Further pathogenicity testing of Verticillium nonalfalfae, a biocontrol agent against the invasive Tree of Heaven (Ailanthus altissima), on non-target tree species in Europe.

Verticillium nonalfalfae is an effective biological control agent against the highly invasive Tree of Heaven (Ailanthus altissima) in Europe, and previous research on ten economically and ecologically important tree species occurring in Austria have so far not revealed undesired non-target effects. In this study, another nine tree species including five native, two non-native as well as two invasive alien tree species were tested for susceptibility to the particular strain of V. nonalfalfae (Vert56) used for biological control of A. altissima. Stem inoculations on potted seedlings revealed that this strain of V. nonalfalfae is generally host-adapted to A. altissima. It induced chlorosis, necrosis and wilting already within two weeks post inoculation on A. altissima and resulted in almost completely defoliated or dead seedlings at the end of the vegetation period. Apart from two species (Quercus rubra and Sorbus aucuparia), that suffered from other abiotic/biotic agents, no mortality was observed on all other tree species tested; however, symptoms caused by other abiotic factors were also found on Prunus avium and Ulmus glabra. All tested tree species exhibited vascular discolorations and the fungus could be re-isolated at varying frequencies (6-100%) from inoculated seedlings of all non-target tree species, although five of these species exhibited no external symptoms. Results confirmed high susceptibility (S) of A. altissima to V. nonalfalfae, whereas Acer platanoides, Castanea sativa, Q. rubra, S. aucuparia and U. glabra were considered as tolerant (T), and A. negundo, P. avium, P. serotina and Q. petraea were rated as possible resistant (PR) due to the low rates of re-isolation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12600-022-01032-z.

Advanced spectroscopy-based phenotyping offers a potential solution to the ash dieback epidemic.

Natural and urban forests worldwide are increasingly threatened by global change resulting from human-mediated factors, including invasions by lethal exotic pathogens. Ash dieback (ADB), incited by the alien invasive fungus Hymenoscyphus fraxineus, has caused large-scale population decline of European ash (Fraxinus excelsior) across Europe, and is threatening to functionally extirpate this tree species. Genetically controlled host resistance is a key element to ensure European ash survival and to restore this keystone species where it has been decimated. We know that a low proportion of the natural population of European ash expresses heritable, quantitative resistance that is stable across environments. To exploit this resource for breeding and restoration efforts, tools that allow for effective and efficient, rapid identification and deployment of superior genotypes are now sorely needed. Here we show that Fourier-transform infrared (FT-IR) spectroscopy of phenolic extracts from uninfected bark tissue, coupled with a model based on soft independent modelling of class analogy (SIMCA), can robustly discriminate between ADB-resistant and susceptible European ash. The model was validated with populations of European ash grown across six European countries. Our work demonstrates that this approach can efficiently advance the effort to save such fundamental forest resource in Europe and elsewhere.

The Ophiostoma clavatum species complex: a newly defined group in the Ophiostomatales including three novel taxa.

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, ßT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

Population structure and diversity of an invasive pine needle pathogen reflects anthropogenic activity.

Dothistroma septosporum is a haploid fungal pathogen that causes a serious needle blight disease of pines, particularly as an invasive alien species on Pinus radiata in the Southern Hemisphere. During the course of the last two decades, the pathogen has also incited unexpected epidemics on native and non-native pine hosts in the Northern Hemisphere. Although the biology and ecology of the pathogen has been well documented, there is a distinct lack of knowledge regarding its movement or genetic diversity in many of the countries where it is found. In this study we determined the global population diversity and structure of 458 isolates of D. septosporum from 14 countries on six continents using microsatellite markers. Populations of the pathogen in the Northern Hemisphere, where pines are native, displayed high genetic diversities and included both mating types. Most of the populations from Europe showed evidence for random mating, little population differentiation and gene flow between countries. Populations in North America (USA) and Asia (Bhutan) were genetically distinct but migration between these continents and Europe was evident. In the Southern Hemisphere, the population structure and diversity of D. septosporum reflected the anthropogenic history of the introduction and establishment of plantation forestry, particularly with Pinus radiata. Three introductory lineages in the Southern Hemisphere were observed. Countries in Africa, that have had the longest history of pine introductions, displayed the greatest diversity in the pathogen population, indicating multiple introductions. More recent introductions have occurred separately in South America and Australasia where the pathogen population is currently reproducing clonally due to the presence of only one mating type.

Do mites phoretic on elm bark beetles contribute to the transmission of Dutch elm disease?

Dutch elm disease (DED) is a destructive vascular wilt disease of elm (Ulmus) trees caused by the introduced Ascomycete fungus Ophiostoma novo-ulmi. In Europe, this DED pathogen is transmitted by elm bark beetles in the genus Scolytus. These insects carry phoretic mites to new, suitable habitats. The aim of this study was to record and quantify conidia and ascospores of O. novo-ulmi on phoretic mites on the three elm bark beetle species Scolytus multistriatus, Scolytus pygmaeus, and Scolytus scolytus. Spores of O. novo-ulmi were found on four of the ten mite species phoretic on Scolytus spp. These included Elattoma fraxini, Proctolaelaps scolyti, Pseudotarsonemoides eccoptogasteri, and Tarsonemus crassus. All four species had spores attached externally to their body surfaces. However, T. crassus carried most spores within its sporothecae, two paired pocket-like structures adapted for fungal transmission. Individuals of Pr. scolyti also had O. novo-ulmi conidia and ascospores frequently in their digestive system, where they may remain viable. While E. fraxini and P. eccoptogasteri rarely had spores attached to their bodies, large portions of Pr. scolyti and T. crassus carried significant numbers of conidia and/or ascospores of O. novo-ulmi. P. scolyti and T. crassus, which likely are fungivores, may thus contribute to the transmission of O. novo-ulmi, by increasing the spore loads of individual Scolytus beetles during their maturation feeding on twigs of healthy elm trees, enhancing the chance for successful infection with the pathogen. Only S. scolytus, which is the most efficient vector of O. novo-ulmi in Europe, carried high numbers of Pr. scolyti and T. crassus, in contrast to S. multistriatus and S. pygmaeus, which are known as less efficient vectors. The high efficiency of S. scolytus in spreading Dutch elm disease may be partly due to its association with these two mites and the hyperphoretic spores of O. novo-ulmi they carry.

Phenotypic and DNA sequence data comparisons reveal three discrete species in the Ceratocystis polonica species complex.

Ceratocystis polonica and C. laricicola are two morphologically similar species that occur on conifers and reside in the Ceratocystis coerulescens species complex. They, however, represent two ecologically distinct entities. C. polonica causes blue stain on Norway spruce (Picea abies) and other spruce species (Picea spp.) in Eurasia and is associated with the bark beetles Ips typographus, I. typographus japonicus, I. amitinus and I. duplicatus. In contrast, C. laricicola lives in a symbiotic relationship with the bark beetles Ips cembrae and I. subelongatus that infest various larch species (Larix spp.). The objective of this study was to consider the phylogenetic relationships of C. polonica and C. laricicola and more specifically to determine the identity of Japanese isolates from both spruce and larch, based on sequences derived from the ITS regions of the rRNA operon, the beta-tubulin gene and the HMG box of the MAT-2 gene. Isolates were also compared based on morphology and cultural characteristics. Comparisons of anamorph and teleomorph structures confirmed that C. polonica and C. laricicola are indistinguishable based on morphology. Both species had an optimal growth temperature of 25 degrees C. However, at temperatures between 31-33 degrees, C. polonica isolates grew slowly or not at all, while C. laricicola isolates grew more actively at these temperatures. Thus, a growth test at 32 degrees can differentiate these species. Phylograms generated using parsimony for the three gene regions were strongly congruent. These showed three distinct clades supported by high bootstrap values. Two of the clades clearly separate C. laricicola from Europe and C. polonica, supporting the view that they represent two discrete taxa. A third clade included isolates obtained from galleries of Ips subelongatus on Larix kaempferi in Japan. This fungus clearly represents a discrete taxon that is closely related to, but distinct from C. laricicola, which is described here as C. fujiensis sp. nov.

Ophiostoma dentifundum sp. nov. from oak in Europe, characterized using molecular phylogenetic data and morphology.

Previous phylogenetic studies based on ITS sequence data have shown that Ophiostoma species with Sporothrix anamorphs include several species complexes. Isolates from oak in Poland and Hungary, which have previously been referred to as O. stenoceras, as well as isolates morphologically similar to S. inflata formed the basis of this study. Identification was based on sequences for the ITS region of rDNA operon and partial beta-tubulin gene. Analyses showed that isolates from Poland and Hungary reside in a well resolved clade, separate from those in the O. stenoceras-complex. The morphology of these isolates was compared with those of strains in the O. stenoceras complex and S. inflata. Morphological differences in teleomorph and anamorph structures were found between the isolates from Poland and Hungary and those in the O. stenoceras-complex. Growth characteristics and the presence of the teleomorph in culture could be used to separate this fungus from isolates in the S. inflata-complex. The fungus from Poland and Hungary is described here as O. dentifundum sp. nov. It is phylogenetically most closely related to isolates of S. inflata, which represent four well defined groups based on morphology and DNA sequence phylogeny.

Two new Ophiostoma species with Sporothrix anamorphs from Austria and Azerbaijan.

The genus Ophiostoma includes numerous species of primarily insect-vectored, wood-staining fungi. Several anamorph genera that differ in their micronematous or macronematous conidiogenous cells have been associated with Ophiostoma species. Among the former group, Sporothrix is associated with many species and is characterized by conidiogenous cells that arise laterally or terminally from any place on the hyphae and produce nonseptate conidia on sympodially developing denticles. The purpose of this study was to characterize ophiostomatoid isolates with Sporothrix anamorphs recently collected in Austria and Azerbaijan. The isolates were characterized based on comparisons of rDNA and ß-tubulin sequence data. Morphology, growth in culture, and sexual reproductive mode were also considered. Phylogenetic analyses of the combined sequence data showed that the isolates formed two distinct groups, one including isolates from Austria and the other isolates from Austria and Azerbaijan. Growth at 25 C and morphology revealed some differences between the two groups, and supported the view that they represent two new species, which we describe here as Ophiostoma fusiforme sp. nov. and Ophiostoma lunatum sp. nov. Both these groups phylogenetically were related to, but distinct from, Ophiostoma stenoceras.

Epitypification of Ophiostoma galeiforme and phylogeny of species in the O. galeiforme complex.

Ophiostoma galeiforme was described first in 1951 from Larix kaempferi in Scotland, where it was found to be associated with the bark beetles Hy-lurgops palliatus, Dryocoetes autographus, and the ambrosia beetle Trypodendron lineatum. The taxonomy of this fungus has been uncertain because of a lack of sexual structures on the type specimen and contamination of a preserved ex-type culture. The aim of this study was to clarify application of the species name, O. galeiforme, by designating an epitype and to consider phylogenetic relationships of the species. Nineteen isolates resembling O. galeiforme from different parts of the world were used, including collections from Pinus sylvestris infested with Tomicus pi-niperda in Scotland and the contaminated ex-type culture. Morphological characteristics of isolates from Sweden, South Africa, Scotland, Chile and Aus-tria corresponded well with those originally described for O. galeiforme, and an isolate from Scotland is designated as the epitype. A detailed description is provided. Results of interfertility tests showed that O. galeiforme is heterothallic. Analysis of ITS rDNA sequences showed that the isolates representing O. galeiforme were distinct from three morphologically similar isolates from the USA and Mexico, which probably represent an undescribed taxon.

Ophiostoma kryptum sp. nov. from Larix decidua and Picea abies in Europe, similar to O. minus.

An unknown species of Ophiostoma was isolated from European larch (Larix decidua) infested by Tetropium gabrieli (Coleoptera: Cerambycidae) and Norway spruce (Picea abies) infested by Tetropium sp. in Austria. The fungus is similar to O. minus, but distinguished from it by the ecology, colony morphologies on OA and MEA, and phylogenetic analysis of aligned DNA sequences of the ITS region of the rDNA operon and the partial beta-tubulin gene. It is described here as O. kryptum sp. nov. The new species readily produces perithecia with short necks and reniform ascospores, and has Hyalorhinocladiella and Leptographium-like anamorphs. Circumstantial evidence suggests that Tetropium spp. act as vectors of O. kryptum. O. minus and O. kryptum represent additional examples of morphologically similar, yet genetically and ecologically distinct species in the genus Ophiostoma. The new combination, O. crenulatum comb. nov. (syn. CeratocYstiopsis crenulata), is also made.

Taxonomic re-evaluation of three related species of Graphium, based on morphology, ecology and phylogeny.

Two fungi associated with bark beetles, Graphium pseudormiticum (described in 1994) and Rhexographium fimbriisporum (described in 1995), have two micromorphological characters in common. Both species produce conidia with conspicuous basal frills, and the conidia align in chains, despite being produced in slime. The association of G. pseudormiticum with the pine bark beetle, Orthotomicus erosus, and the association of R. fimbriisporum with the spruce bark beetle, Ips typographus, suggest ecological differences between the two fungal species. Analyses of micromorphology and phylogenetic analyses of aligned 18S and ITS sequences suggest that these two species are congeneric and should be classified in Graphium but that they represent distinct species. A collection of strains tentatively identified as Graphium spp., isolated from Ips typographus on Picea abies, Ips cembrae on Larix decidua and Tomicus minor on Pinus sylvestris in Austria share the same unusual basal conidial frills and conidial chains. Isolates from spruce were identified as G. fimbriisporum and those from pine as G. pseudormiticum. The strains from Ips cembrae on Larix decidua, distinguished by the reddish color of their colonies, microscopic structures and molecular characteristics, are described as the new species Graphium laricis sp. nov., and the close relationship of this species with the other two species is confirmed.