Pine wilt disease: detection of the pinewood nematode (Bursaphelenchus xylophilus) as a tool for a pine breeding programme

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS-PCR-based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD

Primary Metabolite Adjustments Associated With Pinewood Nematode Resistance in Pinus pinaster

Original ResearchThe pinewood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of the pine wilt disease (PWD) and represents one of the major threats to conifer forests. The detection of the PWN in Portugal, associated with Pinus pinaster, increased the concern of its spread to European forests. Despite its susceptibility to PWD, genetic variability found among P. pinaster populations has been associated with heritable PWD resistance. Understanding the mechanisms underlying tree resistance constitutes a valuable resource for breeding programs toward more resilient forest plantations. This study investigated changes in anatomy, chlorophyll a fluorescence (ChlF), and primary metabolism in susceptible and resistant P. pinaster half-sib plants, after PWN inoculation. Susceptible plants showed a general shutdown of central metabolism, osmolyte accumulation, photosynthetic inhibition, and a decrease in the plant water status. The ChlF transient rise (OJIP curve) revealed the appearance of L- and K-bands, indicators of environmental stress. In contrast, resistant plants revealed a regulated defense response and were able to restrict PWN migration and cellular damage. Furthermore, the accumulation of g-aminobutyric acid (GABA) and succinate suggested a role of these metabolites in PWD resistance and the possible activation of the GABA shunt. Altogether, these results provide new insights to the role of primary metabolism in PWD resistance and in the selection of resistant phenotypes for disease mitigationinfo:eu-repo/semantics/publishedVersio

Molecular Defense Response of Pine Trees (Pinus spp.) to the Parasitic Nematode Bursaphelenchus xylophilus

ReviewPine wilt disease (PWD) is a severe environmental problem in Eastern Asia andWestern Europe, devastating large forest areas and causing significant economic losses. This disease is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, a parasitic migratory nematode that infects the stem of conifer trees. Here we review what is currently known about the molecular defense response in pine trees after infection with PWN, focusing on common responses in different species. By giving particular emphasis to resistance mechanisms reported for selected varieties and families, we identified shared genes and pathways associated with resistance, including the activation of oxidative stress response, cell wall lignification, and biosynthesis of terpenoids and phenylpropanoids. The role of post-transcriptional regulation by small RNAs in pine response to PWN infection is also discussed, as well as the possible implementation of innovative RNAinterference technologies, with a focus on trans-kingdom small RNAs. Finally, the defense response induced by elicitors applied to pine plants before PWN infection to prompt resistance is reviewed. Perspectives about the impact of these findings and future research approaches are discussedinfo:eu-repo/semantics/publishedVersio

Genetic variation in susceptibility to pine wilt disease of maritime pine (Pinus pinaster Aiton) half-sib families

International audienceAbstractKey messageThis paper presents a greenhouse study for assessing the genetic variation in maritime pine (Pinus pinasterAiton) in response to pinewood nematode (Bursaphelenchus xylophilus(Steiner et Buhren) Nickle), which is a causal agent of pine wilt disease. Fifteen out of 96 half-sib families were selected as less susceptible. This experiment is an important first step for creating a resistance breeding program.ContextPine wilt disease is caused by the pinewood nematode (Bursaphelenchus xylophilus (Steiner et Buhren) Nickle), a quarantine pest, and is a concern to maritime pine (Pinus pinaster) in Portugal due to its economic, environmental, and social impacts. This disease is regarded as a major threat to European forests.AimsThis paper aimed to evaluate the genetic variation in maritime pine families that were inoculated with pinewood nematode, identify the most resistant families, and establish the guidelines for a resistance improvement program.MethodsTwo-year-old half-sib progenies obtained from 96 plus trees were inoculated. The plants were monitored for survival on four different dates. The statistical analysis followed the mixed model theory.ResultsGenetic variability of the susceptibility to pine wilt disease was observed. At 157 days after inoculation, the 15 highest genetic ranking families out of 96 were selected, having a predicted survival mean of 15.6% instead of 11.0% on average for the all 96 families.ConclusionThis study allows for the implementation of an improvement program to help control pine wilt disease

SNP Detection in Pinus pinaster Transcriptome and Association with Resistance to Pinewood Nematode

Pinewood nematode (PWN, Bursaphelenchus xylophilus) is the causal agent of pine wilt disease (PWD), which severely affects Pinus pinaster stands in southwestern Europe. Despite the high susceptibility of P. pinaster, individuals of selected half-sib families have shown genetic variability in survival after PWN inoculation, indicating that breeding for resistance can be a valuable strategy to control PWD. In this work, RNA-seq data from susceptible and resistant plants inoculated with PWN were used for SNP discovery and analysis. A total of 186,506 SNPs were identified, of which 31 were highly differentiated between resistant and susceptible plants, including SNPs in genes involved in cell wall lignification, a process previously linked to PWN resistance. Fifteen of these SNPs were selected for validation through Sanger sequencing and 14 were validated. To evaluate SNP-phenotype associations, 40 half-sib plants were genotyped for six validated SNPs. Associations with phenotype after PWN inoculation were found for two SNPs in two different genes (MEE12 and PCMP-E91), as well as two haplotypes of HIPP41, although significance was not maintained following Bonferroni correction. SNPs here detected may be useful for the development of molecular markers for PWD resistance and should be further investigated in future association studiesinfo:eu-repo/semantics/publishedVersio

Quantificação da biomassa florestal residual em povoamentos de pinheiro manso na Mata Nacional do Escaroupim

O projecto Gestão Multifuncional do Pinheiro Manso (Pinus pinea L.) Para a Produção de Fruto, Diminuição de Riscos de Incêndio, Utilização de Biomassa e Recuperação Ambiental, financiado pelo Fundo Florestal Permanente, resulta de uma parceria entre instituições públicas e privadas e tem como um dos objectivos quantificar o material removido em desbastes e desramações tendo em vista a produção de estilha. Aproveitou-se a oportunidade para se instalar um ensaio de desbaste na Mata Nacional do Escaroupim em povoamentos nunca desbastados. Das árvores a desbastar seleccionou-se uma sub-amostra para determinação de biomassa por componentes (folhas, ramos, casca, lenho, pinhas, ramos e folhas secas) o que nos permitiu quantificar, em termos de biomassa seca, as quantidades de estilha obtidas nos respectivos talhões

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.Peer Reviewe

SNP detection in Pinus pinaster transcriptome and association with resistance to pinewood nematode

Pinewood nematode (PWN, Bursaphelenchus xylophilus) is the causal agent of pine wilt disease (PWD), which severely affects Pinus pinaster stands in southwestern Europe. Despite the high susceptibility of P. pinaster, individuals of selected half-sib families have shown genetic variability in survival after PWN inoculation, indicating that breeding for resistance can be a valuable strategy to control PWD. In this work, RNA-seq data from susceptible and resistant plants inoculated with PWN were used for SNP discovery and analysis. A total of 186,506 SNPs were identified, of which 31 were highly differentiated between resistant and susceptible plants, including SNPs in genes involved in cell wall lignification, a process previously linked to PWN resistance. Fifteen of these SNPs were selected for validation through Sanger sequencing and 14 were validated. To evaluate SNP-phenotype associations, 40 half-sib plants were genotyped for six validated SNPs. Associations with phenotype after PWN inoculation were found for two SNPs in two different genes (MEE12 and PCMP-E91), as well as two haplotypes of HIPP41, although significance was not maintained following Bonferroni correction. SNPs here detected may be useful for the development of molecular markers for PWD resistance and should be further investigated in future association studies.SUPPLEMENTARY MATERIALS : Supplementary Figure S1. Type of SNPs identified in P. pinaster RNA-seq analysis; Supplementary Figure S2. Boxplots of the height and diameter at the base of the stem of inoculated plants (half-sib family 440) and t-test results for the comparison of these parameters’ means between susceptible and resistant plants; Supplementary Figure S3. Association analysis of the SNPs in the six sequenced gene fragments under different genetic models with resistance to PWN; Supplementary Figure S4. Genotypes distribution for SNPs associated with phenotype; Supplementary Table S1. Summary of PCR conditions and sequencing results of the 26 SNPs selected for validation; Supplementary Table S2. Summary of mapping statistic per sample and per sequencing lane; Supplementary Table S3. SNPs detected in P. pinaster RNA-seq data; Supplementary Table S4. Details and functional annotation of the SNPs with an Fst ≥ 0.80; Supplementary Table S5. Allele frequencies and Hardy–Weinberg Equilibrium significance values calculated by SNPassoc; Supplementary Table S6. Non-significant results of the haplotype association analysis obtained with SNPassoc. All analyses were performed using a logistics regression model.DATA AVAILABILITY STATEMENT : The sequencing data presented in this study are openly available in European Nucleotide Archive (ENA) at EMBL EBI under accession number PRJEB51636.This work was supported by Fundação para a Ciência e a Tecnologia, I.P. (FCT/MCTES), through Grants GREEN-it (UID/Multi/04551/2013), BioISI (UIDB/04046/2020 and UIDP/04046/2020) and the doctoral fellowship SFRH/BD/111687/2015 (to I.M.). Support was also provided by FCT/MCTES through national funds (PIDDAC) and co-financed by Fundo Europeu de Desenvolvimento Regional (FEDER) of the EU, through Programa Operacional Regional de Lisboa do Portugal 2020 or other programs that may succeed—PTDC/BAA-MOL/28379/2017, LISBOA-01-0145-FEDER-028379.https://www.mdpi.com/journal/forestsBiochemistryGeneticsMicrobiology and Plant Patholog

Pinus pinaster early hormonal defence responses to pinewood nematode (Bursaphelenchus xylophilus) infection

The pinewood nematode (PWN) is the causal agent of pine wilt disease, a pathology that affects conifer forests, mainly Pinus spp. PWN infection can induce the expression of phytohormone-related genes; however, changes at the early phytohormone level have not yet been explored. Phyto-hormones are low-abundance metabolites, and thus, difficult to quantify. Moreover, most method-ologies focus mainly on Arabidopsis or crop species. This work aimed to validate a fast (run time 6.6 min) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) analytical method to quantify 14 phytohormones in Pinus pinaster stem tissues. This method was further applied to evaluate, for the first time, early phytohormone changes in susceptible and resistant phenotypes of P. pinaster 24, 48 and 72 h after inoculation (HAI) with PWN. A significant increase in salicylic acid (SA, 48 and 72 HAI) and jasmonic acid methyl ester (JA-ME, 72 HAI) was observed in susceptible phenotypes. Results indicate that the higher susceptibility of P. pinaster to PWN infection might result from an inefficient trigger of hypersensitive responses, with the involvement of JA and SA pathways. This work provides an important update in forest research, and adds to the current knowledge of Pinus spp. defence responses to PWN infection

An improved reference genome and first organelle genomes of Quercus suber

Cork oak (Quercus suber L.) is an ecologically and economically important evergreen tree species native to the Mediterranean region and widespread in southwest Europe and northwest Africa. An improved genome assembly of cork oak using a combination of Illumina and PacBio sequencing is presented in this study. The assembled genome contains 2351 scaffolds longer than 1000 bp, accounting for 765.7 Mbp of genome size, L90 of 755, and a N50 of 1.0 Mbp, with 40,131 annotated genes. The repetitive sequences constitute 53.6% of the genome. The genome sequences of chloroplast and mitochondrion were determined for the first time, with a genome size of 161,179 bp and 531,858 bp, respectively. Phylogenetic analysis based on complete chloroplast genome sequence showed that Q. suber is closely related to Quercus variabilis, two cork-producing species with commercial use. All data generated are available through the public databases, being ready to be used without restrictions. This study provides an improved nuclear genome assembly together with the organelle genomes of cork oak. These resources will be useful for further breeding strategies and conservation programs and for comparative genomic studies in oak species.info:eu-repo/semantics/publishedVersio