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Diplodia sapinea (Fr.) Fuckel is a widespread fungal pathogen affecting conifers worldwide. Infections can lead to severe symptoms, such as shoot blight, canker, tree death, or blue stain in harvested wood, especially in Pinus species. Its impact on forest health is currently intensified, likely due to climate change, posing an increasing threat to global ecosystems and forestry. Despite extensive and successful research on this pathogen system, fundamental questions about its biology and plant-associated lifestyle remain unanswered. Addressing these questions will necessitate the development of additional experimental tools, including protocols for molecular genetics and cell biology approaches. In this study, we continue to address this need by establishing an Agrobacterium-mediated genetic transformation protocol for D. sapinea, enabling targeted mutagenesis and heterologous gene expression. We utilized this methodology to localize the histone H2B by tagging it with the fluorescent protein mCherry. Additionally, we established a time- and space-efficient laboratory-scale infection assay using two-week-old Pinus sylvestris seedlings. Integrating these tools in a proof-of-concept study enabled the visualization of D. sapinea in planta growth through the fluorescently labeled reporter strain.

Scots pine (Pinus sylvestris) is one of the main sources of timber in the boreal zone of Eurasia. Commercial pine plantations are vulnerable to root and butt rot disease caused by the fungus Heterobasidion annosum. The pathogen affects host growth rate, causes higher mortality and decreases in timber quality, resulting in considerable economic losses to forest owners. Genetic and biochemical factors contributing to Scots pine tolerance against H. annosum infection are not well understood. We assessed the predictive values of a set of potential genetic and chemical markers in a field experiment. We determined the expression levels of 25 genes and the concentrations of 36 terpenoid compounds in needles of 16 Scots pine trees randomly selected from a natural population prior to artificial infection. Stems of the same trees were artificially inoculated with H. annosum, and the length of necrotic lesions was documented 5 months post inoculation. Higher expression level of four genes included in our analysis and encoding predicted a-pinene synthase (two genes), geranyl diphosphate synthase (GPPS), and metacaspase 5 (MC5), could be associated with trees exhibiting increased levels of necrotic lesion formation in response to fungal inoculation. In contrast, concentrations of two terpenoid compounds, β-caryophyllene and α-humulene, showed significant negative correlations with the lesion size. Further studies with larger sample size will help to elucidate new biomarkers or clarify the potential of the evaluated markers for use in Scots pine disease resistance breeding programs.

Continuity in forest habitats is crucial for species diversity and richness. Ancient Scots pine forests are usually under forest management, which disturbs vegetation and causes differentiation in terms of tree stand age. To date, vegetation variability in ancient Scots pine forests has not been examined based on tree stand age classes. In the present study the continuity of a large Scots pine forest complex was investigated, and a system of sampling plots established in five tree stand age classes: initiation stands (4-10 years), young stands (20-35 years), middle-aged stands (45-60 years), pre-mature stands (70-85 years) and mature stands (95-110 years). Species composition, including vascular plants, bryophytes and lichens, on soil, tree trunks, and coarse woody debris, was analyzed. Based on existing classifications systems, forest species and ancient forest species groups were distinguished. In the studied ancient Scots pine forests the species pool and richness were relatively low, and the vegetation consisted mostly of generalist species. Cryptogams, which can grow on diverse substrates, were the most abundant species. Moreover, most species could tolerate both forest and non-forest conditions. Age class forests provided different environmental niches for species. Initiation stands were optimal for terrestrial light-demanding species, and in terms of species composition, initiation stands were most specific. Young stands were most preferred by species on coarse woody debris, and at this stage of stand maturation epiphytic species re-appeared. The oldest stands were not rich in forest specialists, i.e. species of closed forest and ancient forest species. Cryptogams of closed forests inhabited different substrates, and they were not associated only with the oldest stands. The low number of forest specialists in the oldest stands may be a general feature of acidophilus pine forests. However, it may also be a result of the lack of species sources in the vicinity of maturing pine stands. In managed forests a frequent diversity pattern is an increase in a species pool and richness after clear-cut logging. In the present study we obtained higher species pools in initiation and young stands, but richness was similar in all tree stand age classes. This resulted from taking into account species of different substrates (terrestrial, epixylous and epiphytic species) which changed their participation in the vegetation of subsequent stages of tree stand development.

This study aimed to elucidate the relationship between ericaceous understorey shrubs and the diversity and abundance of ectomycorrhizal fungi (EcMF) associated with the invasive Pinus strobus and native Pinus sylvestris . Seedlings of both pines were grown in mesocosms and subjected to three treatments simulating different forest microhabitats: (a) grown in isolation and grown with (b) Vaccinium myrtillus or (c) Vaccinium vitis-idaea . Ericaceous plants did not act as a species pool of pine mycobionts and inhibited the ability of the potentially shared species Meliniomyces bicolor to form ectomycorrhizae. Similarly, Ericaceae significantly reduced the formation of Thelephora terrestris ectomycorrhizae in P. sylvestris . EcMF species composition in the mesocosms was strongly affected by both the host species and the presence of an ericaceous neighbour. When grown in isolation, P. strobus root tips were predominantly colonised by Wilcoxina mikolae , whereas those of P. sylvestris were more commonly colonised by Suillus and Rhizopogon spp. Interestingly, these differences were less evident ( Suillus  +  Rhizopogon spp.) or absent ( W. mikolae ) when the pines were grown with Ericaceae. P. strobus exclusively associated with Rhizopogon salebrosus s.l., suggesting the presence of host specificity at the intrageneric level. Ericaceous plants had a positive effect on colonisation of P. strobus root tips by R. salebrosus s.l. This study demonstrates that the interaction of selective factors such as host species and presence of ericaceous plants may affect the realised niche of the ectomycorrhizal fungi.

Arbutoid mycorrhizae were synthesized in vitro between Arbutus unedo L. and two isolates of Lactarius deliciosus. The fungal isolates were obtained from sporocarps collected under Pinus sylvestris and in a mixed forest stand of Quercus suber and Pinus pinea. Synthesis tubes filled with a mixture of sterilized peat, vermiculite, and perlite imbibed with nutrient solution were used. Two inoculation methods using solid and liquid media were tested. Shoots from an adult selected clone of A. unedo were used after in vitro rooting by auxin dipping. After 3 months of shoots transfer to the substrate, the root systems were examined for arbutoid mycorrhizae formation and later on ex vitro conditions, 9 months after acclimatization. The inoculum treatment with liquid medium improved the mycorrhizal development for both isolates, in vitro. Sterilized substrate for plant acclimatization increased the mycorrhizal development. The arbutoid mycorrhizae were observed in vitro as well as 9 months after acclimatization. Standard arbutoid mycorrhiza features were observed: pale yellow mantle, typical cruciform appearance, Hartig net (HN), and intracellular hyphal complexes, both confined to the epidermis. L. deliciosus mycorrhizae synthetized in vitro persisted 9 months after plant acclimatization. Morphological observations were confirmed by molecular techniques.

Until very recently, complete characterization of the megagenomes of conifers has remained elusive. The diploid genome of sugar pine (Pinus lambertiana Dougl.) has a highly repetitive, 31 billion bp genome. It is the largest genome sequenced and assembled to date, and the first from the subgenus Strobus, or white pines, a group that is notable for having the largest genomes among the pines. The genome represents a unique opportunity to investigate genome “obesity” in conifers and white pines. Comparative analysis of P. lambertiana and P. taeda L. reveals new insights on the conservation, age, and diversity of the highly abundant transposable elements, the primary factor determining genome size. Like most North American white pines, the principal pathogen of P. lambertiana is white pine blister rust (Cronartium ribicola J.C. Fischer ex Raben.). Identification of candidate genes for resistance to this pathogen is of great ecological importance. The genome sequence afforded us the opportunity to make substantial progress on locating the major dominant gene for simple resistance hypersensitive response, Cr1. We describe new markers and gene annotation that are both tightly linked to Cr1 in a mapping population, and associated with Cr1 in unrelated sugar pine individuals sampled throughout the species’ range, creating a solid foundation for future mapping. This genomic variation and annotated candidate genes characterized in our study of the Cr1 region are resources for future marker-assisted breeding efforts as well as for investigations of fundamental mechanisms of invasive disease and evolutionary response.

Forest ecosystems of the Pacific Northwest of the USA are changing as a result of climate change. Specifically, rise of global temperatures, decline of winter precipitation, earlier loss of snowpack, and increased summer drought are altering the range of Pinus contorta. Simultaneously, flux in environmental conditions within the historic P. contorta range may facilitate the encroachment of P. ponderosa into P. contorta territory. Furthermore, successful pine species migration may be constrained by the distribution or co-migration of ectomycorrhizal fungi (EMF). Knowledge of the linkages among soil fungal diversity, community structure, and environmental factors is critical to understanding the organization and stability of pine ecosystems. The objectives of this study were to establish a foundational knowledge of the EMF communities of P. ponderosa and P. contorta in the Deschutes National Forest, OR, USA, and to examine soil characteristics associated with community composition. We examined EMF root tips of P. ponderosa and P. contorta in soil cores and conducted soil chemistry analysis for P. ponderosa cores. Results indicate that Cenococcum geophilum, Rhizopogon salebrosus, and Inocybe flocculosa were dominant in both P. contorta and P. ponderosa soil cores. Rhizopogon spp. were ubiquitous in P. ponderosa cores. There was no significant difference in the species composition of EMF communities of P. ponderosa and P. contorta. Ordination analysis of P. ponderosa soils suggested that soil pH, plant-available phosphorus (Bray), total phosphorus (P), carbon (C), mineralizable nitrogen (N), ammonium (NH₄), and nitrate (NO₃) are driving EMF community composition in P. ponderosa stands. We found a significant linear relationship between EMF species richness and mineralizable N. In conclusion, P. ponderosa and P. contorta, within the Deschutes National Forest, share the same dominant EMF species, which implies that P. ponderosa may be able to successfully establish within the historic P. contorta range and dominant EMF assemblages may be conserved.

Global warming has led to frequent droughts, posing challenges for afforestation in arid and semiarid regions. In search of labor-saving and money-saving methods to improve the survival and growth rates of trees in these regions, we isolated and identified fungal endophytes that can potentially enhance the drought-resistance abilities of seedlings. We isolated 93 endophytic strains from the roots of Pinus tabulaeformis trees grown on an arid cliff. Three isolates increased the drought resistance of the tree seedlings. Using morphological, molecular, and physiological-biochemical methods, we identified three isolates as different clones of a strain of Phoma spp. and studied the strain’s effect on stress resistance-related substances in the seedlings. The results showed that the strain improved drought tolerance and increased the seedlings’ proline levels and antioxidant enzyme activities. The strain also secreted abundant extracellular abscisic acid, which likely triggered the seedlings’ protective mechanisms. This endophytic strain may provide a cheaper labor-saving, sustainable alternative to traditional methods of enhancing drought resistance.

Background A better understanding of non-structural carbohydrate (NSC) dynamics in trees under drought stress is critical to elucidate the mechanisms underlying forest decline and tree mortality from extended periods of drought. This study aimed to assess the contribution of ectomycorrhizal (ECM) fungus ( Suillus variegatus ) to hydraulic function and NSC in roots, stems, and leaves of Pinus tabulaeformis subjected to different water deficit intensity. We performed a continuous controlled drought pot experiment from July 10 to September 10, 2019 using P. tabulaeformis seedlings under 80, 40, and 20% of the field moisture capacity that represented the absence of non-drought, moderate drought, and severe drought stress, respectively. Results Results indicated that S. variegatus decreased the mortality rate and increased height, root biomass, and leaf biomass of P. tabulaeformis seedlings under moderate and severe drought stress. Meanwhile, the photosynthetic rates, stomatal conductance, and transpiration rates of P. tabulaeformis were significantly increased after S. variegatus inoculation. Moreover, the inoculation of S. variegatus also significantly increased the NSC concentrations of all seedling tissues, enhanced the soluble sugars content, and increased the ratios of soluble sugars to starch on all tissues under severe drought. Overall, the inoculation of S. variegatus has great potential for improving the hydraulic function, increasing the NSC storage, and improving the growth of P. tabulaeformis under severe drought. Conclusions Therefore, the S. variegatus can be used as a potential application strain for ecological restoration on arid regions of the Loess Plateau, especially in the P. tabulaeformis woodlands.

The absence of co-evolved mutualists of plants invading a novel habitat is the logical corollary of the more widely recognized 'enemy escape'. To avoid or overcome the loss of mutualists, plants may co-invade with nonnative mutualists, form novel associations with native mutualists or form associations with native cosmopolitan mutualists, which are native but not novel to the invading plant. We tested these hypotheses by contrasting the ectomycorrhizal fungal communities associated with invasive Pinus contorta in New Zealand with co-occurring endemic Nothofagus solandri var. cliffortioides. Fungal communities on Pinus were species poor (14 ectomycorrhizal species) and dominated by nonnative (93%) and cosmopolitan fungi (7%). Nothofagus had a species-rich (98 species) fungal community dominated by native Cortinarius and two cosmopolitan fungi. These results support co-invasion by mutualists rather than novel associations as an important mechanism by which plants avoid or overcome the loss of mutualists, consistent with invasional meltdown.