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 from transcripts, to study both the natural resistance and that induced by Phi. Metabolites extracts were kept at -80°C until they can be analyzed by the Mass Spectrometry Facility (University of Cordoba, Spain).
Objective 3: To integrate all data (physiology and molecular) generated to identify markers of resistance (natural or induced) to pitch canker disease.
The properly integration of physiological and molecular data will allows to identify markers of resistance (natural or induced) to pitch canker disease. After normalization and statistical treatment of generated data the Partial Least Squares (PLS) methodology will be used to integrate physiological and molecular data with the aim to show the interactions between the different organization levels within the cell. For that the platform
https://www.creative-proteomics.com/services/pls-da-service.htm(odnośnik otworzy się w nowym oknie) will be used.
