive years in the period 2010 to 2012. A total of 238 samples (comprising 23,800 tubers) were collected, representing different cultivars and production regions in Norway. Fusarium spp. were detected in 47% of the samples, with one to three species per sample. In total, 718 isolates of Fusarium spp. were recovered and identified to seven species. The most commonly isolated species was Fusarium coeruleum, comprising 59.6% of the total Fusarium isolates and found in 17.2% of the collected samples, followed by Fusarium avenaceum (27.2% of the isolates and found in 27.7% of the samples). Fusarium sambucinum was the third most prevalent species (6.4% in 8.8% of the samples) and Fusarium culmorum the fourth (5.2% in 6.3% of the samples). Less prevalent species included Fusarium cerealis, Fusarium graminearum, and Fusarium equiseti (<1% in 0.4 to 1.3% of the samples). F. coeruleum was the most prevalent species in northern and southwestern Norway, whereas F. avenaceum was dominating in eastern Norway. The potato cultivars Berber and Rutt were susceptible to all Fusarium spp. A new TaqMan real-time PCR assay specific for F. coeruleum was developed, which successfully identified Norwegian isolates. This and other previously developed real-time PCR assays targeting different Fusarium species were evaluated for their ability to detect latent infections in potatoes at harvest. This study provides new information on the current occurrence of different Fusarium species causing Fusarium dry rot in potatoes in Europe including areas far into the arctic in the north of Norway.

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Abstract

Amplified fragment length polymorphism (AFLP) was used to study the genetic variation among 80 F. verticillioides isolates from kernels of Ethiopian maize, collected from 20 different maize growing areas in four geographic regions. A total of 213 polymorphic fragments were obtained using six EcoRI/MseI primer combinations. Analysis of the data based on all 213 polymorphic AFLP fragments revealed high level of genetic variation in the F. verticillioides entities in Ethiopia. About 58% of the fragments generated were polymorphic. The genetic similarity among F. verticillioides isolates varied from 46% to 94% with a mean Dice similarity of 73%. Unweighted Pair Group Method with Arithmetic Average (UPGMA) analysis revealed two main groups and four subgroups. The principal coordinate analysis (PCO) also displayed two main groups that agreed with the results of UPGMA analysis, and there was no clear pattern of clustering of isolates according to geographic origin. Analysis of molecular variance: (AMOVA) showed that only 1.5% of the total genetic variation was between geographic regions, while 98.5% was among isolates from the same geographic regions of Ethiopia. Eighty distinct haplotypes were recognized among the 80 isolates analyzed. Hence, breeding efforts should concentrate on quantitative resistance that is effective against all genotypes of the pathogen.

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Abstract

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar ‘Fagerlin’ based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould.

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Abstract

Fusarium species causing maize kernel rot are major threats to maize production, due to reduction in yield as well as contamination of kernels by mycotoxins that poses a health risk to humans and animals. Two-hundred maize kernel samples, collected from 20 major maize growing areas in Ethiopia were analyzed for the identity, species composition and prevalence of Fusarium species and fumonisin contamination. On average, 38 % (range: 16 to 68 %) of maize kernels were found to be contaminated by different fungal species. Total of eleven Fusarium spp. were identified based on morphological characteristics and by sequencing the partial region of translation elongation factor 1-alpha (EF-1α) gene. Fusarium verticillioides was the dominant species associated with maize kernels (42 %), followed by F. graminearum species complex (22.5 %) and F. pseudoanthophilium (13.4 %). The species composition and prevalence of Fusarium species differed among the areas investigated. Fusarium species composition was as many as eight and as few as four in some growing area. The majority of the maize samples (77 %) were found positive for fumonisin, with concentrations ranging from 25 μg kg−1 to 4500 μg kg−1 (mean: 348 μg kg−1 and median: 258 μg kg−1). Slight variation in fumonisin concentration was also observed among areas. Overall results indicate widespread occurrence of several Fusarium species and contamination by fumonisin mycotoxins. These findings are useful for intervention measures to reduce the impact of the main fungal species and their associated mycotoxins, by creating awareness and implementation of good agricultural practices.

Abstract

The plant pathogenic fungus Fusarium langsethiae produces the highly potent mycotoxins HT-2 and T-2. Since these toxins are frequently detected at high levels in oat grain lots, they pose a considerable risk for food and feed safety in Norway, as well as in other north European countries. To reduce the risk of HT-2/T- 2-contaminated grain lots to enter the food and feed chain, it is important to identify factors that influence F. langsethiae infection and mycotoxin development in oats. However, the epidemiology of F. langsethiae is unclear. A three-year survey was performed to reveal more of the life cycle of F. langsethiae and its interactions with oats, other Fusarium species, as well as insects, mites and weeds. We searched for inoculum sources by quantifying the amount of F. langsethiae DNA in weeds, crop residues, and soil, sampled from a predetermined selection of oat-fields. To be able to define the onset of infection, we analysed the amount of F. langsethiae DNA in oat plant material sampled at selected growth stages (between booting and maturation), as well as the amount of F. langsethiae DNA and HT-2 and T-2 toxins in the mature grain. We also studied the presence of possible insect- and mite vectors sampled at the selected growth stages using Berlese funnel traps. All the different types of materials were also analysed for the presence F. graminearum DNA, the most important deoxynivalenol producer observed in Norwegian cereals, and which presence has shown a striking lack of correlation with the presence F. langsethiae in oat. Preliminary results show that F. langsethiae DNA may occur in the oat plant before heading and flowering. Some F. langsethiae DNA was observed in crop residues and weeds, though at relatively low levels. More results from this work will be presented at the meeting.

Abstract

The plant pathogenic fungus Fusarium langsethiae produces the highly potent mycotoxins HT-2 and T-2. Since these toxins are frequently detected at high levels in oat grain lots, they pose a considerable risk for food and feed safety in Norway, as well as in other north European countries. To reduce the risk of HT-2/T- 2-contaminated grain lots to enter the food and feed chain, it is important to identify factors that influence F. langsethiae infection and mycotoxin development in oats. However, the epidemiology of F. langsethiae is unclear. A three-year survey was performed to reveal more of the life cycle of F. langsethiae and its interactions with oats, other Fusarium species, as well as insects, mites and weeds. We searched for inoculum sources by quantifying the amount of F. langsethiae DNA in weeds, crop residues, and soil, sampled from a predetermined selection of oat-fields. To be able to define the onset of infection, we analysed the amount of F. langsethiae DNA in oat plant material sampled at selected growth stages (between booting and maturation), as well as the amount of F. langsethiae DNA and HT-2 and T-2 toxins in the mature grain. We also studied the presence of possible insect- and mite vectors sampled at the selected growth stages using Berlese funnel traps. All the different types of materials were also analysed for the presence F. graminearum DNA, the most important deoxynivalenol producer observed in Norwegian cereals, and which presence has shown a striking lack of correlation with the presence F. langsethiae in oat. Preliminary results show that F. langsethiae DNA may occur in the oat plant before heading and flowering. Some F. langsethiae DNA was observed in crop residues and weeds, though at relatively low levels. More results from this work will be presented at the meeting.

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Abstract

Delphinella shoot blight (Delphinella abietis) attacks true firs (Abies spp.) in Europe and North America. Especially subalpine fir (A. lasiocarpa), one of the main Christmas tree species in Norway, is prone to the disease. The fungus kills current year needles, and in severe cases entire shoots. Dead needles become covered with black fruiting bodies, both pycnidia and pseudothecia. Delphinella shoot blight has mainly been a problem in humid, coastal regions in the northwestern part of Southern Norway, but, probably due to higher precipitation in inland regions during recent years, heavy attacks were found in 2011 in a field trial with 76 provenances of subalpine fir in Southeastern Norway. However, the amount of precipitation seemed less important once the disease had established in the field. Significant differences in susceptibility between provenances were observed. In general, the more bluish the foliage was, the healthier the trees appeared. The analysis of provenance means indicated that, at least for the southern range, the disease ratings were correlated with foliage color. This study also includes isolation, identification, a pathogenicity test, a seed test and electron microscopy of the wax layer on the needles. The fungus was identified based on the morphology of spores and by sequencing the Internal Transcribed Spacer (ITS) regions of the ribosomal DNA. Koch’s postulates were fulfilled. The fungus was found present on newly harvested seeds and may therefore spread via international seed trade. When comparing the wax layers on green and blue needles, those of the latter were significantly thicker, a factor that may be involved in disease resistance.

Abstract

The woodland strawberry (Fragaria vesca) has become the model plant for the economically important, but genetically complex, octoploid F. × ananassa. Crown rot caused by the oomycete Phytophthora cactorum is a major problem for the strawberry industry and the identification and incorporation of efficient resistance genes into superior cultivars are important for breeding. In the present study, two experimental populations were used in inoculation experiments under controlled greenhouse condition. Studies of a sparse diallel cross between resistant and susceptible F. vesca genotypes concluded that resistance to crown rot is inherited as a dominant trait under nuclear control. Subsequently, an F2 population derived from the grandparents Bukammen (resistant) and Haugastøl 3 (susceptible) collected in Norway, were phenotyped in infection experiments and genotyped using genotyping-by-sequencing. A 416.2-cM linkage map was constructed, and a single major gene locus was identified on linkage group 6 that we attributed to resistance to Phytopthora infection. We propose to name the resistance locus RPc-1 (Resistance to Phytophthora cactorum 1). Gene prediction of the 3.3 Mb QTL recovered 801 genes of which 69 had a potential role in plant disease resistance.

Abstract

Pythium species are fungal-like organisms distributed all over the world. Most Pythium spp. live as saprophytes, but some of them are pathogenic. Here we report on disease incidence in Norway spruce (Picea abies) seedlings caused by Pythium undulatum, and pathogenicity in vitro of Norwegian isolates of P. undulatum and P. anandrum.

Abstract

A survey of nematodes associated with terrestrial slugs was conducted for the first time in Norway. A total of 611 terrestrial slugs were collected from 32 sample sites. Slugs were identified by means of morphological examination, dissection of genitalia and molecular analysis using mitochondrial DNA. Twelve slug species were identified, representing four different slug families. Internal nematodes were identified by means of morphological analysis and the sequencing of the 18S rRNA gene. Of the sample sites studied, 62.5% were found to be positive for nematode parasites, with 18.7% of all slugs discovered being infected. Five nematode species were identified in this study: Alloionema appendiculatum, Agfa flexilis, Angiostoma limacis, Angiostoma sp. and Phasmarhabditis hermaphrodita. Of these species, only one nematode was previously undescribed (Angiostoma sp.). This is the first record of the presence of A. appendiculatum, A. flexilis and A. limacis in Norway.

Abstract

Initial sources of inoculum of Phytophthora infestans were investigated in ten potato fields with early outbreaks of potato late blight. Infected plant samples and isolates from these fields were examined with respect to mating type prevalence, fungicide resistance and genotypes based on microsatellites A high proportion (91 %) of the isolates recovered were of mating type A1. However, both mating types were found in 3 of 9 fields with more than one isolate recovered, and sometimes both mating types were found on the same plant. Most of the isolates recovered from fields treated with metalaxyl-M prior to sampling had reduced sensitivity or were resistant to metalaxyl-M, and most of the isolates recovered form fields without metalaxyl treatment were sensitive. The isolates recovered from fields treated with propamocarb prior to sampling had a higher frequency of reduced sensitivity to propamocarb than isolates from fields without propamocarb treatment. We found that most plants contained more than one P. infestans SSR-genotype. Clustering analysis of the infected samples revealed that most samples clustered together according to fields. By combining information from P. infestans isolates and DNA extracts from the leaf lesions we found examples of both mating type A1 and A2 having the same multilocus genotype. This result indicates that both of these genotypes have a common ancestor, hence the inoculum originates from oospores. Although this a minor study of only 10 fields with a limited amount of isolates and plant samples, the results indicate oospores in the soil is an inoculum source. Hence the forecasting model to predict outbreaks of potato late blight should be modified to include this.

Abstract

In 2008, an epidemic caused by a new Neonectria sp. was discovered on white fir (Abies concolor) in several counties in southern Norway [1]. Later the pathogen was also found on other fir species in Norway and Denmark [2]. Typical symptoms and signs were dead shoots, flagging (dead branches), canker wounds, heavy resin flow, and occasionally red fruiting bodies (perithecia). Pathogenicity tests on several Abies spp. proved the fungus to be very aggressive, which corresponds well with observations of mortality of white fir and subalpine fir (A. lasiocarpa) from different age classes under field conditions. Sequencing of the internal transcribed regions (ITS) of the ribosomal DNA showed that this Neonectria sp. was most similar to N. ditissima (only 5 bp different from isolates in the GenBank), a common pathogen worldwide on broad leaf trees. The ITS sequences were very different (> 20 bp) from N. fuckeliana, a well-known fungus on Norway spruce in Scandinavia and other parts of the world, especially in the northern hemisphere. In 2011, the new Neonectria species was found on diseased trees in a Danish nordmann fir (Abies nordmanniana) seed orchard. Resin flow was seen from mature cones, and tests revealed that the seeds were infected by the Neonectria sp.

Abstract

In order to support functional genomics research in octoploid (Fragaria x ananassa Duch.) and diploid (F. vesca) strawberry, a customized Fragaria microarray chip was developed as a joint collaboration between Graminor Breeding Ltd. and NTNU. F. vesca cDNA sequences were provided by The Center for Genomics and Bioinformatics, Indiana University (an assembly of >3 million reads from GS-FLX Titanium - Roche/454 Life Sciences sequencing), and about 59,000 publicly available Fragaria EST sequences were uploaded from NCBI. In addition, ~190 Mb of preliminary draft genome sequences from F. vesca were provided by the Strawberry Genome Sequencing Consortium (courtesy to V. Shulaev). cDNAs used as templates for probe design were validated by BlastN against the F. vesca draft genome excluding cDNAs of microbial origin. Genes not represented in the cDNA collection were identified by screening F. vesca draft genome against protein sequences from Arabidopsis thaliana, Vitis vinifera, Ricinus communis and Populus trichocarpa. Exon sequences from genes not found in the cDNAs were included. In total, 43723 unique 60-mer probes were designed and the Agilent eARRAY tool was used to produce a 4x44k format microarray chip. Fragaria chip applicability and feasibility for transcriptional profiling was investigated using either abiotic (low temperature) or biotic (pathogenic fungi) stress treatment. Microarray data will be subsequently integrated with other omics data to address gene-regulatory networks and biological functions. Cold acclimation experiments were focused on short- and long-term effects in meristematic tissue, and revealed the up-regulation of ~100 cold-responsive genes (transcription factors, dehydrins, enzymes), and transcripts involved in starch breakdown and raffinose biosynthesis. Beside central metabolism, secondary metabolism was also strongly modulated as seen by changes in the expression of flavonoid biosynthesis-related genes. Time-course studies of transcriptional responses in F. vesca accessions showing contrasting resistance toward the pathogen Phytophthora cactorum are in progress, and will be presented in-depth.

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Abstract

Isolates of Colletotrichum sublineolum were collected from different sorghum-producing regions of Ethiopia and divided into five groups based on their geographic origin. The growth rate of 50 isolates showed considerable variation: 1·7?5·8 mm day?1, mean 3·3 mm day?1. However, the isolates displayed little variation in colony colour and colony margin, except for isolates from the north, which were different from the others. Amplified fragment length polymorphism analysis of 102 isolates revealed much greater variations among the different groups. Dice similarity coefficients ranged from 0·32 to 0·96 (mean 0·78). Cluster analysis and principal coordinate analysis revealed a differentiation of the isolates according to their geographic origin, and both methods clearly indicated a genetic separation between the southern, the eastern and the other isolates. Analysis of molecular variance (amova) indicated a high level of genetic variation both among (42%) and within (58%) the C. sublineolum sampling sites in Ethiopia. The amova also indicated a high level of genetic differentiation (FST = 0·42) and limited gene flow (Nm = 0·343). The results of this study confirmed the presence of a highly diverse pathogen, which is in agreement with the existence of diverse host genotypes and widely ranging environmental conditions in sorghum-producing regions of the country. Such diversity should be taken into account in future breeding programmes to achieve an effective and sustainable disease management strategy.

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Abstract

Conclusion: This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

Abstract

Microdochium nivale (syn. Microdochium nivale var. nivale) and Microdochium majus (syn. Microdochium nivale var. majus) are important pathogens which cause snow mould on grasses and winter cereals. These fungi are also able to cause leaf blotch of oat and seedling blight, foot rot and ear blight in cereals. Although no distinct differences in the host range of M. nivale and M. majus are found, indications for differences in host preferences between these fungal species have previously been discussed. The culture collection at Bioforsk contains about 250 Microdochium sp. isolated from grasses and cereals over the last 20 years. Most of the isolates collected from leaves of cereals displaying snow mould symptoms in spring, were identified as M. nivale (71 %), whereas most of the isolates collected from cereal seeds (mostly wheat) belong to the species M. majus (92 %). All, except one out of the sixty nine Microdochium sp. isolated from grass leaves were identified as M. nivale (99 %). The relatively higher incidence of M. majus vs. M. nivale on cereal seeds (mostly wheat) harvested in Norway, is in agreement with studies in UK (Parry et al. 1995). Parry et al. suggested that higher natural occurrence of M. majus (vs. M. nivale) on seeds of cereals could be partly due to the higher proportion of M. majus isolates producing perithecia and thus, a relatively higher amount of M. majus spores spreading to the ear (Parry et al. 1995). The high frequency of M. nivale (99 %) vs. M. majus on grasses collected in Norway could indicate that M. nivale is more aggressive on certain grass species. Studies in our lab indeed point towards a higher aggressiveness of M. nivale vs. M. majus on perennial ryegrass at low temperature (2?C) (Hofgaard et al 2006). However, the high incidence of M. nivale on grass leaves could also be caused by differences in temperature preferences, saprophytic ability or ability to infect certain plant parts. Isolates of M. nivale display a higher in vitro growth rate compared to isolates of M. majus at 2?C (Hofgaard et al. 2006). In conclusion, the higher natural occurrence of M. nivale vs. M. majus on turf grasses and the relatively higher aggressiveness of M. nivale on perennial ryegrass could indicate that M. nivale somehow is better adapted to infect certain grass species.

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Abstract

Many bacteria produce antimicrobial substances such as nonribosomally synthesized antibiotics and ribosomally synthesized proteinaceous compounds referred to as bacteriocins. Secretion of antimicrobials is generally thought to contribute to the competitiveness of the producing organism, but there are indications that these compounds in some cases may have regulatory roles too. Bacteriocins most often act on closely related species only and are thus of interest for application as targeted narrow-spectrum antimicrobials with few side effects. Although the application of bacteriocins in plant disease control is an attractive option, very little is known about the occurrence and roles of these compounds in plant pathogenic bacteria and their natural competitors occurring in the same biotopes. This study presents an overview of current knowledge of bacteriocins from plant pathogenic bacteria.

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Abstract

The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.

Abstract

We describe the cloning and characterization of a single copy gene from Trichoderma atroviride P1 encoding a novel 30 kDa chitinase, Ech30. Ech30 is a family 18 chitinase showing low sequence similarity to other Trichoderma chitinases. Real-time quantitative RT-PCR studies revealed that expression of the ech30 gene was induced by the presence of Botrytis cinerea in plate confrontation assays, but hardly by chitin in liquid cultures. Studies of Ech30 purified from an Escherichia coli strain overexpressing the ech30 gene devoid of the leader sequence and a predicted intron, showed that the gene encodes an active chitinase, which, as expected for family 18 chitinases, is inhibited by allosamidin.