Forests cover nearly 31% of the total land area globally (4.06 billion ha), representing important economic, environmental, and social assets [Food and Agriculture Organization of the United Nations (FAO), 2020]. In particular, European forests are mainly composed of conifers (46%), with pine species being the greatest growing stock, key for CO₂ sink and a vital source of resin and timber (Forest Europe-Ministerial Conference on the Protection of Forests in Europe, 2020). Forest sustainability is currently threatened by several biotic and abiotic factors that negatively affect its productivity and health. The vulnerability to these stresses is increased by climate change and the global trade of plants and derivates, especially regarding the importation of plant pathogens which may find further opportunities to colonise new environments (Sturrock et al., 2011; Santini et al., 2013; Wingfield et al., 2015).

Pine Pitch Canker (PPC), caused by the ascomycete fungus Fusarium circinatum Nirenberg & O’Donnell (sexual morph Gibberella circinata Nirenberg & O’Donnell), is one of the most important diseases affecting conifers worldwide (Wingfield et al., 2008). Although PPC was first reported in the United States (Hepting and Roth, 1946), phylogenetic analyses indicate that F. circinatum is originally an endemic member of the native Mexican forest (Wikler and Gordon, 2000). Introduction into Europe, Japan, and South Africa seems to have occurred through United States or Mexico (Wikler and Gordon, 2000; Berbegal et al., 2013). PPC has now been reported globally (Figure 1; reviewed by Drenkhan et al., 2020). As the natural spread of F. circinatum via rain, wind, and insects is limited by short spore dispersal and vector-insect flight distances, longer distance dissemination is mainly human-assisted (Zamora-Ballesteros et al., 2019). This occurs mainly through the movement of infected seeds worldwide, and of asymptomatic seedlings, and contaminated substrates, or containers regionally.

After a new introduction occurs, moisture and warm temperatures are crucial for the establishment and spread of PPC (Wingfield et al., 2008; Ganley et al., 2009; Baker et al., 2010; Quesada et al., 2019; Elvira-Recuenco et al., 2021). A large area of European pine forests has been reported to be suitable or optimal for PPC development, with F. circinatum distribution potentially changing towards northern Europe due to the predicted reduction of cold and drought (Ganley et al., 2009; Baker et al., 2010; Watt et al., 2011; Möykkynen et al., 2015). This is of particular concern giving that F. circinatum showed to be able to infect conifers present in these currently disease-free areas such as Pinus sylvestris L. and Picea abies (L.) H. Karst (Martín-García et al., 2017, 2018), and that these areas are populated with a high density of conifers (Figure 1). In nurseries, optimal conditions may be easily achieved for disease development in imported infected seeds or plants independently of its location (Drenkhan et al., 2020).

Pine pitch canker affects more than 60 species of Pinus and Pseudotsuga menziesii (Mirb.) Franco (Bezos et al., 2017) at all stages of tree development infecting seeds, seedlings, and mature trees [European and Mediterranean Plant Protection Organization (EPPO), 2021]. Disease symptoms include excessive pitch flow, resinous canker formation, and branch/canopy dieback in adult trees and tip dieback and seedling damping-off in nurseries (Wingfield et al., 2008; Martín-Rodrigues et al., 2013; Santana et al., 2016). These results in significant economic losses in nurseries and pine plantations/stands (Aegerter et al., 2003; Wingfield et al., 2008; Carrasco et al., 2016). Although natural infections occur through wounds (Iturritxa et al., 2011), the pathogen is also able to colonise seedlings or roots of mature trees without damaging the adjacent tissues allowing plants to remain asymptomatic (Elvira-Recuenco et al., 2015; Swett et al., 2016, 2018; Martín-García et al., 2018; Hernandez-Escribano et al., 2018a). This hemibiotrophic nature represents an additional challenge to manage PPC (Vettraino et al., 2018); as well as its ability to naturally infect grasses surrounding pine species, where it may represent a source of inoculum (Swett and Gordon, 2012; Swett et al., 2014; Hernandez-Escribano et al., 2018b; Herron et al., 2020).

Several studies focused on F. circinatum infection mechanism and virulence. After conidia germination, the pathogen colonises the host stem radially advancing towards the pith, likely releasing cell wall degrading enzymes (e.g. endopolygalacturonase) to liberate nutrients from the host tissues (Chimwamurombe et al., 2001; Martín-Rodrigues et al., 2013). Vertical colonization of the host occurs later through the spread of conidiophores and conidia when the pathogen reaches the phloem or the traumatic resin ducts in the xylem (Martín-Rodrigues et al., 2013). Physical obstruction by F. circinatum growth, generalised cell death in the xylem and increased resin production may restrict water supply and result in plant death (Gordon, 2011; Martín-Rodrigues et al., 2013). Different phenotypical and molecular characteristics have been associated to F. circinatum pathogenicity or virulence (Mullett et al., 2017; Muñoz-Adalia et al., 2018a,b; Phasha et al., 2021a). Also, several compounds have been proposed to allow host colonization, including beauvericin, ergosterol, fusaric acid, and laccases (Fotso et al., 2002; Muñoz-Adalia et al., 2016; Visser et al., 2019; Phasha et al., 2021b).

In general, earlier research on PPC was focused on the characterization of F. circinatum populations and on testing the susceptibility of different hosts to PPC, with information on the mechanisms behind these differential responses remaining scarce. Over the last few years, a new perspective has gained relevance aiming to unveil these mechanisms based on an integrative approach (from physiology to Omics). This review highlights the main advances on PPC knowledge, with a special focus on multi-disciplinary studies exploring host response to F. circinatum and on how these contribute for tree biotic stress response knowledge and for the development of effective disease control measures.

Exploring genetic resistance based on intra- and inter-specific variation has been proposed as a promising environmentally-friendly strategy to avoid the natural spread of PPC (Martín-García et al., 2019; Zamora-Ballesteros et al., 2019). This would allow for the selection/development of resistant trees to reforest high-risk sites. Host susceptibility to F. circinatum is known to vary at the species, population, family, and clone levels (reviewed by Drenkhan et al., 2020). While some species, such as Pinus radiata D. Don and Pinus patula Schltdl. & Cham., are highly susceptible to F. circinatum, others are moderately susceptible (P. pinaster Aiton) or resistant (P. pinea L.). Pinus radiata has been extensively studied as it is the most planted pine worldwide due to its fast growth and wood quality (Mead, 2013). The among-population variation found in some species has been attributed to geographical and environmental gradients (e.g., resistance in low elevation Pinus tecunumanii Eguiluz & J. P. Perry). Moreover, variation in PPC resistance showed to be quantitative and dependent on polygenic mechanism (i.e. results from the integration of small effects of several genes; information mainly based on SNPs associations; Gordon et al., 1998; Kayihan et al., 2005; Quesada et al., 2010), and presents variable heritability estimates (reviewed by Martín-García et al., 2019).

Although resistance to diseases in forest trees has been mostly achieved based on phenotypic selection, advances on genomic techniques and resources contributed for the identification of candidate genes related to biotic stress tolerance (Kovalchuk et al., 2013; Muranty et al., 2014; Sniezko and Koch, 2017). For example, the integration of multiple Omics level data from trees with varying levels of susceptibility to the disease may be used to identify candidate genes or metabolic pathways for manipulation (Naidoo et al., 2019). Innovative bioinformatic and statistical tools allow to build networks that model the dynamics and complexity of a given biological system linking molecular interactions and complex traits (zu Castell and Ernst, 2012; Valledor et al., 2018; Naidoo et al., 2019), such as PPC. This systems biology view may provide foundation for defining specific gene sets relevant for PPC resistance. Despite possible legislative barriers, these may be targeted by novel gene editing tools if the desired combination of genes cannot be achieved through classical breeding (Chang et al., 2018).

The use of high-throughput Omics technologies in forest trees has lagged behind that of model or crop species, especially due to trees long-life cycles, large genome sizes, and the lack of genomic tools (Neale and Kremer, 2011). The availability of Pinus taeda L. whole-sequenced genome allowed for the identification of several single nucleotide polymorphisms (SNPs) associated with PPC resistance (Quesada et al., 2010; Lu et al., 2017; De La Torre et al., 2019; Figure 2). Moreover, the increasing performance and cost reduction of next-generation sequencing technologies make whole-transcriptome sequencing (RNA-Seq) attractive for the evaluation of gene expression in organism for which genome sequencing is not available, in particular for species with megagenomes such as conifers (e.g. Santos et al., 2012). Also, dual RNA-Seq strategies may address changes in both the host and the pathogen transcriptomes at once allowing to better decipher its interaction (Naidoo et al., 2018). RNA-Seq and dual RNA-Seq studies have been performed in pine species with different levels of susceptibility to PPC to unveil host responses to F. circinatum infection and explore how the pathogen is able to overcome them; as well as target transcriptomic studies (check Figure 2 for references).

However, changes at the transcriptional level are not always reflected at the downstream levels of the Omics cascade. Proteomics allow to study the proteins regulating disease development, contributing for the understanding of complex host defence mechanisms (Kaur et al., 2017; Rustagi et al., 2018). Although several of the proteomic studies in forest trees are based on two-dimensional electrophoresis (2-DE) coupled to MS analysis, resulting in low proteome coverage, forest proteomics have evolved, especially regarding LC–MS-based systems for protein identification and quantification using shotgun/bottom-up proteomics (Rodrigues et al., 2021). Early proteomics studies regarding PPC focused only on the role of a particular protein family (chitinases) in response to F. circinatum infection (Davis et al., 2002); while recent studies explored the needle proteome of pine species with contrasting PPC response phenotypes followed by integration with physiological data (Amaral et al., 2021b).

In addition, metabolites, at the end of the Omics cascade, represent a phenotypic signature of genetic variance, and of epigenetic, transcriptomic, and proteomic changes (Fiehn, 2002). As for proteomics, advances in MS approaches currently allow a large-scale profiling of plant metabolites (Rodrigues et al., 2021), allowing to identify biomarkers of forest diseases (e.g. Hantao et al., 2014; Wu et al., 2015) or screen disease resistant trees for breeding programs (e.g. Sambles et al., 2017; Wang et al., 2017). However, to distinguish whether the metabolites analysed are of plant or of pathogen origin may represent a great challenge. Unlike transcripts and proteins, metabolites identification does not depend on known genetic sequences. While some techniques have been proposed to address this issue, these are still associated to several drawbacks (see Tenenboim and Brotman, 2016). Regarding PPC, the quantification of specific metabolites, such as sugars, proline, hormones, terpenoids, phenolics, or lignin has been carried out using mainly traditional biochemical methods (check Figure 2 for references). GC–MS analysis of the needle primary metabolism of pines with different levels of susceptibility to F. circinatum infection were only recently performed together with physiological and targeted gene expression measurements (Amaral et al., 2019a).

Despite phenomics approaches are usually scarce in forest trees, some advances have been reported for PPC (Reynolds et al., 2016; Lee and Lee, 2019). Phenomics combine well-established plant physiology methodologies (e.g., gas exchange, chlorophyll fluorescence, water relations, nutrient uptake, and growth rate measurements) with recent technological advances to develop high-throughput phenotyping capture technologies, such as unmanned aerial vehicle remote sensing, to obtain a large-scale physiological overview of plants under different environmental scenarios (Flexas and Gago, 2018; Naidoo et al., 2019). The integration of different Omics levels with plant physiology theoretical knowledge and techniques is expected to maximise the understanding of plants functioning (Flexas and Gago, 2018); especially given that plant physiology has also proven useful for the understanding of plant-pathogen interaction (Berger et al., 2007). Studying the mechanism behind Pinus-F. circinatum interaction in hosts with varying levels of susceptibility to PPC based on a multidisciplinary approach unveiled defence response associated to each phenotype and is predicted to support innovative disease control measures.

Plants possess a complex innate immune system that enables them to respond to pathogen attack, including non-specific constitutive defences (e.g., lignosuberised trees outer bark, needles cuticular surface, and phytoanticipins, such as phenolics, terpenoids, and alkaloids) and induced defences (Kovalchuk et al., 2013). The latter may be activated by the recognition of Pathogen-Associated Molecular Patterns (PAMPs) by extracellular domains of membrane receptor proteins (pattern-recognition receptors—PRRs) and result in PAMP-triggered immunity (PTI; Chisholm et al., 2006; Jones and Dangl, 2006; Nishad et al., 2020). The activation of PTI in response to F. circinatum infection in P. pinaster has been proposed due to the early upregulation of genes encoding a lysine motif receptor-like kinase (LysM-RLK), which has been associated to the recognition of fungal chitin (Hernandez-Escribano et al., 2020). Several genes coding for PRR containing domains have also been upregulated in the resistant P. pinea but not in the susceptible P. radiata, which may explain the weak downstream signalling in the latter (Zamora-Ballesteros et al., 2021). Regarding effector-triggered immunity (ETI), a second line of defence activated by the recognition of fungal effector proteins by plant resistance (R) proteins (Hammond-Kosack and Parker, 2003), there is still no evidence of its role in PPC.

After pathogen recognition, plants activate several defence responses to avoid its spread. These include morphological modifications (e.g., cytoskeletal reorganization and cell wall fortification); the production of reactive oxygen species (ROS) and secondary metabolites (such as phytohormones, phytoalexins, and plant volatiles), which may interact directly with the pathogen or function as signalling molecules distant from the infection site (Bolton, 2009; Eyles et al., 2010; Rojas et al., 2014); and the induction of pathogenesis-related (PR) proteins (van Loon et al., 2006) and a programmed cell death mechanism known as hypersensitive response (HR; Coll et al., 2011). The production of PR proteins in uninfected tissues is associated to the development of systemic acquired resistance (SAR), a systemic induced resistance (SIR) phenomena mediated by salicylic acid (SA) that protects plants against future infections (Fu and Dong, 2013). SIR is known to occur in trees in response to necrotrophic pathogens, including F. circinatum, although there is no sufficient information on the signalling pathways involved to identify the specific mechanism developed (Bonello et al., 2001, 2006; Eyles et al., 2010; Gordon et al., 2011). The use of chitosan as an elicitor of pine defence against F. circinatum has showed promising results based on the systemic induction of host resistance (e.g., activation of the phenylpropanoids pathway) and/or its fungistatic effect (Reglinski et al., 2004; Fitza et al., 2013; Silva-Castro et al., 2018a,b). The potential of using biotic (pathogens or beneficial microbes) and abiotic (e.g. wounding and chemicals) elicitors to trigger the establishment of induced responses in pines and enhance resistance to F. circinatum has been reviewed by Martín-García et al. (2019). Promising promoters of PPC resistance may be part of an integrated management approach to control PPC in an environmentally friendly manner.

A general reprogramming of host’s metabolism occurs to fuel plant defence responses against pathogens instead of growth, reproduction, and yield; but also to meet the nutritional requirements of the pathogen (accumulation of C and N sources; Berger et al., 2007; Bolton, 2009; Fagard et al., 2014; Rojas et al., 2014). For example, transcriptomic studies evidenced this metabolic trade-off between growth and defence in the P. pinaster-F. circinatum interaction: genes related to growth, morphogenesis, and photosynthesis were downregulated; while genes involved in phytohormone signalling, ROS regulation, oxidative stress, regulation of cell death, signal transduction, synthesis of flavonoids, anthocyanins, and other defence-related secondary metabolites were increased (Hernandez-Escribano et al., 2020). However, the late production of secondary metabolites was insufficient to fight pathogen infection.

The main aspects of pine response to F. circinatum are integrated in the following sections, including changes in plant water relations, signalling pathways (ROS and hormones), primary metabolism, and induction of common conifer defences (resin, phenolics, and PR proteins). These advances were achieved fusing techniques from different disciplines (from physiology to Omics) to explore changes in hosts with different susceptibility to PPC.

The use of a comprehensive number of physiological and Omics techniques to evaluate the response of pine species with different levels of susceptibility to PPC allowed to unravel several aspects of Pinus-F. circinatum interaction (see Figure 3). While susceptible hosts are generally highly responsive to pathogen infection either as a result of pathogenesis or of a late and insufficient activation of plant immune defence; resistant species seem to activate more specific mechanisms that may enable them to fight F. circinatum.

Besides contributing for the identification of potential pathways associated to PPC susceptibility/resistance, the studies herein reviewed pinpoint fields of research that deserve deeper investigation and may contribute for the development of innovative disease control measures. These include exploring the convergence between F. circinatum- and drought-induced responses (including stomata regulation), taking advantage of the advanced knowledge on tree response to water limiting conditions. Also, the importance of ABA-signalling and of several antioxidants on PPC response should be further examined based on the systemic induction of resistance through chemical priming, comprising either its direct application or the application of other compounds that influence its biosynthesis. Different application timings, methods, and concentrations should be tested on seedlings with different ages to assure broad-spectrum and long-lasting resistance against several isolates of F. circinatum.

The recent advent of microbiome studies in hosts with varying levels of susceptibility to PPC (Leitão et al., 2021; Romeralo et al., 2022) also represents an opportunity to identify microorganisms that impact host response with potential to be employed in biocontrol strategies, such as the development of disease suppressive soils. Other advances such as the publication of the chromosome-level assembly and methylome of the 25.4-Gb genome of Pinus tabuliformis (Niu et al., 2022) offer new venues for genomics studies on PPC, as well as for epigenetics which are still absent. In addition, the study of F. circinatum secretome and exometabolome would be key to better understand its interaction with Pinus species allowing to determine the secreted proteins and metabolites that favour tissue penetration and colonization in susceptible hosts. Given the complexity of the next steps proposed for studying PPC and developing effective control measures, it is required that scientists from different disciplines collaborate in order to speed up the understanding of the disease and thus contribute for improving the current forest management policies.

JA and GP designed the article. JA wrote the manuscript and built the figures. All authors contributed to the article and approved the submitted version.

This work was performed under the framework of the URGENTpine project (PTDC/AGR-FOR/2768/2014), which is supported by FEDER through COMPETE (Programa Operacional Fatores de Competitividade; POCI-01-FEDER-016785) and by national funds through the Portuguese Foundation for Science and Technology (FCT), and of the F4F—Forest For Future project (CENTRO-08-5864-FSE-000031) from Programa Operacional Regional do Centro (Centro 2020), Portugal 2020 and Fundo Social Europeu. Thanks are due to FCT/MCTES (Ministry for Science, Technology and Higher Education) for financial support to CESAM (UID/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020) through national funds. FCT also supported JA (SFRH/BD/120967/2016). The Spanish Ministry of Economy and Competitiveness supported LV through the Ramón y Cajal programme (RYC-2015-17871).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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