Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PLIMPine (Pine protection against Pitch canker through genetic resistance and plant immunization)

Teaser

The health of forest trees in Europe is increasingly threatened emerging risks due to pathogens. The new EU plant health policy sets ambitious goals for protection of tree. We explored a new emergent risk that threaten Pine species of European forestry, with potentially huge...

Summary

The health of forest trees in Europe is increasingly threatened emerging risks due to pathogens. The new EU plant health policy sets ambitious goals for protection of tree. We explored a new emergent risk that threaten Pine species of European forestry, with potentially huge production and sustainability losses: Pine pitch canker (caused by the quarantine fungi Fusarium circinatum). More data and experiments are needed to design efficient strategies for integrated pest management. The aim of this proposal was to characterize and identify markers of resistance (natural or induced) to Pine pitch canker disease that later can support selection of resistant plant material.
Phosphites (Phi) used as fertilizers, has been demonstrated to be effective in the control of phytopathogenic fungi, activating plant resistance functioning as elicitors and is a low risk environmental chemical. Our main goal is to explore the molecular mechanisms of natural resistance in pine, and those induced by Phi, with the aim of identify genes and genes products to be used as markers of resistance to F. circinatum.

Work performed

Objective 1: To understand how compounds such as Phi act as inducers of the plant immunity. Taking advantage of protocols used by the host group an experiment was carried out for optimization of Phi treatment for maximum protective effect of Phi. Six-month old P. radiata seedlings were foliarly sprayed with Phi (1%-4% range) until run-off 17 days before inoculation (Cerqueira et al 2017). Morpho-physiological related parameters, such as disease symptoms and gas exchange, were measured. Based in this experience the Phi concentration was fixed on 3% as optimal, to which toxicity was not observed due to the treatment and protection against the pathogen could be appreciated.
To characterize the natural resistance two species, the resistant P. pinea and the susceptible P. radiata, were used. Four groups of 30 plants from each species were treated as follows: control plants (not inoculated) and inoculated with F. circinatum. To study the induced resistance due to Phi treatment the susceptible P. radiata species was used, and four treatments (30 plants each) were performed: a) plants without Phi (control), b) plants sprayed with 3% Phi, c) plants without Phi and inoculated with F. circinatum, d) plants sprayed with 3% Phi and inoculated with F. circinatum. After in vivo morpho-physiological related parameters measurement, needles were collected when 50% of the inoculated plants of P. radiata displayed the initial typical disease symptoms (apical dieback) (Fig. 1), frozen in liquid nitrogen, and stored at -80 °C for molecular analyses.
After 10 days all P. radiata seedlings inoculated with the pathogen showed tip dieback and needle wilting, as well as a significant increase in stem relative internal necrosis. Inoculated P. radiata also suffered a significant decrease on net CO2 assimilation and transpiration rates, stomatal conductance and relative water content, and an increase on sub-stomatal CO2 concentration (Fig. 2). On the contrary, no visual symptoms were detected for P. pinea and only net CO2 assimilation rate significantly increased in the presence of F. circinatum. The data obtained from physiological parameters measured to evaluate the protective effect of Phi in P. radiata against F. circinatum are being analyzed.

Objective 2: To characterize the molecular changes associated to either natural (genotype dependent) or induced (Phi priming) resistance.
RNA, proteins and metabolites extraction was performed using the protocol of Valledor et al. (2010). RNAseq analysis is undergoing using Ilumina platform by the ‘AllGenetics & Biology SL’ company, with the aim of develop a customize database of transcripts and proteins derived from the transcripts. Proteins were analysed by LC-MSMS, and metabolites are been processed to be analysed by GC-MSMS, both on the Proteomics and Mass Spectrometry Facility (University of Córdoba, Spain).
Shotgun proteomics (LC-MSMS) analysis revealed 2988 identified proteins, 1337 of which showed signicant differences (p≤0.05) when species and treatments were compared. Functional categorization of proteins allowed grouping them into 34 categories, with most of them belonging to metabolism of carbohydrate (minor and major CHO metabolism, glycolysis, glyoxylate cycle, TCA/org transformation, OPP groups), transcription/translation (RNA group), protein degradation (protein group) and amino acids and secondary metabolism (Fig. 3). Our results demonstrate that under inoculation P. radiata metabolism is clearly affected showing a significant increase of proteins belonging to carbohydrate and amino acid metabolism, and secondary metabolism, which can be interpreted as a strategy to compensate for the deficit of energy produced during the infection and to cope with the pathogen. On the contrary P. pinea metabolism was less affected, but a significant increase of proteins belonging to the secondary metabolism was observed. A more compressive analysis will be carried out using our customized database f

Final results

The impact of the project has matched the expectations in the section of the considered period, and the communication/dissemination of results has achieved the goals outlined in the original proposal. Several tasks were fulfilled as planned and according the schedule taking advantage of the experience of the host entity in Pinus-F. circinatum pathosystem and the fellow expertise in various molecular approaches. For such sort-term period several objectives were achieved and samples send to sequencing. In addition, the team are commitment to continue working in the results obtained until now and publish it. The collaboration between both parts will continue notwithstanding the early end of the project.

Website & more info

More info: http://www.cesam.ua.pt/.