Hydraulic conductivity in roots of ponderosa pine infected with black-stain (Leptographium wageneri) or annosus (Heterobasidion annosum) root disease

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1 Tree Physiology 18, Heron Publishing----Victoria, Canada Hydraulic conductivity in roots of ponderosa pine infected with black-stain (Leptographium wageneri) or annosus (Heterobasidion annosum) root disease GLADWIN JOSEPH, 1 RICK G. KELSEY 2 and WALTER G. THIES 2 1 Department of Forest Science, Oregon State University, Corvallis, OR 97331, USA 2 USDA Forest Service, Pacific Northwest Research Station, Corvallis, OR 97331, USA Received March 14, 1997 Summary Roots from healthy and diseased mature ponderosa pine, Pinus ponderosa Laws., trees were excavated from a site near Burns, Oregon. The diseased trees were infected with black-stain root disease, Leptographium wageneri Kendrick, or annosus root disease, Heterobasidion annosum (Fr.) Bref., or both. Axial hydraulic conductivity of the roots was measured under a positive head pressure of 5 kpa, and the conducting area was stained with safranin dye to determine specific conductivity (k s ). In diseased roots, only 8--12% of the cross-sectional xylem area conducted water. Resin-soaked xylem completely restricted water transport and accounted for % of the loss in conducting area. In roots with black-stain root disease, 17% of the loss in conducting area was associated with unstained xylem, possibly resulting from occlusions or embolisms. Based on the entire cross-sectional area of infected roots, the k s of roots infected with black-stain root disease was 4.6% of that for healthy roots, whereas the k s of roots infected with annosus root disease was 2.6% of that for healthy roots. Although these low values were partly the result of the presence of a large number of diseased roots (72%) with no conducting xylem, the k s of functional xylem of diseased roots was only 33% of that for healthy roots. The low k s values of functional xylem in diseased roots may be caused by fungus induced occlusions preceding cavitation and embolism of tracheids. The k s of disease-free roots from diseased trees was only 70% of that for healthy roots from healthy trees. The disease-free roots had the same mean tracheid diameter and tissue density as the healthy roots, suggesting that the lower k s in disease-free roots of diseased trees may also have been caused by partial xylary occlusions. Keywords: annosus root disease, black-stain root disease, Pinus ponderosa, root conductivity, specific conductivity, wood density. Introduction Many of the root diseases that commonly infect ponderosa pine, Pinus ponderosa Laws. interfere with axial water conductivity in the stem. Black-stain root disease, Leptographium wageneri var. ponderosum (Harrington & Cobb) Harrington & Cobb, is a vascular wilt fungus that does not decay the xylem (Hansen et al. 1988). Black-stain root disease is thought to inhibit water movement by blocking tracheids with hyphal growth in the lumen and bordered pit pairs (Smith 1967), or by nonresinous (tyloses) and resinous occlusions produced by host cells in response to the infection (Hessburg and Hansen 1987). Annosus root disease, Heterobasidion annosum (Fr.) Bref., unlike black-stain root disease, decays the xylem (Schmitt et al. 1997) and creates nonconducting dry zones in the sapwood of stems by allowing air to enter as bordered pits are enzymatically broken down and water is withdrawn by hydrostatic tension (Coutts 1976). Blue-stain fungi, Ophiostoma spp., which are typically found in the sapwood and phloem of trees attacked by bark beetles (Harrington 1993), disrupt water transport by cavitation and air entry into the tracheids (Mathre 1964). Most studies on diseased conifers have focused on the water relations of infected stems and many of them provide only qualitative descriptions of how the disease affects water conductance. Axial water conductivity in roots of conifers and hardwood trees is several-fold higher than in stems or branches (Gartner 1995). This greater conductivity in roots generally parallels the presence of wider and longer tracheids or vessels in roots than in stems and branches (Zimmerman and Potter 1982, Gartner 1995, Sperry and Ikeda 1997). Although roots have higher water conductivity, root tracheids may be more susceptible to cavitation and embolism (Sperry and Saliendra 1994, Alder et al. 1996, Sperry and Ikeda 1997) than tracheids in stems or branches, making them more susceptible to pathogen-induced embolism. However, little is known about the mechanism or magnitude of decline in water conductivity of diseased conifer roots. In our study of ponderosa pine, roots from healthy and diseased trees infected with black-stain root disease, annosus root disease, or both, were excavated and their hydraulic conductivities measured. Our objectives were to determine the magnitude of decline in specific conductivity as a result of the disease, and to determine the extent and characteristics of nonconducting xylem associated with diseased roots.

2 334 JOSEPH, KELSEY AND THIES Material and methods Plant material The ponderosa pine trees were located 39 km NE of Burns, Oregon ( N; W) in the Malheur National Forest. The site is at 1690 m elevation, with a 6% slope, an eastern aspect, and a mean annual precipitation of 63.5 cm. The soil is a loamy clay type. Most trees in the overstory were less than 100 years old. Two healthy and eight diseased trees with a range of crown symptoms were pushed over with a skidder. Roots of the diseased trees were infected with L. wageneri and H. annosum and some also had blue-stain fungus, Ophiostoma spp. Root segments, 0.3 to 3 cm in diameter and 15 to 25 cm in length, were selected with a range of root disease symptoms. Roots with severe decay were not collected. Disease-free root segments from diseased trees were collected from roots with no apparent symptoms of disease in the portion of the root that was excavated. Seven to 15 roots were sampled from each tree. Root segments were wrapped in moist paper towels, sealed in plastic bags and stored on ice. In the laboratory, root segments were stored at 4 C for a maximum of 10 days during the measurements. Hydraulic conductivity measurements Root hydraulic conductivity was measured with an apparatus that maintained a constant head pressure of 5 kpa. The system consisted of a reservoir with the perfusion fluid connected to a closed loop of flexible tubing (main stem) with four T-junctions and flexible tubing of appropriate diameter to attach to the root segments with a tight seal. A clamp between the root segment and the T-junction regulated the flow. Roots were perfused with degassed distilled water containing prefiltered (0.22 µm nylon membrane) 0.07% HCl (ph 2.6). The water was acidified to inhibit microbial growth on the inner walls of the tubing, which would otherwise cause rapid clogging of the xylem (Sperry et al. 1988). Hydraulic conductivity was measured on a root segment (ranging from 2.7 to 8.2 cm in length) cut from each field sample under distilled water immediately before the measurement. The thin bark around healthy roots was easily peeled. When this occurred the sapwood was wrapped with Parafilm before or after insertion into the tubing to prevent desiccation and possible surface embolism. The bark was not easily removed from diseased roots, so it was shaved from the ends of each segment to expose the sapwood before each segment was inserted in the tubing. The clamps were opened for 10 min allowing four root segments to equilibrate simultaneously with the perfusion solution at 5 kpa of pressure. Preliminary measurements showed no change in flow rates from 10 min to 4 h after pressurization. Clamps were then closed to three of the four segments, and the fluid flowing through the unclamped segment was collected in a preweighed dry beaker for 15 s and weighed to the nearest 0.1 mg. This was repeated four or five times before proceeding to the next root segment. After a segment was measured it was placed in distilled water for approximately 60 min to prevent desiccation while waiting to be perfused with dye. Specific conductivity (k s ) was calculated as (Tyree and Ewers 1991): k s = wl tap = kg s 1 m 1 MPa 1, (1) where w is the weight of the perfusion fluid (kg), l is the segment length (m), t is the duration of perfusion (s), a is the cross-sectional area of the root or conducting xylem (m 2 ), and p is the head pressure (MPa). The cross-sectional area of conducting xylem was determined by perfusing the segments with dye in an apparatus similar to that used for measuring hydraulic conductivity. The dye solution was 0.2% (w/v) safranin in degassed distilled water that had been passed through a 0.22 µm nylon membrane filter. The dye reservoir was placed 1 m above the segments to generate a head pressure of 10 kpa. The root segments were inserted in the T-junctions with appropriately sized tubing and the dye allowed to perfuse for approximately min. Dye emerged from the opposite ends of the segments in a few minutes. Most of the conducting tracheids were stained in this interval, and we assumed that they were functional. Several segments were cut at their midpoint and visually checked to insure that the dye had perfused through the root. Diseased root segments with low conductivity were allowed to perfuse for another min to insure that most of the conducting tracheids were stained. Increasing the dye perfusion time by min for the diseased segments should not have altered staining patterns, because there was no change in k s from 10 min to 4 h of pressurization in preliminary tests. Each root segment was measured to the nearest cm and then stored at --35 C in a sealed plastic bag for later measurements. To measure the stained cross-sectional area, a disk ( cm wide) was cut from the center of each segment and dried at room temperature for 24 h. The disk images were scanned into a computer (Sony, Cypress, CA) and the crosssectional areas of stained and unstained sections were measured with image analyzing software (NIH Image Version 1.52, Rasband and Bright 1995). Disease characterization Nonconducting portions of each root disk not penetrated by dye were sorted into categories described below, and their relative cross-sectional areas measured with the aid of a microscope. Sections with the characteristic black-stain were identified as L. wageneri (Smith 1967). Sections with brown-stain or decayed, yellow spongy tissue were identified as H. annosum (Schmitt et al. 1997). Blue-stained sections with a wedge shape and dry appearance were identified as Ophiostoma spp. (Harrington 1993). A clear stain, of unknown origin, often occurred on the periphery of the black-stain. Resinosis appeared as water-soaked zones, whereas nonconducting-unstained zones, which were nearly healthy in appearance, were probably occluded or embolized. Nonconducting cores of a few healthy roots also had embolized tracheids for reasons that are unknown. TREE PHYSIOLOGY VOLUME 18, 1998

3 AXIAL CONDUCTIVITY OF DISEASED ROOTS 335 Root tissue density A disk cm in width was removed from the mid-section of each root to measure tissue density. The disk was dried at 102 C for 16 h, weighed to the nearest 0.01 g and then immersed in a beaker containing distilled water. The volume of water displaced was measured to the nearest 0.01 g. Density was calculated as the ratio of dry weight to dry volume. Dry volume was used in place of wet volume because the roots had been stored frozen and this might have affected the wet volume measurements. Anatomical measurements Healthy roots, disease-free roots from diseased trees, and diseased roots with some conducting xylem were used to measure tracheid diameters. Free-hand transverse sections were made with a blade and mounted in ethyl glycol. Radial lumen diameters were measured on 40 to 126 tracheids from each cross section with an occular micrometer at 40 magnification. Eight to 16 sectors were randomly located over the entire cross section, except near the root center where tracheids may have been compressed. In each sector, a radial row of tracheids was measured within a growth ring. The percentage of tracheids in 5-µm diameter classes was calculated, as well as the percentage of estimated hydraulic conductance contributed by tracheids in each diameter class. Tracheid hydraulic conductance was assumed proportional to the diameter raised to the fourth power as predicted by the Hagen-Poiseuille equation (Zimmerman 1983). Statistical analysis Separate analyses were conducted for roots classified in two ways: (1) healthy roots, black-stain root disease roots, and annosus root disease roots, and (2) healthy roots from healthy trees, disease-free roots from diseased trees, or diseased roots from diseased trees with some conducting tissue. Differences among mean percent conducting xylem, k s, tissue density, and tracheid diameter for the disease classes were analyzed as a one-way ANOVA. Data were log e transformed when necessary to ensure normal distribution and homogeneity of variance. Back transformed means were presented when data were transformed. For each category of nonconducting xylem (Table 1), the percentage cross-sectional area was rank transformed before analysis. Significantly different means were identified by Fisher s Protected Least Significant Difference (LSD) at α = The relationships between mean tracheid diameter and k s, and tissue density were analyzed by linear regression. Results In roots with black-stain root disease, nonconducting-unstained areas, which were presumably occluded or embolized, occurred adjacent to actively conducting tissue (stained areas), but did not show any particular pattern (Figure 1). Resinosis associated with black-stain root disease often extended from the periphery of the root toward the central core in a wedgeshaped pattern with a water-soaked appearance, rather than the dry appearance of the wedge-shaped blue-stain. In one root section, severe black-stain root disease and resinosis had completely destroyed xylem function. In trees infected with annosus root disease, several roots were completely soaked in resin proximal to the infection and had no functional xylem. Some healthy roots had a central core with little or no functional xylem (Figure 1). In healthy roots with no visible disease, close to 100% of the xylem conducted water, whereas in diseased roots only 8--12% of the xylem conducted water (Table 1). Based on entire cross-sectional areas, the k s of diseased roots was % of that for healthy roots and the tissue densities of diseased roots were significantly lower than those of healthy roots (Table 1). Figure 1. Diseased and healthy root cross sections of ponderosa pine perfused with saffranin dye. Stained xylem (d), black-stain (b), annosus decay (a), nonconducting-unstained xylem (possibly occluded or embolized) (e), resinosis (r), and nonconducting core (p); k s = specific conductivity (kg s 1 m 1 MPa 1 ), f = conducting (functional) area (%). TREE PHYSIOLOGY ON-LINE at

4 336 JOSEPH, KELSEY AND THIES Table 1. Mean (± SE) % conducting xylem, specific conductivity (k s ), tissue density, and characteristics of nonconducting xylem of healthy and diseased (black-stain root disease and annosus root disease) ponderosa pine roots Healthy roots (n = 53) Roots with black-stain root disease (n = 10) Roots with annosus root disease (n = 22) Conducting xylem (%) 96.6 ± 1.7 a 12.0 ± 3.8 b 7.5 ± 2.6 b k s (kg s 1 m 1 MPa 1 ) a c c Tissue density (g ml 1 ) 0.44 ± 0.02 a 0.62 ± 0.04 b 0.53 ± 0.03 b Characteristics of nonconducting xylem (%) Black-stain ± 4.1 a 4.2 ± 2.8 b Decay or brown-stain ± 4.0 a Blue-stain ± 1.8 a 0 Resinosis ± 8.7 b 12.8 ± 5.9 b Clear-stain 0.3 ± 1.0 a 11.2 ± 2.5 b 0.8 ± 1.7 a Unstained 0.05 ± 0.15 a 17.5 ± 3.4 b 0 Nonconducting core 2.6 ± 0.8 a ± 1.2 b 1 The ks was calculated using the entire cross-sectional area of the root. 2 Means (± SE) followed by the same letters are not significantly different at P < 0.05 (Fisher s Protected LSD). 3 Means (± SE) of ks were back-transformed. Twenty-three of the 32 diseased roots had no conducting xylem. Nonconducting unstained tracheids accounted for 17% of the loss in functional xylem in roots with black-stain root disease, whereas there were no nonconducting unstained tracheids in roots with annosus root disease. The loss in xylem conduction in roots with black-stain root disease was probably the result of occlusions or embolism. Many roots with annosus root disease had large areas of advanced decay with the typical yellow spongy appearance (Table 1). Resinosis accounted for % of the nonconducting xylem in roots with either disease. Blue-stain disease was associated with the black-stain root disease and covered 5% of the cross-sectional area. Disease-free roots from diseased trees had a 30% lower k s than healthy roots from healthy trees (Table 2), but they had a similar area of conducting xylem. The k s of diseased roots was 48% of the k s of disease-free roots from diseased trees, and only 33% of the k s of healthy roots from healthy trees. Because the k s values in Table 2 were calculated based on the areas of conducting xylem only, they are higher than the k s values in Table 1. There were no significant differences in mean tracheid diameters among the roots (Table 2), but the distribution of tracheid diameters and estimated hydraulic conductance revealed differences between the diseased roots and the diseasefree and healthy roots (Figure 2). Both disease-free and healthy roots had a higher frequency of larger diameter tracheids and associated hydraulic conductance than the diseased roots. There was no difference in tissue density between disease-free roots from diseased trees and healthy roots, but tissue densities of both root types were lower than the tissue density of diseased roots (Table 2). Mean tracheid diameter was positively related to k s for both disease-free and healthy roots (Figure 3). This relationship was nonsignificant for the diseased roots (data not shown). The relationship for disease-free roots from diseased trees had a slightly smaller slope than that for the healthy roots (0.32 versus 0.47) indicating that k s of disease-free roots may be restricted by xylary occlusions. Root densities were negatively correlated with k s and mean tracheid diameters (Figure 4). Both k s and tracheid diameters decreased rapidly as the tissue density increased from 0.3 to 0.6 g ml 1. This relationship was similar in disease-free, healthy, and diseased roots. Table 2. Mean (± SE) % conducting xylem, specific conductivity (k s ), tracheid diameter, and density of root tissue from healthy and diseased (black-stain root disease and annosus root disease) ponderosa pine trees Healthy trees (n = 2) Diseased trees (n = 9) Healthy roots (n = 16) Disease-free roots (n = 36) Diseased roots (n = 9) Conducting xylem (%) 96.6 ± 2.1 a 96.3 ± 3.2 a 37.6 ± 4.2 b k s (kg s 1 m 1 MPa 1 ) a b c Tracheid diameter (µm) a a a Tissue density (g ml 1 ) 0.43 ± 0.02 a 0.45 ± 0.02 a 0.54 ± 0.04 c 1 The ks was calculated using only the conducting areas of the root, whereas in Table 1 ks was calculated using the cross-sectional area of the entire root. Only diseased roots with some conducting xylem were used to calculate the means for diseased roots. 2 Means (± SE) followed by the same letters are not significantly different at P < 0.05 (Fisher s Protected LSD). 3 Means (± SE) of ks and tracheid diameters were back-transformed. TREE PHYSIOLOGY VOLUME 18, 1998

5 AXIAL CONDUCTIVITY OF DISEASED ROOTS 337 Figure 2. Percent tracheids or % calculated hydraulic conductance versus 5-µm tracheid diameter class for diseased roots (A, n = 9), disease-free roots from diseased trees (B, n = 36), and healthy roots from healthy trees (C, n = 16). Vertical bars are one SE of the mean. Discussion In diseased roots, most of the tracheids could not conduct water because they were structurally altered by decay, plugged by nonresinous or resinous occlusions, or were embolized. Tracheids associated with advanced decay as a result of annosus root disease were probably nonfunctional because they were entirely embolized (Coutts 1976), although annosus root disease may have also plugged some tracheids with hyphae. In black-stain root disease roots, nonresinous occlusions or embolized tracheids probably prevented the xylem from conducting water in both the stained and unstained areas. Black-stain root disease physically plugs tracheid lumens or bordered pits with hyphae, gums, or tyloses (Smith 1967, Hessburg and Hansen 1987). Resinous occlusions blocked about the same proportion of tracheids in roots infected with annosus root as in roots infected with black-stain root disease. Complete resinosis of the root, which inhibited water transport entirely, was Figure 3. Specific conductivity (k s ) as a function of mean tracheid diameter. For healthy roots, y = x, P < 0.04, r 2 = 0.28, and n = 16. For disease-free roots from diseased trees, y = x, P < 0.001, r 2 = 0.68, and n = 36. Figure 4. Specific conductivity (k s ) (A), and mean tracheid diameter (B) as a function of root tissue density (n = 59) for categories of roots. A: y = x x 2, P < 0.001, r 2 = 0.35; B: y = x x 2, P < 0.001, r 2 = more common in roots infected with annosus root disease than in roots infected with black-stain root disease. Pine species have well-defined resin systems that respond rapidly to injury TREE PHYSIOLOGY ON-LINE at

6 338 JOSEPH, KELSEY AND THIES caused by pathogens or other factors (Nebeker et al. 1993). Both constitutive and induced resin can flow into sapwood surrounding the source of injury, thus isolating the injured tissue, but also restricting the pathway for water transport. Tracheids infected with blue-stain probably were disabled by cavitation (Mathre 1964). Specific conductivity (k s ) of diseased roots, calculated on the basis of the entire cross-sectional area of the root, was only 3--5% of that for healthy roots. For those diseased roots that had conducting xylem, the k s was 33% of that for healthy roots. In a similar study, the k s of symptomatic roots (calculated on the basis of the entire cross-sectional area of the root) from Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and grand fir (Abies grandis (D. Don ex Lamb.) Lindl.) infected with a complex of several diseases including annosus root disease and black-stain root disease, was % of that for asymptomatic roots (Baker et al. 1994). Thus, it is apparent that root disease in conifers substantially reduces the k s of roots. The low k s values of diseased ponderosa pine roots in our study was partly because a large number (72%) of diseased roots had no conducting xylem, and perhaps indicates that the ponderosa pine trees were at a more advanced stage of infection than the two species studied by Baker et al. (1994). Based on microscopic examination of cross sections of diseased roots, we conclude that the low k s value of functional xylem in diseased roots (only 33% of that for healthy roots) was not caused by the loss of conductance in larger diameter tracheids. However, diseased roots had a higher percentage of small tracheids and higher tissue density than healthy roots (Figure 2). Slower growth of roots of diseased trees, caused by overall stress from an impaired root system, may result in smaller tracheids and increase the percentage of latewood tracheids that have smaller diameters and thicker cell walls than earlywood tracheids (Gartner 1995), leading to reduced axial and radial hydraulic conductivities (Kramer and Kozlowski 1979). In addition, the presence of partially occluded tracheids and bordered pits probably caused resistance to water flow. This explanation is supported by the finding that, in roots with annosus root disease, most of the tracheids associated with brown-stain showed some conductivity. We do not know why the k s of healthy roots from healthy trees was higher than that of disease-free roots from diseased trees. Because tracheid diameter distributions and tissue densities were similar in disease-free roots and healthy roots (Figure 2), the lower k s in disease-free roots suggests the presence of occlusions, or some other resistance to water flow in the tracheids that are conducting water. For black-stain root disease, occlusions by gum-like tyloses have been observed only in tracheids colonized by the fungus and in adjacent uncolonized tracheids and bordered pits (Hessburg and Hansen 1987). These occlusions have not been observed in tracheids that are some distance from the stain. The occurrence of partially occluded, but functional tracheids in diseased and disease-free roots suggests that plugging of tracheids may precede embolism and also predispose the xylem to stress-induced embolisms. When root k s decreases as a result of plugging, steeper hydraulic gradients are required to transport water through the roots, assuming similar transpiration rates and soil water contents. The resulting increase in hydraulic tension over the air-seeding threshold causes cavitation to occur, resulting in embolism of the tracheids (Zimmerman 1983). In contrast, embolism may precede the physical plugging of vessels in Dutch elm (Ulmus americana L.) infected by Ceratocystis ulmi (Buism.) C. Moreau (Newbanks et al. 1983), because the fungus degrades cell walls allowing air to enter the tracheids (Dimond and Husain 1958). Although fungal infections cause the formation of occluded or embolized xylem in ponderosa pine, the mechanism allowing air to enter these tracheids is not known. Diseased roots reduce water conductivity and this can lead to increased water stress in the trees, as indicated by decreased stem water content (unpublished data). In conifers, the entire sapwood functions in axial water transport (Lassoie et al. 1977), whereas in ring-porous hardwoods, such as Dutch elm, 90% of the fluid flow occurs through the outermost ring (Ellmore and Ewers 1985). Therefore, in conifers, fungal infections and any associated effects may have to extend over a large cross-sectional area of the conducting sapwood before significant disruption of tree water balance occurs. In addition, most conifers have well developed resin systems that can isolate the fungal infection in roots by resinosis, which could restrict the hydraulic damage. Although roots are more vulnerable to embolism than shoots (Sperry and Ikeda 1997), embolized tracheids in conifer roots may readily refill when water is available, even in the absence of positive pressures (Borghetti et al. 1991, Sperry et al. 1994). On the other hand, in hardwoods, embolized vessels refill only under positive pressures, or they require new xylem production to restore hydraulic conductance (Sperry et al. 1994). Consequently, the differences in hydraulic characteristics between conifers and hardwoods may result in a slower development of water stress in diseased conifers than in diseased hardwoods. In summary, the reduction in conducting area of diseased roots by occlusions, embolisms, and resinosis, combined with a large drop in k s of conducting xylem can significantly disrupt root water transport, and may also decrease water uptake in diseased trees. Additionally, disease-free roots of diseased trees have reduced k s which may further affect the water balance of such trees. The subsequent development of water stress in diseased trees can affect their leader growth (unpublished data) and possibly weaken their defence system, making them more susceptible to insect attack and colonizaton (Nebeker 1993). Acknowledgments We thank Max Ollieu (USDA Forest Service, Region Six, Forest Insect and Disease) and Mark Loewen (USDA Forest Service, Burns Ranger District) for arranging the excavation of trees, Dr. B. Gartner for use of the image analyzing equipment, Dr. J. Zaerr and Dr. B. Yoder for helpful reviews of the manuscript, and Dr. L. Ganio for assistance with the statistics. TREE PHYSIOLOGY VOLUME 18, 1998

7 AXIAL CONDUCTIVITY OF DISEASED ROOTS 339 References Alder, N.N., J.S. Sperry and W.T. Pockman Root and stem xylem embolism, stomatal conductance, and leaf turgor in Acer grandidentatum populations along a soil moisture gradient. Oecologia 105: Baker, F.A., K. Kavanagh and J.B. Zaerr Root disease reduces hydraulic conductivity of mature Douglas-fir and grand fir roots. In Proc. Eighth International Conference on Root and Butt Rots. Eds. M. Johansson and J. Stenlid. IUFRO Working Party, Wik, Sweden and Haikko, Finland, pp Borghetti, M., W.R.N. Edwards, J. Grace, P.G. Jarvis and A. Raschi The refilling of embolized xylem in Pinus sylvestris L. Plant Cell Environ. 14: Coutts, M.P The formation of dry zones in the sapwood of conifers. I. Induction of drying in standing trees and logs by Fomes annosus and extracts of infected wood. Eur. J. For. Pathol. 6: Dimond, A.E. and A. Husain Role of extracellular enzymes in pathogenesis of Dutch elm disease. Science 127:1059. Ellmore, G.S. and F.W. Ewers Hydraulic conductivity in trunk xylem of elm, Ulmus americana. IAWA Bull. 6: Gartner, B.L Patterns of xylem variation within a tree and their hydraulic and mechanical consequences. In Plants Stems: Physiology and Functional Morphology. Eds. B.L. Gartner. Academic Press, San Diego, pp Hansen, E.M., D.J. Goheen, P.F. Hessburg, J.J. Witcosky and T.D. Schowalter Biology and management of black-stain root disease in Douglas-fir. In Leptographium Root Diseases on Conifers. Eds. T.C. Harrington and F.W. Cobb, Jr. APS Press, The American Phytopathol. Soc., St. Paul, MN, pp Harrington, T.C Biology and taxonomy of fungi associated with bark beetles. In Beetle--Pathogen Interactions in Conifer Forests. Eds. T.D. Schowalter and G.M. Filip. Academic Press, San Diego, pp Hessburg, P.F. and E.M. Hansen Pathological anatomy of blackstain root disease of Douglas-fir. Can. J. Bot. 65: Kramer, P.J. and T.T. Kozlowski Physiology of woody plants. Academic Press, Inc., Orlando, FL, 811 p. Lassoie, J.P., D.R.M. Scott and L.J. Fritschen Transpiration studies in Douglas-fir using the heat pulse technique. For. Sci. 23: Mathre, D.E Pathogenicity of Ceratocystis ips and Ceratocystis minor to Pinus ponderosa. Contrib. Boyce Thompson Inst. 22: Nebeker, T.E., J.D. Hodges and C.A. Blanche Host response to bark beetle and pathogen colonization. In Beetle--Pathogen Interactions in Conifer Forests. Eds. T.D. Schowalter and G.M. Filip. Academic Press, London, pp Newbanks, D., A. Bosch and M.H. Zimmerman Evidence for xylem dysfunction by embolization in Dutch elm disease. Phytopathology 73: Rasband, W.S. and D.S. Bright A public domain image processing program for the Macintosh. Microbeam Anal. Soc. J. 4: Schmitt, C.L., J.R. Parmeter, Jr. and J.T. Kliejunas Annosus root disease of western conifers. Forest Insect and Disease Leaflet, USDA Forest Service, Washington, DC. In Press. Smith, R.S., Jr Verticicladiella root disease of pines. Phytopathology 57: Sperry, J.S. and T. Ikeda Xylem cavitation in roots and stems of Douglas-fir and white fir. Tree Physiol. 17: Sperry, J.S. and N.Z. Saliendra Intra- and inter-plant variation in xylem cavitation in Betula occidentalis. Plant Cell Environ. 17: Sperry, J.S., J.R. Donnelly and M.T. Tyree A method for measuring hydraulic conductivity and embolism in xylem. Plant Cell Environ. 11: Sperry, J.S., K.L. Nichols, J.E.M. Sullivan and S.E. Eastlack Xylem embolism in ring-porous, diffuse porous, and coniferous trees of northern Utah and interior Alaska. Ecology 75: Tyree, M.T. and F.W. Ewers The hydraulic architecture of trees and other woody plants. New Phytol. 119: Zimmerman, M.H Xylem structure and the ascent of sap. Springer-Verlag, New York, 143 p. Zimmerman, M.H. and D. Potter Vessel-length distribution in branches, stem and roots of Acer rubrum L. IAWA Bull. 3: TREE PHYSIOLOGY ON-LINE at

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