Fusarium oxysporum Schl. f. sp. pisi Snyd. and Hans. (Fop) is an important pathogen causing vascular wilt of field pea (Pisum sativum L.) worldwide (Rubiales et al., 2015). Four different races of Fop, named 1, 2, 5, and 6, have been described so far (Infantino et al., 2006). Races 1 and 2 are widely distributed, while races 5 and 6 are, to date, only important in western Washington State (Infantino et al., 2006). Control of soil-borne fungal diseases is mainly achieved by the integration of different disease management procedures. Among these methods, the use of resistant cultivars is widely recognized as the safest, most economical and most effective crop protection method (Ciancio and Mukerji, 2008; Rubiales et al., 2015). Resistance to Fop races 1, 5, and 6 is conferred by single dominant genes whereas resistance to race 2 is quantitative (Bani et al., 2012; McPhee et al., 2012).

Similarly to other formae speciales of F. oxysporum, Fop behaves as a classical soil-borne pathogen with propagules that can survive in soil during extended periods of time in the absence of suitable host (Roncero et al., 2003). The infection cycle of F. oxysporum is initiated by spore germination and its directed elongation toward host plant root in response to specific plant signals (Nelson, 1991; Turrà et al., 2015). Then the growing, infective hyphae adhere to host roots and penetrate it through wounds or by piercing through the epidermis (Nelson, 1981; Bishop and Cooper, 1983a; Benhamou and Garand, 2001; Zvirin et al., 2010). The mycelium then advances intercellularly through the root cortex, until it reaches the xylem vessels and colonize them through the pits (Bishop and Cooper, 1983b; Beckman, 1987). Then, the fungus progresses vertically, through xylem vessels, to invade the shoot (Bishop and Cooper, 1983b; Beckman, 1987). After wilting and plant death, the fungus reaches the plant surface where it produces chlamydospores that are dispersed onto the soil for a second cycle of infection (Kraft et al., 1994). During root invasion and colonization, Fop is exposed to various plant defense mechanisms (Beckman, 1987; Michielse and Rep, 2009).

Several histological studies have characterized the infection processes and resistance mechanisms in different plant species (Bishop and Cooper, 1983a,b; Beckman, 1987; Baayen et al., 1989; Tessier et al., 1990; Pereira et al., 2013). Successful host invasion by F. oxysporum depends on a multitude of factors, which vary according to the pathosystem. Similarly, hosts develop different physical and chemical barriers to block the pathogen progressions at different levels depending on host species or cultivar. The most efficient mechanisms are production of antifungal compounds, formation of papillae at penetration sites, suberization and lignification of cell walls, accumulation of gums, gels or tyloses within xylem cells and vessel crushing by proliferation of adjacent parenchyma cells (Bishop and Cooper, 1983a; Baayen and Elgersma, 1985; Beckman, 1987; Charchar and Kraft, 1989; Ouellette et al., 1999; Grayer and Kokubun, 2001; Yadeta and Thomma, 2013; Pouralibaba et al., 2017). These mechanisms contributes to block pathogen progression and to kill invading hypha. Although several of these mechanisms are often described simultaneously within host root, their relative contribution to resistance varies depending on the host and cultivar. In pea, previous reports demonstrated that the main determinant of resistance to Fop was the occlusion of xylem cells and to a lesser extent to xylem cell wall strengthening (Bishop and Cooper, 1983a, 1984; Tessier et al., 1990; Benhamou and Garand, 2001). Interestingly, occluding pea cells accumulated gels composed of a mixture of carbohydrates, proteins and pectins instead of tylose commonly detected in other F. oxysporum-host interaction (Bishop and Cooper, 1984; Tessier et al., 1990). Papilla formation at epidermis and cortex, and endodermis suberization was also reported in pea. However, they were shown to play only little role in resistance against Fop, if any, since they were also detected in susceptible accessions (Bishop and Cooper, 1983a).

Recently, new sources of quantitative resistance have been identified in pea and Medicago truncatula (Bani et al., 2012; Rispail and Rubiales, 2014). Although some reports have described the histopathology of resistant and susceptible pea cultivars (Bishop and Cooper, 1983a,b; Tessier et al., 1990), detailed studies of the infection process and of the resistance mechanisms of genotypes with different levels of resistance are scarce. In this work, we aimed to determine in detail the infection process of Fop race 2 in eight pea accessions showing different levels of quantitative resistance and to characterize the resistance mechanisms established in these accessions.

Fop is one of the major constraints of pea worldwide. Defining the defense mechanisms acting in sources of resistance is crucial for a more targeted and efficient breeding. Reaching a deep understanding of Fop infection process is also important to define efficient control strategies. Thus, we performed a detailed histological study on eight pea accessions with differential responses to Fop race 2 isolates (Bani et al., 2012). Although several histological studies has characterized the mechanisms of resistance to F. oxysporum in several plant species including pea (Bishop and Cooper, 1984; Baayen et al., 1989; Tessier et al., 1990; Shi et al., 1992; Ouellette et al., 1999; Zvirin et al., 2010; Pereira et al., 2013), our study is the first comparative histological study on several resistant accessions. This revealed a large variety of responses taking place throughout the fungal colonization sites from root epidermis to vascular stele and indicated the importance of the outer root region and of lignification in Fop resistance.

Successful host infection by F. oxysporum is a complex process that can be divided in four main steps, (1) recognition of host root, (2) attachment and superficial colonization of host root, (3) penetration of root epidermis and progression through cortex and (4) colonization of xylem vessel. Although the histology of F. oxysporum infection has been studied in details by light, confocal and electron microscopy, several steps of this process remain under debate including how F. oxysporum penetrates host root, colonizes cortical cells and spreads within xylem vessels. Our observation in pea may contribute to clarify some of these steps.

It is generally accepted that F. oxysporum penetrates root directly without the formation of specialized appresorium-like structures (Bishop and Cooper, 1983b). Several reports further evidenced that epidermal cell wall was breached by the differentiation of constricted hyphae or infective hyphae that recover their normal size upon cell wall penetration (Bishop and Cooper, 1983a; Rodríguez-Gálvez and Mendgen, 1995). By contrast, several recent studies in Arabidopsis and watermelon observed the swelling of infective hyphae forming rudimentary appressorium-like penetration structures (Czymmek et al., 2007; Lü et al., 2014). In pea, our observations suggested the direct penetration of host root (Figure 1B). Swelling of infective hypha before infection was not observed in our root preparations. Instead, the constriction of the penetrating hypha at the site of cell penetration was recurrently observed at the epidermis, exodermis and in the cortex (Figures 1B,D) supporting the process of penetration previously described by electron microscopy in tomato, pea and cotton (Bishop and Cooper, 1983a; Rodríguez-Gálvez and Mendgen, 1995; Benhamou and Garand, 2001).

Once inside the root, most studies described the intercellular progression of F. oxysporum through the cortex (Kroes et al., 1998; Jiménez-Fernández et al., 2013; Niño-Sánchez et al., 2015). This exclusively apoplastic progression was also reported in pea following root dipping inoculation (Bishop and Cooper, 1983a). In this species, the intracellular progression of Fop through cortex was also reported following inoculation of Ri-transformed pea root culture (Benhamou and Garand, 2001). Here, following the root dipping inoculation, an abundant colonization of cortical cells by Fop was detected in susceptible accessions. As reported in Figure 1D, numerous infective hyphae were observed moving in and out of cortical cells. In most cases cell-to-cell movement was mediated by constricted hypha as previously reported (Figure 1D; Benhamou and Garand, 2001). Recent studies in cabbage and lentil also reported the intracellular colonization of cortical cells (Li et al., 2015; Pouralibaba et al., 2017). Intracellular progression of F. oxysporum through the cortex may thus be more common than previously though. Interestingly, Fop progression in root of resistant and partially resistant pea accessions was strictly intercellular due to the establishment of efficient defense reactions (Figure 1E). This indicates that plant genotype is an important determinant of F. oxysporum progression. Therefore the difference in root cortex colonization might reflect differences in the susceptibility of the pea accessions used. Colonization of susceptible accessions was also associated with the degradation of the cell layers surrounding endodermis (Figures 1, 6A,B) as previously shown for several formae speciales of F. oxysporum (Tessier et al., 1990; Kroes et al., 1998; Olivain and Alabouvette, 1999; Ouellette et al., 1999; Benhamou and Garand, 2001). Cell wall degrading enzymes (CWDE) have been recently identified as key pathogenicity factors that contribute to successful F. oxysporum infection in tomato (Bravo Ruiz et al., 2016). The intensive cell degradation detected here suggested that CWDE might also be involved in the pathogenesis of Fop race 2.

The last steps of F. oxysporum infection are the colonization of xylem vessels and its subsequent spreading to the hypocotyl. Traditionally, it is though that F. oxysporum infect xylem vessel through the pits or by breaching cell walls of immature xylem elements (Bishop and Cooper, 1983a,b). In susceptible pea accession, Fop was detected entering immature and mature xylem vessel alike by direct penetration of xylem cell wall. Abundant inter- and intra-cellular colonization of fiber and parenchyma cells was also often observed in these pea accessions (Figure 1F). By contrast, penetration of mature xylem vessel was not observed in resistant and partially resistant accessions. Nevertheless, the restricted presence of Fop cells in xylem vessels in partially resistant accessions inoculated without root trimming indicates that in these accessions the fungus can overcome plant defense by infecting immature vascular tissue. Altogether these observations also support that Fop infection process depends at least partly on the level of susceptibility of its host. Once the F. oxysporum enter within xylem vessels, it colonizes them by growing longitudinally in both directions. According to literature, intra-tracheary colonization is either accomplished by germination of microconidia through xylem perforation plates (Beckman, 1987) or by hypha elongation of the growing F. oxysporum colonies (Czymmek et al., 2007; Michielse and Rep, 2009). Observation of numerous longitudinal sections of inoculated pea roots revealed that intra-tracheary movement was made mainly by elongation of infective hypha (Figure 1G) although germination of microconidia through xylem perforation plates was also detected in a few instances (Figure 1H). Our observations also showed that the fungus spread in vascular tissue by colonizing adjacent vessels through elongation of infective hypha breaching their cell walls (Figure 1G). These observations would support the minor role of microconidia in the colonization of xylem vessels and hypocotyl previously reported (Czymmek et al., 2007; Michielse and Rep, 2009).

Resistance to fusarium wilt has been associated to several defensive mechanisms. Three main areas were differentiated, the outer root section, the endodermis and the vascular stele. Previous studies in flax, tomato and watermelon demonstrated the key role of the outer root region (epidermis, exodermis and cortex) in the resistance (Kroes et al., 1998; Olivain and Alabouvette, 1999; Olivain et al., 2003; Lü et al., 2014). Other studies, performed in pea or carnation showed that the main discriminating factor between susceptibility and resistance localized at the endodermis (Bishop and Cooper, 1983b; Baayen et al., 1989). Here, we observed the coordinated establishment of different mechanisms across pea root tissues to efficiently block the pathogen progression before vascular stele. Significant differences in the intensity of defense reaction at each root area were detected between accessions (Figure 6A). This suggests that the contribution of each root area to resistance depends on the accessions. Despite these differences, the outer root region, including epidermis and exodermis, was identified as the major determinant of resistance since defense reactions established at this region was highly correlated with disease severity and their frequency discriminates between resistant and susceptible accessions (Figure 7A and Table 2). Interestingly, this region did not discriminate partially resistant from susceptible accessions (Figure 7A) suggesting that the main discriminating defense element might take place in another location in these accessions. Strong disease reaction at endodermis was detected in both partially resistant accessions tested (Figures 2B, 6A) that were also characterized by the massive accumulation of fungal cell at this level (Figure 4A). Endodermis would thus appear as the main discriminant factor of partially resistant accessions while it only plays a minor role in resistant accessions since fungal progression was most often blocked before this region. Despite the absence of fungus in this area, endodermis strengthening may have a secondary role in the resistant accessions by impeding the fungus growing out of infected xylem vessels.

A battery of cell defense reactions have been previously related to F. oxysorum resistance including, cell wall strengthening, papilla formation, production of antifungal compounds, accumulation of gums, gels or tyloses within xylem cells and vessel crushing (Bishop and Cooper, 1983a; Baayen and Elgersma, 1985; Beckman, 1987; Charchar and Kraft, 1989; Ouellette et al., 1999; Grayer and Kokubun, 2001; Yadeta and Thomma, 2013). The importance of each of these cell reactions in resistance remains unclear. Our thorough histological analysis revealed that resistance was mainly based on three defense mechanisms, papilla formation, cell wall strengthening and accumulation of (poly)phenolics and carbohydrates. Although all accessions combined these mechanisms, differences in the frequency of these defensive reactions, were detected between accessions (Figures 2B, 6B) suggesting that each accessions develop its own defense strategy. Despite these variations, cell wall strengthening mechanisms through lignification was identified as the main determinant of resistance. Indeed, lignification was the unique defense mechanism significantly correlated with resistance and the sole mechanism discriminating between (partially) resistant and susceptible accessions (Figure 7B). Lignification might contribute to resistance through different actions. Lignification can not only strengthen cell wall making them more resistant to the compressive forces exerted by fungal pathogen during infection attempts but it might also shield cell wall components preventing their degradation (Vance et al., 1980; Lattanzio et al., 2006). The intense cell wall lignification detected here might thus form a physical barrier to the pathogen entry. Lignification has also been postulated to restrict diffusion of fungal secreted enzymes or toxins. In addition, lignins and its derivatives can bind and inhibit CWDE (Walters et al., 2007; Vidhyasekaran, 2008). Thus, the intense lignification and suberisation of cortical and endodermal cell wall detected in resistant pea accessions (Figures 3, 4) might contribute to neutralize fungal CWDE.

In addition to the extensive lignification of cell walls of resistant accessions, we observed formation of papillae at sites of attempted hyphae penetration at the exodermis and cortex. This localized cell wall strengthening mechanism was particularly effective in some resistant accessions such as JI1412 and P42 impeding pathogen entry within cell and its intracellular progression through root. Accumulation of (poly)phenolics and carbohydrates was also detected at epidermis, exodermis, cortex, endodermis and the vascular stele forming five spatially differentiated chemical barriers within the roots. Altogether, these different mechanisms contributed to create physical and chemical barriers that efficiently blocked and killed the invading pathogen before reaching the stele.

Several mechanisms have been shown to limit fungal growth within xylem vessel and to block its vertical spread in several species including pea (Beckman et al., 1972; Baayen et al., 1989; Tessier et al., 1990; Shi et al., 1992; Zvirin et al., 2010; Pereira et al., 2013). In order to detect these defense mechanisms, we performed a series of inoculation experiments with root trimming that favor direct fungal entry in the vascular tissues. In this way, most of these mechanisms could be detected in resistant accessions that otherwise would had completely blocked fungal invasion at outer cell layers. These mechanisms were also detected in partially resistant accessions independently to the inoculation method. These mechanisms consisted mainly in vessel clogging or occlusion with materials, usually denoted as gels, gums or mucilages. Mucilage production has been previously reported as a defensive response against the parasitic plant Orobanche crenata (Pérez-De-Luque et al., 2005, 2006) indicating that it might be a general mechanism to prevent spreading of invading organisms through xylem. The main mucilage components are carbohydrates (specifically pectins) and polyphenols although they can also contain phytoalexins, lignin-like compounds or lipoidal substances (Niemann et al., 1990; Shi et al., 1992; Baayen et al., 1996; Pérez-De-Luque et al., 2006; Hall et al., 2011). Upon conducive conditions, we observed the important production of carbohydrates, (poly)phenolics and additional undetermined substances by vascular parenchyma cells and their progressive accumulation in the lumen of xylem cells, which supports the origin and composition of mucilage previously reported. Our data shows that accumulation of phenolic compounds and carbohydrates was higly correlated, supporting the relevance of both sustances as resistance response at xylem vessels. Although the nature of the additional occluding substances are unknown, their staining pattern and mode of accumulation suggest that they might be formed by other carbohydrates and/or lipidic substances known to be also present in mucilages (Niemann et al., 1990; Shi et al., 1992). Apart from mucilage, we detected the thickening of vessel cell walls controlling lateral spreading of the pathogen. Vessel wall thickening has been reported as a typical defense reaction against F. oxysporum in many species (Tessier et al., 1990; Shi et al., 1992; Baayen et al., 1996; Ouellette et al., 2002; Pereira et al., 2013). The phenolic and lignin nature of these coatings may contribute to chemical inhibition and physical restriction of the fungus within vessels (Shi et al., 1992; Hilaire et al., 2001).

Our observation supports a key function of phenolic compounds in pea defense against Fop, although further studies would be needed to identify the phenolic compounds accumulating in the resistant pea roots and confirm their role. Beside the intense lignification that might contribute to strengthen cell walls and neutralize CWDE action, our study revealed the frequent accumulation of (poly)phenolic compounds both inter and intracellular near fungal cells (Figures 3–5). Phytoalexins such as pisatin and lignans are well-known defense metabolites due to their potent anti-fungal activities and their capacity to inhibit secreted fungal enzymes (Lattanzio et al., 2006). Accumulation of (poly)phenolics in response to Fop was previously evidenced. A recent proteomic analysis of the resistant pea-Fop interaction revealed the accumulation of several enzymes of the phenylpropanoid pathways involved in the synthesis of (poly)phenolic compounds (Castillejo et al., 2015). In addition, Fop inoculation was found to induce pisatin accumulation in resistant pea roots suggesting a role of pisatin in quantitative resistance in pea against Fop (Bani et al., 2018). Although their function is still unclear, our results further supports the important role of (poly)phenolic compounds in Fop resistance.

Altogether, our results clarified the infection process of Fop in susceptible and resistant pea accessions. More importantly, our results indicated that resistant accessions developed different mechanisms creating physical and chemical barriers to impede the pathogen progression toward the vascular stele. Interestingly, it showed quantitative rather than qualitative differences between accessions that differed between each other in the frequency and the spatial distribution of the established defense reactions. This study contributes to our understanding of the resistance mechanisms that efficiently block F. oxysporum infection and it advances our understanding of the mechanisms acting in pea against Fop race 2. Potentiating the different defense mechanisms detected in these pea accessions in a single resistant cultivar through breeding would contribute to control this important pea disease and improve the durability of existing resistance levels in the field.

MB, NR, and DR conceived and designed the experiments. MB performed the experiments. MB, NR, and AP-D-L discussed and interpreted the data. MB and NR drafted and wrote the manuscript. AP-D-L and DR also participated in the critical reading, writing, and correction of the manuscript.

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.