Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.

Looking for relationships between the populations of Dothistroma septosporum in northern Europe and Asia.

Dothistroma septosporum, a notorious pine needle pathogen with an unknown historical geographic origin and poorly known distribution pathways, is nowadays found almost in all areas inhabited by pines (Pinus spp.). The main aim of this study was to determine the relationship between North European and East Asian populations. In total, 238 Eurasian D. septosporum isolates from 11 countries, including 211 isolates from northern Europe, 16 isolates from Russian Far East and 11 isolates from Bhutan were analysed using 11 species-specific microsatellite and mating type markers. The most diverse populations were found in northern Europe, including the Baltic countries, Finland and European Russia. Notably, D. septosporum has not caused heavy damage to P. sylvestris in northern Europe, which may suggest a long co-existence of the host and the pathogen. No indication was obtained that the Russian Far East or Bhutan could be the indigenous area of D. septosporum, as the genetic diversity of the fungus there was low and evidence suggests gene flow from northern Europe to Russian Far East. On the western coast of Norway, a unique genetic pattern was observed, which differed from haplotypes dominating other Fennoscandian populations. As an agent of dothistroma needle blight, only D. septosporum was documented in northern Europe and Asia, while D. pini was found in Ukraine and Serbia.

Chemical Content and Cytotoxic Activity on Various Cancer Cell Lines of Chaga (Inonotus obliquus) Growing on Betula pendula and Betula pubescens.

Chaga mushroom (Inonotus obliquus) is a pathogenic fungus that grows mostly on birch species (Betula pendula Roth and B. pubescens Ehrh.) and has traditionally been used as an anticancer medicine. This study aimed to compare the chemical composition and cytotoxic activity of chagas growing on both Betula spp. on various cancer cell lines. The freeze-dried extracts contained triterpenes inotodiol, lanosterol betulin, and betulinic acid typical to conks growing on Betula species. The cytotoxic activity of chaga growing on Betula pendula and B. pubescens 80% ethanolic extracts against 31 human cancer cell lines was evaluated by a sulforhodamine B assay. Chaga extract showed moderate activity against all cancer cell lines examined; it did not result in high cytotoxicity (IC50 ≤ 20 µg/mL). The strongest inhibitions were observed with chaga (growing on B. pendula) extract on the HepG2 and CAL-62 cell line and with chaga (from B. pubescens) extract on the HepG2 cell line, with IC50 values of 37.71, 43.30, and 49.99 µg/mL, respectively. The chaga extracts from B. pendula exert somewhat stronger effects on most cancer cell lines studied than B. pubescens extracts, which can be attributed to a higher content of inotodiol in B. pendula extracts. This study highlights the potential of chaga as a source of bioactive compounds with selective anticancer properties. To the best of our knowledge, this study is the first investigation of the chemical composition of I. obliquus parasitizing on B. pubescens.

Oomycete Soil Diversity Associated with Betula and Alnus in Forests and Urban Settings in the Nordic-Baltic Region.

This study aimed to determine the differences and drivers of oomycete diversity and community composition in alder- and birch-dominated park and natural forest soils of the Fennoscandian and Baltic countries of Estonia, Finland, Lithuania, Norway, and Sweden. For this, we sequenced libraries of PCR products generated from the DNA of 111 soil samples collected across a climate gradient using oomycete-specific primers on a PacBio high-throughput sequencing platform. We found that oomycete communities are most affected by temperature seasonality, annual mean temperature, and mean temperature of the warmest quarter. Differences in composition were partly explained by the higher diversity of Saprolegniales in Sweden and Norway, as both total oomycete and Saprolegniales richness decreased significantly at higher longitudes, potentially indicating the preference of this group of oomycetes for a more temperate maritime climate. None of the evaluated climatic variables significantly affected the richness of Pythiales or Peronosporales. Interestingly, the relative abundance and richness of Pythiales was higher at urban sites compared to forest sites, whereas the opposite was true for Saprolegniales. Additionally, this is the first report of Phytophthora gallica and P. plurivora in Estonia. Our results indicate that the composition of oomycetes in soils is strongly influenced by climatic factors, and, therefore, changes in climate conditions associated with global warming may have the potential to significantly alter the distribution range of these microbes, which comprise many important pathogens of plants.

Climate, host and geography shape insect and fungal communities of trees.

Non-native pests, climate change, and their interactions are likely to alter relationships between trees and tree-associated organisms with consequences for forest health. To understand and predict such changes, factors structuring tree-associated communities need to be determined. Here, we analysed the data consisting of records of insects and fungi collected from dormant twigs from 155 tree species at 51 botanical gardens or arboreta in 32 countries. Generalized dissimilarity models revealed similar relative importance of studied climatic, host-related and geographic factors on differences in tree-associated communities. Mean annual temperature, phylogenetic distance between hosts and geographic distance between locations were the major drivers of dissimilarities. The increasing importance of high temperatures on differences in studied communities indicate that climate change could affect tree-associated organisms directly and indirectly through host range shifts. Insect and fungal communities were more similar between closely related vs. distant hosts suggesting that host range shifts may facilitate the emergence of new pests. Moreover, dissimilarities among tree-associated communities increased with geographic distance indicating that human-mediated transport may serve as a pathway of the introductions of new pests. The results of this study highlight the need to limit the establishment of tree pests and increase the resilience of forest ecosystems to changes in climate.

Comparative Analyses of Bioactive Compounds in Inonotus obliquus Conks Growing on Alnus and Betula.

Inonotus obliquus grows in the Northern Hemisphere on some living broadleaved tree species as a pathogen, causing stem rot. In Estonia, the fungus is well known in the Betula species but can also be found on Alnus. Sterile conks of I. obliquus contain different bioactive compounds, but the quantitative and comparative research of these compounds in conks on different host species is limited. In the current work, I. obliquus was isolated and, evidently, determined from Alnus incana (L.) Moench., Alnus glutinosa (L.) Gaertn., and Betula pendula Roth, and the content of bioactive compounds in conks on these hosts were analysed. All the analysed conks sampled from A. incana and B. pendula contained betulin that varied from 111 to 159 µg/g. A significantly (p < 0.05) higher betulinic acid content was found in conks sampled from A. incana when compared with B. pendula: 474−635 and 20−132 µg/g, respectively. However, the conks from Betula were richer in total polyphenols, flavonols, and glucans. The content of inotodiol was quite similar in the conks from A. incana (7455−8961 µg/g) and B. pendula (7881−9057 µg/g). Also, no significant differences in the lanosterol content were found between the samples from these two tree species. To the best of our knowledge, this study is the first investigation of the chemical composition of I. obliquus parasitizing on Alnus. The results demonstrate that the bioactive compounds are promising in conks of I. obliquus growing not only on Betula but also on the Alnus species. It supports the opportunity to cultivate I. obliquus, also on the Alnus species, thus increasing the economic value of growing this tree species in forestry.

Diversity, migration routes, and worldwide population genetic structure of Lecanosticta acicola, the causal agent of brown spot needle blight.

Lecanosticta acicola is a pine needle pathogen causing brown spot needle blight that results in premature needle shedding with considerable damage described in North America, Europe, and Asia. Microsatellite and mating type markers were used to study the population genetics, migration history, and reproduction mode of the pathogen, based on a collection of 650 isolates from 27 countries and 26 hosts across the range of L. acicola. The presence of L. acicola in Georgia was confirmed in this study. Migration analyses indicate there have been several introduction events from North America into Europe. However, some of the source populations still appear to remain unknown. The populations in Croatia and western Asia appear to originate from genetically similar populations in North America. Intercontinental movement of the pathogen was reflected in an identical haplotype occurring on two continents, in North America (Canada) and Europe (Germany). Several shared haplotypes between European populations further suggests more local pathogen movement between countries. Moreover, migration analyses indicate that the populations in northern Europe originate from more established populations in central Europe. Overall, the highest genetic diversity was observed in south-eastern USA. In Europe, the highest diversity was observed in France, where the presence of both known pathogen lineages was recorded. Less than half of the observed populations contained mating types in equal proportions. Although there is evidence of some sexual reproduction taking place, the pathogen spreads predominantly asexually and through anthropogenic activity.

Worldwide diversity of endophytic fungi and insects associated with dormant tree twigs.

International trade in plants and climate change are two of the main factors causing damaging tree pests (i.e. fungi and insects) to spread into new areas. To mitigate these risks, a large-scale assessment of tree-associated fungi and insects is needed. We present records of endophytic fungi and insects in twigs of 17 angiosperm and gymnosperm genera, from 51 locations in 32 countries worldwide. Endophytic fungi were characterized by high-throughput sequencing of 352 samples from 145 tree species in 28 countries. Insects were reared from 227 samples of 109 tree species in 18 countries and sorted into taxonomic orders and feeding guilds. Herbivorous insects were grouped into morphospecies and were identified using molecular and morphological approaches. This dataset reveals the diversity of tree-associated taxa, as it contains 12,721 fungal Amplicon Sequence Variants and 208 herbivorous insect morphospecies, sampled across broad geographic and climatic gradients and for many tree species. This dataset will facilitate applied and fundamental studies on the distribution of fungal endophytes and insects in trees.

Seasonal Dynamics of Fungi Associated with Healthy and Diseased Pinus sylvestris Needles in Northern Europe.

The relationship between the ecological success of needle pathogens of forest trees and species richness of co-inhabiting endophytic fungi is poorly understood. One of the most dangerous foliar pathogens of pine is Dothistroma septosporum, which is a widely spread threat to northern European forests. We sampled two Pinus sylvestris sites in Estonia and two in Norway in order to analyse the relations between the abundance of D. septosporum and overall fungal richness, specific fungal species composition, time of season, needle age and position in the canopy. In both countries, the overall species richness of fungi was highest in autumn, showing a trend of increase with needle age. The overall species richness in the second-year needles in Estonia and third-year needles in Norway was similar, suggesting that a critical colonization threshold for needle shed in P. sylvestris is breached earlier in Estonia than in Norway. The fungal species richness in P. sylvestris needles was largely affected by Lophodermium conigenum. Especially in older needles, the relative abundance of L. conigenum was significantly higher in spring compared to summer or autumn. The timing of recruitment and colonization mechanisms of different foliage endophytes are shortly discussed.

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia.

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain.

Worldwide Genetic Structure Elucidates the Eurasian Origin and Invasion Pathways of Dothistroma septosporum, Causal Agent of Dothistroma Needle Blight.

Dothistroma septosporum, the primary causal agent of Dothistroma needle blight, is one of the most significant foliar pathogens of pine worldwide. Its wide host and environmental ranges have led to its global success as a pathogen and severe economic damage to pine forests in many regions. This comprehensive global population study elucidated the historical migration pathways of the pathogen to reveal the Eurasian origin of the fungus. When over 3800 isolates were examined, three major population clusters were revealed: North America, Western Europe, and Eastern Europe, with distinct subclusters in the highly diverse Eastern European cluster. Modeling of historical scenarios using approximate Bayesian computation revealed the North American cluster was derived from an ancestral population in Eurasia. The Northeastern European subcluster was shown to be ancestral to all other European clusters and subclusters. The Turkish subcluster diverged first, followed by the Central European subcluster, then the Western European cluster, which has subsequently spread to much of the Southern Hemisphere. All clusters and subclusters contained both mating-types of the fungus, indicating the potential for sexual reproduction, although asexual reproduction remained the primary mode of reproduction. The study strongly suggests the native range of D. septosporum to be in Eastern Europe (i.e., the Baltic and Western Russia) and Western Asia.

The Relationship between Fungal Diversity and Invasibility of a Foliar Niche-The Case of Ash Dieback.

European ash (Fraxinus excelsior) is threatened by the invasive ascomycete Hymenoscyphus fraxineus originating from Asia. Ash leaf tissues serve as a route for shoot infection but also as a sporulation substrate for this pathogen. Knowledge of the leaf niche partitioning by indigenous fungi and H. fraxineus is needed to understand the fungal community receptiveness to the invasion. We subjected DNA extracted from unwashed and washed leaflets of healthy and diseased European ash to PacBio sequencing of the fungal ITS1-5.8S-ITS2 rDNA region. Leaflets from co-inhabiting rowan trees (Sorbus aucuparia) served as a reference. The overlap in leaflet mycobiomes between ash and rowan was remarkably high, but unlike in rowan, in ash leaflets the sequence read proportion, and the qPCR-based DNA amount estimates of H. fraxineus increased vigorously towards autumn, concomitant with a significant decline in overall fungal richness. The niche of ash and rowan leaves was dominated by epiphytic propagules (Vishniacozyma yeasts, the dimorphic fungus Aureobasidion pullulans and the dematiaceous hyphomycete Cladosporium ramotenellum and H. fraxineus), and endophytic thalli of biotrophs (Phyllactinia and Taphrina species), the indigenous necrotroph Venturia fraxini and H. fraxineus. Mycobiome comparison between healthy and symptomatic European ash leaflets revealed no significant differences in relative abundance of H. fraxineus, but A. pullulans was more prevalent in symptomatic trees. The impacts of host specificity, spatiotemporal niche partitioning, species carbon utilization profiles and life cycle traits are discussed to understand the ecological success of H. fraxineus in Europe. Further, the inherent limitations of different experimental approaches in the profiling of foliicolous fungi are addressed.

Regional-Scale In-Depth Analysis of Soil Fungal Diversity Reveals Strong pH and Plant Species Effects in Northern Europe.

Soil microbiome has a pivotal role in ecosystem functioning, yet little is known about its build-up from local to regional scales. In a multi-year regional-scale survey involving 1251 plots and long-read third-generation sequencing, we found that soil pH has the strongest effect on the diversity of fungi and its multiple taxonomic and functional groups. The pH effects were typically unimodal, usually both direct and indirect through tree species, soil nutrients or mold abundance. Individual tree species, particularly Pinus sylvestris, Picea abies, and Populus x wettsteinii, and overall ectomycorrhizal plant proportion had relatively stronger effects on the diversity of biotrophic fungi than saprotrophic fungi. We found strong temporal sampling and investigator biases for the abundance of molds, but generally all spatial, temporal and microclimatic effects were weak. Richness of fungi and several functional groups was highest in woodlands and around ruins of buildings but lowest in bogs, with marked group-specific trends. In contrast to our expectations, diversity of soil fungi tended to be higher in forest island habitats potentially due to the edge effect, but fungal richness declined with island distance and in response to forest fragmentation. Virgin forests supported somewhat higher fungal diversity than old non-pristine forests, but there were no differences in richness between natural and anthropogenic habitats such as parks and coppiced gardens. Diversity of most fungal groups suffered from management of seminatural woodlands and parks and thinning of forests, but especially for forests the results depended on fungal group and time since partial harvesting. We conclude that the positive effects of tree diversity on overall fungal richness represent a combined niche effect of soil properties and intimate associations.

A look into the genetic diversity of Lecanosticta acicola in northern Europe.

For northern Europe Lecanosticta acicola is an emerging pine needle pathogen. This study gives a first look into the population genetics of the pathogen in Estonia, the first population documented in that region. The main aim of this study was to investigate the genetic diversity and population structure of the pathogen in this new region for the fungus. For this purpose, 104 isolates from 2010 to 2017 were analysed with 11 microsatellite and mating type markers. The stand where the pathogen's jump from an exotic host to the native Scots pine was recorded was also involved in this analysis. The analysis revealed low genetic diversity and a high number of clones that indicated L. acicola is an invasive species in northern Europe. Results suggest that several separate introductions have taken place and anthropogenic activity has apparently affected the spread of the pathogen. Clonal reproduction is dominating and although sexual reproduction is possible, it probably takes place infrequently.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Diplodia Tip Blight on Its Way to the North: Drivers of Disease Emergence in Northern Europe.

Disease emergence in northern and boreal forests has been mostly due to tree-pathogen encounters lacking a co-evolutionary past. However, outbreaks involving novel interactions of the host or the pathogen with the environment have been less well documented. Following an increase of records in Northern Europe, the first large outbreak of Diplodia sapinea on Pinus sylvestris was discovered in Sweden in 2016. By reconstructing the development of the epidemic, we found that the attacks started approx. 10 years back from several isolated trees in the stand and ended up affecting almost 90% of the trees in 2016. Limited damage was observed in other plantations in the surroundings of the affected stand, pointing to a new introduced pathogen as the cause of the outbreak. Nevertheless, no genetic differences based on SSR markers were found between isolates of the outbreak area and other Swedish isolates predating the outbreak or from other populations in Europe and Asia Minor. On a temporal scale, we saw that warm May and June temperatures were associated with higher damage and low tree growth, while cold and rainy conditions seemed to favor growth and deter disease. At a spatial scale, we saw that spread occurred predominantly in the SW aspect-area of the stand. Within that area and based on tree-ring and isotope (δ13C) analyses, we saw that disease occurred on trees that over the years had shown a lower water-use efficiency (WUE). Spore traps showed that highly infected trees were those producing the largest amount of inoculum. D. sapinea impaired latewood growth and reduced C reserves in needles and branches. D. sapinea attacks can cause serious economic damage by killing new shoots, disrupting the crown, and affecting the quality of stems. Our results show that D. sapinea has no limitations in becoming a serious pathogen in Northern Europe. Management should focus on reducing inoculum, especially since climate change may bring more favorable conditions for this pathogen. Seedlings for planting should be carefully inspected as D. sapinea may be present in a latent stage in asymptomatic tissues.