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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.

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.

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus , threatens Pinus seriously. Pinus koraiensis is one of the most important pine species in China and is the host for PWN. However, our understanding of the defence-regulating process following infection by B. xylophilus at the molecular level remains limited. To understand the mechanisms that P. koraiensis responds to B. xylophilus invasion, P. koraiensis was inoculated with B. xylophilus solutions and observed no obvious symptoms during the early stage; symptoms began to appear at 5 dpi. Therefore, we conducted comparative transcriptomic, metabonomic and proteomic analyses between P. koraiensis 5dpi and 0 dpi. In infected plants, 1574 genes were significantly up-regulated, including 17 terpenoid-, 41 phenylpropanoid- and 22 flavonoid-related genes. According to GO and KEGG enrichment analyses of significantly up-regulated genes, 86 GO terms and 16 KEGG pathways were significantly enriched. Most terms and pathways were associated with terpenoid-, phenylpropanoid-, flavonoid- and carbohydrate-related events. Similarly, the abundance of 36 and 30 metabolites, significantly increased in positive and negative polarity modes, respectively. Among them, naringenin and 3-methyl-2-oxovaleric acid exhibited significant toxic effects on B. xylophilus . According to functional analysis of significantly up-regulated metabolites, most terms were enriched in above pathways, in addition to alkaloid biosynthesis. Although the abundance of few proteins changed, response to stress term was significantly enriched in significant up-regulated proteins. Furthermore, plant receptor-like serine/threonine kinases, pectin methylation modulators, pinosylvin O-methyltransferase and arabinogalactan/proline-rich proteins were significantly up-regulated in the infected P. koraiensis compared to healthy plants. These proteins were not abundant in the healthy plant. Overall, these results indicate that P. koraiensis can actively response to PWN via various defense strategies, including events related to terpenoids, flavonoids, phenylpropanoids, lipids and alkaloids. Particularly, terpenoids and flavonoids are required for the early defence of P. koraiensis against B. xylophilus infection.

The migratory plant‐parasitic nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which causes serious damage to pine forests in China. Plant immunity plays an important role in plant resistance to multiple pathogens. Activation of the plant immune system is generally determined by immune receptors, including plant pattern recognition receptors, which mediate pattern recognition. However, little is known about molecular pattern recognition in the interaction between pines and B. xylophilus. Based on the B. xylophilus transcriptome at the early stages of infection and Agrobacterium tumefaciens‐mediated transient expression and infiltration of recombinant proteins produced by Pichia pastoris in many plant species, a novel molecular pattern (BxCDP1) was characterized in B. xylophilus. We found that BxCDP1 was highly up‐regulated at the early infection stages of B. xylophilus, and was similar to a protein in Pararhizobium haloflavum. BxCDP1 triggered cell death in Nicotiana benthamiana when secreted into the apoplast, and this effect was dependent on brassinosteroid‐insensitive 1‐associated kinase 1, but independent of suppressor of BIR1‐1. BxCDP1 also exhibited cell death‐inducing activity in pine, Arabidopsis, tomato, pepper, and lettuce. BxCDP1 triggered reactive oxygen species production and the expression of PAMP‐triggered immunity marker genes (NbAcre31, NbPTI5, and NbCyp71D20) in N. benthamiana. It also induced the expression of pathogenesis‐related genes (PtPR‐3, PtPR‐4, and PtPR‐5) in Pinus thunbergii. These results suggest that as a new B. xylophilus molecular pattern, BxCDP1 can not only be recognized by many plant species, but also triggers innate immunity in N. benthamiana and defence responses of P. thunbergii. BxCDP1 can not only trigger innate immunity of the nonhost plant Nicotiana benthamiana, which depends on co‐receptors NbBAK1, but also induces defence responses of the host Pinus thunbergii.

Dothistroma needle blight, Cyclaneusma needle blight and red needle cast are devastating foliar pine diseases caused by the fungi Dothistroma septosporum and Cyclaneusma minus and the oomycete Phytophthora pluvialis, respectively. These pathogens colonise the host apoplast, secreting effector proteins to promote infection and disease. If these effectors are recognised by corresponding host resistance proteins, they activate the plant immune system to stop pathogen growth. We aimed to identify and characterise effectors that are common to all three pathogens. Using D. septosporum as a starting point, three candidate effectors (CEs) were investigated: Ds69335 (a CAP protein) and Ds131885, both of which have sequence and structural similarity to CEs of C. minus and P. pluvialis, and Ds74283, which adopts a β-trefoil fold and has structural rather than sequence similarity to CEs from all three pathogens. Notably, of the CEs investigated, Ds74283 and Ds131885, as well as their homologues from C. minus and P. pluvialis, elicited chlorosis or cell death in Nicotiana species, with Ds131885 and its homologues also triggering cell death in Pinus radiata. In line with these observed responses being related to activation of the plant immune system, the chlorosis triggered by Ds131885 and its homologues was compromised in a Nicotiana benthamiana mutant lacking the extracellular immune system co-receptor, SOBIR1. Such cross-kingdom, plant immune system-activating effectors, whether similar in sequence or structure, might ultimately enable the selection or engineering of durable, broad-spectrum resistance against foliar pine pathogens

If carbon (C) sinks withdraw carbohydrates as they are transported along tree stems, carbohydrate availability may depend on local sink strength and distance from sources. Defenses, including monoterpenes – a major component of resin – limit the invasibility of pines. Since carbohydrate reserves fund monoterpene synthesis, we hypothesized that monoterpene concentrations in pine stems would decrease from the crown to the lower stem, and susceptibility to fungal infection would increase. Here, we measured carbohydrate and monoterpene concentrations along the stems of lodgepole pine trees (Pinus contorta var. latifolia) before inoculating with a blue-stain fungus at different heights. After 6 wk, we assessed tree responses to fungal infection based on lesion length and carbohydrate mobilization. Concentrations of carbohydrates and monoterpenes in the phloem before inoculation decreased with distance from the crown, whereas lesion lengths after inoculation increased. However, trees mobilized sugars in response to fungal infection such that carbohydrate reserves near lesions were similar at all heights. Despite C mobilization, the lower stem was more vulnerable than the upper stem. Consistent with predictions based on sink–source relationships, vulnerability occurred where carbohydrates were less available, and likely resulted from C withdrawal by sinks higher in the supply chain.

Forests are under threat from pests, pathogens, and changing climate. A major forest pathogen worldwide is the hemibiotroph Dothistroma septosporum , which causes dothistroma needle blight (DNB) of pines. While D. septosporum uses effector proteins to facilitate host infection, it is currently unclear whether any of these effectors are recognised by immune receptors to activate the host immune system. Such information is needed to identify and select disease resistance against D. septosporum in pines. We predicted and investigated apoplastic D. septosporum candidate effectors (DsCEs) using bioinformatics and plant-based experiments. We discovered DsCEs that trigger cell death in the angiosperm Nicotiana spp., indicative of a hypersensitive defence response and suggesting their recognition by immune receptors in non-host plants. In a first for foliar forest pathogens, we developed a novel protein infiltration method to show that tissue-cultured pine shoots can respond with a cell death response to a DsCE, as well as to a reference cell death-inducing protein. The conservation of responses across plant taxa suggests that knowledge of pathogen–angiosperm interactions may also be relevant to pathogen–gymnosperm interactions. These results contribute to our understanding of forest pathogens and may ultimately provide clues to disease immunity in both commercial and natural forests.

The mechanisms and conditions affecting expression of systemic induced resistance (SIR) in pine are not clearly understood. Two hypotheses were tested here: that SIR against a pathogen induced by either a pathogen or an insect involves coordinated shifts in phloem secondary metabolism; and that fertility affects the production of these compounds. To test these hypotheses, a tripartite system was used comprising Austrian pine (Pinus nigra) grown under three different fertility regimes, the fungal pathogen Diplodia pinea, and the defoliator Neodiprion sertifer. Fungal induction led to systemic accumulation of lignin, phenolic glycosides and stilbenes, whereas insect defoliation led to an increase in germacrene D concentration in branch phloem. Fertility affected the concentrations of only the phenolic glycosides. Multivariate analyses showed coregulation of compounds within at least three consistent groupings: phenolic glycosides, stilbenes and monoterpenes. As groups and as individual compounds, accumulation of phenolic glycosides and stilbenes was negatively correlated with disease susceptibility. The experimental manipulation of the phenolics and terpenoids metabolic networks achieved in this study by biotic induction and changes in nutrient availability suggests that lignin, phenolic glycosides and stilbenes are important biochemical factors in the expression of SIR against the pathogen in this system.

Background Many conifer breeding programs are paying increasing attention to breeding for resistance to needle disease due to the increasing importance of climate change. Phenotyping of traits related to resistance has many biological and temporal constraints that can often confound the ability to achieve reliable phenotypes and consequently, reliable genetic progress. The development of next generation sequencing platforms has also enabled implementation of genomic approaches in species lacking robust reference genomes. Genomic selection is, therefore, a promising strategy to overcome the constraints of needle disease phenotyping. Results We found high accuracy in the prediction of genomic breeding values in the disease-related traits that were well characterized, reaching 0.975 for genotyped individuals and 0.587 for non-genotyped individuals. This compared well with pedigree-based accuracies of up to 0.746. Surprisingly, poorly phenotyped disease traits also showed very high accuracy in terms of correlation of predicted genomic breeding values with pedigree-based counterparts. However, this was likely caused by the fact that both were clustered around the population mean, while deviations from the population mean caused by genetic effects did not appear to be well described. Caution should therefore be taken with the interpretation of results in poorly phenotyped traits. Conclusions Implementation of genomic selection in this test population of Pinus radiata resulted in a relatively high prediction accuracy of needle loss due to Dothistroma septosporum compared with a pedigree-based approach. Using genomics to avoid biological/temporal constraints where phenotyping is reliable appears promising. Unsurprisingly, reliable phenotyping, resulting in good heritability estimates, is a fundamental requirement for the development of a reliable prediction model. Furthermore, our results are also specific to the single pathogen mating-type that is present in New Zealand, and may change with future incursion of other pathogen varieties. There is no doubt, however, that once a robust genomic prediction model is built, it will be invaluable to not only select for host tolerance, but for other economically important traits simultaneously. This tool will thus future-proof our forests by mitigating the risk of disease outbreaks induced by future changes in climate.

Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.