Fungi associated with Ips acuminatus (Coleoptera: Curculionidae) in Ukraine with a special emphasis on pathogenicity of ophiostomatoid species

Conifer bark beetles are well known to be associated with fungal complexes, which consist of pathogenic ophiostomatoid fungi as well as obligate saprotroph species. However, there is little information on fungi associated with Ips acuminatus in central and eastern Europe. The aim of the study was to investigate the composition of the fungal communities associated with the pine engraver beetle, I. acuminatus, in the forest-steppe zone in Ukraine and to evaluate the pathogenicity of six associated ophiostomatoid species by inoculating three-year-old Scots pine seedlings with these fungi. In total, 384 adult beetles were collected from under the bark of declining and dead Scots pine trees at two different sites. Fungal culturing from 192 beetles resulted in 447 cultures and direct sequencing of ITS rRNA from 192 beetles in 496 high-quality sequences. Identification of the above revealed that the overall fungal community was composed of 60 species. Among these, the most common were Entomocorticium sp. (24.5%), Diplodia pinea (24.0%), Ophiostoma ips (16.7%), Sydowia polyspora (15.1%), Graphilbum cf rectangulosporium (15.1%), Ophiostoma minus (13.8%) and Cladosporium pini-ponderosae (13.0%). Pathogenicity tests were done using six species of ophiostomatoid fungi, which were inoculated into Scots pine seedlings. All ophiostomatoid fungi tested successfully infected seedlings of Scots pine with varying degrees of virulence. Ophiostoma minus was the only fungus that caused dieback in inoculated seedlings. It is concluded that I. acuminatus vectors a species-rich fungal community including pathogens such as D. pinea and O. minus. The fungal community reported in the present study is different from that reported in other regions of Europe. Pathogenicity tests showed that O. minus was the most virulent causing dieback in seedlings of Scots pine, while other fungi tested appeared to be only slightly pathogenic or completely non-pathogenic

Fungal Communities Vectored by Ips sexdentatus in Declining Pinus sylvestris in Ukraine: Focus on Occurrence and Pathogenicity of Ophiostomatoid Species

Drought-induced stress and attacks by bark beetle Ips sexdentatus currently result in a massive dieback of Pinus sylvestris in eastern Ukraine. Limited and fragmented knowledge is available on fungi vectored by the beetle and their roles in tree dieback. The aim was to investigate the fungal community vectored by I. sexdentatus and to test the pathogenicity of potentially aggressive species to P. sylvestris. Analysis of the fungal community was accomplished by combining different methods using insect, plant, and fungal material. The material consisted of 576 beetles and 96 infested wood samples collected from six sample plots within a 300 km radius in eastern Ukraine and subjected to fungal isolations and (beetles only) direct sequencing of ITS rDNA. Pathogenicity tests were undertaken by artificially inoculating three-to-four-year-old pine saplings with fungi. For the vector test, pine logs were exposed to pre-inoculated beetles. In all, 56 fungal taxa were detected, 8 exclusively by isolation, and 13 exclusively by direct sequencing. Those included nine ophiostomatoids, five of which are newly reported as I. sexdentatus associates. Two ophiostomatoid fungi, which exhibited the highest pathogenicity, causing 100% dieback and mortality, represented genera Graphium and Leptographium. Exposure of logs to beetles resulted in ophiostomatoid infections. In conclusion, the study revealed numerous I. sexdentatus-vectored fungi, several of which include aggressive tree pathogens

Potential Westward Spread of Emerald Ash Borer, Agrilus planipennis Fairmaire, 1888 (Coleoptera: Buprestidae) from Eastern Ukraine

Emerald ash borer (EAB), Agrilus planipennis, is a phloem-boring beetle, native to East Asia that has become a serious invasive pest of ash (Fraxinus spp.) trees in North America and European Russia since the early 2000s. In 2019, EAB was detected in Ukraine. It had spread over 300 km from the entry point over two years and killed hundreds of Fraxinus excelsior and F. pennsylvanica trees. EAB poses a threat to the ash forests of neighboring European countries, which have already been damaged by the invasive fungus Hymenoscyphus fraxineus. The purpose of this research was (i) to reveal the traits of EAB and the climatic variables that affect its survival; (ii) to predict the EAB expansion range in Ukraine and westward; and (iii) to compare the most significant bioclimatic variables in the native, invasive ranges of EAB, as well as outside these ranges. The results demonstrated the following: (i) in all ranges, EAB has adapted to the seasonal temperature variations; (ii) the MaxEnt model predicted the potential distribution of EAB with high accuracy (AUC = 0.988); the predicted area of EAB invasion covered 87%, 48%, and 32% in Luhansk, Kharkiv, and Donetsk regions, respectively; and (iii) the ranges of climatic variables in EAB-inhabited regions demonstrated the high ecological plasticity of this pest. However, the predictions could be improved by considering forest structure, as well as the localization of roads

Identifying Fraxinus excelsior tolerant to ash dieback: Visual field monitoring versus a molecular marker

Ash dieback (ADB) caused by the pathogen Hymenoscyphus fraxineus is the cause of massive mortality of Fraxinus spp. in Europe. The aim of this work was to check for the presence of the molecular marker for ADB tolerance in mapped healthy-looking F. excelsior trees, and to compare its occurrence in trees exhibiting severe ADB symptoms. Monitoring of 135 healthy-looking F. excelsior on the island of Gotland, Sweden, showed that after 3-4 years 99.3% of these trees had 0%-10% crown damage, thus remaining in a similar health condition as when first mapped. After 5-6 years, 94.7% of these trees had 0%-10% crown damage. Molecular analysis of leaf tissues from 40 of those showed the presence of the molecular marker in 34 (85.0%) trees, while it was absent in 6 (15.0%) trees. Analysis of leaf tissues from 40 severely ADB-diseased trees showed the presence of the molecular marker in 17 (42.5%) trees, but its absence in 23 (57.5%) trees (p < .0001). The results demonstrated that monitoring of healthy-looking F. excelsior is a simple and straightforward approach for the selection of presumably ADB-tolerant ash for future breeding. The cDNA-based molecular marker revealed moderate capacity on its own to discriminate between presumably ADB-tolerant and susceptible F. excelsior genotypes

Comparative analyses of the Hymenoscyphus fraxineus and Hymenoscyphus albidus genomes reveals potentially adaptive differences in secondary metabolite and transposable element repertoires

Background The dieback epidemic decimating common ash (Fraxinus excelsior) in Europe is caused by the invasive fungus Hymenoscyphus fraxineus. In this study we analyzed the genomes of H. fraxineus and H. albidus, its native but, now essentially displaced, non-pathogenic sister species, and compared them with several other members of Helotiales. The focus of the analyses was to identify signals in the genome that may explain the rapid establishment of H. fraxineus and displacement of H. albidus. Results The genomes of H. fraxineus and H. albidus showed a high level of synteny and identity. The assembly of H. fraxineus is 13 Mb longer than that of H. albidus', most of this difference can be attributed to higher dispersed repeat content (i.e. transposable elements [TEs]) in H. fraxineus. In general, TE families in H. fraxineus showed more signals of repeat-induced point mutations (RIP) than in H. albidus, especially in Long-terminal repeat (LTR)/Copia and LTR/Gypsy elements. Comparing gene family expansions and 1:1 orthologs, relatively few genes show signs of positive selection between species. However, several of those did appeared to be associated with secondary metabolite genes families, including gene families containing two of the genes in the H. fraxineus-specific, hymenosetin biosynthetic gene cluster (BGC). Conclusion The genomes of H. fraxineus and H. albidus show a high degree of synteny, and are rich in both TEs and BGCs, but the genomic signatures also indicated that H. albidus may be less well equipped to adapt and maintain its ecological niche in a rapidly changing environment

Population structure and diversity of the needle pathogen Dothistroma pini suggests human-mediated movement in Europe

Dothistroma needle blight (DNB) is an important disease of Pinus species that can be caused by one of two distinct but closely related pathogens; Dothistroma septosporum and Dothistroma pini. Dothistroma septosporum has a wide geographic distribution and is relatively well-known. In contrast, D. pini is known only from the United States and Europe, and there is a distinct lack of knowledge regarding its population structure and genetic diversity. The recent development of 16 microsatellite markers for D. pini provided an opportunity to investigate the diversity, structure, and mode of reproduction for populations collected over a period of 12 years, on eight different hosts in Europe. In total, 345 isolates from Belgium, the Czech Republic, France, Hungary, Romania, Western Russia, Serbia, Slovakia, Slovenia, Spain, Switzerland, and Ukraine were screened using microsatellite and species-specific mating type markers. A total of 109 unique multilocus haplotypes were identified and structure analyses suggested that the populations are influenced by location rather than host species. Populations from France and Spain displayed the highest levels of genetic diversity followed by the population in Ukraine. Both mating types were detected in most countries, with the exception of Hungary, Russia and Slovenia. Evidence for sexual recombination was supported only in the population from Spain. The observed population structure and several shared haplotypes between non-bordering countries provides good evidence that the movement of D. pini in Europe has been strongly influenced by human activity in Europe

The increasing threat to European forests from the invasive foliar pine pathogen, Lecanosticta acicola

European forests are threatened by increasing numbers of invasive pests and pathogens. Over the past century, Lecanosticta acicola, a foliar pathogen predominantly of Pinus spp., has expanded its range globally, and is increasing in impact. Lecanosticta acicola causes brown spot needle blight, resulting in premature defoliation, reduced growth, and mortality in some hosts. Originating from southern regions of North American, it devastated forests in the USA's southern states in the early twentieth century, and in 1942 was discovered in Spain.Derived from Euphresco project 'Brownspotrisk,' this study aimed to establish the current distribution of Lecanosticta species, and assess the risks of L. acicola to European forests. Pathogen reports from the literature, and new/ unpublished survey data were combined into an open-access geo-database (http://www.portaloff orestpathology.com), and used to visualise the pathogen's range, infer its climatic tolerance, and update its host range. Lecanosticta species have now been recorded in 44 countries, mostly in the northern hemisphere. The type species, L. acicola, has increased its range in recent years, and is present in 24 out of the 26 European countries where data were available. Other species of Lecanosticta are largely restricted to Mexico and Central America, and recently Colombia.The geo-database records demonstrate that L. acicola tolerates a wide range of climates across the northern hemisphere, and indicate its potential to colonise Pinus spp. forests across large swathes of the Europe. Pre-liminary analyses suggest L. acicola could affect 62% of global Pinus species area by the end of this century, under climate change predictions.Although its host range appears slightly narrower than the similar Dothistroma species, Lecanosticta species were recorded on 70 host taxa, mostly Pinus spp., but including, Cedrus and Picea spp. Twenty-three, including species of critical ecological, environmental and economic significance in Europe, are highly susceptible to L. acicola, suffering heavy defoliation and sometimes mortality. Variation in apparent susceptibility between reports could reflect variation between regions in the hosts' genetic make-up, but could also reflect the signif-icant variation in L. acicola populations and lineages found across Europe. This study served to highlight sig-nificant gaps in our understanding of the pathogen's behaviour.Lecanosticta acicola has recently been downgraded from an A1 quarantine pest to a regulated non quarantine pathogen, and is now widely distributed across Europe. With a need to consider disease management, this study also explored global BSNB strategies, and used Case Studies to summarise the tactics employed to date in Europe

Forewarned is forearmed : harmonized approaches for early detection of potentially invasive pests and pathogens in sentinel plantings

This work was supported by COST Action Global Warning (FP1401). DLM and YB contribution was also supported by the Russian Foundation for Basic Research (Grant No. 17-04-01486). MG was supported by Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant III43002. MKA was supported by the Ministry of Science and Higher Education of the Republic of Poland. NK was supported by Le Studium foundation (France) and RFBR (Grant No. 19-04-01029). RE, IF and MK contribution was also supported by CABI with core financial support from its member countries (see http://www.cabi.org/about-cabi/who-we-work-with/key-donors/ for details). IF contribution was further supported through a grant from the Swiss State Secretariat for Science, Education and Research (Grant C15.0081, awarded to RE).Peer reviewedPublisher PD

Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Funding: This study was financially supported by COST Action FP1406 (PINESTRENGTH), the Estonian Science Foundation grant PSG136, the Forestry Commission, United Kingdom, the Phytophthora Research Centre Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000453, a project co-financed by the European Regional Development Fund. ANSES is supported by a grant managed by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” programme (ANR-11-LABX-0002-01, Laboratory of ExcellenceARBRE). SW was partly supported by BBSRC Grant reference BB/L012251/1 “Promoting resilience of UK tree species to novel pests & pathogens: ecological & evolutionary solutions (PROTREE)” jointly funded by BBSRC, Defra, ESRC, the Forestry Commission, NERC and the Scottish Government, under the Tree Health and Plant Biosecurity Initiative. Annual surveys in Switzerland were financially supported by the Swiss Federal Office for the Environment FOEN. Acknowledgments: Andrea Kunova and Cristina Pizzatti are acknowledged for the assistance in the sampling. Thanks are due to Dina Ribeiro and Helena Marques from ICNF-Portuguese Forest Authority for providing location coordinates. We thank three anonymous reviwers for valuable corrections and suggestions.Peer reviewedPublisher PD