(This article can be cited as: Biggs, A.R. 1992. Responses of angiosperm bark tissues to fungi causing cankers and canker rots. In:  Defense Mechanisms of Woody Plants Against Fungi. Blanchette, R.A. and A.R. Biggs, Eds. Springer-Verlag (Berlin) pp. 41-61.)

1. Introduction

2. Infection Courts for Canker Pathogens

3. Colonization and Establishment of Canker Pathogens

    3.1 Valsa and Leucostoma Canker of Peach and Various Hardwoods

    3.2 Nectria Canker of Apple and Various Hardwoods

    3.3 Hypoxylon Canker of Aspen

    3.4 Cryphonectria Canker of American Chestnut

    3.5 Eutypella Canker of Maple

    3.6 Botryosphaeria Canker of Peach

    3.7 Cerrena unicolor Canker Rot of Maple and Birch

4. Delimitation of Canker Pathogens in Bark Tissues

5. Breaching of Host Boundaries by Canker Pathogens

6. Expression of Resistance to Canker Pathogens in Bark Tissues

7. Molecular Regulation of Plant Response: Normal Development, Wounding, and Pathogenesis

1. Introduction

Cankers on trees are the visible manifestation of necrotic periderm, cortex, phloem and vascular cambium tissues (Figs 1- 4). In most shade tree or forest pathology textbooks, cankers are classified according to types or classes to facilitate instruction and discussion (Boyce 1961, Tattar 1978, Manion 1981). Generally, cankers can be either annual or perennial in occurrence and, if perennial, can be either diffuse or target shaped. In reality, canker symptomatology is extremely diverse, often with gradations among the distinct types. The biological basis for the varying expression of disease symptoms is due to the interaction of several factors, including pathogen virulence, host resistance, influence of environment, and time.

Cankers may be incited by fungi from the Ascomycetes, Deuteromycetes, Basidiomycetes, and, less commonly, Oomycetes. Generally, stem cankers are caused by Ascomycetes, Deuteromycetes, and Oomycetes, whereas canker rots are caused by Basidiomycetes. Most fungi that cause stem cankers are restricted to bark and xylem tissues that succumb due to the effects of toxins or secreted enzymes. Such organisms are termed necrotrophic or facultative parasites and include many of the more extensively studied canker pathogens (e.g. Nectria galligena, Cryphonectria parasitica, Leucostoma persoonii). Many stem canker pathogens invade and colonize xylem tissues in the vicinity of the canker without causing decay. Basidiomycetous fungi that cause cankers and which also extensively invade the xylem, simultaneously causing wood decay, are termed canker rot pathogens (e.g. Cerrena unicolor). This distinction between stem cankers and canker rots is, in many cases, artificial because many ascomycetous fungi that cause cankers can also extensively degrade the xylem (e.g. Strumella, Xylaria, Hypoxylon, and many others).

In evaluating abnormal plant tissues, not only a comparison of normal and abnormal forms is of great importance, but also a comparison of the abnormal with one another is essential. With this premise in mind, this chapter provides points of comparison and contrast with several of the various stem cankers and canker rots caused by fungi. Normal tissues are not discussed extensively in this chapter given that this information is summarized elsewhere. The terminology of bark tissues discussed by Trockenbrodt (1990) is utilized in this chapter.

2. Infection Courts for Canker Pathogens

Most fungi causing stem cankers and canker rots colonize the host plant via open wounds, dead branches, branch stubs, twigs, leaf scars or, less commonly, through leaves. The importance of dead branches and branch stubs is based not so much on quantitative data, although the limited data available do confirm these observations (Biggs 1989b), rather it is based on the observation of the dead branch or stub in the center of the canker (Fig. 3). Much is known about the anatomy of bark wounds, however considerably less is known about the anatomy of senescing and dead, but still attached, branches (Shigo 1985). Direct penetration of intact bark or epidermis has not been demonstrated for any of the facultative parasites that cause most stem cankers. However, some pathogens (e.g. Macrophoma tumefaciens on poplar (Kaufert 1937, Zalasky 1964), Apiosporina morbosa on plum (Koch 1935)) which initiate galls and other hypertrophic tissues on stems may penetrate directly the epidermis of current years' shoots or via lenticels. A possible exception that has been reported recently, although not confirmed histologically, is the direct penetration of peach bark lenticels by Botryosphaeria dothidea when trees had been exposed to water stress conditions following inoculation (Pusey 1989). One potentially significant observation that deserves further investigation is the often superficial location of the phellogen in branch/trunk junctions (Shigo 1985). This raises the possibility of direct penetration of bark tissues by any or all of the canker-causing fungi. Accumulation of snow and ice also may wound easily this apparently sensitive area which could help explain the high frequency of canker initiation by Nectria galligena in branch axils of New England hardwoods observed by Grant and Spaulding (1939).

3. Colonization and Establishment of Canker Pathogens

Most anatomical studies of the colonization and establishment of canker fungi have used artificial wounds and massive amounts of hyphal inoculum, on agar plugs or infested wood dowels or grain, to initiate infection. Under these conditions, the frequency of infection is nearly 100%, and colonization of bark tissues proceeds at a rapid rate. Typically, a portion of bark is pulled back or removed with a scalpel or cork borer and the inoculum is put in place and covered with the original bark, and the site is sealed with tape or a plastic wrapping. Very few detailed histopathological studies on the initial events of host colonization have been conducted with spore inoculations because of the generally low frequency of successful infection. The following examples, however, provide some information on the early stages of infection.

3.1 Valsa and Leucostoma Canker of Peach and Various Hardwoods (in-depth article!)

Butin (1955) and Biggs et al. (1983) reported that the advancing margin of Cytospora chrysosperma in poplar bark (Figs. 5 and 6) is associated with an intercellular aggregation of hyphae that appears in the outer cortex and inner periderm or, more rarely, in the phloem region (Figs. 7 and 8). Behind the margin and in the obviously necrotic area, hyphae become less tightly woven, smaller in diameter and more diffuse (Fig. 9). Colonization in this area is by both intercellular and intracellular hyphae that are present in the cortex, phloem, xylem, and ray cells. In cankers sampled 6 to 12 months after inoculation, mycelial aggregates are observed adjacent to phloem fiber bundles (Biggs et al. 1983). The authors suggested that the fungus could penetrate into living tissues through the fibers and/or along the interface of fiber bundles and necrophylactic periderm.  

Single hyphae of Valsa ceratosperma, the causal fungus of apple canker in Japan, invade bark slowly during the dormant season. As temperatures increase in the summer, the fungus forms mycelial aggregates which penetrate and destroy wound periderm tissues (Tamura and Saito 1982). In peach bark inoculated with Leucostoma persoonii, fungal colonization was initially diffuse although mycelial aggregations formed within 3 weeks (Wisniewski et al. 1984). These authors also observed the penetration of wound periderm tissues by mycelial aggregates. Other researchers have reported the presence of mycelial aggregations of L. persoonii (Tekauz and Patrick 1974, Biggs 1984b, 1986a) and L. cincta in peach bark (Tekauz and Patrick 1974, Biggs 1986a).

3.2 Nectria Canker of Apple and Various Hardwoods

Nectria galligena is able to enter the host through wounds inflicted by pruning, frost injury, breakage caused by ice and snow (Lortie 1964), and small dead branch stubs (Grant and Childs 1940, Zalasky 1968). In apple, N. galligena enters the host through pruning wounds, frost injury, infections caused by Venturia inaequalis and Neofabrea malicorticis, and leaf scars (Wiltshire 1921, Crowdy 1949). It is not known if forest trees are infected through leaf scars. For the fungus to become well established, wounds must be to the depth of the vascular cambium. The host is able to confine the pathogen to the cortex when wounds are shallow. The fungus is able to colonize all of the tissues which it penetrates. In the cortex and phloem, hyphae are intercellular in the early stages of infection but intracellular hyphae were observed during the later stages. The fungus grows vigorously in the phloem region and enters the xylem via the medullary rays. Although the pathogen is unable to penetrate suberized cell walls (Krähmer 1980), it grows freely in the vessels and tracheids and spreads in the xylem through the pits. Intracellular phenolic compounds, gum in the xylem, and wound lignin at the levels observed in this host/pathogen interaction do not deter fungal colonization.

3.3 Hypoxylon Canker of Trembling Aspen

Hypoxylon mammatum enters aspen trees through wounds caused by insects, axe cuts, snow and ice breakage, and dead branches. The periderm is not invaded, but the hyphae are found in the phloem, cortex, and cambium tissues (Bier 1940). Hyphae penetrate the cell walls directly, forming bore holes, and spread within the tissue is intracellular. Cambial cells, sieve tubes, and phloem parenchyma cells are destroyed by the fungus, however, phloem fibers and other sclerified cells are not colonized (Rogers and Berbee 1964). These authors described two hyphal types associated with bark colonization; primary hyphae averaging 1.2 m in diameter were observed immediately beyond the advancing margin of the canker, while the secondary hyphae, averaging 2-5 m in diameter, were present in greatest abundance in previously killed tissue. Hypoxylon cankers are one of the stem cankers caused by ascomycetous fungi that cause extensive decay of the xylem, often resulting in breakage of the trunk at the canker.

3.4 Cryphonectria Canker of American Chestnut (Chestnut Blight)

The fungus Cryphonectria parasitica can become established through any type of wound in the bark that is deeper than the outer green cortex (Boyce 1961). The initial penetration of chestnut bark tissue by the fungus also is accomplished by mass action of hyphae (Keefer 1914, Bramble 1934). The fungus mycelium accumulates in the wound, expanding the initial lesion for about 9 days (Hebard et al. 1984). Lesion enlargement ceases and then resumes at about 26 days after inoculation, persisting until the tree is girdled or until environmental factors limit growth. The fungus makes its initial penetration into surrounding tissue by means of flattened masses of mycelium termed "mycelial-fans" (Bramble 1934). Mycelial fans cleave and partially destroy cells during penetration of the bark. Secondary invasion of individual cells occurs behind the advancing margin, similar to that described for C. chrysosperma. The advancing margin of hyphae may invade just beneath the periderm, as described for C. chrysosperma, and also into the internal cortical tissues. Individual hyphae are not found in advance of mycelial fans in living bark, however, host cells in advance of the fungus appear dead before being colonized by the fungus (Hebard et al. 1984).

3.5 Eutypella Canker of Maple

The fungus Eutypella parasitica enters its host mostly through dead branch stubs and, less often, through dead twigs and other wounds deep enough to penetrate the bark (French 1969). The colonization of bark tissues occurs initially in the functional phloem. The first indication of infection is a zone of necrotic tissue in advance of the mycelium, suggesting the action of a toxin or other secreted substance. The necrotic cells are then invaded by hyphae and then mycelial fans form in the necrotic zone that was initially infected. As the fungus ramifies in the phloem, hyphae also spread radially into the phelloderm, cambium, and xylem tissues. The fungus can degrade completely sclerified cells in bark. The fungus enters xylem and phloem cells through the pits, later causing wall erosion and forming bore holes. In thick bark, multiple mycelial fans often form and may coalesce to form larger mycelial aggregates. The bark of Eutypella cankers remains firmly attached to the face of the canker, unlike that of Nectria cankers.

3.6 Botryosphaeria Canker of Peach

Inoculation of peach bark wounds, inflicted with a cork borer, with spores of B. obtusa and B. dothidea provided an interesting contrast to the mycelial inoculation studies described above. Initial colonization by the fungus occurred on the wound surface, in necrotic bark tissue on the wound margin, and in necrotic xylem tissue to a depth of about 200 m (Biggs and Britton 1988). In inoculated trees, few macroscopic symptoms of infection were present during the eight week course of the study, and normal wound healing responses appeared macroscopically and microscopically to be proceeding similar to the noninoculated wounds. Histologically, no differences in lignin and suberin deposition, periderm formation, and callus formation were observed between noninoculated wounds and wounds inoculated with either fungus after one and two weeks incubation. However, by 28 days after inoculation for both fungi, new callus tissue was being colonized by fungal hyphae located on the surface of the xylem beneath the nonsuberized ventral callus surface. The nonsuberized ventral region of callus was primarily parenchymatous with a ligno-suberized outer layer, and was the focal point for fungal pathogenesis. By eight weeks, the breakdown of tissue in this region was directly associated with fungal hyphae in close ventral proximity. Direct fungal penetration of the callus, although only occasionally observed, was not required to induce gum pockets, cambial alterations, and tissue breakdown. Intracellular hyphae of both Botryosphaeria spp. were observed colonizing cortical and callus parenchyma, and xylem ray parenchyma, vessels and tracheids. Intercellular hyphae were observed in phloem fibers, necrotic cortical parenchyma external to wound periderm, and necrotic ligno-suberized parenchyma on the callus surface.

3.7 Cerrena unicolor Canker Rot of Maple and Birch

Little is known about how canker rot pathogens penetrate and become established in their hosts. Following mycelial inoculations, Cerrena unicolor, the cause of canker rot and decay in sugar maple and paper birch, colonized both phloem and xylem tissues (Enebak and Blanchette 1989). In phloem observed 6 months after inoculation, the fungus had formed hyphal wedges which grew intercellularly resulting in splitting and subsequent death of the tissue. Similar patterns of colonization occur for other canker rot fungi (Fig. 10) (Shigo 1969).

4. Delimitation of Canker Pathogens in Bark Tissues

The delimitation of fungal pathogens in tree bark ultimately occurs via the formation of ligno-suberized boundary tissue derived from cells extant at inoculation and necrophylactic periderm derived from newly differentiated phellogen (Mullick 1977, Biggs 1984a,b, Biggs et al. 1984). For annual cankers, these barriers appear to be effective for preventing continued colonization by the fungus and diseased tissues are usually sloughed. For perennial cankers, bark boundary tissues, which also may include extensive xylem formation (Figs. 11 - 14) are either directly penetrated or circumvented annually resulting in a series of concentric callus ridges. The direct involvement of the ligno-suberized layer and/or wound periderm, or by-products generated by the tree during the differentiation of these tissues, in the resistance of woody plants to fungi has never been demonstrated conclusively. Several lines of evidence indicate that periderms function to prevent desiccation of internal layers, prevent inward movement of pathogen toxins or enzymes, and serve as physical barriers to colonization. The process of generating new periderm also may result in lignification of pathogen hyphae (Vance et al. 1980, Biggs 1986a) or in the production of fungistatic compounds which act as phytoalexins. Additional discussion of resistance in tree bark to canker pathogens is presented in section 3.6. 

Several recent papers have described the formation of necrophylactic periderm and associated tissues in response to pathogens in considerable detail (Biggs 1984b, 1986a, Biggs and Britton 1988). The formation of ligno-suberized boundary tissue and necrophylactic periderm in inoculated bark is similar to that in noninoculated wounds   although there are many notable differences.  

Pathogens affect the position or location of periderm and related tissues in wounded bark relative to their location in the absence of pathogens. In wounds, new tissues are regenerated usually within 1-2 mm of the wound surface (Biggs 1984a). When wounds are colonized by aggressive pathogens, the formation of new tissues may be several centimeters from the infection court.  

Pathogens affect the morphology and differentiation of new tissues and may retard or inhibit the formation of periderm and/or the deposition of suberin in wound periderm and xylem barrier zones (Goring 1975, Biggs 1986a, Biggs and Britton 1988). Necrophylactic periderm that formed in peach bark cortex inoculated with L. persoonii and L. cincta occasionally exhibited phellem cells with altered morphology (Fig. 3.14). Altered cells appeared thin-walled and rounded compared with the relatively thick, rectangular phellem produced at control wounds. Bramble (1934) also reported that wound periderms in American chestnut inoculated with C. parasitica varied in their structure from that of normal periderm. In wound periderms, as many as ten layers of thin-walled phellem formed prior to the formation of thick-walled phellem in comparison to normal periderm which formed a minimum of three layers of thin-walled cells. Wound periderm in chestnut also possessed additional phelloderm layers relative to normal periderm (Bramble 1934, Hebard et al. 1984).  

Wounds inoculated with Leucostoma spp. and Botryosphaeria spp. both showed delayed or inhibited suberin formation, beyond that which occurred naturally, in the ventral callus surface (Biggs 1986a, Biggs and Britton 1988). This region was often a site for continued colonization of bark and xylem tissues by these pathogens. When xylem tissues were examined for suberin formation following wounding, large areas of suberized ray parenchyma were observed in the wall 3 region based on the CODIT model of compartmentalization (Shigo 1979). Such regions were absent in inoculated xylem (Biggs 1986a).  

Few hosts are able to wall out the initial invasion of a pathogen with the formation a single ligno-suberized boundary zone and necrophylactic periderm. Instead, the tree often forms a series of incompletely formed periderms within a single season in response to the advancing fungus (Fig. 15). The fungus will continue to spread in the host tissue until conditions become more favorable for the host to form a complete periderm and less favorable for the pathogen to penetrate further. This is why many canker lesions on juvenile stems have visible concentric areas of discoloration during the initial establishment phase. For perennial target-shaped cankers, the tree also produces annual rings of callus tissue which represent the successful formation of necrophylactic periderm and callus tissue for that year but which gives no clue as to the number of attempts during that particular year.  

In bark of peach cultivars inoculated with L. persoonii and L. cincta, necrophylactic periderm formed at 21-28 days after inoculation and was associated with an effectively delimited lesion in both cultivars inoculated with L. cincta, the less pathogenic fungus (Fig. 16). For cultivars inoculated with L. persoonii, the wound periderm was not as thick, had fewer cells, and was associated with only a slight inhibition of lesion expansion followed by a second rapid increase in canker length as the periderm was breached between 28-35 days after inoculation (Biggs 1986a). In chestnut, Hebard et al. (1984) observed that periderms were produced by the host and then breached by the fungus at 18 or 28 days after inoculation depending on the virulence of the isolate. In resistant chestnuts, some virulent isolates continued to expand lesions up to 30 days after inoculation (Russin and Shain 1984).

5. Breaching of Host Boundaries by Canker Pathogens

Very few studies on hardwood cankers have presented evidence to show conclusively how the fungus invades the phellogen layer to reinfect the phloem after the canker has stopped expanding for the season. For many cankers, the main mode of reinfection is by hyphal penetration of the wound periderm. Penetration may be either direct via mass action of hyphae or indirect via hyphal penetration of fibers or sclereids which occasionally bridge wound periderms and connect diseased with healthy tissue. Penetration of periderms by mass action has been described for Cytospora canker of peach (Wisniewski et al. 1984), Nectria canker of apple (Crowdy 1949), and canker rot of maple and birch caused by C. unicolor (Enebak and Blanchette 1989). Cryphonectria parasitica, which forms diffuse cankers, grows under wound periderm before it forms (Bramble 1934) and thus can rapidly girdle chestnut stems. Hebard et al. (1984) showed that mycelial fans of the fungus also could penetrate developing wound periderms directly.  

Other avenues of reinfection occur in natural and pathogen-induced discontinuities along the wound periderm and new callus tissue produced by the host in response to invasion. For example, Biggs (1986a) observed L. persoonii to circumvent wound periderm by colonizing the nonsuberized ventral callus region (Figs. 17 and 18) or by growing between the original periderm and wound periderm external to the cortex, or between the wound periderm and phloem fibers. Similar routes of reinfection were described for Valsa canker of apple (Tamura and Saito 1982). 

The lumens of phloem fibers also may provide a means of entry for fungi into healthy tissues delimited by wound periderm. Poplar bark colonized by C. chrysosperma (Biggs et al. 1983) and apple colonized by N. galligena (Crowdy 1949) may be reinvaded in this manner. Similarly, Eutypella parasitica may reinvade maple bark through sclereids or other discontinuities in the wound periderm (French 1969). 

Ashcroft (1934) proposed that fungi such as N. galligena which colonize the xylem may reinvade the bark by growing through the medullary rays and into the wound wood (CODIT wall 4) and ray parenchyma into healthy bark that was not delimited by wound periderm. This has never been demonstrated although French (1969) mentions it as a possibility for E. parasitica.

6. Expression of Resistance to Canker Pathogens in Bark Tissues

Discussions of resistance include two basic concepts: constitutive, or passive defense, and induced, or active, counter-attack defense (Cowling and Horsfall 1980). Constitutive defenses are critical in the protection of trees against fungal pathogens. These defenses encompass the plethora of structures and chemicals that trees have evolved over the millennia to exclude pathogens and avoid or tolerate herbivory (Berryman 1988), and include leaf trichomatous structures, resin-covered or resin-filled structures in conifers, cell walls impregnated with lignin or phenolic compounds, cells containing phenolic compounds or other preformed toxic substances (Tattar and Rich 1973, Parker 1977), and heavily cutinized or suberized tissues. Fungi that cause canker diseases most often encounter the latter in the form of cutinized epidermis and suberized exophylactic (first) or necrophylactic (sequent) periderm. Given that the majority of trees are free of canker infections, it appears that constitutive defenses are very effective.  

Preformed or constitutive defenses against fungi that cause cankers are breached by wounds thus initiating the processes of induced defense. This type of defense consists of the production of chemicals and/or reestablishment of barriers (wound periderms) to pathogen ingress in response to infection. Phytoalexins have been detected in the bark of woody angiosperms, although these substances have not been as extensively characterized as those found in herbaceous plants. Their production in the barks of Populus tremuloides (Flores and Hubbes 1980) and Morus alba (Shirata 1978) may have a defensive function prior to the formation of new periderms. The new periderms are distinct from the preformed constitutive barriers based on 1) their dissimilar histochemical reactions to lignin reagents (Biggs 1984b, Rittinger et al 1987), 2) the formation of a ligno-suberized boundary as a prerequisite to periderm differentiation (Mullick 1977, Soo 1977, Biggs 1984a,b), and 3) biochemical differences in phellem cell contents (Mullick 1977, Soo 1977).  

The induction of defensive reactions in plants is multifaceted. Two types of induced defensive reactions, localized and systemic, have been described for plants and several hypotheses have been proposed as possible mechanisms for their induction (Sequeria 1983, Darvill and Albersheim 1984, Ryan 1988). The localized response occurs in the vicinity of the site of wounding or infection and is characterized by the necrosis of plant cells and the accumulation of phytoalexins. Phytoalexins, substances produced de novo which act nonspecifically to restrict colonization by microorganisms, are accumulated and released by plant cells in response to various elicitors released from pathogen or plant cell walls (Hahn et al. 1981, Nothnagle et al. 1983, Darvill and Albersheim 1984, Ryan 1988, Bostock and Stermer 1989). The localized response is expressed in variable intensity depending upon the extent of host cell necrosis triggered by the wound itself and the colonization of the wound by microorganisms. 

Systemic defense reactions are observed in the plant at sites other than the point of injury or infection and may be present well after its induction. Studies have shown that the systemic reaction in bean, which is not induced by wounding alone, makes a plant more resistant to subsequent inoculations following the original inducing inoculation (Kuc 1983). It is not certain what role, if any, systemic resistance (sensu Kuc) has in defense against canker pathogens. Other investigations of systemic resistance (sensu Ryan and others) have shown the induction of various defensive chemicals by wounding, pathogen colonization, and fungal elicitors (see below). Indeed, the discussion of intraplant systemic communication takes on much broader implications with the recent discovery of interplant communication and the possible role of airborne volatile substances, such as methyl jasmonate (Farmer and Ryan 1990), acting to induce defensive genes in nonwounded plants located adjacent to wounded plants. 

The occurrence of wounds and the reaction of the healthy plant to wounds are part of the natural life process for trees. The plant's response to wounding leads to the restoration of its natural permeability barriers after they are broken. Plant responses to wounding occur as part of normal developmental processes and responses to infection are essentially similar or identical to wound responses. Given the greater likelihood of ingress furnished by wounding, the delimitation of pathogens by the same or similar processes that restore the plant's natural permeability barriers and meristems is strategic, from an evolutionary point of view, in its conservation of response repertoires. What are the differences then between the tree's response to wounding and infection, and what factors regulate these differences?  

Induction of the wound response is characterized by chemical biosynthesis regulated by a relatively small number of cells in which relatively small quantities of defensive chemicals are produced by living cells in close proximity to the point of injury. Cell death is minimal because toxic substances within the cells are membrane bound, limited to intercellular spaces, impregnated into cell walls, or are synthesized in quantities that are nontoxic. Some induction of systemic responses may occur following wounding in woody plants (Parsons et al. 1989, Farmer and Ryan 1990). Simple wound response may be triggered by host cell wall oligosaccharides, oligosaccharides from nonpathogenic fungi, lipids, peptides, or toxins secreted or released during the host/pathogen interaction, or wound responses may be related to hormones (e.g. ethylene, ABA) transported via the vascular tissue and regulated by barriers to outward diffusion (Li and Cui 1985, Yev-Ledun and Aloni 1990).  

Wound periderm formation by itself is an effective resistance mechanism which is highly dependent on suberin biosynthesis (see Wound Response Chapter). This assertion is based on the strong statistical correlation between the amount of suberin in the infection court at the time of inoculation and resistance in peach cultivars to L. persoonii (Biggs and Miles 1988, Biggs 1989a). Relationships between resistance and lignin accumulation, rate of periderm formation, thickness of periderm, and number of cells in periderm were not significant in these experiments. It is likely that suberin accumulation prior to the arrival of inoculum at the infection court is only one component of resistance. It functions epidemiologically as a type of rate limiting resistance and should be considered as a form of partial resistance. Suberin accumulation in wounds is also a type of active resistance characterized by chemical biosynthesis regulated by a relatively small number of cells.  

When canker-causing fungi are placed into a wound, induction of defense becomes characterized by chemical biosynthesis regulated by a relatively large number of cells which leads to a hypersensitive reaction. Large quantities of defensive chemicals are produced as a result of the autonomous process of cell death triggered by pathogen metabolites or by the induction of the host's own self-destruct mechanisms (eg. deregulation of host metabolism by pathogen products that are not directly toxic). The reactions of living cells away from the site of pathogen ingress are characterized by the rapid conversion of starches and sugars into molecules with multiple functions, i.e. lignin, suberin, their precursors, and breakdown products act as physical and chemical barriers leading to reduced water loss, restricted toxin diffusion, restricted nutrient availability, and increased antibiotic potential. In addition, processes are triggered which lead to cell dedifferentiation, redifferentiation, and the eventual formation of heavily suberized periderm tissues. Effective defense against fungi most likely occurs from combinations of responses acting in concert to reestablish the continuity of tissues and restrict pathogen ingress.  

Only a few studies have been conducted to explore the elicitation of defense reactions in woody species (Miller et al. 1986, Lieutier and Berryman 1988, Parsons et al. 1989, Biggs and Peterson 1990, Farmer and Ryan 1990). In studies on the defense reactions of seven conifer species, Lieutier and Berryman (1988) made inoculations with Ceratocystis clavigera and two chemical elicitors, chitosan and the solanaceous proteinase inhibitor-inducing factor (PIIF). Chitosan is a glucosamine polymer from fungal cell walls that has been shown to trigger lignin accumulation (Pearce and Ride 1982) and PIIF is a pectic oligomer from plant cell walls that has been shown to trigger a systemic response in herbaceous plants (Green and Ryan 1972, Ryan 1988, Farmer et al 1989). Inoculation with fungus or chitosan induced similar reactions including resin soaking in the phloem and sapwood. The plant reaction to PIIF and the buffer control were similar. Although PIIF induces hypersensitive reactions in some herbaceous plants (Nothnagel et al. 1983), it did not do so in conifers.  

In studies on the elicitation of the defense reaction in peach bark, Biggs and Peterson (1990) exposed bark wounds to several potential trigger chemicals including cell wall extract from L. persoonii and L. cincta, cell wall extract from peach leaf cells, leaf and fungal cell wall extracts combined, chitosan, calcium ion, chitosan + calcium, and the disaccharide cellobiose. Only the fungal cell wall extract and cellobiose induced a significantly greater defensive reaction than the control, based on thioglycolic acid analyses of lignin produced after wounding and treatment with elicitor. Although treatment with wall extracts from both fungi stimulated lignin production and resulted in smaller canker size after inoculation, only treatment with the extract from L. persoonii resulted in decreased percent infection rates. These results suggest that a mechanism(s) in addition to lignification is involved in the resistance of peach to these two fungi. It is likely that suberin may be more important than lignin (Biggs and Miles 1988, Biggs 1989a), however a correlation with suberization could not be established in the present study due its relatively short time course. Although the involvement of other factors, such as the production of phytoalexins, has not been investigated for the peach system, their presence is highly likely, given that a level of increased resistance to inoculations with various fungi has been observed in bark of Prunus spp. prior to the formation of ligno-suberized tissue and new periderm (Biggs 1986b, Bostock and Middleton 1987, Doster and Bostock 1988b).  

One of the more promising chemical treatments in terms of reducing both infection frequency and canker size was cellobiose. It stimulated lignin production in both the field and laboratory experiments. This suggests that small saccharides may be responsible for triggering the processes that lead to the observed differences in degree of lignin deposition in wounds versus infections (see Biggs 1984b). Another chemical treatment included in these experiments was calcium. Although this ion did not stimulate the production of lignin or suberin, it was the most effective chemical for reducing both infection frequency and size of cankers. The mode of action of calcium could be related to the stimulation of the synthesis of phytoalexins and/or phenols as suggested by Kohle et al. (1985), although our analyses did not confirm this hypothesis. A second possibility is that calcium may reduce fungal polygalacturonase activity by forming cation cross bridges between pectic acids in the plant, thus making the plant cell walls more resistant to degradation. The latter possibility is supported by the observation that breakdown of the pectins present in peach bark cell walls and the sequestration of calcium by fungal oxalic acid have been shown to occur during pathogenesis by L. persoonii (Biggs 1984b, Traquair 1987).

7. Molecular Regulation of Plant Response: Normal Development, Wounding, and Pathogenesis

There is a large body of literature on the regulation of wound responses, and responses to herbivores and pathogens, by wound-responsive genes in plant systems. The production of phytoalexins, hydroxyproline-rich glycoproteins, pathogenesis-related (PR) proteins, chitinases, glucanases, and proteinase inhibitors may all be subject to the expression of defensive genes in plants. A greater understanding of the molecular regulation of the defensive chemicals listed above could lead to augmentation of natural defense mechanisms via direct manipulation of the tree genome to produce trees with increased resistance to pathogens. 

In hybrid poplar, Parsons et al. (1989) have demonstrated the existence of two wound-inducible mRNAs present in unwounded upper leaves following mechanical wounding of the lower leaves. Several classes of transcripts accumulated in response to repeated wounding, although the two cDNAs that were characterized and sequenced showed a high degree of similarity to gene families which encode chitinases from bean, tobacco, and barley. Wargo (1975) has demonstrated the presence of chitinases in phloem tissues of Acer saccharum and several Quercus spp. Pathogenesis-related proteins identified as chitinases are known to be induced by pathogen colonization, fungal elicitors, ethylene, and mechanical wounding in Phaseolus vulgaris (Broglie et al. 1986). Systemic occurrence after wounding also is characteristic of the proteinase inhibitors in solanaceous plants (Ryan 1988). In this system, systemic gene activation can be elicited by host cell wall oligogalturonides, although it is not likely that these substances move systemically within the plant because of their relatively large size (Dixon and Lamb 1990). Their function may be as local elicitors, either potentiating or transducing the systemic signal. Furthermore, their presence may be germane to nonsystemic wound reactions and/or responses which lead to the delimitation of lesions. Plant hormones are well known to mediate effects in plants over long distances and it is well known that ethylene (Broglie et al. 1986) and ABA (Sánchez-Serrano et al. 1990) promote gene activation of some defense relate processes. Recently, a heat stable non-carbohydrate substance has been isolated which acts to phosphorylate specific plasma membrane polypeptides (Farmer et al. 1989). These workers suggest that protein kinases may play an important role in the signalling mechanism for proteinase inhibitor synthesis in response to wounding.

From the above, it appears that a broad array of defensive chemicals are induced by a diversity of signal events or molecules, and it is possible that the initiation of transcripts stimulated by wounding or infection is initiated at different genomic sites regulated by different promoters. For example, the activation of the genes for cell wall hydroxyproline-rich glycoproteins (HRGP) show markedly different patterns of transcript accumulation under conditions of wounding or infection (Corbin et al. 1987). The presence of several distinct intercellular stress systems would provide plants with the machinery to activate similar arrays of defensive responses under different sets of circumstances related to the expression of different forms of resistance, including periderm regeneration and prevention of infection after mechanical injury, induced systemic resistance, and resistance involved in the delimitation of fungal pathogens. 

Interestingly, systemic wound-inducible and developmental (i.e. preformed in seeds and tubers) expression of potato proteinase inhibitor II was shown to be mediated by a single member of the proteinase inhibitor II gene family. This finding contrasts well with multiple genomic hydroxyproline-rich glycoprotein induction described above and suggests a possible linkage of wound responses with normal plant developmental processes. Indeed, one common feature appears to be that the phloem tissue serves as the transmitting organ used in both developmental and wound-induced gene activation (Keil et al. 1989).

The above examples describe single components that comprise a portion of the massive changes in gene expressing following wounding or pathogen invasion. These changes underlie the induction of an array of defensive responses, including phytoalexin accumulation, synthesis of cell wall HRGP, and lignin and suberin biosynthesis and deposition. In trees, it is likely that many defensive responses act in concert or in tandem to prevent colonization of wounds and stimulate generation of new tissues. Given the varied nature of fungal canker and canker rot diseases, it is surprising to find a high degree of similarity among pathosystems in the resistance and boundary setting processes. Future research on the resistance in tree bark to fungal pathogens should include well-designed time course studies on the host-pathogen interaction. Noninoculated wounds should be included as controls. Studies with biochemical inhibitors will help identify the pathways that are critical to resistance in trees. Genetic studies on the inheritance of nonspecific types of resistance are needed to determine the potential for success in conventional breeding programs. Studies in molecular biology are needed to determine the potential for in vitro approaches to augmenting natural mechanisms of defense in trees.

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