(This article can be cited as: Biggs, A.R. 1992. Anatomical and physiological responses of bark tissues to mechanical injury. In:  Defense Mechanisms of Woody Plants Against Fungi. Blanchette, R.A. and A.R. Biggs, Eds. Springer-Verlag (Berlin) pp. 13-40.)

1 Introduction

2 Bark

    2.1 Tissues of the Inner Bark

    2.2 Natural Periderms and Wound Periderms

3 Anatomy of Wound Response in Bark

    3.1 Light Microscopy

    3.2 Electron Microscopy

4 Physiological and Biochemical Aspects of Wounding

5 External Factors Influencing the Generation of New Bark Tissues

6 Regeneration of Vascular Cambium and Wound Closure

7 Wounds as Infection Courts

8. Proposed Model for Wound Responses in Woody Plants

1 Introduction

Studies of the defense systems of trees have focused mostly on xylem tissues because of their direct economic importance to the forest industry. Because bark tissues shield the xylem from the environment, containment of mechanical injuries and infectious microorganisms by bark tissues is of primary importance. The integrity of normal periderm and the ability of plants to form new periderms at wounds or injuries are essential characteristics for normal plant growth and development. However, in comparison to xylem tissues, responses of periderm and other bark tissues to injury and infection are inadequately defined.

Research on wound responses of trees is required in order to understand the processes that favor or impede the development of fungal infections in wood and bark. Many important and serious diseases of trees are caused by pathogens that initiate infections at wounds caused by insects, humans, fire, lightning, wind, hail, animals, and nutritional and physiological disorders. Therefore, it is possible that more precise information about wound responses could lead to innovative control measures based on a better understanding of the chronology of the wound response, how wound response may be influenced by external factors, or how the wound response could be modified for improved disease control. There have been many recent advances in the study of signal molecules exchanged between host and pathogen in wound tissues, and on the regulation of wound metabolism (Van Sambeek and Pickard 1976, Davies and Schuster 1981, Halverson and Stacey 1986). These and other studies have shown that plant responses to wounding and infection are often similar, suggesting the presence of common signal molecules. In trees, it is likely that the tissue regeneration process following wounding is also the defense process, given that structural responses often coincide with physiological processes that contribute to the biochemical foundation of resistant structures. In this context, the effort to distinguish between structural and physiological responses becomes misguided. Any distinctions made between physiological processes and structural wound responses in this chapter are done merely for convenience and for the purpose of discussion. It is the goal of this chapter to discuss recent findings on the anatomical and metabolic consequences of wounding in the bark of trees and to draw attention to investigations that are relevant to host-pathogen interactions.

2 Bark

The term " bark" is used most often in a nontechnical context and refers to all tissues external to the vascular cambium (Srivastava 1964, Esau 1965). Accordingly, the bark is an aggregation of organs and tissues that includes phloem and secondarily thickened tissues from the secondary plant body, as well as epidermis, cortex and phloem derived from the primary plant body (Esau 1965). The term bark was used by earlier authors in a technical context in reference to all dead tissues exterior to a deep-seated periderm (de Bary 1884, Büsgen and Münch 1929). Contemporary authors refer to this aggregate of dead tissues, which consists of alternating layers of periderms and associated tissues, as "rhytidome", a term often considered synonymous with the term "outer bark" (Eames and MacDaniels 1947, Esau 1965). The living organs of the bark consist of the phloem and the living tissues of the innermost periderm, the phellogen and phelloderm. All living tissues have been collectively termed the "inner bark" (Eames and MacDaniels 1947). Borger (1973) provided an excellent discussion of the development and shedding of characteristic bark types. Trockenbrodt (1990) has provided an informative survey and discussion of terminology used in the bark anatomy literature.

Most pathogens are unable to penetrate directly the corky, suberized tissues of most outer bark and rhytidome. These outer layers represent constitutive defenses or preformed anatomical barriers to pathogen ingress. Given that the cutinized epidermis or suberized periderms of trees are the first tissues that potential pathogens encounter (Kolattukudy and Kohler 1983), and given that the majority of trees remain alive for decades or centuries, these barriers are apparently very effective. This chapter will address anatomical aspects of wound response as it occurs in reaction to injuries of the living tissues of the inner bark and the role of wound response in resistance to fungal pathogens of trees.

2.1 Tissues of the Inner Bark

The living inner bark of a tree is composed of secondary phloem and periderm tissues (Figs. 1 and 2). In addition, cortical tissues fulfil an important role in bark of young stems. Periderm, the term first used by von Mohl (1845), is a protective tissue of secondary origin which replaces the epidermis in stems and roots that have continual secondary growth. Detailed descriptions of periderm formation are available (Srivastava 1964, Esau 1965, Fahn 1967). Roots, stems, and branches of gymnosperms, most dicotyledons, and a few monocotyledons develop periderm (Esau 1965). Herbaceous dicotyledons may form periderm, usually in the roots or oldest portions of the stem. In most coniferous and dicotyledonous trees, a periderm replaces the epidermis as the protective layer within the first year of growth. As trees age, sequent periderms may arise at successively greater depths thus causing an accumulation of dead tissues on the surface of the stem or root and contributing to the formation of rhytidome on rough-barked species or simply outer bark on smooth-barked species.

Briefly, the periderm consists of the following as originally described by de Bary (1884): phellogen (cork cambium), the lateral meristem which produces the periderm; the phellem (cork), the suberized protective tissue formed outwardly by the phellogen; and the phelloderm, a living parenchyma formed inwardly by the phellogen. Phellogen cells generally appear oblong in transverse and radial sections and appear polygonal or irregularly shaped when examined in tangential section (Schneider 1955, Srivastava 1964, Esau 1965). Phellogen cells are characteristically thin-walled, have protoplasts, are vacuolated to varying degrees, and may contain ergastic substances, starch, and chloroplasts. The phellogen may consist of only one layer, as in Fraxinus pennsylvanica, or may consist of a zone of meristematic cells, as in Ailanthus altissima (Borger and Kozlowski 1972a).

Phelloderm cells resemble cortical parenchyma cells in shape and content, although their radial arrangement makes them easily distinguished from cortical cells. Walls of phelloderm cells may be thickened and intercellular spaces may be abundant. Phelloderm cells of Populus tremuloides are chlorophyllous and photosynthetically active (Strain and Johnson 1963).

There are two main types of phellem cells, suberized cork cells and lignified phelloid cells. Cork cells are radially shortened with relatively thick walls and phelloid cells are usually thin walled and radially elongate (Grozditz et al. 1984). Both cells are dead at maturity and generally lack intercellular spaces (Esau 1965). The arrangement of phellem cells in bark varies according to species (Esau 1965).

Cortical tissues are found primarily in the bark of young stems. The first periderm in the stems of most species arises in the cortex, which is ultimately shed as new periderms arise. Phloem tissues are intimately involved in development of bark structure (Borger and Kozlowski 1972a,b,c,d). The inner bark of smooth-barked species consists largely of phloem tissues. In addition, patterns of phloem element deposition in conjunction with particular patterns of periderm development are responsible for the structure of ring barks, scale barks, and furrowed barks (Kozlowski 1971). The living cells of the outer phloem give rise to deep-seated periderms.

2.2 Natural Periderms and Wound Periderms

According to Esau (1965), natural (including first and sequent periderms) and wound periderms are basically alike in method of origin and growth. The difference between them is mainly in timing of origin and restriction of wound periderm to the place of injury. Also, wound periderm is believed to differ from natural periderms in that the former is induced by a stimulus or injury, or by factors other than those responsible for the induction of natural periderms (Bloch 1941, 1952, 1953, Akai 1959, Srivastava 1964).

Mullick's studies of periderm formation in conifer bark revealed differences in pigment composition in different periderms and provided the first evidence that natural and wound periderms may be biochemically distinct (Mullick 1977). Based on these biochemical differences, he proposed a new nomenclature for woody plant periderms in order to distinguish the two types: 1) exophylactic periderms, which includes the first periderm and sequent periderms containing similar pigments, are thought to provide protection of living tissues from the environment, and 2) necrophylactic periderms, which includes wound periderms, certain sequent periderms, and other periderms which are always found adjacent to dead tissue (nonsuberized impervious tissue or NIT), are thought to protect living tissues from the adverse effects of cell death. In angiosperms, wound periderms are distinct from the first periderms based on their dissimilar histochemical reactions to lignin reagents (Biggs 1984b, Rittinger et al 1987) and the formation of a ligno-suberized boundary as a prerequisite to barrier differentiation (Mullick 1997, Soo 1977, Biggs 1984a,b).

3 Anatomy of Wound Response in Bark

Much is known about wound anatomy in trees and there is good documentation in the literature on the anatomical events that lead to boundary zone and wound periderm formation (Hartig 1894, Bramble 1934, Crowdy 1949, Bloomberg and Farris 1962, Butin 1955, Mullick 1977, Biggs et al. 1984). The concepts regarding the chronology of wound responses in plants were developed initially with sweet potato (Artschwager and Starrett 1931), and later studies confirmed and elaborated upon earlier observations (Morris and Mann 1955, Strider and McCombs 1958, McClure 1960).

3.1 Light microscopy

Generally, the first indications of a tree's response to mechanical wounding of the bark may be viewed with the light microscope as early as 24 hours after wounding although there may be species differences in the timing of events and other specific features or there may be differences due to environmental effects on plant response. In injured peach bark tissues, for example, there may be extensive degradation of starch granules within the first 12 to 24 hours. By 96 hours these granules will have disappeared entirely in the area of tissue that is undergoing dedifferentiation although they may still be visible in the desiccated area near the wound surface and in the internal tissues some distance from the wound (Biggs 1984b). Cells in the incipient boundary zone undergo changes in the nucleus and the ability of the cytoplasm to take up morphological stains. In most cells of the incipient boundary zone, metaphase nuclei are easily seen and the nucleoli are prominent 48 hr after wounding (Fig. 3).

In some species, including Acer saccharum, Prunus persica, and other Prunus spp., the deposition of a polysaccharide substance has been detected with periodic acid-Schiff's reagent in the walls of cells located in a zone about 300 um from the wound surface (Biggs 1984b). Polysaccharide deposition occurred prior to the formation of a visible lignified zone. Although increased lignin can be detected with biochemical methods within the first 24 hours after wounding (Doster and Bostock 1988a), the first signs of lignification detectable with histochemical reagents (phloroglucinol/HCl) are apparent within 72 hours (Fig. 4) and occur internal to the area of polysaccharide deposition if the latter occurs. Reports of lignification as a response to wounding in tree bark are numerous (Bramble 1934, Butin 1955, Mullick 1977, Soo 1977, Krähmer 1980, Biggs et al. 1983a, Biggs 1984a, 1985a,b). The first lignified cells can be detected in areas of the wound in closest proximity to the vascular cambium. Although most cell types exhibit lignification in response to wounding, the parenchymatous cells in phloem ray tissue often are the first to stain visibly with phloroglucinol/HCl.

Mullick, in a series of elegant studies (see Mullick 1977), described a nonsuberized impervious tissue in conifer bark that was located in a position similar to the lignified zones reported in some of the earlier literature on tree wounds and infections. Soo (1977), a student of Mullick, reported a similar nonsuberized impervious tissue in angiosperm bark. However, results of studies reported from our laboratory on both angiosperms and gymnosperms have shown that the impervious layer that is formed prior to periderm regeneration is closely related to the formation of intracellular suberin linings in cells present at the time of wounding, and which have become lignified following wounding (Biggs 1984a, 1985a). Similar processes occur in wounded xylem parenchyma (Biggs 1987) and wounded tissues of various herbaceous and woody plant species and organs (Rittinger et al. 1987). Suberin deposition in lignified cells occurs within 24 to 48 hours after visible lignification.

The ligno-suberized boundary zone is most often located approximately 0.8 to 1.0 mm internal to the wound surface. Initial cells of this tissue can be detected within 4 to 7 days in wounds on actively growing trees in mid-summer (Fig. 5) (Biggs 1985b, 1986c) and usually occur in an area of the wound with closest proximity to the vascular cambium. A meristematic layer forms immediately internal to and abutting the primary ligno-suberized tissue and is usually detected 24-48 hr after the formation of the latter tissue (Figs. 6 - 8). Wound (necrophylactic) periderm may be well formed by 10 days postwounding (Figs. 9 and 10). Complete formation of the boundary zone and new periderm around the entire wound may take up to 28 days under ideal conditions (Figs. 11 and 12), however as boundary tissues continue to form in an outward direction, new phellogen cells form immediately internal to the established boundary tissue. As phellem is produced in an outward direction, the ligno-suberized boundary is crushed and diminishes in thickness (Biggs 1986c).

It is important to note that the presence of tissues in various stages of wound response can be observed by sampling wounded tissues at any one time, i.e. 7 to 24 days after wounding, depending upon species and inherent regenerative capacity. Ligno-suberized cells do not form synchronously into a distinct boundary zone (Biggs 1985b). The cells and subsequent tissues form first between the wound surface and the vascular cambium and last in the region of the original phellogen. One can often view wound tissues ranging from no visible reaction to those exhibiting complete periderm regeneration within the same histological section (Biggs 1985b).

3.2 Electron microscopy

At the ultrastructural level, dramatic changes are evident within the first 24 hours in cells adjacent to the wound. Generally, these changes reflect the subcellular alterations visible with light and fluorescence microscopy described above. The most noticeable subcellular modifications are in the nucleus and include changes in nucleolar fine structure and chromatin organization, both of which suggest rRNA synthesis and active production of ribosomes. In the cytoplasm, there are increases in rough endoplasmic reticulum (ER), free ribosomes, and polysomes. Cells in the region of activity of the new phellogen show increases in the amount of cytoplasm, smooth ER and dictyosomes (Barckhausen R 1978, Biggs and Stobbs 1986). All these changes reflect the intensified transcriptional, translational, and secretory activities of the responding cells. After 24 hours, ultrastructural changes are limited to those cells either undergoing dedifferentiation to form the ligno-suberized boundary zone or those undergoing redifferentiation to form the new phellogen and its derivatives.

Ultrastructural evidence for cell wall suberization in wounded peach bark was observed at 8 days after wounding (Figs. 13 - 20). Boundary zone cell walls were completely lined on the inside with an electron lucid material corresponding to cell wall linings with the histochemical and autofluorescence characteristics of suberin. The suberin portion of the cell walls appeared, at first, electron lucid, followed by formation of many light and dark lammelations. The suberin lining in individual cells appeared uniform in thickness, although thickness of the lining varied from cell to cell (ca 40-120 nm). Suberized cells in the boundary zone contained senescing cytoplasm with fragments of undifferentiated dense material that formed a thin, discontinuous granular deposit inside the suberin layer. The granular, electron dense materials likely resulted from the disintegration of the cytoplasmic ground substance and the various cell organelles. Between 8 and 12 days after wounding, the primary walls and the middle lamella in the boundary zone exhibited an increase in electron density. This is due probably to the deposition of phenolic polysaccharide material in the wall. In peach, these substances, usually referred to as gum, are produced nonspecifically in response to wounds or infections. Transmission electron microscopy of the boundary zone revealed that suberin linings were discontinuous over pit areas (Figs. 21 and 22) (Biggs and Stobbs 1986). Therefore, it is likely that the impermeable nature of these primary ligno-suberized boundaries is due, to some extent, to both lignin and suberin. Vesicles of varying proportions were frequently associated with the periphery of the senescent cytoplasm (Fig. 23).

Cells of the new necrophylactic phellem possess dense, granular cytoplasm with few distinct organelles (Figs. 24 - 26). In mature phellem, cell contents appear as a compact mass of electron-dense amorphous material interspersed with electron-lucid deposits and dark bodies of various dimensions. Numerous vesicular elements were observed, the membrane elements appearing to embedded randomly throughout the granular matrix. The plasmalemma was typically separated from the cell wall. Phellem cells possessed a compound middle lamella with an amorphous fine structure. This portion of the cell wall appeared red when stained with phloroglucinol/HCl. Suberin comprised the largest portion of the secondary wall and displayed fine light and dark lammelations. Thickness of phellem suberin layers (ca. 60-350 nm) increased with distance from the phellogen. Cell wall pits and plasmodesmata canals were not observed in the phellem. Phellem cells with intact organelles were detected infrequently and, when detected (Fig. 2.26), were characterized by abundant mitochondria, rough endoplasmic reticulum, dictyosomes, and associated vesicles.

4 Physiological and Biochemical Aspects of Wounding

In comparison to herbaceous plants, there is little information available on biochemical aspects of wounding in tree species, perhaps due to the inherent difficulty of conducting biochemical analyses of woody tissues. Much of our knowledge about trees in this area comes from histochemical investigations and is thus limited in scope. Generally, when a plant is injured, a complex array of physiological and biochemical responses are elicited. Bostock and Stermer (1989) categorized these into immediate or rapid responses (e.g. depolarization of cell membranes, release of host or pathogen cell wall fragments) which occur within seconds or minutes after wounding and slow responses (e.g. complex biosynthetic reactions, formation of boundary tissues) which occur over a period of hours, days, or even weeks. For convenience, wound responses can be separated among the cellular compartments where they occur, i.e. membranes, cytosol, and cell wall (Table 1). The separation of events according to spacial and temporal criteria is artificial given the intimate contact and free exchange of materials among compartments. What emerges following an injury is a series of predictable and coordinated events which concludes, in the bark of woody plants, with formation of a primary ligno-suberized boundary layer, cell division leading to the formation of wound periderm, the production of callus tissue, new vascular cambium, and eventual closure of the wound.

Stimulation of the shikimic acid pathway leading to enhanced production of phenolic derivatives is a ubiquitous response among plants when injured or infected (Vance et al. 1980). Phenolic substances derived from the shikimic acid pathway may be directly toxic to potential pathogens or may form the foundation for large molecular weight polymers, such as lignin, that are incorporated into walls of extant or newly differentiated cells. Fatty acid metabolism is involved in the synthesis of suberin and waxes (Kolattukudy 1984), and these substances are found also in extant and newly differentiated cells.

In trees, the rapid production of high levels of suberin may be a determining factor in resistance against fungal pathogens (Biggs and Miles 1988, Biggs 1989). Since suberization appears to be triggered by wounding, it is likely that some chemical signal generated soon after the wound is inflicted initiates the process which leads to suberin deposition. Experiments with potato tubers have shown that suberization can be inhibited by washing the wound with water during the first 72 hours (Soliday et al. 1978). Abscisic acid is removed by these washes and exogenous ABA applied to washed tissues partially restored suberin production. Abscisic acid presumably is an important intermediate in the signal transduction pathway leading to the induction of suberin (Kolattkudy 1984). Abscisic acid is probably not the direct inducer of suberin because suberin deposition can still be inhibited beyond the time that ABA can be washed out of the tissue. Kolattukudy maintains that ABA produced during the first 24-48 hours after wounding triggers the formation of an as yet unidentified suberin inducing factor at about 72 hours postwounding. This is followed by the appearance of enzymes involved in suberin biosynthesis within 96 hours and the presence of suberin soon thereafter.

There has been much recent interest in the stimulation of defense reactions in plants by polysaccharides of animal, plant and fungal origin (Darvill and Albersheim 1984). Albersheim and his associates maintain that the synthesis of phytoalexins in some plants is elicited by cell wall oligosaccharides. When oligosaccharides from both the fungal and plant walls are used as a treatment, their effect on the production of phytoalexins is synergistic. Many other substances of host or microbial origin may be involved in the elicitation of defense reactions in plants. Traumatin, trans-2-dodecenedioic acid, or the oxidation product of its aldehyde, 12-oxo-trans-10-dodecenoic acid, have been found in healthy and wounded bean cells (Zimmerman and Coudron 1979). The eicosapolyenoic acids, arachidonic acid and eicosapentaenoic acid, are abundant in the lipids of Phytophthora infestans and related Oomycota (Bostock and Stermer 1989). When applied to potato tuber disks, these fatty acids cause marked changes in isoprenoid metabolism, especially when combined with fungal -glucans, thus demonstrating that fungal substances can enhance and redirect wound metabolism.

Chitinase and -1,3 glucanase activity increases in some plants after wounding or infection (Boller 1987). Indeed, Wargo (1975) extracted these enzymes from the phloem of Acer saccharum and several Quercus spp. and demonstrated that the enzymes could lyse the hyphal walls of the root rotting fungus Armillaria mellea. Hydrolase enzymes may act to inhibit fungal growth and degrade fungal cell walls, thus releasing elicitors during the early stages of colonization (Mauch et al. 1988). The release of vacuolar hydrolases in combination with secondary compounds and proteinase inhibitors (see Chapter 3) may act in the initial stages of a multicomponent system of defense.

If oligosaccharins, hormones, cations, or other substances are responsible for triggering wound responses, it may be possible to facilitate wound processes by treating tissues with putative messenger compounds. In experiments with peach bark, Biggs and Peterson (1990) applied 14 different chemical treatments to wounds including acid extracts from peach leaf cell walls and fungal cell wall extracts (from L. persoonii) alone and in combination, ethylene, abscisic acid, chitosan, calcium ion, and cellobiose. Fungal cell wall extract and cellobiose stimulated bark lignin 10 fold over the control. None of the other treatments affected lignin production significantly. Given the quantities of lignin observed in these treatments, it is possible that these polysaccharide substances play a role in triggering the processes that lead to the observed differences in degree of lignin deposition in wounds versus infections (Biggs 1984b, Chapter 3). None of the treatments stimulated suberin production and, in fact, many appeared inhibitory.

Cell division is associated with both periderm formation and callus production leading to wound closure. Yev-Ladun and Aloni (1990) hypothesized that auxin and ethylene are the major factors controlling first periderm formation in woody stems. They suggest that moderate auxin flow retards periderm formation whereas high auxin levels promote ethylene production which indirectly results in periderm formation. Their hypothesis is based on observations of the patterns of first periderm ontogeny in young woody stems. They observed that first periderm formation is inhibited below leaves and buds, probably due to polar auxin transport. In addition they maintain that effects of light, humidity, oxygen, wounding, and pressure on periderm formation are related to the effects of these factors on relative levels of auxin and ethylene in the tissues. Furthermore, they suggest the presence of a positive feedback control mechanism which promotes phellogen activity and rhytidome formation. The mechanism is based on the assumption that the first-formed periderm constitutes a barrier to the outward movement of ethylene from the inner tissues of the plant. Therefore, the bark inside the first formed phellem accumulates relatively high ethylene concentrations that increases phellogen activity. This promotes the initiation of deeper phellogen and ultimately gives rise to rhytidome and the formation of visible bark scales. This interesting hypothesis awaits the actual determination of critical hormone levels in tissues undergoing these developmental changes. Refer to Chapter 3 for additional discussion of the regulation of plant development, wound response and resistance to pathogens in bark.

5 External Factors Influencing the Generation of New Bark Tissues

Many discussions on the role of wound healing in the resistance of plants to pathogens neglect the importance of external factors as determinants of wound response rate and, indirectly, the host/pathogen interaction. Environmental factors may influence any or several of the factors listed in Table 1 and thereby alter quality and quantity of wound related events. Additional factors other than those discussed below probably affect the wound response either directly or indirectly, including plant nutrient status, level of herbivory and history of defoliation (Wargo 1977), carbon/nitrogen ratio, acid rain, and air pollution.

Temperature has a strong influence on the rate of wound healing in both herbaceous and woody plant species (Bloch 1952, Krähmer 1980, Biggs and Northover 1985, Biggs 1986b, Morris et al. 1989). With apple, sweet cherry and peach, a significant correlation exists between temperature and rate of boundary zone and periderm regeneration in wounded bark (Biggs 1986b). Trees wounded at various times during the growing season were examined for complete formation of the primary ligno-suberized zone and new periderm. Although the tissues could be detected with a 7 to 21 day time period after wounding, degree-days (base = 0° C) accumulated during the postwounding period explained over 80% of the observed variation in wound response. Tree phenological stage did not appear to exert significant influence on the wound responses measured in this study.

In experiments to determine the formation of the primary ligno-suberized layer and phellogen following leaf abscission in peach, Biggs and Northover (1985) reported that plants maintained at 7.5, 12.5, and 17.5° C showed first indications of the primary ligno-suberized layer at 18, 9, and 6 days, respectively. Subsequent generation of phellogen and the appearance of the first phellem cells were observed at 30, 18, and 12 days, respectively. Earlier research on the influence of temperature on wound-induced cell division has shown that, within limits, the time required for the first cell division is linearly and inversely related to temperature (Lipetz 1970). Maximum and minimum temperature limits for wound responses have not been established for any tree species, however, in potato tubers, maximum suberization occurs at 20-25° C (Artschwager 1927, Wigginton 1974, Thomas 1982, Morris et al. 1989). Krähmer (1980) found that periderm could not be detected microscopically in leaf scars of apple at temperatures below 8° C. Accordingly, fruit scars remained susceptible to infection by Nectria galligena for more than 4 weeks at 6° C.

When trees were wounded during the dormant stage, lignin content of the bark, determined with a thioglycolic acid assay, increased although an increase in lignin detectable with the histochemical reagent phloroglucinol/HCl was not observed (Doster and Bostock 1988b). Wounds became more resistant to inoculation with Phytophthora syringae, however, thus suggesting resistance associated with lignin at very low tissue levels or mechanisms of resistance in addition to those associated with wound repair. Wound studies during the dormant season require long observation and sampling schedules, and it is possible that epiphytic microorganisms could colonize the wound site and contribute to altered rates of pathogen colonization independent of the responses of the host.

Plants under water stress are generally considered more susceptible to invasion by weak pathogens (Schoeneweiss 1981), however, few studies have been able to demonstrate the mechanism by which water stress increases the susceptibility of trees to fungal invasion (see Schoeneweiss (1981) for a review of this literature). Water deficit influences numerous physiological processes and pathways, and wound responses are no exception. Plant water status has been shown to influence the formation of the boundary zone, affect cell division in the wound periderm, and contribute to increased susceptibility of wounds to fungal pathogens.

Biggs and Cline (1986) examined the effects of irrigation treatments on the rate of boundary zone and wound periderm formation in wounds on peach limbs. No differences could be determined at 7 and 10 days after wounding between irrigated and nonirrigated trees for lignin autofluorescence, the intensity of suberin autofluorescence, or in the numbers of boundary zone cells; thus irrigation did not influence the formation of the primary ligno-suberized layer. Significant differences in suberin autofluorescence due to irrigation were measured 14 days after wounding and were related to increased numbers of suberized phellem cells in the wound periderm of irrigated trees relative to nonirrigated trees. Water stress, therefore, in the range of - 0.65 to - 0.80 MPa can inhibit periderm formation by diminishing the rate of cell division. Puritch and Mullick (1975) reported that formation of nonsuberized impervious tissue (NIT) (i.e., the primary ligno-suberized layer) in Abies grandis was inhibited by water stress less than - 1.5 MPa. Where formation of NIT was retarded, subsequent generation of wound periderm was thought to be slowed, also. Butin (1955) also determined that wound periderm formation was related to tissue water content. He found that susceptibility of poplar to Cytospora chrysosperma was increased where callusing of wounds was inhibited by increased water loss.

Relative humidity has an influence on the wound repair process in potatoes (Wigginton 1974, Morris et al. 1989) and, presumably on that of woody species, also. In general, wound healing proceeds most rapidly at 20-25 C with relative humidities between 70 and 100% and, at 10 C, between 80 and 100%. As relative humidity approaches 100%, cell proliferation, rather than periderm formation, may occur. Relative humidities below 50% may inhibit wound healing, although it is possible that as humidity declines, the depth at which the plant forms new periderm increases as desiccation of wounded tissues occurs.

Other external factors also may influence the expression of wound responses in plants. Suberin deposition in primary ligno-suberized tissue in wounded Pachysandra terminalis was affected adversely by both exposure to solar radiation and deicing salts (Hudler et al. 1990). The influence of microorganisms as irritants (Kaufert 1936) or stimulating factors at the wound site (Blanchette and Sharon 1975) has received little attention. Studies in this area could reveal significant new information about fundamental aspects of wound responses in nature.

6 Regeneration of Vascular Cambium and Wound Closure

Given that many of the tissues involved in callus regeneration are part of the barrier zone in the process of compartmentalization, only a limited discussion will be presented here. Refer to Chapter 5 for additional discussion on barrier zones. The production of callus tissue, the differentiation of new vascular cambium within callus, and the eventual closure of the wound, usually leading to the reestablishment of vascular cambium continuity, occurs following wounds inflicted to the depth of the xylem. The amount and rate of callus production following wounding varies according to tree species and selections within species (Gallagher and Sydnor 1983, Martin and Sydnor 1987), the size of the wound, and location of the wound on the tree (Wensley 1966). The source of callus tissue also varies to some extent. In scoring experiments with poplar, silver maple, pear, and apple, callus formation is contributed mainly by living cells of vascular rays in the proximity of the cut, and, to a lesser extent, the longitudinal parenchyma of the phloem and xylem (Soe 1959). In linden, callus tissue forms from any active, newly formed cambial derivative rather than from any one particular cell type (Barker 1954). Generally, there is a diversity of opinion on the source of callus in the regeneration of new bark (Bloch 1941), although most researchers agree that the original vascular cambium does not contribute significantly to the formation of callus tissue. The initiation of phellogen regeneration in callus always precedes that of vascular cambium. The ventral region of growing callus, where the differentiating phellogen and vascular cambium are in close proximity, is nonlignified, nonsuberized, and is especially susceptible to disruption by pathogenic fungi (Biggs 1986a, Biggs and Britton 1988).

Formation of callus over large diameter wounds may occur in a distinctive pattern (Fig. 27) (Shigo 1986). Usually, the first year's callus production is the greatest after which it declines within 3 to 5 years. Factors influencing the anatomical co-mingling of the new vascular cambia as the callus ribs converge on the center of the wound from the perimeter are not understood. It appears that complete closure is prevented if the vascular cambium turns inward. The periderm on the callus surface may prevent physically the co-mingling of vascular cambia in this situation, thus leading to a "bark inclusion", visible as a fine crack between the converged callus ribs. An incompletely closed wound may provide an excellent environment for fungal pathogens and it is possible that pathogenic microorganisms influence the direction of growth of new vascular cambia.

When wounds close via the co-mingling and junction of converging vascular cambia (Figs. 28 and 29), active growth of microorganisms ceases in that area (Shigo 1986), probably due to altered gaseous regimes within the wound site. It is not clear whether organisms within closed sites actually succumb or remain latent, able to resume pathogenesis when conditions conducive to their growth re-occur.

7 Wounds as Infection Courts

In many host/pathogen interactions, the boundary setting process in the wound (the infection court) confers resistance to infection. Many researchers have demonstrated that wounds become increasingly less susceptible to infection with age (Butin 1955, Cline and Neely 1983, Russin and Shain 1984, Riffle and Peterson 1986, Bostock and Middleton 1987). This type of resistance to infection is thought to be related to nonspecific plant responses leading up to and including formation of primary ligno-suberized tissues and secondary wound (or necrophylactic) periderm. The major structural components of these tissues are lignin and suberin (Kolattukudy 1984, Biggs 1985a). Definitive proof of the role and importance of ligno-suberized tissue and wound periderm in resistance to disease in trees has never been presented.

When an infection court is created for a wound pathogen, disease is most severe when the inoculum arrives at the infection court immediately. If the inoculum arrives later, disease frequency and severity decline with time until the wounded tissues express resistance comparable to that of noninjured bark (Biggs 1986c,1989). The time it takes for complete resistance to become reestablished is dependent upon the factors discussed above as well as the pathogenic capabilities of the fungus. Histological studies with peach and the canker pathogen L. persoonii showed that wounds resistant to inoculation possessed a minimum of three phellem cells in the new periderm. At earlier stages, wounds were susceptible to the fungus, although severity of the symptoms declined beginning about 3 days after wounding (Biggs 1986c). The presence of periderm was more critical for inhibition of the pathogen than primary ligno-suberized tissues.

The relative number of cells in periderm tissue or the thickness of the suberized layers in wounded potato tubers generally reflect the relative resistance to pathogens (i.e. wounds with thicker suberized layers are more resistant to pathogens). Generally, this is true for wound periderms in woody plants when comparisons are being made within a single genotype (Biggs 1986c) or among different species over a time course study (Biggs 1986b). However, when comparing genotypes within a species, it is unlikely that numbers of wound phellem cells or the thickness of the suberized layer is of major importance in determining the resistance or susceptibility to wound pathogens. This statement is based upon the work of Biggs (1989) which demonstrated no significant correlation of these anatomical parameters with the field performance of various peach cultivars, canker length after inoculations, or the amount of accumulated suberin measured photometrically. For peach, resistance to Leucostoma spp. was correlated with increased rate of suberin accumulation (Biggs and Miles 1985, 1988, Biggs 1989). Lignin accumulation, which is thought to play an important role in many host/pathogen interactions (Craft and Audia 1962, Vance et al. 1980), appears to serve a less important role than suberin in peach bark and perhaps the bark of other tree species. Periderm regeneration is probably only one of many possible types of resistance in peach bark to Leucostoma spp., and can be described as a type of rate-limiting or partial resistance.

Fungi inoculated into older wounds can survive in nature without causing immediate infection. Mycelium of Hypoxylon mammatum survived in wounds of aspen for periods of up to two years prior to development of symptoms (Ostry and Anderson 1983). Similarly, Russin and Shain (1984) reported that Chryphonectria parasitica remained viable for more than 36 weeks in inoculated wounds. Wounded peach bark inoculated with spores of Botryosphaeria spp. did not develop visible macroscopic symptoms until eight weeks after inoculation (Biggs and Britton 1988). Formation of a ligno-suberized boundary zone, generation of new periderm and callus tissue do not guarantee that wounds will not become infected, although the risk of infection is reduced greatly. Aspects of wound closure and microenvironment may help explain the apparent latent colonization of canker-causing fungi.

8 Proposed Anatomical Model for Wound Responses in Woody Plants

The regeneration of periderm, callus tissue and new vascular cambium is an energy intensive process that serves three purposes: 1) replace tissue and regenerate lateral meristems, 2) reestablish control over gas exchange and desiccation, and 3) prevent or restrict the ingress of pathogens. The model presented in Fig. 30 was originally developed by Mullick (1977) and was revised for this presentation to account for the presence of suberin in the primary ligno-suberized boundary zone which forms from extant cells prior to the formation of new phellem. The model describes, at the anatomical level, the nonspecific host responses associated with wounding, pathogen invasion, or insect injury. Part (a), the simplest response, occurs following any disruption of the living phellogen. Part (b), a more complex reaction than (a), occurs following any injury or irritation to the bark tissues that also disrupts the vascular cambium. Part (c) occurs following any injury or irritation which is inclusive of the bark tissues, the vascular cambium, and the functional sapwood.

Understanding the basis for wound responses in woody plants could lead to innovative control measures for tree diseases. Research in this area should include basic studies on the molecular regulation of wound metabolism in trees, pathological studies on the role of wound responses in resistance to bark and xylem pathogens, the role of nonpathogenic microorganisms on properties of wound tissues, and genetic experiments to determine the heritability of favorable wound response characters. Well designed time-course studies are required in order to completely characterize wound related phenomena.

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