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Graphium species form a well-supported monophyletic lineage within the Microascales (Ascomycota). Members of this genus can be found in association with bark beetles, as well as on tree wounds and in soils. During surveys of bark and ambrosia beetle-associated fungi and cavities made by woodpeckers on hardwood trees in Poland, many isolates with an affinity to Graphium were recovered. They were identified based on their morphological characters and sequence data for the internal transcribed spacer (ITS), 28S rDNA, β-tubulin ( TUB2 ), and translation elongation factor 1-α ( TEF1 ) gene regions. The results revealed five new species, described here as G. brachiatum, G. longistipitatum, G. polonicum, G. radicatum , and G. trypophloei .

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

High biodiversity is regarded as a barrier against biological invasions. We hypothesized that the invasion success of the pathogenic ascomycete Hymenoscyphus fraxineus threatening common ash in Europe relates to differences in dispersal and colonization success between the invader and the diverse native competitors. Ash leaf mycobiome was monitored by high-throughput sequencing of the fungal internal transcribed spacer region (ITS) and quantitative PCR profiling of H. fraxineus DNA. Initiation of ascospore production by H. fraxineus after overwintering was followed by pathogen accumulation in asymptomatic leaves. The induction of necrotic leaf lesions coincided with escalation of H. fraxineus DNA levels and changes in proportion of biotrophs, followed by an increase of ubiquitous endophytes with pathogenic potential. H. fraxineus uses high propagule pressure to establish in leaves as quiescent thalli that switch to pathogenic mode once these thalli reach a certain threshold - the massive feedback from the saprophytic phase enables this fungus to challenge host defenses and the resident competitors in mid-season when their density in host tissues is still low. Despite the general correspondence between the ITS-1 and ITS-2 datasets, marker biases were observed, which suggests that multiple barcodes provide better overall representation of mycobiomes., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Background: NB-LRR resistance proteins are involved in recognizing pathogens and other exogenous stressors in plants. Resistance proteins are the first step in induced defence responses and a better understanding of their regulation is important to understand the mechanisms of plant defence. Much of the post-transcriptional regulation in plants is controlled by microRNAs (miRNA). We examined the expression of five Norway spruce miRNA that may regulate NB-LRR related transcripts in secondary phloem (bark) of resistant Norway spruce after wounding and inoculation with the necrotrophic blue stain fungus Ceratocystis polonica., Results: The plants of this clone recovered from both the pathogen inoculations and wounding alone. We found local and systemic induction of the resistance marker genes PaChi4, PaPAL and PaPX3 indicative of an effective induced host defence response. There were minor local and systemic changes in the expression of five miRNAs and 21 NB-LRRs between healthy and treated plants. Only five putative NB-LRRs (PaLRR1, PaLRR3, PaLRR14, PaLRR15 and PaLRR16) showed significant increases greater than two-fold as a local response to C. polonica. Of all NB-LRRs only PaLRR3, the most highly differentially regulated NB-LRR, showed a significant increase also due to wounding. The five miRNAs showed indications of an initial local and systemic down-regulation at day 1, followed by a later increase up to and beyond the constitutive levels at day 6. However, the initial down-regulation was significant only for miR3693 and miR3705., Conclusions: Overall, local and systemic expression changes were evident only for the established resistance marker genes and PaLRR3. The minor expression changes observed both for the followed miRNAs and their predicted NB-LRR targets suggest that the expression of most NB-LRR genes are maintained close to their constitutive levels in stressed and healthy Norway spruce plants.

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce (Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC-MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-alpha-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (-)-beta-pinene and (-)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (-)-alpha-pinene and (-)-limonene 1 month after treatment, whereas MJ only increased that of (-)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (-)-beta-pinene, (-)-beta-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (-)-alpha-pinene was significantly higher and the relative proportions of (-)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica., (Copyright 2010. Published by Elsevier Ltd.)

The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nursery-grown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.

To identify chemical resistant markers induced by fungal or mechanical injury, young trees of Scots pine (Pinus sylvestris) were subjected to inoculations of blue stain fungi associated with the pine shoot beetles Tomicus piniperda and T. minor. Among the 20 trees selected for chemical analyses, 16 were divided into four groups: one as control and three were pretreated by wounding only, or by inoculation with either the blue stain fungus Leptographium wingfieldii or Ophiostoma canum. Four wk after pretreatment, all 16 pretreated trees were mass-inoculated with L. wingfieldii. The absolute and relative amounts, as well as the enantiomeric compositions of monoterpene hydrocarbons in the phloem, were determined via a small sample of the phloem before and after the pretreatment and mass inoculation, by using two-dimensional gas chromatography (2D GC) and GC-mass spectrometry (MS). After mass inoculation, the absolute amounts of most of the monoterpenes decreased in the phloem sampled >20 cm from the fungal infection, and were higher in the phloem sampled within the infected reaction zone. The relative amounts of both (-)-beta-pinene and (-)-limonene increased in phloem samples taken >20 cm above the fungal inoculation in the preinoculated trees compared with phloem sampled from the remaining four control trees. The enantiomeric compositions of beta-pinene and limonene changed, after fungal growth, at defined distances from the inoculation site: the proportion of the (-)-enantiomers was highest in the phloem sampled >20 cm from the fungal inoculation. Four wk after pretreatment, monoterpene production in the phloem at the site of inoculation was more enhanced by L. wingfieldii than by O. canum. However, the different virulence levels of the fungi did not affect the enantiomeric composition of the monoterpenes. The biosynthesis of monoterpene enantiomers is discussed in relation to induced pathogen resistance.

We investigated the cellular responses of stem tissues of mature Scots pine (Pinus sylvestris L.) trees to inoculations with two fungal pathogens. The bark beetle vectored fungus, Leptographium wingfieldii Morelet, induced longer lesions in the bark, stronger swelling of polyphenolic parenchyma cells, more polyphenol accumulation and increased ray parenchyma activity compared with the root rot fungus, Heterobasidion annosum (Fr.) Bref., or mechanical wounding. Axial resin ducts in the xylem are a general feature of the preformed defenses of Scots pine, but there was no clear induction of additional traumatic axial resin ducts in response to wounding or fungal infection. The anatomical responses of Scots pine to pathogen infection were localized to the infection site and were attenuated away from bark lesions. The responses observed in Scots pine were compared with published studies on Norway spruce (Picea abies (L.) Karst.) for which anatomically based defense responses have been well characterized.

The genus Leptographium was described in 1927 and currently includes 48 species, with L. lundbergii as the type species. In recent years, the taxonomic status of L. lundbergii has not been uniformly agreed upon and it has been the topic of considerable debate. The problem was compounded by the absence of a type specimen, and the species was epitypified at a later stage. Unfortunately, the whereabouts of the epitype is now unknown. In 1983, Wingfield & Marasas described L. truncatum, which is morphologically similar to L. lundbergii. Based on DNA comparisons and similarities in their morphology, this fungus was reduced to synonymy with L. lundbergii. The loss of the type specimen as well as variation in the morphology of strains identified as L. lundbergii prompted us to re-examine the taxonomic status of this species. A number of strains from various geographic areas were studied. These include a strain of L. lundbergii deposited at CBS by Melin in 1929 (CBS 352.29) as well as the ex-type strain of L. truncatum. The strains were compared based on morphology and comparison of multiple gene sequences. Three genes or genic regions, ITS2 and part of the 28S gene, partial beta-tubulin and partial elongation factor 1-alpha were compared. Strains currently identified as L. lundbergii, represented a complex of species. Strains initially described as L. truncatum clustered separately from other L. lundbergii strains, could be distinguished morphologically and should be treated as a distinct taxon. L. lundbergii is provided with a new and expanded description based on a neotype designated for it. A third group was also identified as separate from the main L. lundbergii clade and had a distinct Hyalorhinocladiella-type anamorph, described here as H. pinicola sp. nov.

Genetic variation in three multiallelic loci was analysed with Temperature Gradient Gel Electrophoresis in order to assess the genetic population structure of Venturia tremulae var. tremulae in order to understand the evolutionary potential of the pathogen against resistance breeding. Also the identification of the fungus was verified with molecular analysis of reference isolates. The Fst and Gst values were very low indicating no substructuring or restrictions to gene flow between Fennoscandian populations of V. tremulae. The results imply high epidemiological efficiency of the fungus and therefore continuous breeding programme should be implemented for Venturia resistance of aspen.

Norway spruce (Picea abies (L.) Karst.) trees were preinoculated with a sublethal dose of the blue-stain fungus Ceratocystis polonica Siem. (C. Moreau) 1 to 52 weeks before they were mass inoculated with the same fungus. Trees pretreated 1 week before mass inoculation had similar, severe symptoms of fungal infection as the control trees. Pretreatment 3, 6 or 9 weeks before mass inoculation resulted in effective protection of the trees, reducing pathogenic symptoms by 63-90% relative to the control trees, whereas pretreatment 52 weeks before mass inoculation gave intermediate protection (44-71% reduction in symptoms). Thus, pretreatment induced resistance to the blue-stain fungus in Norway spruce by a process that requires more than 1 week to become activated and protects trees for at least one year after pretreatment. Pretreatment induced formation of traumatic resin ducts (TDs) in the sapwood and swelling and proliferation of polyphenolic parenchyma cells (PP cells) in the phloem. Trees pretreated 3-9 weeks before mass inoculation had more TDs and showed greater swelling of existing PP cells than control trees or trees pretreated 1 week before mass inoculation. We conclude that induced disease resistance in Norway spruce is probably associated with PP cell activation and TD induction, because resistance was enhanced within the same time frame as the induction of these defense responses.