First report of Truncatella angustata causing leaf blight on Thuja plicata Donn ex D. Don in Canada.

Western redcedar (Thuja plicata Donn ex D. Don) is one of the most important commercial tree species in British Columbia, generates more than $1 billion in economic activity annually and about 8-10 million trees are planted in reforestation efforts (Gregory et al. 2018). It has been selected as the provincial tree of British Columbia (BC) because of its tremendous economic, ecoogical and cultural value. However, foliar diseases such as leaf blights have serious impact on redcedar growth and may cause significant loss of tree volume (Russell, 2007). Our 2014 - 2015 surveys of western redcedar forests in coastal areas of BC indicated high incidence of a distinctive type of blight. We observed the incidence of this disease on more than 80% of western redcedar (approximately 493) trees from late May to early December. Early symptoms appeared as circular to oval, brownish to black spots (2-3 mm), 1-5 spots per branch tip, scattered at the tip margins. Sequentially, the spots enlarged and developed into necrotic lesions on both young and old leaves. More than 50 symptomatic leaves from 10 different trees were collected and rinsed in distilled water then surface-sterilized with three times washing in Tween 20 (%5 solution) for 2 minutes (each time) and %70 ethanol for 30 second (3 times repeat). Tissues from under lesions were placed on MEA (Malt Extract Agar; Phyto Tech® labs-Product ID: M498) and PDA (Potato Dextrose Agar; Phyto Tech® Labs-Product ID: P772). The plates were incubated at 21°C in the dark. They developed distinct dull white to brown, cottony colonies with each black acervuli approximately 450-500µm. The isolates produced fusiform conidia with four cells. They didn't have any distinct color. The conidiophore size was approximately 23-24 x 2-3 µm with mostly hyaline to light brown color, branched and conidiogenous was hyaline and not branched and simple. The spore size was approximately 15-20µm by 7-10µm with three transverse septa and endogenous papillae with hyaline apical appendages. Next, we collected spores and replated them on fresh MEA media culture and placed back in the incubator to produce pure cultures. We studied conidia from leaves of trees mentioned above using light and electron microscopy using Hitachi S-3500N Scanning Electron Microscope (Noshad et al. 2023). After morphological study, further identification to the species level conducted using Zambounis and Wenneker's approach (Zambounis 2019; Wenneker,2017). Genomic DNA from two single-spore isolates were isolated and sequenced. Sequences of ITS (Internal Transcribed Spacer) region amplified using primers ITS1/ITS4 and sequenced. Final sequences were deposited in Genbank and published (accession numbers OP086244 and OP086251). Blast analysis of these sequences showed 99% and 99% resemblances with T. angustata sequence (Sutton 1980). To verify its pathogenicity, we performed a comprehensive pathogenicity test to fulfill Koch's postulates. We collected their distinctive spores in an aseptic environment and standardized them (5000/ml) using a haemocytometer. Then we inoculated 100 western redcedar seedlings (three years old) by injecting standardized spore suspension solution (inoculum) using ultra-fine 0.3ml, 31G, 8mm syringes (approximately 0.1ml per inoculation site). Ten positive control seedlings were inoculated with distilled water and ten negative control seedlings were not inoculated at all. All inoculated (experimental) seedlings demonstrated same symptoms (black spots and characteristic spores) after eight weeks. None of the control seedlings showed any similar symptoms. In the next stage, we isolated and cultured spores from inoculated seedlings and studied them. The identity of reisolates confirmed using DNA sequencing. We used these spores for our next set of disease screening which was successful again. We identified Truncatella angustata (Pers.) Hughes as the causal agent for shoot-tip blight (STB) on western redcedar by examining morphological and molecular characteristics of the pathogen. This is the first report of T. angustata as a primary pathogen on western redcedar in British Columbia, Canada.

Genetic architecture of terpene chemistry and growth traits and the impact of inbreeding on these traits in western redcedar (Thuja plicata).

Western redcedar (WRC; Thuja plicata) is a conifer of the Pacific Northwest of North America prized for its durable and rot-resistant wood. WRC has naturally low outcrossing rates and readily self-fertilizes in nature. Challenges faced in WRC breeding and propagation involve selecting trees for accelerated growth while also ensuring enhanced heartwood rot resistance and resistance to ungulate browsing, as well as mitigating potential effects of inbreeding depression. Terpenes, a large and diverse class of specialized metabolites, confer both rot and browse resistance in the wood and foliage of WRC, respectively. Using a Bayesian modelling approach, we isolated single nucleotide polymorphism (SNP) markers estimated to be associated with three different foliar terpene traits and four different heartwood terpene traits, as well as two growth traits. We found that all traits were complex, being associated with between 1700 and 3600 SNPs linked with putatively causal loci, with significant polygenic components. Growth traits tended to have a larger polygenic component while terpene traits had larger major gene components; SNPs with small or polygenic effect were spread across the genome, while larger-effect SNPs tended to be localized to specific linkage groups. To determine whether there was inbreeding depression for terpene chemistry or growth traits, we used mixed linear models for a genomic selection training population to estimate the effect of the inbreeding coefficient F on foliar terpenes, heartwood terpenes and several growth and dendrochronological traits. We did not find significant inbreeding depression for any assessed trait. We further assessed inbreeding depression across four generations of complete selfing and found that not only was inbreeding depression not significant but that selection for height growth was the only significant predictor for growth during selfing, suggesting that inbreeding depression due to selfing during operational breeding can be mitigated by increased selection intensity.

Genomic selection reveals hidden relatedness and increased breeding efficiency in western redcedar polycross breeding.

Western redcedar (WRC) is an ecologically and economically important forest tree species characterized by low genetic diversity with high self-compatibility and high heartwood durability. Using sequence capture genotyping of target genic and non-genic regions, we genotyped 44 parent trees and 1520 offspring trees representing 26 polycross (PX) families collected from three progeny test sites using 45,378 SNPs. Trees were phenotyped for eight traits related to growth, heartwood and foliar chemistry associated with wood durability and deer browse resistance. We used the genomic realized relationship matrix for paternity assignment, maternal pedigree correction, and to estimate genetic parameters. We compared genomics-based (GBLUP) and two pedigree-based (ABLUP: polycross and reconstructed full-sib [FS] pedigrees) models. Models were extended to estimate dominance genetic effects. Pedigree reconstruction revealed significant unequal male contribution and separated the 26 PX families into 438 FS families. Traditional maternal PX pedigree analysis resulted in up to 51% overestimation in genetic gain and 44% in diversity. Genomic analysis resulted in up to 22% improvement in offspring breeding value (BV) theoretical accuracy, 35% increase in expected genetic gain for forward selection, and doubled selection intensity for backward selection. Overall, all traits showed low to moderate heritability (0.09-0.28), moderate genotype by environment interaction (type-B genetic correlation: 0.51-0.80), low to high expected genetic gain (6.01%-55%), and no significant negative genetic correlation reflecting no large trade-offs for multi-trait selection. Only three traits showed a significant dominance effect. GBLUP resulted in smaller but more accurate heritability estimates for five traits, but larger estimates for the wood traits. Comparison between all, genic-coding, genic-non-coding and intergenic SNPs showed little difference in genetic estimates. In summary, we show that GBLUP overcomes the PX limitations, successfully captures expected historical and hidden relatedness as well as linkage disequilibrium (LD), and results in increased breeding efficiency in WRC.

The western redcedar genome reveals low genetic diversity in a self-compatible conifer.

We assembled the 9.8-Gbp genome of western redcedar (WRC; Thuja plicata), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity.

Factors Affecting Foliar Oregonin and Condensed Tannin in Red Alder (Alnus rubra Bong.): Phytochemicals Implicated In Defense Against Western Tent Caterpillar (Malacosoma californicum Packard).

Past work shows a significant negative correlation between foliar oregonin concentration and western tent caterpillar (Malacosoma californicum Packard) feeding on red alder (Alnus rubra Bong.). Above an oregonin threshold of 20% leaf dry weight, little feeding by caterpillars is observed. Concentrations of defensive chemicals are influenced by plant genotype, environmental conditions, insect feeding, and the interactions of these factors. Our objective was to measure the effects of nitrogen (N) availability and wounding on foliar oregonin and condensed tannin concentrations in red alder genotypes. One-year-old seedlings from 100 half-sib red alder families were treated with two levels of ammonium nitrate (NH4NO3) for two growing seasons in a common garden. In the second year, leaves from 50 families from the fertilization experiment were used in a bioassay feeding experiment to determine the effects of N fertilization and genotype on WTC damage, and to identify a subset of 20 families with a range of damage to analyze for phytochemical composition. In separate experiments, wound-induction treatments were conducted outdoors and, in a greenhouse using the N treated trees in their third and fourth year, respectively. Foliar condensed tannin, oregonin and N concentrations were measured and ranked among the plant genotypes, and between the two N treatments and two wounding treatments. Results showed that oregonin and condensed tannin concentrations varied among the alder genotypes. Leaf N concentration was negatively correlated with concentration of oregonin. Neither of the measured phenolic compounds responded to wounding. The results suggest that red alder foliar oregonin and condensed tannin are likely constitutive defenses that are largely determined by genotype, and that the negative correlation of defense compounds with plant internal N status holds in this N-fixing tree.

Improvement of non-key traits in radiata pine breeding programme when long-term economic importance is uncertain.

Diameter at breast height (DBH), wood density (DEN) and predicted modulus of elasticity (PME) are considered as 'key traits' (KT) in the improvement in radiata pine breeding programmes in New Zealand. Any other traits which are also of interest to radiata pine breeders and forest growers are called 'non-key traits' (NKTs). External resin bleeding (ERB), internal checking (IC), number of heartwood rings (NHR) are three such non-key traits which affect wood quality of radiata pine timber. Economic importance of the KTs and NKTs is hard to define in radiata pine breeding programmes due to long rotation period. Desired-gain index (DGIs) and robust selection were proposed to incorporate NKTs into radiata pine breeding programme in order to deal with the uncertainty of economic importance. Four desired-gain indices A-D were proposed in this study. The desired-gain index A (DGI-A) emphasized growth and led to small decrease in ERB and small increase in IC and NHR. The expected genetic gains of all traits in the desired-gain index B (DGI-B) were in the favourable directions (positive genetic gains in the key traits and negative genetic gains in the non-key traits). The desired-gain index C (DGI-C) placed emphasis on wood density, leading to favourable genetic gain in the NKTs but reduced genetic gains for DBH and PME. The desired-gain index D (DGI-D) exerted a bit more emphasis on the non-key traits, leading large favourable reduction in the non-key traits and lower increase in the key traits compared with the other DGIs. When selecting both the key traits and the non-key traits, the average EBVs of six traits were all in the same directions as the expected genetic gains except for DBH in the DGI-D. When the key traits were measured and selected, internal checking always had a negative (favourable) genetic gain but ERB and NHR had unfavourable genetic gain in the most of time. After removing some individuals with high sensitivity to the change of economic weights, robust desired-gain selection made genetic gains of all the key and non-key traits to move a little bit toward unfavourable directions in the four indices. It is concluded that desired-gain index combined with robust selection concept is an efficient way for selecting the key and non-key traits in radiata pine breeding programmes.

Improved white spruce (Picea glauca) genome assemblies and annotation of large gene families of conifer terpenoid and phenolic defense metabolism.

White spruce (Picea glauca), a gymnosperm tree, has been established as one of the models for conifer genomics. We describe the draft genome assemblies of two white spruce genotypes, PG29 and WS77111, innovative tools for the assembly of very large genomes, and the conifer genomics resources developed in this process. The two white spruce genotypes originate from distant geographic regions of western (PG29) and eastern (WS77111) North America, and represent elite trees in two Canadian tree-breeding programs. We present an update (V3 and V4) for a previously reported PG29 V2 draft genome assembly and introduce a second white spruce genome assembly for genotype WS77111. Assemblies of the PG29 and WS77111 genomes confirm the reconstructed white spruce genome size in the 20 Gbp range, and show broad synteny. Using the PG29 V3 assembly and additional white spruce genomics and transcriptomics resources, we performed MAKER-P annotation and meticulous expert annotation of very large gene families of conifer defense metabolism, the terpene synthases and cytochrome P450s. We also comprehensively annotated the white spruce mevalonate, methylerythritol phosphate and phenylpropanoid pathways. These analyses highlighted the large extent of gene and pseudogene duplications in a conifer genome, in particular for genes of secondary (i.e. specialized) metabolism, and the potential for gain and loss of function for defense and adaptation.

Insights into conifer giga-genomes.

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world's forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20-30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes.

Assembling the 20 Gb white spruce (Picea glauca) genome from whole-genome shotgun sequencing data.

UNLABELLED: White spruce (Picea glauca) is a dominant conifer of the boreal forests of North America, and providing genomics resources for this commercially valuable tree will help improve forest management and conservation efforts. Sequencing and assembling the large and highly repetitive spruce genome though pushes the boundaries of the current technology. Here, we describe a whole-genome shotgun sequencing strategy using two Illumina sequencing platforms and an assembly approach using the ABySS software. We report a 20.8 giga base pairs draft genome in 4.9 million scaffolds, with a scaffold N50 of 20,356 bp. We demonstrate how recent improvements in the sequencing technology, especially increasing read lengths and paired end reads from longer fragments have a major impact on the assembly contiguity. We also note that scalable bioinformatics tools are instrumental in providing rapid draft assemblies. AVAILABILITY: The Picea glauca genome sequencing and assembly data are available through NCBI (Accession#: ALWZ0100000000 PID: PRJNA83435). http://www.ncbi.nlm.nih.gov/bioproject/83435.

Genetic variation of lodgepole pine, Pinus contorta var. latifolia, chemical and physical defenses that affect mountain pine beetle, Dendroctonus ponderosae, attack and tree mortality.

Plant secondary chemistry is determined by both genetic and environmental factors, and while large intraspecific variation in secondary chemistry has been reported frequently, the levels of genetic variation of many secondary metabolites in forest trees in the context of potential resistance against pests have been rarely investigated. We examined the effect of tree genotype and environment/site on the variation in defensive secondary chemistry of lodgepole pine, Pinus contorta var. latifolia, against the fungus, Grosmannia clavigera (formerly known as Ophiostoma clavigerum), associated with the mountain pine beetle, Dendroctonus ponderosae. Terpenoids were analyzed in phloem samples from 887, 20-yr-old trees originating from 45 half-sibling families planted at two sites. Samples were collected both pre- and post-inoculation with G. clavigera. Significant variation in constitutive and induced terpenoid compounds was attributed to differences among families. The response to the challenge inoculation with G. clavigera was strong for some individual compounds, but primarily for monoterpenoids. Environment (site) also had a significant effect on the accumulation of some compounds, whereas for others, no significant environmental effect occurred. However, for a few compounds significant family x environment interactions were found. These results suggest that P. c. latifolia secondary chemistry is under strong genetic control, but the effects depend on the individual compounds and whether or not they are expressed constitutively or following induction.