Genomic Comparisons of Two Armillaria Species with Different Ecological Behaviors and Their Associated Soil Microbial Communities

Armillaria species show considerable variation in ecological roles and virulence, from mycorrhizae and saprophytes to important root pathogens of trees and horticultural crops. We studied two Armillaria species that can be found in coniferous forests of northwestern USA and southwestern Canada. Armillaria altimontana is considered as a weak, opportunistic pathogen of coniferous trees, but it also appears to exhibit in situ biological control against A. solidipes, formerly North American A. ostoyae, which is considered a virulent pathogen of coniferous trees. Here, we describe their genome assemblies and present a functional annotation of the predicted genes and proteins for the two Armillaria species that exhibit contrasting ecological roles. In addition, the soil microbial communities were examined in association with the two Armillaria species within a 45-year-old plantation of western white pine (Pinus monticola) in northern Idaho, USA, where A. altimontana was associated with improved tree growth and survival, while A. solidipes was associated with reduced growth and survival.

Potential Use of the Pigments from Scytalidium cuboideum and Chlorociboria aeruginosa to Prevent ‘Greying’ Decking and Other Outdoor Wood Products

UV-light degradation of wood is one of the top reasons for consumer replacement of outdoor wooden structures. This type of degradation is seldom mechanical, and is instead often motivated by loss of aesthetics (graying). There are numerous commercial products available on the market that deal with this loss of color, many of which contain added pigments to ‘rejuvenate’ or ‘revitalize’ greyed wood. These pigments are almost uniformly synthetic. In contrast, pigments from wood decay fungi (spalting), which have been used in woodworking since the 1400s (intarsia), have remarkable optical (UV-light resistance) properties due to their naphthoquinonic configuration. In recent years the pigments made from these fungi have been extracted and tested across numerous substrates, from solar cells to textile dyes. In this work, researchers extracted pigments from Scytalidium cuboideum (red pigmentation) and Chlorociboria aeruginosa (blue-green pigmentation), solubilized the pigments in raw linseed oil, and tested the resulting solution on samples of Douglas-fir (Pseudotsuga menziesii) and western white pine (Pinus monticola). These mixtures were compared against a ‘stain and coat’ treatment (utilizing an aniline stain and coated with raw linseed oil), raw linseed oil, and untreated wood. The wood samples were then placed in an accelerated weathering machine (Q-UV) following the ASTM G154 standard, for 500 and 1000 h. The results showed that while no visible color change occurred to the wood when the pigmented oil was applied, the red pigment oil significantly lowered the coating degradation for both wood types at an exposure of 500 h. The results show the potential applications for fungal pigments in the wood coating industry, as it offers an increased coating service life. As there is a shift to renewable products, the pigments from wood decay fungi show potential as additives for wood coatings.

Comparative Transcriptomics and RNA-Seq-Based Bulked Segregant Analysis Reveals Genomic Basis Underlying Cronartium ribicola vcr2 Virulence

Breeding programs of five-needle pines have documented both major gene resistance (MGR) and quantitative disease resistance (QDR) to Cronartium ribicola (Cri), a non-native, invasive fungal pathogen causing white pine blister rust (WPBR). WPBR is one of the most deadly forest diseases in North America. However, Cri virulent pathotypes have evolved and can successfully infect and kill trees carrying resistance (R) genes, including vcr2 that overcomes MGR conferred by the western white pine (WWP, Pinus monticola) R gene (Cr2). In the absence of a reference genome, the present study generated a vcr2 reference transcriptome, consisting of about 20,000 transcripts with 1,014 being predicted to encode secreted proteins (SPs). Comparative profiling of transcriptomes and secretomes revealed vcr2 was significantly enriched for several gene ontology (GO) terms relating to oxidation-reduction processes and detoxification, suggesting that multiple molecular mechanisms contribute to pathogenicity of the vcr2 pathotype for its overcoming Cr2. RNA-seq-based bulked segregant analysis (BSR-Seq) revealed genome-wide DNA variations, including about 65,617 single nucleotide polymorphism (SNP) loci in 7,749 polymorphic genes shared by vcr2 and avirulent (Avcr2) pathotypes. An examination of the distribution of minor allele frequency (MAF) uncovered a high level of genomic divergence between vcr2 and Avcr2 pathotypes. By integration of extreme-phenotypic genome-wide association (XP-GWAS) analysis and allele frequency directional difference (AFDD) mapping, we identified a set of vcr2-associated SNPs within functional genes, involved in fungal virulence and other molecular functions. These included six SPs that were top candidate effectors with putative activities of reticuline oxidase, proteins with common in several fungal extracellular membrane (CFEM) domain or ferritin-like domain, polysaccharide lyase, rds1p-like stress responsive protein, and two Cri-specific proteins without annotation. Candidate effectors and vcr2-associated genes provide valuable resources for further deciphering molecular mechanisms of virulence and pathogenicity by functional analysis and the subsequent development of diagnostic tools for monitoring the virulence landscape in the WPBR pathosystems.

In-vitro anti-fungal assay and association analysis reveal a role for the Pinus monticola PR10 gene (PmPR10-3.1) in quantitative disease resistance to white pine blister rust

Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of conifer trees. Here we identified multiple novel members in the western white pine (Pinus monticola) PmPR10 family by bioinformactic mining available transcriptomic data. Phylogenetic analysis of protein sequences revealed four PR10 and two PR10-like clusters with a high synteny across different species of five-needle pines. Of ten PmPR10 genes, PmPR10-3.1 was selected and expressed in Escherichia coli. The purified recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens C. ribicola, Phoma exigua and P. argillacea by in-vitro antifungal analysis. Genetic variation analysis detected a total of 21 single nucleotide polymorphisms (SNPs) within PmPR10-3.1 in a collection of P. monticola seed families. A nonsynonymous SNP (t178g) showed significant association with relative levels of quantitative disease resistance (QDR), explaining about 8.7% of phenotypic variation as the peak value across all SNPs. Our results provide valuable insight into the genetic architecture underlying P. monticola QDR, and imply that PmPR10-3.1 may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.

Evaluation of Stem-Injected TREE-äge® (4% Emamectin Benzoate) for Protecting Western White Pines (Pinus monticola) from Mountain Pine Beetle (Dendroctonus ponderosae Hopkins)(Coleoptera: Curculionidae: Scolytinae)

The protection of high-value trees against bark beetles and the development of alternatives to bole sprays is a priority for the tree manager. The objective of this study was to evaluate stem-injected TREE-äge® (emamectin benzoate [EB]) as a protective treatment for western white pines (Pinus monticola Dougl. ex D. Don) against mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins). Treatment efficacy was based solely on tree mortality as per Shea protocols (i.e., ≥ 60% check vs. ≤ 20% treated tree mortality). Our first experiment was installed in 2007 and included trees stem-injected with TREE-äge and untreated controls. Bole application of S-(-)-verbenone and green leaf volatile (GLV) blend was included for observational comparison. Pressure from MPB was heavy, as indicated by the number and timing of control tree mortality (90%). Strip attacks by MPB in TREE-äge trees indicated that the impacts of EB, and by inference its distribution, were inconsistent. In 2009, the injection protocol was revised to improve EB distribution in the phloem via closer injection points. In the 2009 TREE-äge-treated trees, adult beetle mining stopped when they contacted phloem and was insufficient to cause tree death by girdling. Blue-stain fungi colonized the sapwood of trees in both studies. Isolates from autopsied trees treated with TREE-äge alone were subsequently identified as Grosmannia clavigera and Leptographium longiclavatum (Ophiostomatales: Ascomycota), species that can incite tree mortality. In 2013, we revised our protocol to include GLV plus verbenone or propiconazole with TREE-äge, wherein these treatments proved effective in protecting trees against MPB and their associated pathogenic fungi.

Multidecadal Growth of Western White Pine and Interior Douglas-Fir Following Site Preparation

Site preparation is used to favor seedling regeneration and establishment by enhancing growing conditions and increasing resource availability, yet few studies have compared different site preparation techniques on growth and yield of trees over multiple decades. We destructively sampled 34-year old trees of western white pine (Pinus monticola Douglas ex D. Don) and Interior Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. glauca (Beissn.) Franco planted at two sites using a replicated experiment to test the effectiveness of different site preparation treatments: (1) no site preparation, (2) scalping, (3) bedding, and (4) bedding plus three years of competition control with herbicide. Growth and yield were compared among the treatments, and models of growth were developed for each species and treatment combination. The herbicide treatment was the only treatment that consistently improved growth and yield of both species resulting in 19%–30% gains in height, 43%–63% gains in diameter, and 31%–109% gains in stem volume by age 34. Height growth response to herbicide was sustained until age 14 for white pine and age 12 for Douglas-fir, while the diameter response was sustained until age 23 for white pine and 20 for Douglas-fir. The later peak in growth for white pine suggests a better response to treatment and that the species was able to maintain higher growth following crown closure. Both species exhibited a Type 2 growth response to herbicide, suggesting competition control resulted in sustained gains over time with associated age shifts of 8.5 and 9.7 years for white pine and 7.1 and 10.2 years for Douglas-fir, height and diameter, respectively. This compares to scalping and bedding which produced no detectable difference in growth compared to the control, and in some instances, reduced growth. In the Northern Rocky Mountains, moisture is most limiting. This is likely why trees showed the greatest response to competition control. Interestingly, this growth was sustained well beyond seedling establishment.

Six co-occurring conifer species in northern Idaho exhibit a continuum of hydraulic strategies during an extreme drought year

As growing seasons in the northwestern USA lengthen, on track with climate predictions, the mixed conifer forests that dominate this region will experience extended seasonal drought conditions. The year of 2015, which had the most extreme drought for the area on record, offered a potential analogue of future conditions. During this period, we measured the daily courses of water potential and gas exchange as well as the hydraulic conductivity and vulnerability to embolism of six dominant native conifer species, Abies grandis, Larix occidentalis, Pinus ponderosa, Pinus monticola, Pseudotsuga menziesii and Thuja occidentalis, to determine their responses to 5 months of record-low precipitation. The deep ash-capped soils of the region allowed gas exchange to continue without significant evidence of water stress for almost 2 months after the last rainfall event. Midday water potentials never fell below −2.2 MPa in the evergreen species and −2.7 MPa in the one deciduous species. Branch xylem was resistant to embolism, with P50 values ranging from −3.3 to −7.0 MPa. Root xylem, however, was more vulnerable, with P50 values from −1.3 to −4.6 MPa. With predawn water potentials as low as −1.3 MPa, the two Pinus species likely experienced declines in root hydraulic conductivity. Stomatal conductance of all six species was significantly responsive to vapour pressure only in the dry months (August–October), with no response evident in the wet months (June–July). While there were similarities among species, they exhibited a continuum of isohydry and safety margins. Despite the severity of this drought, all species were able to continue photosynthesis until mid-October, likely due to the mediating effects of the meter-deep, ash-capped silty-loam soils with large water storage capacity. Areas with these soil types, which are characteristic of much of the northwestern USA, could serve as refugia under drier and warmer future conditions.

Characterization of the western white pine TIR-NBS-LRR (PmTNL2) gene by transcript profiling and promoter analysis

Proteins with nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) have been reported to play important roles in plant disease resistance, growth, and development. However, no comprehensive analysis of this protein family has been performed in conifers. Here we report that the Pinus monticola PmTNL2 gene is a member of the NBS-LRR superfamily. Quantitative reverse transcription-PCR (qRT-PCR) analysis revealed that the PmTNL2 transcript was expressed in a tissue-specific pattern with extensive regulation by various environmental stimuli in western white pine seedlings, suggesting its wide involvement in stress defense and diverse developmental processes. In silico analysis of the PmTNL2 promoter region revealed multiple cis-regulatory elements characterized with potential functions for development-, light-, and stress-regulated transcript expression. Expression patterns were largely confirmed by PmTNL2 promoter-directed reporter gene expression using stable transgenic Arabidopsis plants. Notably, the PmTNL2 promoter activity was highly expressed in shoot apical and floral meristems and was induced strongly with vascular specificity by pathogen infection. Our data has provided a fundamental insight into both expression regulation and putative functions of the PmTNL2 gene in the context of plant growth and development, as well as in responses to environmental stressors. Promoter application as a potential tool for tree improvement was further discussed.

Association analysis and in-vitro anti-fungal assay reveal a role for the Pinus monticola PR10 gene (PmPR10-3.1) in quantitative resistance to white pine blister rust

Background Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of forest conifer trees. Results To elucidate the biological roles of PR proteins of class 10 (PR10) in tree resistance to pathogens, in the present study we identified multiple novel members in the complex PmPR10 family of western white pine (Pinus monticola Douglas ex D. Don). Phylogenetic analysis revealed that PmPR10 genes were grouped into clusters and sub-clusters, showing a relatively high synteny between the same cluster/sub-cluster across different species of five-needle pines. RNA-seq-based global gene expression study demonstrated the PmPR10 genes were differentially expressed in response to infection by Cronartium ribicola, an exotic fungus causing white pine blister rust (WPBR) across North America. Among all family members, PmPR10-3.1 transcripts were significantly up-regulate at both early and late stages post rust infection. This gene was further revealed to have significant contribution to host quantitative resistance to WPBR by an association study. Moreover, PmPR10-3.1 recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens C. ribicola, Phoma exigua and P. argillacea by in-vitro antifungal analysis. Conclusion Our results suggest that PmPR10-3.1 may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.