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A delayed response in phytohormone signaling and production contributes to pine susceptibility to Fusarium circinatum

BMC Plant Biology volume 24, Article number: 727 (2024) Cite this article

Abstract

Background

Fusarium circinatum is the causal agent of pine pitch canker disease, which affects Pinus species worldwide, causing significant economic and ecological losses. In Spain, two Pinus species are most affected by the pathogen; Pinus radiata is highly susceptible, while Pinus pinaster has shown moderate resistance. In F. circinatum-Pinus interactions, phytohormones are known to play a crucial role in plant defense. By comparing species with different degrees of susceptibility, we aimed to elucidate the fundamental mechanisms underlying resistance to the pathogen. For this purpose, we used an integrative approach by combining gene expression and metabolomic phytohormone analyses at 5 and 10 days post inoculation.

Results

Gene expression and metabolite phytohormone contents suggested that the moderate resistance of P. pinaster to F. circinatum is determined by the induction of phytohormone signaling and hormone rearrangement beginning at 5 dpi, when symptoms are still not visible. Jasmonic acid was the hormone that showed the greatest increase by 5 dpi, together with the active gibberellic acid 4 and the cytokinin dehydrozeatin; there was also an increase in abscisic acid and salicylic acid by 10 dpi. In contrast, P. radiata hormonal changes were delayed until 10 dpi, when symptoms were already visible; however, this increase was not as high as that in P. pinaster. Indeed, in P. radiata, no differences in jasmonic acid or salicylic acid production were found. Gene expression analysis supported the hormonal data, since the activation of genes related to phytohormone synthesis was observed earlier in P. pinaster than in the susceptible P. radiata.

Conclusions

We determine that the moderate resistance of P. pinaster to F. circinatum is in part a result of early and strong activation of plant phytohormone-based defense responses before symptoms become visible. We suggest that jasmonic acid signaling and production are strongly associated with F. circinatum resistance. In contrast, P. radiata susceptibility was attributed to a delayed response to the fungus at the moment when symptoms were visible. Our results contribute to a better understanding of the phytohormone-based defense mechanism involved in the Pinus-F. circinatum interactions and provide insight into the development of new strategies for disease mitigation.

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Background

Pine pitch canker (PPC) disease, caused by the fungus Fusarium circinatum Nirenberg & O´Donnell, is known to affect more than 60 Pinus species [1], causing considerable ecological and economic losses in nurseries and forest plantations worldwide [1,2,3]. In Europe, the disease was officially reported in Spain in 2005 [4] and thereafter spread throughout its Atlantic coast, representing a serious threat to P. radiata and P. pinaster plantations. Symptomatic infected mature trees exhibit needle wilting and chlorosis, stem cankers, canopy dieback and mortality, while infected seedlings show wilting and damping-off in nurseries [2, 3]. The susceptibility of different Pinus species to the pathogen varies widely [1, 2, 5]. The exotic P. radiata, which is highly susceptible to the pathogen [5, 6], is widely planted in northern Spain because of its fast growth and good wood quality [7], contributing positively to the region’s economy. In contrast, P. pinaster, which is native to the Mediterranean area and economically significant for resin, timber and paper production, has shown moderate resistance to F. circinatum [6]. Despite efforts to find effective management measures, the pathogen has not yet been controlled in nurseries or forest plantations [8, 9].

In recent decades, research on PPC has focused on phenotypic studies in species with different degrees of susceptibility to fungal infection and on isolate pathogenicity [6, 10,11,12,13,14,15]. However, in recent years, advances in molecular techniques and bioinformatic tools have improved the genetic resources available in forest areas, representing great advances in the knowledge and management of tree diseases [16, 17]. Several transcriptomic studies highlighting the roles of pathogenesis-related (PR) proteins, reactive oxygen species (ROS) production, phytohormone signaling and secondary metabolism (phenylpropanoid pathway and terpenes) related to pine defense against PPC have been published [18,19,20,21,22,23].

Phytohormones act as signaling molecules that trigger molecular responses to biotic and abiotic stresses [24,25,26]. These phytohormones also play important roles in regulating plant responses, and cross-talk among different phytohormones is essential for establishing a balanced defenses response [26]. Defense against pathogens is known to be primarily coordinated by jasmonic acid (JA) and ethylene (ET), along with salicylic acid (SA) [27, 28]. This regulatory network has expanded over time to include additional hormones, such as indole acetic acid (IAA), abscisic acid (ABA), cytokinins (Ck) and gibberellic acid (GA), all of which have been implicated in plant defense mechanisms [26]. Regarding PPC, Visser et al. [22] suggested an earlier transcriptomic response based on JA, SA, ET and auxins in the more resistant P. tecunumanii and a delayed response in the susceptible P. patula. The susceptible P. radiata has also shown a weaker induction of phytohormone-related transcripts compared to the resistant P. pinea, possibly due to impaired perception of the fungus [21]. The upregulation of ABA signaling-related genes also occurs in P. radiata under F. circinatum challenge, and its involvement in stomatal opening and closure has been discussed [18]. Davis et al. [29] reported SA and JA induction of chitinases of the PR3 family in P. elliotti-resistant genotypes inoculated with F. circinatum.

In a previous study [20], we determined the transcriptome profile of P. pinaster under F. circinatum challenge by a dual RNA sequencing (RNAseq) assay. From this study, we hypothesized that the moderate resistance shown by P. pinaster may be explained by recognition of the pathogen from as early as 3 dpi, the induction of pathogenesis-related genes and the activation of complex phytohormone signaling pathways that include mainly SA, JA and ET. We also suggested that F. circinatum manipulates the host phytohormone balance to its own benefit through the expression of genes related to hormone biosynthesis. We hypothesized three key steps of host manipulation: perturbing ET signaling via the fungal expression of genes related to ET biosynthesis, blocking JA signaling via coronatine insensitive 1 (COI1) suppression, and disturbing SA biosynthesis from the chorismate pathway via the synthesis of isochorismatase family hydrolase (ICSH) genes. The blocking of JA signaling by F. circinatum has also been proposed by Zamora-Ballesteros et al. [21]. Taken together, these findings suggest the importance of a phytohormone-based defense response against F. circinatum infection in Pinus species at the transcriptomic level.

In addition to transcriptomics, other ‘omic sciences provide a massive amount of information at various molecular levels, including information about DNA, proteins and metabolites. Together, they provide a holistic view of the biological system under study. However, changes such as epigenetic or posttranslational regulatory mechanisms affect different layers of the ‘omic cascade, altering gene expression and ultimately metabolite production [17, 30]. Therefore, only through an ‘omic-based integrative approach exploring the variation of genetic resistance of pine species with different degrees of susceptibility to F. circinatum we will reveal the biological complexity of the interaction. Metabolomic analysis of the response of Pinus to F. circinatum revealed that ABA catabolism is a key defense mechanism, with the accumulation of inactive dihydrophaseic acid (DPA) in the needles of susceptible P. radiata and weakly ABA-like active phaseic acid (PA) in the resistant P. pinea [31]. The ABA concentration increased in both P. radiata and P. pinaster seedlings upon inoculation [32, 33]. By immunolocalization, ABA and JA accumulation were detected in needles of P. radiata- and P. pinea-inoculated seedlings, respectively [34]. In addition to hormones, the quantification of other metabolites in response to F. circinatum revealed an increase in monoterpenes and diterpenes in P. radiata, whereas P. pinaster showed no significant changes [35]. Several amino acids accumulate in both P. radiata and P. pinaster upon inoculation, which is attributed to the induction of secondary metabolite production involved in the defense response [32].

In the present study, we combined gene expression and metabolomic phytohormone analyses to investigate variations in two pine species exhibiting different susceptibility levels to F. circinatum: P. pinaster (with moderate resistance) and P. radiata (highly susceptible). Through an integrated approach encompassing gene expression and metabolomics, we will provide a comprehensive understanding of the genetic and hormonal foundations underlying resistance to F. circinatum in these pine species. Based on our findings from a previous transcriptomic study [20], we focused our current research on the activation of the defense response at 5 and 10 days post inoculation (dpi).

Results

Differences in susceptibility to F. circinatum among Pinus species

At the time of sampling (5 dpi), no lesions were observed, while all the seedlings showed lesions at the inoculation site by 10 dpi and needle wilting in P. radiata seedlings. Significant differences were detected between species at 21 dpi, with lesion lengths of 3.27 ± 0.31 cm (mean ± standard deviation) and 2.38 ± 0.48 cm for P. radiata and P. pinaster, respectively (p value = 0.024).

Hormonal response of P. pinaster and P. radiata to infection and wounding

Principal component analysis (PCA), based on the normalized hormone abundances, was performed to determine differences between treatments (Fig. 1). The first two components (PC1 and PC2) explained 79.7% of the total variance, with a major effect associated with PC1 (63.6% of the variance), while PC2 explained 16.1% of the total variance. Separation according to PC1 clustered samples based on their treatment as follows: all unwounded (UW) samples from both pine species were grouped together in one extreme axis, and the other axis was P. pinaster inoculated at 10 dpi. Mock-inoculated (MI) seedlings were grouped together and localized close to the inoculated P. radiata seedlings at 5 dpi. Hierarchical clustering of hormone content in response to treatments (Fig. 2) classified the seedlings into three clusters: (1) all UW seedlings at 5 and 10 dpi; (2) all MI seedlings, and the seedlings inoculated with P. radiata at 5 dpi; and (3) the groups inoculated with P. pinaster at 5 and 10 dpi and those inoculated with P. radiata at 10 dpi. This plot revealed a delayed response of the hormone abundance of infected P. radiata, which clustered at 5 dpi with the MI seedlings, and it was within the group of inoculated P. pinaster only at 10 dpi. The highest hormone content was detected in inoculated P. pinaster at 10 dpi and for JA at 5 dpi (as indicated by the relatively high red color intensity).

Fig. 1
figure 1

Principal component analysis (PCA) of P. pinaster (P) and P. radiata (R) seedlings inoculated with F. circinatum (I), mock-inoculated (MI) and unwounded (UW) at 5 and 10 days post inoculation. Classes: I5 and I10 for inoculation at 5 and 10 dpi; MI5 and MI10 for mock inoculation at 5 and 10 dpi; and UW5 and UW10 for unwounding at 5 and 10 dpi; P and R for P. pinaster and P. radiata, respectively

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Fig. 2
figure 2

Hormone quantification heatmap of the P. pinaster and P. radiata datasets. Classes: I5 and I10 for inoculation at 5 and 10 dpi; MI5 and MI10 for mock inoculation at 5 and 10 dpi; and UW5 and UW10 for unwounding at 5 and 10 dpi; P and R for P. pinaster and P. radiata, respectively. GA4: gibberellic acid 4; ABA: abscisic acid; JA: jasmonic acid; IAA: indole acetic acid; SA: salicylic acid; DHZ: dehydrozeatin; tZ: trans-zeatin; iP: isopentenyladenine base

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The PCA-based biplot for P. pinaster and P. radiata highlights the separation of the UW seedlings inoculated at 10 dpi for both Pinus species by PC1, representing 70% and 58%, respectively, of the total variance (Fig. 3). Unlike those of P. radiata, the inoculated seedlings of P. pinaster at 5 dpi did not show a well-formed group, but they were clearly separated from the MI seedlings. GA4, dehydrozeatin (DHZ) and JA were the hormones that most influenced the inoculated separation of the P. pinaster-inoculated seedlings, as did ABA but to a lesser extent. P. radiata inoculation at 10 dpi was strongly correlated with GA4, DHZ and JA based on PC1.

Fig. 3
figure 3

Principal component analysis (PCA) biplots derived from the relative abundances of phytohormones in P. pinaster (P) and P. radiata (R) seedlings inoculated with F. circinatum (I), mock-inoculated (MI) and unwounded (UW) at 5 and 10 days post inoculation. GA4: gibberellic acid 4; ABA: abscisic acid; JA: jasmonic acid; IAA: indole acetic acid; SA: salicylic acid; DHZ: dehydrozeatin; tZ: trans-zeatin; iP: isopentenyladenine base. A: P. pinaster; B: P. radiata

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The effects of F. circinatum infection and wounding on P. pinaster and P. radiata seedlings at 5 and 10 dpi were analyzed with a mixed model (Table 1). The results showed that the effects due to ‘treatment’, ‘species’ and ‘dpi’ were significant for all phytohormones, with the exception of ‘dpi’ for the JA content and ‘species’ for tZ. Pairwise comparisons of the phytohormone contents among species and treatments revealed an earlier response to the pathogen in P. pinaster than in P. radiata. In P. pinaster, the JA, GA4 and DHZ metabolite contents increased in response to fungal inoculation as early as 5 dpi, while in P. radiata, only GA4 and DHZ increased but at 10 dpi (Fig. 4C_JA, GA4, Cks). At this time, the GA4 and DHZ contents were 2- and 4-fold greater, respectively, in P. pinaster. The ABA content increased in both Pinus species but increased 2-fold in P. pinaster (Fig. 4C_ABA). Notably, the JA content in the inoculated P. pinaster at 5 dpi was 65- and 268-fold greater than that in the MI and UW treatments, respectively (Fig. 4C_JA). The SA content increased in the P. pinaster-inoculated samples by 10 dpi (3-fold greater than the MI and UW seedlings), while no differences were found in the SA content of P. radiata (Fig. 4C_SA). None of these phytohormones showed any significant change in response to wounding (no significant differences were observed between the UW and MI treatments). However, a significant effect of wounding was observed for indole acetic acid (IAA), isopentenyladenine base (iP) and trans-zeatin (tZ) (Fig. 5). Both pine species reduced the IAA content in response to wounding at 5 and 10 dpi, while tZ increased in the MI seedlings at 10 dpi. iP increased at 5 and 10 dpi in P. radiata MI seedlings but only at 5 dpi in P. pinaster.

Fig. 4
figure 4

Abscisic acid (ABA), cytokinins (CKs), gibberellic acid (GAs), jasmonic acid (JA), and salicylic acid (SA) production and related gene expression in Pinus pinaster and Pinus radiata under Fusarium circinatum infection. Figure 4A: Heatmap representing differentially expressed genes in P. pinaster under F. circinatum challenge at 3, 5 and 10 days post inoculation (dpi) as determined by RNA-seq. Figure 4B: The relative expression of candidate genes in P. pinaster and P. radiata seedlings inoculated with F. circinatum at 5 dpi as determined by RT‒qPCR. LOX: lipoxygenase; COI1: coronatine-insensitive protein 1; NCED: 9-cis-epoxy-carotenoid dioxygenase; ASR: abscisic acid-stress-ripening; SN: Snakin/GASA Gibberellin regulated protein; GID: gibberellin receptor; CkGT: cytokinin-O-glucosyltransferase; CkZb: cytokinin hydroxylase; PAD4: lipase; ICS: isochorismatase synthase family. Significant differences (p value < 0.05) between inoculated and mock-inoculated seedlings are marked with *. Figure 4C: Phytohormone content (ng of hormone per g of fresh weight) in P. pinaster and P. radiata seedlings unwounded (UW), mock-inoculated (MI) and inoculated with F. circinatum (I) at 5 and 10 dpi. JA, jasmonic acid; ABA, abscisic acid; GA, gibberellic acid; CKs, cytokinins; DHZ, dehydrozeatin; SA, salicylic acid. The different letters above the columns indicate significant differences between treatments (p value < 0.05)

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Table 1 Type 3 tests for fixed effects in a mixed model including species, treatment and days postinoculation
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Fig. 5
figure 5

Indole acetic acid (IAA), isopentenyladenine base (iP), and trans-zeatin (tZ) phytohormone contents in P. pinaster and P. radiata seedlings at 5 and 10 dpi. The data are expressed as ng of hormone per g of fresh weight. The different letters indicate significant differences between samples (p value < 0.05)

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A further comparison between the inoculated and MI treatments at each dpi for each Pinus species highlighted the quick activation of the P. pinaster phytohormone-based response to F. circinatum. The log2−fold change (FC) values of GA4, ABA, DHZ and JA were significantly greater in the inoculated P. pinaster seedlings compared to MI from as early as 5 dpi (Fig. 6A). Notably, JA significantly increased by 5 dpi, with a log2(FC) value of 6 (Fig. 6A). Among these phytohormones, only DHZ increased in P. radiata at this time point, with a value similar to that of P. pinaster (Fig. 6B). At 10 dpi, DHZ, JA, GA4 and ABA increased due to fungal infection in both Pinus species, with higher values in P. pinaster, except for GA4 (Fig. 6A and B). Compared with those in the MI seedlings, the SA and iP contents in the inoculated P. pinaster samples increased by 10 dpi (Fig. 6A).

Fig. 6
figure 6

Hormone content (represented as Log2FC) in infected seedlings relative to mock-inoculated seedlings of P. pinaster (A) and P. radiata (B) at 5 and 10 dpi. An asterisk (*) indicates a fold change (FC) greater than |1.5| that is also statistically significant (p value < 0.05).value GA4: gibberellic acid 4; ABA: abscisic acid; JA: jasmonic acid; IAA: indole acetic acid; SA: salicylic acid; DHZ: dehydrozeatin; tZ: trans-zeatin; iP: isopentenyladenine base. A: P. pinaster; B: P. radiata

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The constitutive hormone content refers to the hormone content in UW seedlings, and both Pinus species had similar compositions. ABA, IAA and SA were the most abundant, followed by JA, with Ck (DHZ, iP and tZ) and GA4 being less abundant (Figs. 4C and 5). Significant differences were found only for iP in P. pinaster, with a greater difference at 5 dpi (Fig. 5).

Updating P. pinaster transcriptomic hormone response to F. circinatum

We revisited the transcriptome dataset of P. pinaster under F. circinatum infection at 3, 5 and 10 dpi determined previously using a dual RNAseq assay [20]. In this work, we reported that JA, ET and SA play major roles in the P. pinaster defense response. A deeper study focused on hormones revealed new differentially expressed genes (DEGs) related to GA, ABA and Ck phytohormones. A total of 45 new DEGs were identified and added to the set of 234 DEGs initially identified (Fig. 7, Additional Table 1). With respect to ABA signaling, eight ASR genes (abscisic stress ripening proteins) and seven abscisic acid receptor genes were DEGs and were mostly upregulated at 5 and 10 dpi. Several GA-regulated proteins (GASA, SN2) and a GA receptor (GID1), as well as several GA20 oxidase genes, were also upregulated at 5 and 10 dpi. Within Cks, some zeatin biosynthesis-related genes were DEGs and were up- and downregulated at 5 and 10 dpi. These newly identified DEGs suggest that not only JA, SA, and ethylene but also ABA and GA are key players in the P. pinaster transcriptomic response to F. circinatum (Fig. 4A).

Fig. 7
figure 7

Total number of differentially expressed genes (DEGs) from dual RNA sequencing analysis of P. pinaster under F. circinatum infection at 3, 5 and 10 days post inoculation (dpi) (updated from Hernandez-Escribano et al. [20]). Up- or downregulated genes were determined by comparing the expression levels in inoculated samples with those in the mock-inoculated samples at each dpi. BR: brassinosteroid; CKs: cytokinins; GA: gibberellin; ABA: abscisic acid; Aux: auxins; SA: salicylic acid; ET: ethylene; JA: jasmonic acid

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Relative expression of genes related to phytohormones by RT‒qPCR

Two candidate genes for each of the quantified hormones (JA, ABA, GA, Cks and SA) were selected for relative expression analysis in P. pinaster and P. radiata. The expression of these genes differed between inoculated and MI samples at 5 dpi according to the pine species considered (Fig. 4B). In P. pinaster, F. circinatum infection led to an increase in the abundance of transcripts related to JA (LOX, COI1), ABA (ASR, NCED), GA4 (GID, SN) and DHZ (CkZb) signaling. In P. radiata, only the relative expression of LOX, ASR, SN and CkZb increased due to fungal infection. For both pine species, SN was the gene with the greatest fold change, at 417-fold for P. pinaster, while its expression in P. radiata was observed only in inoculated samples (with almost no expression in MI samples) (Fig. 4B_GAs). The relative expression of all other genes was greater in P. pinaster. No significant differences were found in the relative expression of SA-related genes at 5 dpi in either of the pine species (Fig. 4B_SA).

Stomatal conductance of needles

Fusarium circinatum did not significantly alter the stomatal conductance of the pine seedlings (Fig. 8). Nevertheless, both the inoculated and mock-inoculated P. pinaster and P. radiata seedlings exhibited reduced stomatal conductance compared to the unwounded treatment, but were not significant at the 0.05 statistical level.

Fig. 8
figure 8

Stomatal conductance (expressed in mmol/m2 s) of P. pinaster and P. radiata needles at 10 days after treatment. UW: unwounded; MI: mock-inoculated; I: inoculated with F. circinatum

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Discussion

In this work, we studied the hormone content in P. pinaster and P. radiata as a defense response to F. circinatum. The combination of gene expression and hormonal analyses indicated that the moderate resistance of P. pinaster to F. circinatum is associated with the prompt activation of a phytohormone-based defense response, based mainly on JA. This phenomenon becomes evident as early as 5 dpi, preceding the onset of initial symptoms. In contrast, the susceptibility exhibited by P. radiata could be attributed at least in part to a delayed response to fungal infection, mainly at the moment when symptoms are visible (10 dpi). The phytohormone abundance data based on the clustering analysis and PCA support this hypothesis since the P. radiata-inoculated samples at 5 dpi clustered together with the MI samples, suggesting that the response to wounding was greater than that to the pathogen at this time point. Indeed, the heatmap color intensity of the P. pinaster-inoculated group indicated that a substantial increase in hormone content had already occurred in response to F. circinatum at 5 dpi and increased from 5 to 10 dpi.

JA is often associated with defense against herbivores and necrotrophic pathogens and usually acts in synergy with ET [36]. Fusarium circinatum is a hemibiotrophic fungus with a necrotrophic phase when infecting Pinus seedlings [37]. Interestingly, the increase in JA in inoculated P. pinaster seedlings at 5 dpi and the slight increase in the susceptible P. radiata seedlings by 10 dpi (Fig. 6) is in agreement with previous results. The JA concentration increased in the needles of P. pinaster seedlings infected with F. circinatum by 17 dpi [32], the time at which at least 50% of the inoculated seedlings display symptoms, while no changes were observed in the inoculated P. radiata seedlings by 10 dpi [32]. However, our results revealed an earlier increase in JA in the stems of inoculated P. pinaster seedlings by 5 dpi, before symptoms became visible (Fig. 4C_JA). In a recent study, using the hormone immunolocalization technique, the JA signal was shown to increase rapidly in the needles of resistant P. pinea under F. circinatum infection from 2 h post inoculation to 10 dpi and was maintained during the time course [34]. JA accumulation as a plant response to other pathogens has also been reported, as in needles and roots of P. sylvestris under infection by the pathogen Diplodia pinea [38].

The transcriptomic data also supported the involvement of JA in the moderate resistance of P. pinaster to PPC. We previously reported the upregulation of several genes related to JA biosynthesis (LOX, OPR, AOC, AOS) and signaling (COI1, TPL, NINJA, JMT) from the early stage at 3 dpi [20]. In the present study, the RT‒qPCR results showed an increase in COI1 and LOX transcripts in P. pinaster seedlings at 5 dpi (Fig. 4B_JA). COI1 is the receptor that perceives the active form of JA, jasmonoyl-isoleucine (JA-Ile), and LOX is a lipoxygenase enzyme that catalyzes the essential steps in JA biosynthesis [39, 40]. In contrast, COI1 expression did not change in the susceptible P. radiata at 5 dpi, suggesting that no signaling occurred at this time point. Similarly, Zamora-Ballesteros et al. [21] determined that the number of genes involved in JA signaling was considerably greater in resistant P. pinea than in susceptible P. radiata during F. circinatum infection. Taken together, these findings emphasize the importance of JA in the response of P. pinaster to PPC beginning in the early stages of infection, even before the first symptoms become visible. We propose that JA is a key hormone in the early defense response of P. pinaster against F. circinatum that contributes to pine resistance.

In contrast to JA, SA is commonly involved in the response to biotrophic or hemibiotrophic fungi [36, 41,42,43]. The antagonistic effects of JA and SA have been largely reported [41, 44,45,46], but crosstalk between these two phytohormones and cooperation rather than antagonism have also been reported [47,48,49]. Previous studies did not report any changes in the SA content in P. pinea, P. pinaster or P. radiata under F. circinatum infection at 64, 17 and 10 dpi, respectively, when 50% of the seedlings displayed symptoms [32, 34]. Our findings do not support these results, possibly because of factors such as the sampling time or seedling age. We showed that the SA content increased at 10 dpi in P. pinaster due to pathogen infection, which was the time at which we detected the first symptoms at the inoculation site (Fig. 4C_SA). By RT‒qPCR, we detected no differences in the relative expression of the two genes related to SA at 5 dpi in any of the pine species (Fig. 4B_SA). However, in a deeper study based on transcriptomic data analysis, we found several DE genes related to SA biosynthesis and signaling in P. pinaster, mainly at 5 and 10 dpi [20]. This could explain the accumulation of SA we detected in P. pinaster at 10 dpi. We previously reported the upregulation of the isochorismatase family hydrolase (ICSH) gene in F. circinatum following P. pinaster infection, and proposed that F. circinatum could manipulate SA signaling through the chorismatase pathway [20]. In this context, the susceptibility of P. radiata may be due in part to its inability to overcome this imbalance. However, whether F. circinatum manipulates host hormone homeostasis requires further studies using knockout mutants. Taken together, these findings collectively indicate that SA likely plays a role in the defense response of P. pinaster but not in the initial stage.

The role of ABA as a positive [50] or negative [51, 52] regulator of plant defense has been reported. ABA accumulation in P. pinaster and P. radiata was detected when symptoms were visible upon F. circinatum infection [32], which is consistent with the results presented here. Furthermore, we found that the increase in ABA content at 10 dpi was more pronounced in P. pinaster than in P. radiata (Fig. 4C_ABA), and the ABA content in inoculated P. radiata seedlings did not increase significantly until 10 dpi compared to the MI seedlings, contributing to an earlier response in P. pinaster compared to P. radiata (Fig. 6). RNA-seq analysis [20] suggested that ABA signaling does not seem to play a major role in the P. pinaster response to F. circinatum. However, reinspection of the data revealed new upregulated transcripts related to ABA biosynthesis and signaling (Additional Table 1; Fig. 7). Similarly, ABA signaling-related genes were DEGs in P. radiata and P. pinea upon F. circinatum inoculation [18, 21]. The results of the RT‒qPCR analysis at 5 dpi revealed the induction of both the ASR and NCED genes in the inoculated P. pinaster seedlings, while only ASR was induced in the P. radiata seedlings, and the relative expression of these genes was decreased (Fig. 4B_ABA). These results suggest a stronger ABA-mediated response in P. pinaster than in P. radiata.

A delayed hormone response in P. radiata compared to P. pinaster under F. circinatum challenge was also detected for GA4 (Fig. 4C_GAs). In the metabolomics analysis of plant-pathogen interactions, the difficulty of discriminating between plant and pathogen metabolites poses a particular challenge [53]. Indeed, some fungi-produced hormones, such as ABA, GA and ET, are known to contribute to pathogenicity [54]. Fusarium circinatum belongs to the F. fujikuroi species complex and is known to produce GA, resulting in host hormone imbalance and contributing to plant disease [55, 56]. Unlike metabolites, transcripts depend on a known genetic sequence and allow the identification of the organism of origin [57]. We identified genes related to GA biosynthesis and signaling via in silico analysis of P. pinaster and F. circinatum transcriptomes during the infection process, suggesting that GA is produced not only by the plant but also by the fungus [20]. By RT‒qPCR at 5 dpi, we found that the relative expression of both SN (Snakin Gibberellin regulated protein) and GID (Gibberellin receptor) increased in the moderately resistant P. pinaster, while the expression of only SN was increased in P. radiata. Remarkably, the relative expression of SN in both Pinus species increased in response to the fungus at this time point. Therefore, gene expression analysis related to GA supports the hormonal data and the delayed response observed in P. radiata. Whether a portion of the quantified metabolite GA4 originates from fungal metabolite production needs further investigation.

Cks are adenine derivatives with isoprenoid side chains that can be categorized as isopentenyl adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ) or dihydrozeatin (DHZ)-type derivatives depending on the hydroxylation of the isoprenoid side chains. Cks regulate numerous processes involved in plant growth and development [58, 59], including plant responses to abiotic and biotic stresses [60]. Among the three zeatin-type Cks (DHZ, tZ and iP) quantified in this study, the DHZ content increased due to fungal infection at both time points in P. pinaster but only at 10 dpi in P. radiata, indicating a delay in the hormonal response (Fig. 5). The difference in the iP content was not significant until 10 dpi and was greater in P. pinaster (Fig. 5). By RT‒qPCR, we found that the relative expression of CkZb increased in both Pinus species at 5 dpi (Fig. 4C_CKs). Although the role of Cks in plant defense is not clear, crosstalk with other phytohormones, such as SA and JA, has been proposed [60,61,62]. For example, Cks are needed for JA-mediated resistance of Arabidopsis to the necrotroph Alternaria alternata [63]. We observed an effect of wounding on Ck production: the iP content increased in P. radiata MI seedlings but decreased in P. pinaster seedlings at 5 dpi (Fig. 5), and the tZ content increased in response to wounding in both Pinus species at 10 dpi (Fig. 5). We also observed an inhibitory effect of wounding on IAA production in both Pinus species beginning at 5 dpi (Fig. 5). Regarding infection, the results showed that IAA does not play a major role in the response to F. circinatum.

Physiological changes regulated by hormonal metabolism have also been investigated in Pinus-F. circinatum interactions. Dieback and girdling cankers in branches with symptoms of PPC are known to occur as a result of water flow obstruction after F. circinatum infection [10, 64]. Under pathogen infection, plants are known to control transpirational water loss by regulating stomatal opening and closure, which is mainly controlled by ABA signaling [65]. Stomatal closure is also mediated by JA [66,67,68], and a role for Ck in preinvasive defense via the induction of stomatal closure has been documented [69]. Stomatal opening and increased transpiration rates in the highly resistant P. pinea upon inoculation with F. circinatum were reported [32], while stomatal closure and photosynthesis impairment were found in P. radiata and P. pinaster [32, 33, 70]. Although our data suggest a decrease in stomatal conductance in inoculated and MI P. pinaster and P. radiata seedlings by 10 dpi, no significant differences were found. Similarly, Zamora-Ballesteros et al. [21] reported that stomatal conductance was not influenced by the pathogen in P. radiata and P. pinea seedlings, although differences were found between species, with stomatal conductance being greater in P. pinea. The connection of ABA accumulation with stomatal conductance and its correlation with PPC disease has been a subject of discussion [34, 70].

The constitutive content of the studied phytohormones did not determine the outcome of the interaction, as indicated by the fact that no differences were found between P. pinaster and P. radiata in the unwounded seedlings, neither wounded or inoculated with F. circinatum. This suggests that the induction of these phytohormones is more closely related to fungal resistance/susceptibility than to the basal hormone content.

Methods

Plant material and fungal culture

Eight-month-old P. pinaster and P. radiata seedlings were used for the experiment and were obtained from Valladolid (Castilla y León, Spain) and Basque Country (Northern Spain) nurseries, respectively. Seedlings were maintained at 22 °C under a 14/10 h light/dark photoperiod and inoculated after one month of acclimation. Plants were irrigated regularly when needed.

Pine seedlings were inoculated with a virulent strain of F. circinatum isolated from an infected P. radiata tree located in Basque Country (isolate CECT20759, [13]). The fungus was grown on potato dextrose agar (PDA, Condalab, Madrid, Spain) plates at 22 °C in the dark for 6 days. A spore suspension was prepared by using a hemocytometer and adjusted to a final concentration of 5 × 105 spores/mL with sterile distilled water.

Inoculation and tissue sampling

Pinus pinaster and P. radiata seedlings were distributed into three groups: those inoculated with F. circinatum, those mock-inoculated with sterile distilled water (MI), and those neither wounded nor inoculated (UW). For inoculation, the first two cm of the shoot tip of each seedling in the inoculated or MI group was excised [20]. For inoculated seedlings, a 2 µl drop of the spore suspension was deposited in the wound, while sterile distilled water was used for the MI seedlings. Plants were covered with plastic bags during the first 24 h after inoculation to promote fungal infection.

Tissues were collected at 5 and 10 dpi. We selected these time points based on a previous transcriptomic assay in which the greatest changes in the expression of phytohormone-related genes were detected [20]. The experiment consisted of six different groups for each Pinus species: those inoculated at 5 and at 10 dpi, those mock-inoculated at 5 and at 10 dpi and those unwounded at 5 and at 10 dpi; the groups were named I5 and I10, MI5 and MI10, and UW5 and UW10, respectively. We used three biological replicates for each class, and each biological replicate consisted of a pool of eight seedlings. The top 1.5 cm of shoot tissue, from which the needles were removed, was harvested and immediately frozen in liquid nitrogen. For the UW seedlings, the first 2 cm was removed. The tissue was ground into powder with a mortar and pestle and maintained at -80 °C until use.

A set of five seedlings for each species was maintained for 21 days for visual observation to assess disease symptoms. At this time point, the lesion length was measured, and the shoot tips of all the seedlings were cultured in PDA media to verify the efficacy of the inoculation.

Plant hormone quantification in P. pinaster and P. radiata

Hormone quantification of P. pinaster and P. radiata samples was performed at the Institute for Plant Molecular and Cellular Biology (IBMCP-UPV-CSIC, Valencia, Spain) using a metabolomics platform. Eight hormones were quantified: jasmonic acid (JA), salicylic acid (SA), active gibberellic acid 4 (GA4), abscisic acid (ABA), indole acetic acid (IAA), the active cytokinins dehydrozeatin (DHZ), trans-zeatin (tZ) and isopentenyladenine base (iP).

Ground tissue (approximately 50–200 mg fresh weight) was suspended in 80% methanol/1% acetic acid containing internal standards and mixed by shaking for one hour at 4 °C. The extract was kept at -20 °C overnight and then centrifuged, and the supernatant was dried in a vacuum evaporator. The dry residue was dissolved in 1% acetic acid and passed through a reversed-phase column (HLB Oasis 30 mg, Waters) [71]). The extracts were additionally passed through an Oasis MCX (cation exchange). For GA4, IAA, ABA, SA and JA quantification, the dried eluates were eluted with 100% methanol/1% acetic acid to recover the acid fraction. In cases where the resolution of the peaks was poor, the extracts were further purified by passing through an Oasis WAX (ion exchange) column eluted with 80% methanol/1% acetic acid. Cytokinins (Cks) were eluted with 60% methanol/5% NH4OH from the Oasis MCX column to obtain the basic fraction containing Cks.

The final residues were dried and dissolved in 5% acetonitrile/1% acetic acid, and the hormones were separated by UHPLC with a reverse Accucore C18 column (2.6 μm, 100 mm length; Thermo Fisher Scientific) with an acetonitrile gradient containing 0.05% acetic acid at 400 µL/min for the acid hormones. For GA4, IAA, ABA, SA and JA, the gradient was 2 to 55% acetonitrile over 21 min. For Cks, the acetonitrile gradient was 2 to 25% over 13 min.

The hormones were analyzed with a Q-Exactive mass spectrometer (Orbitrap detector; Thermo Fisher Scientific) by targeted selected ion monitoring (tSIM; capillary temperature 300 °C, S-lens RF level 70, resolution 70.000) and electrospray ionization (spray voltage 3.0 kV, heater temperature 150 °C, sheath gas flow rate 40 µL/min, auxiliary gas flow rate 10 µL/min) in negative mode for acidic hormones or positive mode for Cks.

The concentrations of hormones in the extracts were determined using embedded calibration curves and the Xcalibur 4.0 and TraceFinder 4.1 SP1 programs. The internal standards for the quantification of each of the different plant hormones were deuterium-labeled hormones, except for JA, for which the compound dhJA was used (purchased from OlChemim Ltd., Olomouc, Czech Republic). For each sample, the final metabolite content was expressed as ng of hormone per g of fresh weight tissue.

Relative gene expression by RT?qPCR in P. pinaster and P. radiata

To characterize the DEGs in P. pinaster and P. radiata, ten candidate hormone-related genes were selected on the basis of the P. pinaster transcriptome under F. circinatum infection ( [20]; BioProject accession number PRJNA543723). The selected transcript IDs are found in Additional Table 1. Total RNA from inoculated and MI seedlings at 5 dpi was extracted using a Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Thorold, Ontario) and treated with a TURBO DNA-free kit (Thermo Fisher Scientific) following manufacturer’s instructions. The integrity and concentration of the RNA were measured using a Nanodrop (NanoDrop 2000, Thermo Fisher Scientific, MA, USA), and the RNA was stored at -80 °C until further use. cDNA was synthesized from 1 µg of total RNA using an iScript cDNA synthesis kit (Bio-Rad, Barcelona, Spain) following the manufacturer’s instructions.

Primer3Plus v.3.3.0 software [72] was used to design specific primers. Actin (ACT) [19] and ubiquitin (UBQ) [73] were selected as reference genes for normalizing the relative expression profiles of P. radiata and P. pinaster, respectively. The efficiency of all primers was checked, and primers with an efficiency less than 90% were discarded. The reactions were run on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA) with the SYBR Green (NZYSpeedy qPCR Green Master Mix 2x; NZYTech, Lisbon, Portugal) detection method. A 1:10 dilution of 2 µl of cDNA was added to 10 µl of SYBR Green mix. The PCR program consisted of an initial denaturation step (2 min at 97 °C) followed by 40 cycles of denaturation (5 s at 95 °C) and annealing (30 s at 60 °C). After amplification, a melting step was performed at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Three technical replicates of each of the three biological replicates were used. The FC of the relative gene expression between the inoculated and MIMI seedlings was calculated by the 2–∆∆Ct method [74].

Stomatal conductance of needles

The stomatal conductance (expressed in mmol/m2 s) of the needles was measured with a leaf porometer (Leaf Porometer Model SC-1, Decagon Devices) at 10 dpi. For each pine species, three seedlings for each treatment (UW, inoculated or MI) were measured. Each replicate consisted of three measurements of the needles of the apex. The back side of a pool of 5 needles was measured each time to cover the measurement surface of the porometer.

Statistical analysis

A mixed model was used to analyze the effect of ‘species’ (P. radiata, P. pinaster), ‘days post inoculation’ (5 dpi, 10 dpi) and ‘treatment’ (inoculated, MI or UW) on the total hormone content. All factors were considered fixed, and the model included two- and three-order interaction factors. Pairwise comparisons of the least square means for all effects were performed using Tukey’s post hoc test at a significance level of 0.05. Analysis was performed with SAS Studio 3.8 (SAS Institute Inc., Cary, NC, USA) (Table 1).

Metaboanalyst 5.0 software (https://www.metaboanalyst.ca) was used for the metabolome analysis. The data was previously normalized to adjust for systematic differences via logarithmic base 10 transformation and Pareto scaling [75]. The data were subjected to PCA to reveal the structure of the samples. A heatmap clustering analysis was conducted using each metabolite abundance based on the Euclidean distance and performed using the Ward clustering method. To better discriminate which phytohormones are produced in response to F. circinatum, the log2 FC of each phytohormone between the inoculated and MI groups was calculated at each dpi for each Pinus species. For this purpose, we set a log2 FC threshold of |1.5| and a significance level of 0.05.

For the RT‒qPCR data, Student’s’ t test was performed to estimate the significance difference between the normalized Ct values of the inoculated and MI samples for each gene in each pine species (p ≤ 0.05). Student’s t test was also used to determine significant differences in the final lesion length between the P. pinaster- and P. radiata-inoculated seedlings.

For the stomatal conductance data analysis, a mixed model was fitted that included ‘species’ (P. radiata, P. pinaster) and ‘treatment’ (inoculated, MI or UW) as fixed factors with two-factor interaction terms. Pairwise comparisons of the least square mean for all effects were performed using Tukey’s test at a significance level of 0.05.

Conclusions

We conclude that moderate P. pinaster resistance is, at least in part, a result of rapid and strong activation of phytohormone-based defense responses. In contrast, we propose that P. radiata susceptibility to F. circinatum is explained by a delayed response in gene activation associated with phytohormone biosynthesis and signaling, which ultimately leads to a postponed production of hormone metabolites. The selection of pine species exhibiting different susceptibility levels to PPC enables a comparative analysis of the molecular mechanisms associated with resistance and susceptibility. This valuable information can contribute to the identification of key factors involved in resistance, enhancing our understanding of Pinus-F. circinatum interactions. Such insights are crucial for developing effective and innovative disease control measures, thereby aiding in disease mitigation strategies related to PPC.

Data availability

Raw sequence data were accessed at NCBI BioProject accession PRJNA543723.

Abbreviations

SA:

salicylic acid, GAs: gibberellins, ET: ethylene

JA:

jasmonic acid

CKs:

cytokinins

IAA:

indole acetic acid

ABA:

abscisic acid

DHZ:

dehydrozeatin

GA4:

gibberellic acid 4

tZ:

trans-zeatin

iP:

isopentenyladenine base, dpi: days postinoculation

PPC:

pine pitch cankerMI: mock-inoculated

UW:

unwounded

PCA:

principal component analysis

FC:

fold change

DEG:

differentially expressed genes

PC:

principal component

LOX:

lipoxygenase

COI1:

coronatine-insensitive protein 1

NCDE:

9-cis-epoxy-carotenoid dioxygenase

ASR:

abscisic acid-stress-ripening

SN:

snakin/GASA

GID:

gibberellin receptor

CkGT:

Cytokinin-O-glucosyltransferase

CkZb:

cytonikin hydroxylase

PAS4:

lipase

ICS:

isochorismatase synthase family

ACT:

activin

UBQ:

ubiquitin

PR:

pathogenesis-related

ICSH:

isochorismatase family hydrolase

DPA:

dihydrophaseic acid: PA: phaseic acid

References

  1. Drenkhan R, Ganley B, Martín-García J, Vahalík P, Adamson K, Adamcíková K, et al. Global geographic distribution and host range of Fusarium Circinatum, the causal agent of pine pitch canker. Volume 11. Forests. MDPI AG; 2020. https://doi.org/10.3390/F11070724

  2. Wingfield MJ, Hammerbacher A, Ganley RJ, Steenkamp ET, Gordon TR, Wingfield BD, et al. Pitch canker caused by Fusarium circinatum - A growing threat to pine plantations and forests worldwide. Australas Plant Pathol. 2008;37(4):319–34. https://doi.org/10.1071/AP08036

    Article  Google Scholar 

  3. Martín-García J, Paraschiv M, Flores-Pacheco JA, Chira D, Diez JJ, Fernández M. Susceptibility of several northeastern conifers to Fusarium Circinatum and strategies for biocontrol. Forests. 2017;8(9). https://doi.org/10.3390/f8090318

  4. Landeras E, García P, Fernández Y, Braña M. Outbreak of pitch canker caused by Fusarium circinatum on Pinus spp. North Spain Plant Dis. 2005;89(9):1015. https://doi.org/10.1094/PD-89-1015A

    Article  CAS  Google Scholar 

  5. Iturritxa E, Mesanza N, Elvira-Recuenco M, Serrano Y, Quintana E, Raposo R. Evaluation of genetic resistance in Pinus to pitch canker in Spain. Australas Plant Pathol. 2012;41(6):601–7. https://doi.org/10.1007/s13313-012-0160-4

    Article  Google Scholar 

  6. Iturritxa E, Ganley RJ, Raposo R, García-Serna I, Mesanza N, Kirkpatrick SC, et al. Resistance levels of Spanish conifers against Fusarium Circinatum and Diplodia pinea. Pathol. 2013;43(6):488–95. https://doi.org/10.1111/efp.12061

    Article  Google Scholar 

  7. Mead DJ, editor. Mead. Sustainable management of Pinus radiata plantations. Rome: Italy; 2013. p. 246.

    Google Scholar 

  8. Bezos D, Martínez-Álvarez P, Fernández M, Diez JJ. Epidemiology and management of Pine Pitch Canker disease in Europe - A Review. Balt for. 2017;23(1):279–93.

    Google Scholar 

  9. Gordon TR, Swett CL, Wingfield MJ. Management of Fusarium diseases affecting conifers. Crop Prot. 2015;73:28–39. https://doi.org/10.1016/j.cropro.2015.02.018

    Article  Google Scholar 

  10. Martin-Rodrigues N, Espinel S, Sanchez-zabala J, Ort A, Gonz C. Spatial and temporal dynamics of the colonization of Pinus radiata by Fusarium Circinatum, of conidiophora development in the pith and of traumatic resin duct formation. 2013; https://doi.org/10.1111/nph.12222

  11. Swett CL, Gordon TR. Latent infection by Fusarium Circinatum influences susceptibility of Monterey Pine Seedlings to Pitch Canker. 2011;159–61.

  12. Gordon TR, Kirkpatrick SC, Aegerter BJ, Fisher a J, Storer, Wood aJ. DL. Evidence for the occurrence of induced resistance to pitch canker, caused by Gibberella circinata (anamorph Fusarium circinatum), in populations of Pinus radiata. For Pathol. 2011; 41(3):227–32. https://doi.org/10.1111/j.1439-0329.2010.00678.x

  13. Iturritxa E, Ganley RJ, Wright J, Heppe E, Steenkamp ET, Gordon TR, et al. A genetically homogenous population of Fusarium circinatum causes pitch canker of Pinus radiata in the Basque Country, Spain. Fungal Biol. 2011;115(3):288–95. https://doi.org/10.1016/j.funbio.2010.12.014

    Article  PubMed  Google Scholar 

  14. Hernandez-Escribano L, Iturritxa E, Aragon A, Mesanza N, Berbegal M, Raposo R, et al. Root infection of canker pathogens, Fusarium Circinatum and Diplodia Sapinea, in asymptomatic trees in Pinus radiata and Pinus pinaster plantations. Forests. 2018;9(3):128. https://doi.org/10.3390/f9030128

    Article  Google Scholar 

  15. Elvira-Recuenco M, Pando V, Berbegal M, Muñoz AM, Iturritxa E, Raposo R. Influence of temperature and moisture duration on pathogenic life history traits of predominant haplotypes of Fusarium circinatum on Pinus spp. Spain Phytopathol. 2021;111(11):2002–9. https://doi.org/10.1094/PHYTO-10-20-0445-R

    Article  Google Scholar 

  16. Naidoo S, Visser EA, Zwart L, Du Toit Y, Bhadauria V, Shuey LS. Dual RNA-seq to elucidate the Plant– Pathogen Duel. Curr Issues Mol Biol. 2018;27:127–42. https://doi.org/10.21775/cimb.027.127

    Article  PubMed  Google Scholar 

  17. Amaral J, Valledor L, Alves A, Martín-García J, Pinto G. Studying tree response to biotic stress using a multi-disciplinary approach: the pine pitch canker case study. 13, Frontiers in Plant Science. Frontiers Media S.A.; 2022. https://doi.org/10.3389/fpls.2022.916138

  18. Carrasco A, Wegrzyn JL, Durán R, Fernández M, Donoso A, Rodriguez V, et al. Expression profiling in Pinus radiata infected with Fusarium Circinatum. Tree Genet Genomes. 2017;13(2). https://doi.org/10.1007/s11295-017-1125-0

  19. Donoso A, Rodriguez V, Carrasco A, Ahumada R, Sanfuentes E, Valenzuela S. Relative expression of seven candidate genes for pathogen resistance on Pinus radiata infected with Fusarium Circinatum. Physiol Mol Plant Pathol. 2015;92:42–50. https://doi.org/10.1016/j.pmpp.2015.08.009

    Article  CAS  Google Scholar 

  20. Hernandez-Escribano L, Visser EA, Iturritxa E, Raposo R, Naidoo S. The transcriptome of Pinus pinaster under Fusarium circinatum challenge. BMC Genomics. 2020;21(1). https://doi.org/10.1186/s12864-019-6444-0

  21. Zamora-Ballesteros C, Pinto G, Amaral J, Valledor L, Alves A, Diez JJ, et al. Dual RNA-sequencing analysis of resistant (Pinus pinea) and susceptible (Pinus radiata) hosts during Fusarium circinatum challenge. Int J Mol Sci. 2021;22(10). https://doi.org/10.3390/ijms22105231

  22. Visser EA, Wegrzyn JL, Steenkamp ET, Myburg AA, Naidoo S. Dual RNA-Seq analysis of the Pine-Fusarium Circinatum Interaction in resistant (Pinus tecunumanii) and susceptible (Pinus patula) hosts. Microorganisms. 2019;7(9):315. https://doi.org/10.3390/microorganisms7090315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Visser EA, Kampmann TP, Wegrzyn JL, Naidoo S. Multispecies comparison of host responses to Fusarium circinatum challenge in tropical pines show consistency in resistance mechanisms. Plant Cell Environ. 2022. https://doi.org/10.1111/pce.14522

    Article  PubMed  Google Scholar 

  24. Waadt R. Phytohormone signaling mechanisms and genetic methods for their modulation and detection. Current opinion in Plant Biology. Volume 57. Elsevier Ltd; 2020. pp. 31–40. https://doi.org/10.1016/j.pbi.2020.05.011

  25. Miller RNG, Alves GSC, Van Sluys MA. Plant immunity: unravelling the complexity of plant responses to biotic stresses. Ann Bot. 2017;119(5):681–7. https://doi.org/10.1093/aob/mcw284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bari R, Jones JDG. Role of plant hormones in plant defence responses. Plant Mol Biol. 2009;69(4):473–88. https://doi.org/10.1007/s11103-008-9435-0

    Article  CAS  PubMed  Google Scholar 

  27. Pieterse CMJ, Van der Does D, Zamioudis C, Leon-Reyes A, Van Wees SCM. Hormonal modulation of Plant Immunity. Annu Rev Cell Dev Biol. 2012;28(1):489–521. https://doi.org/10.1146/annurev-cellbio-092910-154055

    Article  CAS  PubMed  Google Scholar 

  28. Kunkel BN, Brooks DM. Cross talk between signaling pathways in pathogen defense. Current opinion in Plant Biology. Volume 5. Elsevier Ltd; 2002. pp. 325–31. https://doi.org/10.1016/S1369-5266(02)00275-3

  29. Davis JM, Wu H, Cooke JEK, Reed JM, Luce KS, Michler CH. Pathogen Challenge, Salicylic Acid, and Jasmonic Acid regulate expression of Chitinase Gene homologs in Pine. 2002;15(4):380–7. https://doi.org/10.1094/MPMI.2002.15.4.380

  30. Fiehn O. Metabolomics-the link between genotypes and phenotypes. Volume 48. Plant Molecular Biology; 2002.

  31. Amaral J, Ribeyre Z, Vigneaud J, Sow MD, Fichot R, Messier C et al. Advances and promises of epigenetics for forest trees. Vol. 11, Forests. MDPI AG; 2020. https://doi.org/10.3390/f11090976

  32. Amaral J, Correia B, António C, Rodrigues AM, Gómez-Cadenas A, Valledor L, et al. Pinus susceptibility to pitch canker triggers specific physiological responses in symptomatic plants: an integrated approach. Front Plant Sci. 2019;10. https://doi.org/10.3389/fpls.2019.00509

  33. Amaral J, Pinto G, Flores-Pacheco JA, Díez-Casero JJ, Cerqueira A, Monteiro P, et al. Effect of Trichoderma Viride pre-inoculation in pine species with different levels of susceptibility to Fusarium Circinatum: physiological and hormonal responses. Plant Pathol. 2019;68(9):1645–53. https://doi.org/10.1111/ppa.13080

    Article  CAS  Google Scholar 

  34. Amaral J, Correia B, Escandón M, Jesus C, Serôdio J, Valledor L, et al. Temporal physiological response of pine to Fusarium circinatum infection is dependent on host susceptibility level: the role of ABA catabolism. Tree Physiol. 2021;41(5):801–16. https://doi.org/10.1093/treephys/tpaa143

    Article  CAS  PubMed  Google Scholar 

  35. Lombardero MJ, Solla A, Ayres MP. Pine defenses against the pitch canker disease are modulated by a native insect newly associated with the invasive fungus. Ecol Manage. 2019;437:253–62. https://doi.org/10.1016/j.foreco.2019.01.041

    Article  Google Scholar 

  36. Glazebrook J. Contrasting Mechanisms of Defense Against Biotrophic and Necrotrophic Pathogens. Annu Rev Phytopathol. 2005;43(1):205–27. https://doi.org/10.1146/annurev.phyto.43.040204.135923. http://www.annualreviews.org/doi/

    Article  CAS  PubMed  Google Scholar 

  37. Swett CL, Kirkpatrick SC, Gordon TR. Evidence for a Hemibiotrophic Association of the Pitch Canker Pathogen Fusarium circinatum with Pinus radiata. Plant Dis. 2016;100(1):79–84.

    Article  CAS  PubMed  Google Scholar 

  38. Hu B, Mithöfer A, Reichelt M, Eggert K, Peters FS, Ma M, et al. Systemic reprogramming of phytohormone profiles and metabolic traits by virulent Diplodia infection in its pine (Pinus sylvestris L.) host. Plant Cell Environ. 2021;44(8):2744–64. https://doi.org/10.1111/pce.14061

    Article  CAS  PubMed  Google Scholar 

  39. Feys BJ, Benedetti CE, Penfold CN, Turner JG. Arabidopsis Mutants Selected for Resistance to the Phytotoxin Coronatine Are Male Sterile, lnsensitive to Methyl Jasmonate, and Resistant to a Bacterial Pathogen. Vol. 6, The Plant Cell. 1994. https://doi.org/10.1105/tpc.6.5.751

  40. Mueller MJ. Enzymes involved in jasmonic acid biosynthesis. Physiol Plant. 1997;100(3):653–63. https://doi.org/10.1111/j.1399-3054.1997.tb03072.x

    Article  CAS  Google Scholar 

  41. Truman W, Bennett MH, Kubigsteltig I, Turnbull C, Grant M. Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates. Proceedings of the National Academy of Sciences. 2007; 104(3):1075–80. http://www.pnas.org/lookup/doi/https://doi.org/10.1073/pnas.0605423104

  42. Ton J, Van Pelt JA, Van Loon LC, Pieterse CMJ. Differential Effectiveness of Salicylate-Dependent and Jasmonate/Ethylene-Dependent Induced Resistance in Arabidopsis. Mol Plant Microbe Interact. 2002;15(1):27–34. https://doi.org/10.1094/MPMI.2002.15.1.27

    Article  CAS  PubMed  Google Scholar 

  43. Vlot AC, Dempsey DA, Klessig DF. Salicylic acid, a multifaceted hormone to Combat Disease. Annu Rev Phytopathol. 2009;47(1):177–206. https://doi.org/10.1146/annurev.phyto.050908.135202

    Article  CAS  PubMed  Google Scholar 

  44. Loake G, Grant M. Salicylic acid in plant defence-the players and protagonists. Curr Opin Plant Biol. 2007;10(5):466–72. https://doi.org/10.1016/j.pbi.2007.08.008

    Article  CAS  PubMed  Google Scholar 

  45. Norton G, Pappusamy A, Yusof F, Pujade-Renaud V, Perkins M, Griffiths D, et al. Characterisation of recombinant Hevea brasiliensis allene oxide synthase: effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity. Plant Physiol Biochem. 2007;45(2):129–38. https://doi.org/10.1016/j.plaphy.2007.01.003

    Article  CAS  PubMed  Google Scholar 

  46. Thaler JS, Humphrey PT, Whiteman NK. Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci. 2012;17(5):260–70. https://doi.org/10.1016/j.tplants.2012.02.010

    Article  CAS  PubMed  Google Scholar 

  47. Arnerup J. Induced defence responses in Picea abies triggered by Heterobasidion annosum s. l. Vol. 46. 2011.

  48. Engelberth J, Viswanathan S, Engelberth MJ. Low concentrations of salicylic acid stimulate insect elicitor responses in Zea mays seedlings. J Chem Ecol. 2011;37(3):263–6. https://doi.org/10.1007/s10886-011-9926-3

    Article  CAS  PubMed  Google Scholar 

  49. Berrocal-Lobo M, Molina A. Ethylene Response factor 1 mediates Arabidopsis Resistance to the Soilborne Fungus Fusarium oxysporum. Mol Plant Microbe Interact. 2004;17(7):763–70. https://doi.org/10.1094/MPMI.2004.17.7.763

    Article  CAS  PubMed  Google Scholar 

  50. Mauch-Mani B, Mauch F. The role of abscisic acid in plant-pathogen interactions. Curr Opin Plant Biol. 2005;8(4):409–14. https://doi.org/10.1016/j.pbi.2005.05.015

    Article  CAS  PubMed  Google Scholar 

  51. Anderson JP. Antagonistic Interaction between Abscisic Acid and Jasmonate-Ethylene Signaling pathways modulates Defense Gene expression and Disease Resistance in Arabidopsis. Plant Cell Online. 2004;16(12):3460–79. https://doi.org/10.1105/tpc.104.025833. http://www.plantcell.org/cgi/doi/

    Article  CAS  Google Scholar 

  52. Asselbergh B, Achuo AE, Höfte M, Van Gijsegem F. Abscisic acid deficiency leads to rapid activation of tomato defence responses upon infection with Erwinia chrysanthemi. Mol Plant Pathol. 2008;9(1):11–24. https://doi.org/10.1111/j.1364-3703.2007.00437.x

    Article  CAS  PubMed  Google Scholar 

  53. Rodrigues AM, Ribeiro-Barros AI, António C. Experimental design and sample preparation in forest tree metabolomics. Vol. 9, Metabolites. MDPI AG; 2019. https://doi.org/10.3390/metabo9120285

  54. Chanclud E, Morel JB. Plant hormones: a fungal point of view. Molecular plant pathology. Volume 17. Blackwell Publishing Ltd; 2016. pp. 1289–97. https://doi.org/10.1111/mpp.12393

  55. Bömke C, Tudzynski B. Diversity, regulation, and evolution of the gibberellin biosynthetic pathway in fungi compared to plants and bacteria. Phytochemistry. 2009;70(15–16):1876–93. https://doi.org/10.1016/j.phytochem.2009.05.020

    Article  CAS  PubMed  Google Scholar 

  56. Studt L, Humpf HU, Tudzynski B. Signaling governed by G Proteins and cAMP is crucial for growth, secondary metabolism and sexual development in Fusarium fujikuroi. PLoS ONE. 2013;8(2). https://doi.org/10.1371/journal.pone.005818

  57. Tenenboim H, Brotman Y. Omic relief for the Biotically stressed: metabolomics of plant biotic interactions. Trends in Plant Science. Volume 21. Elsevier Ltd; 2016. pp. 781–91. https://doi.org/10.1016/j.tplants.2016.04.009

  58. Kieber JJ, Schaller GE. Cytokinin signaling in plant development. Dev (Cambridge). 2018;145(4). https://doi.org/10.1242/dev.149344

  59. Zürcher E, Müller B. Cytokinin Synthesis, Signaling, and function-advances and New insights. International Review of Cell and Molecular Biology. Elsevier Inc.; 2016. pp. 1–38. https://doi.org/10.1016/bs.ircmb.2016.01.001

  60. Cortleven A, Leuendorf JE, Frank M, Pezzetta D, Bolt S, Schmülling T. Cytokinin action in response to abiotic and biotic stresses in plants. Plant Cell and Environment. Volume 42. Blackwell Publishing Ltd; 2019. pp. 998–1018. https://doi.org/10.1111/pce.13494

  61. Igari K, Endo S, Hibara KI, Aida M, Sakakibara H, Kawasaki T, et al. Constitutive activation of a CC-NB-LRR protein alters morphogenesis through the cytokinin pathway in Arabidopsis. Plant J. 2008;55(1):14–27. https://doi.org/10.1111/j.1365-313X.2008.03466.x

    Article  CAS  PubMed  Google Scholar 

  62. O’Brien JA, Benková E. Cytokinin cross-talking during biotic and abiotic stress responses. 4, Frontiers in Plant Science. Frontiers Research Foundation; 2013. https://doi.org/10.3389/fpls.2013.00451

  63. Choi J, Huh SU, Kojima M, Sakakibara H, Paek KH, Hwang I. The cytokinin-activated transcription factor ARR2 promotes plant immunity via TGA3/NPR1-dependent salicylic acid signaling in Arabidopsis. Dev Cell. 2010;19(2):284–95. https://doi.org/10.1016/j.devcel.2010.07.011

    Article  CAS  PubMed  Google Scholar 

  64. Gordon TR, Storer AJ, Wood DL. The Pitch Canker Epidemic in California. Plant Dis. 2001;85(11):1128–39.

    Article  CAS  PubMed  Google Scholar 

  65. Lim CW, Baek W, Jung J, Kim JH, Lee SC. Function of ABA in stomatal defense against biotic and drought stresses. International Journal of Molecular Sciences. Volume 16. MDPI AG; 2015. pp. 15251–70. https://doi.org/10.3390/ijms160715251

  66. Yin Y, Adachi Y, Nakamura Y, Munemasa S, Mori IC, Murata Y. Involvement of OST1 protein kinase and PYR/PYL/RCAR receptors in methyl jasmonate-induced stomatal closure in Arabidopsis guard cells. Plant Cell Physiol. 2016;57(8):1779–90. https://doi.org/10.1093/pcp/pcw102

    Article  CAS  PubMed  Google Scholar 

  67. Yan S, McLamore ES, Dong S, Gao H, Taguchi M, Wang N, et al. The role of plasma membrane H+-ATPase in jasmonate-induced ion fluxes and stomatal closure in Arabidopsis thaliana. Plant J. 2015;83(4):638–49. https://doi.org/10.1111/tpj.12915

    Article  CAS  PubMed  Google Scholar 

  68. Ghorbel M, Brini F, Sharma A, Landi M. Role of jasmonic acid in plants: the molecular point of view. Plant Cell Rep Springer Sci Bus Media Deutschland GmbH. 2021. https://doi.org/10.1007/s00299-021-02687-4

    Article  Google Scholar 

  69. Arnaud D, Lee S, Takebayashi Y, Choi D, Choi J, Sakakibara H, et al. Cytokinin-mediated regulation of reactive oxygen species homeostasis modulates stomatal immunity in Arabidopsis. Plant Cell. 2017;29(3):543–59. https://doi.org/10.1105/tpc.16.00583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Cerqueira A, Alves A, Berenguer H, Correia B, Gómez-Cadenas A, Diez JJ, et al. Phosphite shifts physiological and hormonal profile of Monterey pine and delays Fusarium circinatum progression. Plant Physiol Biochem. 2017;114:88–99. https://doi.org/10.1016/j.plaphy.2017.02.020

    Article  CAS  PubMed  Google Scholar 

  71. Seo M, Jikumaru Y, Kamiya Y. Profiling of hormones and related metabolites in seed dormancy and germination studies. Methods in Molecular Biology. Humana Press Inc.; 2011. pp. 99–111. https://doi.org/10.1007/978-1-61779-231-1_7

  72. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 2007;35(SUPPL2):71–4. https://doi.org/10.1093/nar/gkm306

    Article  Google Scholar 

  73. Sánchez M, Gianzo C, Sampedro J, Revilla G, Zarra I. Changes in a-Xylosidase during Intact and Auxin-Induced Growth of Pine Hypocotyls. 44, Plant Cell Physiol. 2003. https://academic.oup.com/pcp/article/44/2/132/1844176

  74. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-Tim e quantitative PCR and the 2 C T method. Methods. 2001;2(5):402–8.

    Article  Google Scholar 

  75. van den Berg RA, Hoefsloot HCJ, Westerhuis JA, Smilde AK, van der Werf MJ. Centering, scaling, and transformations: improving the biological information content of metabolomics data. BMC Genomics. 2006;7. https://doi.org/10.1186/1471-2164-7-142

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Acknowledgements

The authors thank Vivero Forestal Central, Valladolid, Junta de Castilla y León, for supplying the Pinus pinaster seedlings.

Funding

This work was supported by the Agencia Estatal de Investigación MCIN/AEI/https://doi.org/10.13039/501100011033 (reference project PID2020-118734RR-C21). Laura Hernandez-Escribano was supported by project PID2020-118734RR-C21, and David Fariña-Flores was supported by a fellowship from the Agencia Estatal de Investigación, PRE2018-086768.

Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.

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  1. Instituto de Ciencias Forestales (ICIFOR-INIA), CSIC, Carretera Coruña km 7.5, Madrid, 28040, Spain

    Laura Hernandez-Escribano, M. Teresa Morales Clemente, David Fariña-Flores & Rosa Raposo

  2. Departamento de Biotecnología-Biología Vegetal, E.T.S. de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, 28040, Spain

    David Fariña-Flores

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  4. Rosa Raposo
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Contributions

LHE participate in conception and design of the study, performed the experimental work, data analysis and drafted the manuscript. MM and DF participate in experimental work. RR participated in the conception and experimental design of the study, assisted in biological interpretation and with critical evaluation of the manuscript. All authors have read and approved the final version of the manuscript.

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Correspondence to Laura Hernandez-Escribano or Rosa Raposo.

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Hernandez-Escribano, L., Morales Clemente, M., Fariña-Flores, D. et al. A delayed response in phytohormone signaling and production contributes to pine susceptibility to Fusarium circinatum. BMC Plant Biol 24, 727 (2024). https://doi.org/10.1186/s12870-024-05342-8

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BMC Plant Biology

ISSN: 1471-2229

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