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Fusarium circinatum Nirenberg & O'Donnell (teleomorph=Gibberella circinata Nirenberg & O'Donnell 1998) is the causal agent of pitch canker, one of the most serious pine diseases world-wide. The lifestyle of this filamentous fungus is typical of that of necrotrophic pathogens, which do not have specialized infection structures to derive nutrients from sacrificed cells (Lewis, 1973). The pathogen causes cankers which girdle the shoots, branches and even trunks of susceptible pine species. The characteristic feature of the cankers is excessive pitch flow resulting in complete impregnation of the wood inside the canker (Hepting & Roth, 1946). Multiple branch infections may cause severe crown dieback and lead to the death of the tree. However, the most significant loss from an economic perspective is caused by reduced growth of adult trees in forest plantations (Dwinell & Phelps, 1977; Arvanitis et al., 1984), as well as the detrimental effects of the pathogen in seedling nurseries (Dwinell & Barrows-Broaddus, 1978; Viljoen et al., 1994; Wingfield et al., 2002).
Pitch canker is known to affect over 50 Pinus species which vary widely in susceptibility (Dwinell, 1978; Gordon et al., 1998a; Mitchell et al., 2012). Pinus radiata D. Don is one of the most economically important pine species in the world, as a consequence of its fast growth rate, and is also one of the most susceptible species (Hodge & Dvorak, 2000). However, even for species considered to be susceptible, the degree to which individual trees develop pitch canker varies widely (Storer et al., 1999a). In addition, the degree of tolerance in an individual may change over time as a result of induced resistance, associated with the activation of natural defense mechanisms of the host plant, resulting in changes which diminish the effects of subsequent biotic attack (Eyles et al., 2010). This phenomenon, first described in gymnosperms by Christiansen et al. (1999), has been demonstrated to occur in radiata pine trees as a response to infection by F. circinatum (Bonello et al., 2001; Gordon et al., 2011).
Conifers are organisms that live for many years and part of their success is attributable to their capacity to integrate multiple constitutive and inducible defense mechanisms into an effective coordinated response. Sometimes the same defense traits can be both constitutively expressed and inducible (Franceschi et al., 2005). This is the case for oleoresin, the most familiar of the conifer defenses. Oleoresin is a complex mixture of terpenoids which is known to deter insect pests and their symbiotic fungal pathogens (Phillips & Croteau, 1999). Terpenoid resin is constitutively synthesized and stored in specialized secretory structures. Pinus spp. have developed a complex network of highly branched radial and axial resin ducts which are interconnected by anastomoses. In this genus, resin is synthesized by the thin-walled, long-lived secretory epithelial cells lining the lumen of the resin ducts. Resin ducts are formed as a normal feature of development, linked to secondary growth, or their formation may be induced by external factors, in which case they are referred to as traumatic resin ducts (TRDs). In contrast to constitutive axial ducts, which generally occur alone and have a scattered distribution, TRDs are normally found in tangential series of one or two rows within an annual ring of xylem (Bannan, 1936). The resin formed by TRDs can be more fungistatic than primary resin, because of altered terpene content and additional phenolics (Nagy et al., 2000; Krokene et al., 2003). Several studies have analyzed this defense mechanism in the pine response to fungal pathogen invasions (Reid et al., 1967; Hudgins et al., 2005; Luchi et al., 2005; Nagy et al., 2005). However, little is known about the role of the constitutive resin ducts or TRDs during pitch canker development, despite the fact that copious pitch flow is one of its principal symptoms, as evidenced by the disease denomination.
In this sense, a more integral analysis of the spatiotemporal events that occur during the process of infection by F. circinatum is needed. Conventional light microscopy (Barrows-Broaddus & Dwinell, 1983, 1984) and, more recently, advanced microscopy technologies (Thoungchaleun et al., 2008; Kim et al., 2009) have provided valuable insights into this pathogenic interaction. Confocal laser scanning microscopy (CLSM) and the use of green fluorescent protein (GFP)-tagged fungal transformants have been shown to be new and powerful tools with which to discover novel aspects of the in vivo pathogenic interaction between organisms (see e.g. Lagopodi et al., 2002; Jansen et al., 2005; Zvirin et al., 2010). Moreover, quantitative real-time polymerase chain reaction (q-RT-PCR) is currently the most accurate method for quantifying fungal colonization of host tissues (Stephens et al., 2008; Maciá-Vicente et al., 2009). Although rapid and sensitive protocols have been developed for the specific detection of F. circinatum DNA (Schweigkofler et al., 2004; Ioos et al., 2009), to date these have not been optimized for and applied to the monitoring of colonization of the natural substrate of this pathogen.
In the present work, we have applied the above-mentioned methodologies to systematically investigate the spatial and temporal dynamics of the infection process in planta during compatible interactions between P. radiata and F. circinatum which lead to pitch canker development. q-RT-PCR was used to estimate fungal DNA as an indirect measure of fungal biomass during infection, followed by histological CLSM analysis of stem colonization of P. radiata by a GFP-labeled F. circinatum strain. The role of the constitutive resin ducts and the formation of TRDs associated with pathogen invasion were also characterized, as well as the efficacy of this induced reaction to counteract fungal pathogen challenge.
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Using advanced confocal microscopy and molecular q-RT-PCR analyses, novel features of infection have been revealed which extend our understanding of infection dynamics that occur during compatible interactions between F. circinatum and susceptible P. radiata seedlings. Fusarium circinatum exhibited a homogeneous spatial pattern in colonizing the pine stem. Initially there appeared to be two predominant pathways for fungal advance which were taken simultaneously: a radial advance toward the pith via medullary rays; and a tangential invasion of the outermost layers of the stem through the phloem and the cortex. These tendencies during the initial stages of colonization may reflect fungal foraging for host nutrients, as food reserves in the form of starch and fat are stored principally in the pith and cortex, but also in the ray parenchyma and resin canal epithelium in the wood (Sinnot, 1918). We observed what appeared to be starch grains inside these parenchymatous tissues (Fig. 6e). Our findings indicate that initial colonization of the cortex and pith by F. circinatum is through growth in intercellular spaces (Fig. 4a,e). It is likely that the fungus releases cell wall-degrading enzymes into the apoplast which are required to liberate large amounts of nutrients from these plant tissues. Indeed, Chimwamurombe et al. (2001) have sequenced and characterized a gene encoding an F. circinatum endopolygalacturonase. This enzyme is involved in the digestion of polysaccharides present in primary cell walls. Subsequent stages of pitch canker disease involve vertical colonization of the pine stem by three principal pathways: through the cortex and the phloem in the stem periphery (consistent with the external visual necrotic lesion); through the xylem via axial tracheids and resin ducts, and through the inner parenchymatous pith tissue.
We also analyzed the temporal progression of pathogenic invasion of the host. Three phases of colonization from the inoculation zone can be distinguished. The exponential growth phase, during the first week following inoculation, correlates with germination of spores and hyphal development across the parenchymatous cortex from the inoculation zone to the phloem. Rapid proliferation of the pathogen may be attributable to its initial invasion of host tissues which present more available and nutrient-rich resources, that is, the phloem. The lack of a lag phase, typically characteristic of an adaptation period before exponential growth, suggests prompt adaptation of the pathogen to the new growth milieu. Then, F. circinatum took 2 wk, from 7 to 21 dpi, to reach the stationary phase. This period was coincident with the colonization of the xylem and the pith, and also with the production of conidiophora in the pith. Stephens et al. (2008) showed a 1-wk transition phase in the growth curve of Fusarium graminearum during crown rot disease of wheat (Triticum aestivum) using a similar fungal biomass assay. In our study, the slowdown in the fungal growth during the large transition period is probably a result of the large energy investment required for the morphogenic switch from vegetative mycelium to conidia. This requires an alteration in the pattern of gene expression (Iida et al., 2006; Garzia et al., 2013) and is likely to occur intensively once the fungus appropriates the required starch reserved in the parenchyma cells of the pith.
We report here for the first time the capacity of F. circinatum to complete its asexual cycle inside the host by 14 dpi, producing conidiophora orientated toward the hollow cavities of the pith at the moment when the first pitch canker symptoms appeared. Up to now, F. circinatum sporodochia (1–3 mm in diameter) and microscopic sporodochia (0.06–0.2 mm in diameter) have been known to be produced on the surface of the dead shoots or needles of infected plants, serving as an inoculum source to spread the fungus to neighboring host plants (Blakeslee et al., 1978; Barrows-Broaddus & Dwinell, 1984). To the best of our knowledge, sporulating hyphae deep within the host have never been reported in gymnosperm plants. In angiosperm crops, the production of conidia in the inner tissues inside the host has been reported to be related to the spread of the pathogen to different parts of the same plant, as occurs with vascular wilt parasites (Di Pietro et al., 2003), or to perpetuation of the fungus over time, as a reservoir of inoculum, as has been reported for some endoparasites, such as the rice blast fungus Magnaporthe grisea (Terui, 1940). In this regard, we did not observe conidia within the vascular tissues which could be carried upward in the xylem or downward in the sap stream. Neither did we observe translocation of conidia to other tissues. However, the development of F. circinatum conidia in the cavities of the pith may lead to dispersion through these gaps and the generation of new infection foci distal to the IP within the pith. As spore dissemination is passive, spores will be subject to the force of gravity. Thus, the germination of secondary mycelium from new infecting foci may partially explain the preponderance of fungus in the pith at locations below the IP as seen at stages later than 21 dpi, and also the slowdown of fungal progression through this tissue. Indeed, the colonization of the pith in positions below the IP appears to be intrinsic, as fungus was absent in the immediate vicinity of this tissue (Fig. 7e–g). By contrast, the colonization of the pith in upper locations appears to be associated with invasion of the pathogen from the xylem (Fig. 7a–c).
Symptoms of dieback began to appear at 21 dpi and were apparent in the majority of seedlings by 28 dpi. Dieback consists mainly of chlorosis, wilting of needles and desiccation of the upper part of the seedling tip, symptoms that were consistent with those described in pine seedlings by other authors (Solel & Bruck, 1990; Correll et al., 1991; Viljoen et al., 1994). Solel & Bruck (1990) attribute wilting of young shoots to the pathogen's ability to obstruct water flow through the vascular bundle. In our case, q-RT-PCR analysis showed that fungal biomass around the inoculation zone had reached carrying capacity at 21 dpi (Fig. 2) and confocal microscopy revealed that xylem colonization at the IP was almost complete by 21–28 dpi (Fig. 3c,d). However, by this time, extensive invasion of the three-dimensional network of living cells in the xylem as well as the highest amounts of exuded resin (Fig. 9a) were both observed. These data together indicate that F. circinatum may play an active role not only in the physical obstruction of the tracheid pits, but also in the generalized collapse of the network of living cells in the xylem and in the stimulation of resin production, all of which combined may ultimately produce plant death.
CLSM analysis showed that, in general, fungal colonization was predominantly upward when dieback symptoms were still not apparent and then, after wilting of the seedlings, fungal colonization occurred predominantly in a downward direction. On this basis, we propose that upper or lower colonization along the stem probably relies on the direction of water flow and on the hydration state of the host. Thus, analysis of the colonization pattern during the consummated stage of infection represented in Fig. 7 suggests an initial upward trend due to the transpiration stream, which leads to intense colonization of upper locations close to the inoculation zone (Fig. 7c) and which may also explain the scattered distribution of the pathogen seen in xylem tissue above the IP (Fig. 7b,c). After disruption of water flow, this ascending invasion loses protagonism, in favor of a descending invasion via phloem and pith through lower positions in the stem which are still alive and have more available succulent tissues (Fig. 7e–g).
Barrows-Broaddus & Dwinell (1983) suggested that reddening and wilting of needles was the result of invasion by F. circinatum hyphae from the infected cortex in the shoot to the adjacent needle. We also observed invasion of the vascular tissue of needle traces arising from the infected pith, as well as an extensive reaction zone in the cortex adjacent to the needle fascicle (Figs 7b, 8d). This reaction zone exhibited a different fluorescence pattern which was probably a result of secreted plant substances such as phenolic compounds with different autofluorescence properties (Hutzler et al., 1998). Resin synthesized by TRDs is considered to be enriched with phenolics (Nagy et al., 2000) and we indeed observed an intense yellow fluorescence emission (530–600 nm) when blue excited (488 nm) from the secretory epithelial cells lining resin ducts (Figs 3b, 5d, 6a–c, 8b).
Despite the general effectiveness of the enhanced biosynthesis and accumulation of oleoresin as an induced chemical defense mechanism in many conifer species (Eyles et al., 2010), our results suggest that the opposite may also be the case, that is, that resin production following F. circinatum infection may be proportional to the degree of susceptibility to the pathogen. Evidence for this possibility can also be found in the data provided by Enebak & Stanosz (2003) and Kim et al. (2008, 2010), who evaluated tolerance to infection by F. circinatum in different pine species. Indeed, Barrows-Broaddus & Dwinell (1983) affirmed that resin production did not appear to be a protective advantage against F. circinatum. Our study indicates that F. circinatum tolerates resin and even stimulates its production, initially in the epithelial cells lining preformed resin ducts (Fig. 3b) and then indirectly by inducing the reprogramming of the cambial zone to produce TRDs (Fig. 7b,c,g). We visualized new TRDs as early as 14 dpi (Fig. 8e), in keeping with observations by others in Picea abies (Nagy et al., 2000; Krekling et al., 2004). Xylem resin duct density increased during pitch canker development in radiata pine seedlings (Fig. 9b) with the subsequent significant increase in resin exudation 1 wk later (Fig. 9a).
Fusarium circinatum appears to exploit to its benefit one of the classical conifer defense mechanisms, that is, enhanced formation of TRDs and associated increase in resin production. On the one hand, the pathogen uses resin ducts for vertical colonization of new zones in the pine stem. Indeed, Barrows-Broaddus & Dwinell (1984) had suggested that resin ducts may be being used as portals for the vertical spread of F. circinatum. On the other hand, high amounts of resin accumulation are thought to restrict the supply of water (Gordon, 2011), thereby contributing to plant death. Moreover, epithelial cells lining the resin ducts and surrounding parenchyma cells contain starch grains (Hudgins et al., 2005; Nagy et al., 2005) which provide the pathogen with additional food. Supporting this idea, Krekling et al. (2004) found that susceptible clones of P. abies produced more TRDs in response to Heterobasidion annosum inoculations than resistant clones; Hudgins et al. (2005) reported that the accumulated resin produced by constitutive resin ducts and by TRDs in Pinus monticola did not directly affect Cronartium ribicola development; and Luchi et al. (2005) concluded that resin flow may not be a good predictor of host resistance to Sphaeropsis sapinea and Diplodia scrobiculata canker pathogens. The fact that F. circinatum induced a defense mechanism which appeared to damage pine health by favoring fungal establishment is quite intriguing. However, others have also suggested that some alleged defense-related molecules, such as stilbenes, function more as markers of disease or are autotoxic to the plant (Bonello et al., 1993).
To conclude, the present findings extend our knowledge of the mechanisms underlying the interaction between fungal pathogens and the most successful gymnosperm plants, the conifers. Today, coniferous forests are dominant in eastern North America, Europe and Siberia, so a more precise characterization and understanding of the mechanisms that underlie disease development is very important in order to improve management systems for minimizing the damage or loss from both an ecological and economic point of view. In this sense, our results support the idea proposed by Lombardero et al. (2006) that increased resin flow in conifers following fungal inoculation should not be interpreted exclusively as an effective inducible defense mechanism against some fungal pathogens. In addition, with respect to pitch canker disease, our findings of conidiophora inside the pine stem at early stages point to the importance of preventing primary infection from this source to other plants as a consequence of incorrect management procedures. Moreover, we have also identified signs of disease before the manifestation of external symptomatology, using confocal microscopy and q-RT-PCR techniques. This provides the groundwork for the detection of compatible and incompatible plant–pathogen interactions during the first week post-inoculation, although the optimization of specific protocols for this purpose will require further studies.