Beyond the surface: exploring the mycobiome of Norway spruce under drought stress and with Heterobasidion parviporum.

The mycobiome, comprising fungi inhabiting plants, potentially plays a crucial role in tree health and survival amidst environmental stressors like climate change and pathogenic fungi. Understanding the intricate relationships between trees and their microbial communities is essential for developing effective strategies to bolster the resilience and well-being of forest ecosystems as we adopt more sustainable forest management practices. The mycobiome can be considered an integral aspect of a tree's biology, closely linked to its genotype. To explore the influence of host genetics and environmental factors on fungal composition, we examined the mycobiome associated with phloem and roots of Norway spruce (Picea abies (L.) Karst.) cuttings under varying watering conditions. To test the "mycobiome-associated-fitness" hypothesis, we compared seedlings artificially inoculated with Heterobasidion parviporum and control plants to evaluate mycobiome interaction on necrosis development. We aimed to 1) identify specific mycobiome species for the Norway spruce genotypes/families within the phloem and root tissues and their interactions with H. parviporum and 2) assess stability in the mycobiome species composition under abiotic disturbances (reduced water availability). The mycobiome was analyzed by sequencing the ribosomal ITS2 region. Our results revealed significant variations in the diversity and prevalence of the phloem mycobiome among different Norway spruce genotypes, highlighting the considerable impact of genetic variation on the composition and diversity of the phloem mycobiome. Additionally, specific mycobiome genera in the phloem showed variations in response to water availability, indicating the influence of environmental conditions on the relative proportion of certain fungal genera in Norway spruce trees. In the root mycobiome, key fungi such as Phialocephala fortinii and Paraphaeosphaeria neglecta were identified as conferring inhibitory effects against H. parviporum growth in Norway spruce genotypes. Furthermore, certain endophytes demonstrated greater stability in root ecosystems under low water conditions than ectomycorrhizal fungi. This knowledge can contribute to developing sustainable forest management practices that enhance the well-being of trees and their ecosystems, ultimately bolstering forest resilience.

Genetic variation of Picea abies in response to the artificial inoculation of Heterobasidion parviporum.

Norway spruce Picea abies is one of Europe's most economically important tree species. However, it is highly susceptible to the root rot fungus Heterobasidion parviporum. Climate change will benefit the pathogen as the tree host is weakened by, e.g., extended drought. Breeding can improve forest health, and several root rot genetic markers are suggested to improve the resistance of Norway spruce. This study aimed to compare the resistance (here defined as necrosis length) of Norway spruce families and genotypes against two strains of H. parviporum under different water availabilities. Our results show that the family and the genotype within the family have an impact on the necrosis length that is related to the aggressiveness of the fungal strains. Under low water conditions, the necrosis increased only in horizontal directions in phloem and sapwood. Similarly, the growth (seedling height) was not disturbed by abiotic stress (less water), indicating that the stress level (drought) was too low in this setting. The knowledge gained in this study could improve forest health in the changing climate by understanding the response of Norway spruce to pathogenic attacks under additional stress at the family level. This knowledge could be strategically used in forest breeding to improve the resistance of Norway spruce trees against root rot. Supplementary Information: The online version contains supplementary material available at 10.1007/s10342-023-01534-3.

Diplodia tip blight pathogen's virulence empowered through host switch.

Increased drought combined with emerging pathogens poses an increased threat to forest health. This is attributable to the unpredictable behaviour of forest pathosystems, which can favour fungal pathogens over the host under persistent drought stress conditions. Diplodia sapinea (≡ Sphaeropsis sapinea) is one of the most severe pathogens in Scots pine (Pinus sylvestris) causing Diplodia tip blight (conifer blight) under certain environmental conditions. Recently, the fungus has also been isolated from non-conifer hosts, indicating that it has a broader host range than previously known. In this study we compared the impact of different levels of water availability on necrosis length caused by D. sapinea strains isolated as endophytes (eight strains isolated from asymptomatic Scots pine) and pathogens (five strains isolated from symptomatic Scots pine) and five strains isolated from symptomatic non-pine hosts. For all strains the decreased water availability increased the necrosis length in Scots pine shoots. The isolates from non-pine hosts caused the most severe reactions under all water availabilities. The results of the study indicate the likelihood that effects of climatic changes such as drought will drive D. sapinea damage in Scots pine-dominated forests and increase mortality rates in affected trees. Further, the higher necrosis in the Scots pines caused by strains that had performed a host switch are concerning with regard to future scenarios thus increasing infection pressure on Scots pine from unknown sources.

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.

The Diplodia Tip Blight Pathogen Sphaeropsis sapinea Is the Most Common Fungus in Scots Pines' Mycobiome, Irrespective of Health Status-A Case Study from Germany.

The opportunistic pathogen Sphaeropsis sapinea (≡Diplodia sapinea) is one of the most severe pathogens in Scots pine, causing the disease Diplodia tip blight on coniferous tree species. Disease symptoms become visible when trees are weakened by stress. Sphaeropsis sapinea has an endophytic mode in its lifecycle, making it difficult to detect before disease outbreaks. This study aims to record how S. sapinea accumulates in trees of different health status and, simultaneously, monitor seasonal and age-related fluctuations in the mycobiome. We compared the mycobiome of healthy and diseased Scots pines. Twigs were sampled in June and September 2018, and filamentous fungi were isolated. The mycobiome was analyzed by high-throughput sequencing (HTS) of the ITS2 region. A PERMANOVA analysis confirmed that the mycobiome community composition significantly differed between growth years (p < 0.001) and sampling time (p < 0.001) but not between healthy and diseased trees. Sphaeropsis sapinea was the most common endophyte isolated and the second most common in the HTS data. The fungus was highly abundant in symptomless (healthy) trees, presenting in its endophytic mode. Our results highlight the ability of S. sapinea to accumulate unnoticed as an endophyte in healthy trees before the disease breaks out, representing a sudden threat to Scots pines in the future, especially with increasing drought conditions experienced by pines.

New R-Based Methodology to Optimize the Identification of Root Endophytes against Heterobasidion parviporum.

Many root fungal endophytes inhabiting forest trees have potential impact on the health and disease progression of certain tree species. Hence, the screening of root endophytes for their biocontrol abilities is relevant for their potential to protect their hosts against invaders. The aim of this research is to screen for the potential inhibitory effects of selected conifer root endophytes during interaction, in vitro, with the root rot pathogen, Heterobasidion parviporum. Here, we introduce a guideline that facilitates the use of root fungal endophytes as biocontrol agents. We isolated fungal root endophytes from eight different conifers. These root fungal endophytes were evaluated for their antagonism against the root rot pathogen, H. parviporum, by means of paired-culture antagonism assays. We determined the antagonism of the isolated root fungal endophytes to elucidate potential biocontrol applications. For the analysis, a software package in R was developed. Endophyte candidates with antagonistic potential were identified.

Phenotype MicroArrays as a complementary tool to next generation sequencing for characterization of tree endophytes.

There is an increasing need to calibrate microbial community profiles obtained through next generation sequencing (NGS) with relevant taxonomic identities of the microbes, and to further associate these identities with phenotypic attributes. Phenotype MicroArray (PM) techniques provide a semi-high throughput assay for characterization and monitoring the microbial cellular phenotypes. Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique. We found that the PM approach enables effective screening of substrate utilization by endophytes. However, the technical limitations are multifaceted and the interpretation of the PM data challenging. For the best result, we recommend that the growth conditions for the fungi are carefully standardized. In addition, rigorous replication and control strategies should be employed whether using pre-configured, commercial microwell-plates or in-house designed PM plates for targeted substrate analyses. With these precautions, the PM technique is a valuable tool to characterize the metabolic capabilities of individual endophyte isolates, or successional endophyte communities identified by NGS, allowing a functional interpretation of the taxonomic data. Thus, PM approaches can provide valuable complementary information for NGS studies of fungal endophytes in forest trees.

Resistance to Dutch elm disease reduces presence of xylem endophytic fungi in Elms (Ulmus spp.).

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.