Pinus Susceptibility to Pitch Canker Triggers Specific Physiological Responses in Symptomatic Plants: An Integrated Approach

Abstract

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is an emergent and still understudied risk that threatens Pinus forests worldwide, with potential production and sustainability losses. In order to explore the response of pine species with distinct levels of susceptibility to PPC, we investigated changes in physiology, hormones, specific gene transcripts, and primary metabolism occurring in symptomatic Pinus pinea, Pinus pinaster, and Pinus radiata upon inoculation with F. circinatum. Pinus radiata and P. pinaster exhibiting high and intermediate susceptibility to PPC, respectively, suffered changes in plant water status and photosynthetic impairment. This was associated with sink metabolism induction, a general accumulation of amino acids and overexpression of pathogenesis-related genes. On the other hand, P. pinea exhibited the greatest resistance to PPC and stomatal opening, transpiration increase, and glycerol accumulation were observed in inoculated plants. A stronger induction of pyruvate decarboxylase transcripts and differential hormones regulation were also found for inoculated P. pinea in comparison with the susceptible Pinus species studied. The specific physiological changes reported herein are the first steps to understand the complex Pinus-Fusarium interaction and create tools for the selection of resistant genotypes thus contributing to disease mitigation.

Keywords: biotic stress response; disease differential susceptibility; forest tree disease; gene expression; plant hormones; plant physiology; plant primary metabolism.

FIGURE 1

Progression of pitch canker disease in Pinus. (A) Time course of the percentage of plants inoculated with F. circinatum showing symptoms for each pine species. Sampling points were carried out individually for each species when at least 50% of the inoculated plants presented symptoms (red points). Images representative of the symptoms at each sampling point are presented. Days post-infection (d.p.i.). (B) Stem relative internal necrosis length of non-inoculated controls (black bars) and of plants inoculated with F. circinatum (grey bars) when 50% of the inoculated plants of each species expressed disease symptoms. Data are presented as mean ± SE. Asterisks on grey columns indicate significant differences between treatments for the correspondent species (p ≤ 0.05). (C) Stem internal necrosis representative of non-inoculated controls (left column) and plants inoculated with F. circinatum (right column) of each pine species observed using a zoom stereomicroscope when 50% of the inoculated plants of each species expressed disease symptoms.

FIGURE 2

Plant water status of non-inoculated controls (black bars) and plants inoculated with F. circinatum (grey bars) when 50% of the inoculated plants of each species expressed disease symptoms. (A) Midday water potential. (B) Relative water content. Data are presented as mean ± SE. Asterisks on the grey bars indicate significant differences between treatments for the correspondent species (p ≤ 0.05).

FIGURE 3

Needle gas exchange-related parameters of non-inoculated controls (black bars) and plants inoculated with F. circinatum (grey bars) when 50% of the inoculated plants of each species expressed disease symptoms. (A) Stomatal conductance. (B) Transpiration rate. (C) Net CO₂ assimilation rate. (D) Sub-stomatal CO₂ concentration. Data are presented as mean ± SE. Asterisks on grey bars indicate significant differences between treatments for the correspondent species (p ≤ 0.05).

FIGURE 4

Hormones concentration of non-inoculated controls (black bars) and plants inoculated with F. circinatum (grey bars) when 50% of the inoculated plants of each species expressed disease symptoms. (A) Abscisic acid. (B) Salicylic acid. (C) Jasmonic acid. Data are presented as mean ± SE. Asterisks on grey bars indicate significant differences between treatments for the correspondent species (p ≤ 0.05).

FIGURE 5

Relative abundance of primary metabolism- and pathogenesis-related genes in Pinus inoculated with F. circinatum with respect to their non-inoculated controls when 50% of the inoculated plants of each species expressed disease symptoms. (A) Ribulose 1,5-biphosphate carboxylase/oxygenase small subunit (RuBisCO). (B) Cell wall invertase (cwINV). (C) (Cytosolic) Glucose-6-phosphate dehydrogenase (G6PDH). (D) Pyruvate decarboxylase (PDC). (E) Glycolate oxidase (GOX). (F) Sucrose non-fermenting 1-related protein kinase 2.6 (SnRK2.6). (G) Phenylalanine ammonia lyase (pal). (H) Thaumatin-like protein (pr5). (I) Chitinase (pr3). The abundance of transcripts in plants inoculated with F. circinatum for each pine species is represented considering its correspondent non-inoculated controls. Data are presented as mean ± SE. Asterisks indicate significant differences between non-inoculated controls and plants inoculated with F. circinatum for each species (p ≤ 0.05).

FIGURE 6

Primary metabolite changes occurring in Pinus inoculated with F. circinatum with respect to their non-inoculated controls when 50% of the inoculated plants of each species expressed disease symptoms. Relative values are normalised to the internal standard (ribitol) and dry weight (DW) of the samples. Significant changes are indicated as ° P < 0.05, with respect to controls for each one of the species represented in the three different squares. False-colour imaging was performed on log₁₀-transformed GC-TOF-MS data. Grey-colour square represents not detected (n.d.) value.

FIGURE 7

Integrated data analysis. (A) Sparse partial least squares (sPLS) regression analysis of the complete dataset of physiological, hormonal, gene expression and primary metabolism alterations occurring in Pinus inoculated with F. circinatum and their respective non-inoculated controls when 50% of the inoculated plants of each species expressed disease symptoms. First two components are plotted in the graph. (B) Correlation circle plot of the components represented in the sPLS regression analysis. Gene expression levels (blue) were used as predictor matrix for metabolite and physiological responses (orange).