Charting the isophasic endophyte of dwarf mistletoe (Arceuthobium) in host apical buds

Charting the isophasic endophyte of dwarf mistletoe (Arceuthobium) in host apical buds

David Lye

B.Sc.

University of Victoria, 1997

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE In the Department of Biology

We accept this thesis as conforming to the required standard

O David Lye, 2004 University of Victoria

Ail rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author.

Supervisors; Dr. J, Kuijt Dr. N. Turner

ABSTRACT ^& I

Dwarf mistletoes (Arceuthobium, Viscaceae) are highly derived dioecious angiosperms parasitic on many gymnosperm hosts worldwide. Several mistletoe species are capable of inducing an unusual form of isophasic infection in which the internal (endophytic) system proliferates even into the apical buds of its hosts. Studies of

the internal endophytic system have for the most part focused on the parasite within host secondary tissues. The present study characterizes the growth pattern of the isophasic endophytic system of Arceuthobium dou~lasii within the dormant apical buds of Pseudotsuna menziesii, documenting a consistent pattern of growth occurring even into the preformed leaves of the host. The apparently non-intrusive growth of the parasite appears to be developmentally synchronized with that of the host. I describe the

. _{ultrastructure of the parasitelhost interface within apical buds of}

P.

_{menziesii parasitized}

by

A.

dounlasii and of Pinus contorta parasitized by

A.

americanum; no symplastic connections were observed.

TABLE OF CONTENTS Title page Abstract Table of contents List of Tables List of Figures Acknowledgements Introduction Mistletoe Infection

Dwarf Mistletoe Shoot Emergence in lsophasic Brooms The Dwarf Mistletoe Endophyte

The Douglas fir Apical Bud Materials and Methods

Collections

Sample Preparation Sample Imaging Results and Discussion

Morphology of Shoot Emergence Endophyte Identification

Preliminary findings

Lipid Distribution and Plasmolysis Endophyte Distribution

Ultrastructural Observations Suggestions for Future Studies Literature Cited

Figures Appendices:

Appendix A: Spreadsheet of Infected Preformed Leaves Appendix B: PCR Testing

Appendix C: Copyright Permission for Material by Others

i

ii

iii

LIST OF TABLES

Table 1. Arceuthobium species which cause isophasic brooming 6 and their hosts.

Table 2. Details of field collections included in this study. 16 Table 3. Quantitative estimate of the number of mistletoe infected 31

needles expressed as a percentage of the total needles examined.

LIST OF FIGURES Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14.

ACKNOWLEDGEMENTS

The following individuals are acknowledged for their support and assistance during the period of this research project.

Graduate Supervisors, Dr. Job Kuijt and Dr. Nancy Turner. Graduate Committee Departmental Member, Dr. Louise Page. Graduate Committee External Examiner, Dr. David Dobbins.

Vivienne Wilson of Royal Roads University for collection assistance, sample preparation and initial instruction.

Tom Gore and Heather Down of the University of Victoria Advanced Imaging Laboratory for technical assistance with digital imaging.

Dr. C. Singla of the University of Victoria Electron Microscopy Laboratory for instruction in the use of Scanning and Transmission Electron Microscopes. Brent Gowen of the University of Victoria Electron Microscopy Laboratory for

additional Electron Microscopy training and for the production of many of the electron micrographs used in this study.

From the University of Victoria herbarium, Dr. Geraldine Allen for providing the PCR primers and Erica Wheeler for performing the initial DNA extractions.

Ms. Eleanore Floyd, Graduate Secretary, Department of Biology, University of Victoria for assistance with administrative details.

Dr. William Lanterman and Dr. Delano James, Canadian Food Inspection Agency, Centre for Plant Health, Sidney, B.C. for allowing the educational leave required to complete this study.

Funding for this research was provided by the National Science and Engineering Research Council through Dr. Job Kuijt.

Introduction

The biological concept of parasitism holds, for many, a curious mix of fascination and repulsion. The Oxford English Dictionary defines a parasite as "an animal or plant which lives in or upon another organism (technically called its host) and draws its nutriment directly from it." It is not surprising that the evolution of parasitism has

coincided with increasingly sophisticated methods and organs of obtaining such nutrients from hosts. In the case of higher plants parasitism is defined by an often complex, specialized mechanical and physiological bridge, the haustorium, largely composed of living tissues, through which water and nutrients are transported from one organism to another (Kuij t, 1969, 1979).

Among plants, parasites, as defined by the presence of a haustorium, occur only in about 15 families of Dicotyledons (Kuijt, 1979). The largest number of these families is in the order Santalales which includes the four major mistletoe families,

Eremolepidaceae, Loranthaceae, Misodendraceae, and Viscaceae. The haustoria of these and several other families of parasitic Angiosperms are very poorly known.

In Viscaceae, the haustorial organ shows a distinct tendency to fragment within host tissues, and this has given rise to the term endophytic system. In contrast to that of the other mistletoe families, the endophyte in Viscaceae tends to consist of two

distinctive types of organs (Calvin, 1967; Calvin et al., 199 1 ; SallC, l976a, 1 W6b, 1978, 1979). First of all, cortical strands extend parallel to the host cambium and somewhat external to it, radiating outward from the base of the plant and frequently branching. Cortical strands may reach several cm in length, and at maturity consist of a central core of xylem and weakly developed phloem (except in Arceuthobium, where no phloem is

present) surrounded by a cylinder of parenchyma. Especially near the base of the plant, cortical strands sometimes give rise to additional aerial shoots. Secondly, cortical strands give rise to radially oriented sinkers which appear to penetrate host xylem but in all probability are passively encased by it. Sinkers frequently assume the morphology of host rays and consist of parenchyma and some xylem, the latter often establishing direct contact with host xylem.

Arceuthobium (dwarf mistletoes), a well defined genus of the family Viscaceae, limited almost entirely to the Northern Hemisphere, is parasitic on Pinaceae in the Old and New Worlds and also on Cupressaceae in the Old World. It is a dioecious genus and is currently recognized to contain 42 species worldwide with 34 indigenous to the northern hemisphere of the New World (Hawksworth & Wiens, 1996). Some recent molecular studies have proposed the possibility of increased species numbers (Jerome & Ford, 2002a, 2002b), while other workers have suggested it may be more appropriate to decrease the number from 42 to 26 (Nickrent et al., 2004). Undoubtedly these numbers will continue to change as more sophisticated molecular tools are applied to this fascinating genus.

The dwarf mistletoes are widely recognized as the most highly specialized and evolutionarily advanced genus within the Viscaceae (Hawksworth & Wiens, 1996; Thoday & Johnson, 1 Boa) and this specialization applies particularly to the structure and growth of its haustorium.

Many species are also damaging forest pathogens that suppress growth, reduce wood quality and cause reduced seed production thus limiting reproductive capacity of their hosts (Bhandari & Nanda, 1970; Hawksworth & Wiens, 1996). They thus

predispose trees to other damaging forest pests both directly, by creating entry points for insects and fungi, and indirectly by stressing trees and reducing their vigor. They are of immense economic importance as they are probably the single most destructive pathogen of commercially valuable conifers in Canada, Mexico and the United States (Hawksworth & Wiens, 1996). In British Columbia the estimate of annual losses caused by

Arceuthobium

m.

range from 2.5 million cubic meters (Anonymous, 1990) to 3.7 million cubic meters (Shamoun, 1997). Baranyay & Smith (1972) state that the wood volume loss caused by dwarf mistletoes on western hemlock and lodgepole pine is about

150 million cubic feet (4.25 million cubic meters), a quarter of the annual cut of these species.

Virtually all genera of aerial mistletoes except Arceuthobium rely on birds for the dissemination of seeds. The word "mistletoe" comes from the Anglo-Saxon words meaning "dung-on-a-twig" (Calder & Bernhardt, 1983) and although mistletoe plants do not arise spontaneously from bird droppings as was once believed, the name indicates a recognition of birds as dispersal agents (Aukema, 2003). Arceuthobium is unique among mistletoe genera in its development of a highly effective mechanism of explosive seed dispersal. Dwarf mistletoes are capable of dispersion distances of 10 meters or more and their seed's viscin coating readily adheres to any object they strike (Hawksworth & Wiens, 1996).

MISTLETOE INFECTION

Dwarf mistletoe infection begins after a seed contacts a host branch and

germinates, forming a disk-like holdfast. From the lower portion of the holdfast a wedge of tissue forms which penetrates the outer layer of the branch and establishes a

connection with the living tissue of the host. Soon after this connection is established the external portion of the parasite withers and falls off, leaving little external evidence of the infection. The first sign of infection is a very slight swelling of the host branch.

Eventually three types of infection may be recognized (Kuijt, 1960; Hawksworth & Wiens, 1996).

The first type of infection is characterized by a localized swelling of the host branch into a spindle

-

or fusiform - shaped structure caused by hypertrophy of host tissues (Hawksworth & Wiens, 1972). The amount and extent of this swelling varies according to both host and parasite (Kuijt, 1960). The internal or endophytic portion of the mistletoe is generally restricted to the swollen portion of the host and external shoots are of the tufted type (Baranyay et al., 1971). Sometimes establishment of the parasite takes place on somewhat older host growth where the endophyte fails to reach an apical meristem and no brooming response and very little or no swelling follows (Figure 1A).

Witches' brooms are induced by biotic agents including dwarf mistletoes and are characterized by physiological changes in the host distinguished by a partial or complete loss of apical dominance and the resulting stimulation of lateral buds which produces an unusually dense mass of host branches. The second and third types of infection involve broom-like malformations, which develop only where the original site of infection is in close proximity to one or more host apical meristems. In both types of brooms the growth pattern of the host is affected at an early stage in that profuse localized branching takes place.

Anisovhasic (non-systemic) brooms begin with a localized infection like that described above. The infection spreads out from the initial site of infection and is

characterized by a series of swollen host branches radiating outwards from a common centre. The irregular shoot emergence of the mistletoe may be observed on the younger swollen branches but the endophyte does not extend into the host branches beyond the swelling. In contrast, isophasic (systemic) brooms (Figs. lB,D) lack the characteristic swelling seen in anisophasic brooms and shoot emergence occurs in a regular predictable pattern.

Isophasic brooms only occur in specific parasite / host pairs (Kuijt, 1960). Three species capable of isophasic growth are native to temperate North America; a fourth, endemic to the Himalayas of Pakistan, India, Nepal and Bhutan, also produces isophasic brooming (See Table 1). Hawksworth & Wiens (1 996) also report the occurrence of isophasic brooms induced by a single species from Central America,

8.

guatemalense

Hawksw. & Wiens. Two species from China, _A. chinense Lecomte and

8.

sichuanense

(H.S. Kui) Hawksw. & Wiens (Kiu, 1984), are also reported to cause isophasic infections. Additionally, A. vini Hawksw. & Wiens (Hawksworth & Wiens, 1996) is reported to induce isophasic brooming but the only known occurrence is on a single branch of the type specimen (Forrest 10169) at Kew (Hawksworth & Wiens, 1972).

A.

abietis-reliniosae Hie1 has also been reported to occasionally induce isophasic brooming (Hawksworth & Wiens, 1996), but as detailed morphological descriptions of these species have not been made it is difficult to state definitively that they do indeed induce this type of infection. Species that cause isophasic brooming do so only on specific hosts (Table 1); when they infect secondary or occasional hosts they may produce only local, anisophasic infections. The key to recognizing an isophasic pattern is a regular,

predictable pattern of shoot emergence as seen in Figs. 47a and 48a of Kuijt (1960); such a pattern is documented only in the 4 species in Table 1.

Table 1. Arceuthobium species which cause isophasic brooming and their hosts.

8.

americanum Nutt. ex Engelm. A. douulasii

-

Engelmann

A.

pusillum Peck

A.

minutissimum J.D. Hooker Principal lsophasic host Pinus contorta Doug. ex Loud. Pseudotsusa menziesii (Mirb.) Franco Picea ulauca (Moench) Voss Pinus excelsa complex Wall. ex Lambert Abies Secondary lsophasic host Pinus banksiana Lamb.

Pinus jeffrevi Balfour Pinus ~onderosa Dougl. ex Laws.

Anisophasic host Picea glauca (Moench) Voss

Two of these primary host / parasite associations occur in British Columbia, Pinus Picea mariana (Mill.)

BSP.

Picea rubens Sarg.

--

contorta /

A.

americanum and Pseudotsuaa menziesii 1

A.

dounlasii.

Larix laricina (Du Roi) K. Koch

DWARF MISTLETOE SHOOT EMERGENCE IN ISOPHASIC BROOMS

When young tissue of a susceptible host is infected by a dwarf mistletoe species capable of causing isophasic brooming a very different pattern of shoot emergence than that described above for local infections occurs.

A complete description of the emergence pattern of

A.

americanum parasitic on Pinus contorta has been made by Kuijt (1960) and is represented diagrammatically in Fig.

2A. When the branches of an isophasic broom are examined it is possible to observe the

The brief description that follows provides a generalized and simplified version of Kuijt's detailed discussion.

Observed during dormancy, the terminal year's growth, immediately proximal to the apical bud, bears no evidence of the presence of the parasite. At the base of the terminal segment is a helical arrangement of tightly spaced bud scales; a few of the lowest of these subtend from one to three very small mistletoe buds. The second segment from the apical bud again shows no evidence of the infection but the bud scales at its base support several small mistletoe shoots bearing flower buds. The third segment is also devoid of emergences but the bud scale region subtending it often supports dense clusters of two different types of mistletoe shoots. Larger shoots contain flower buds and

immature fruits (in the case of pistillate plants) and smaller shoots resemble those observed at the base of the second segment. It is on the fourth and fifth segments

proximal to the apical bud that mistletoe shoot emergences are observed; the fourth with small shoots containing flower buds and the fifth with the two shoot types observed at the third bud scale region. Below the fifth segment emergences are rare except at the bud scale regions which may support mistletoe shoots for 10 or more years (Fig. 2A).

The pattern of shoot emergence observed when

A.

dounlasii infects Pseudotsuga menziesii, as reported by Kuijt (1960), is somewhat different from the

8.

americanum /

P. contorta association described above. A branch from a vigorous Douglas fir broom -

observed during dormancy reveals a similar lack of evidence of the mistletoe's presence only on the first segment proximal to the apical bud. One to several of the oldest bud scales at the base of the first segment contains a single minute mistletoe bud, often difficult to observe with the naked eye. The presence of the mistletoe is evidenced in the

second segment by the occurrence of several small bulges in the young bark; later in the growing season, splits occur in the bark at these bulges and the tips of mistletoe shoots can be observed within these splits. A few small shoots with flowers are visible at the bud scale region at the base of the second segment. Along the third segment many shoots at the same developmental stage as those at the base of the second segment can be observed. The bud scale region at the base of the third segment and the entire fourth segment are often crowded with shoots (Fig. 1C) bearing flower buds and maturing year old h i t s (in pistillate brooms). Proximal to the base of the fourth segment there is a noted reduction in the number of mistletoe shoot emergences; beyond the fifth bud scale region below the terminal bud the shoot emergences are almost entirely absent. The pattern described by Kuijt (1960) is illustrated diagrammatically in Fig. 2B.

The anatomy of the aerial system of Arceuthobium has been extensively

described in the literature; Gill (1935), Thoday & Johnson (1930b), Kuijt (1960), Calvin & Wilson (1996) and many others have dealt extensively with the largely reproductive, external structures of the dwarf mistletoes. The aerial mistletoe system is not the subject of this study.

THE DWARF MISTLETOE ENDOPHYTE

The endophytic or internal portion of the dwarf mistletoe, derived fiom the penetration wedge, initially grows radially into host tissues to reach the vascular

cambium. From this position, series of discrete strands called "bark strands" or "cortical strands" grow circumferentially, acropetally and basipetally representing the early haustorium. The cortical strands produce radial "sinkers" which grow radially ultimately becoming embedded in the xylem of the host. In local infections the dwarf mistletoe

shoots, which are initiated from the cortical strands, tend to cluster near the point of infection (Calvin & Wilson, 1996). The cortical strands are believed to terminate in uniseriate filaments, presumably located near the periphery of the swelling (Kuijt, 1964).

Several species are capable of causing a more diffuse pattern of infection and a peculiar broom-like habit of host growth quite different from that described above. This host response, among other things, involves what appears to be a partial or complete interference with apical dominance allowing numerous lateral buds to proliferate. Two distinct brooming responses may be recognized, each characteristic of specific host 1

parasite combinations. Anisophasic (non-systemic) brooming occurs when the host branches near the centre of the broom become swollen; these portions contain all of the endophyte and bear the parasite's shoots; some remnant of the host's apical dominance is retained. In isophasic (systemic) brooming, very little swelling of the host branches develops and the endophyte's growth keeps pace with that of the host. A regular,

predictable, annual, pattern of shoot emergence characterizes isophasic brooms, initially concentrated in the axils of bud scales produced during the previous year's growth (Fig. 2). As stated by Kuijt (1964), "the ultimate filaments of the endophyte are in an intimate relationship with the apical meristem of the host shoot; and that host and parasite seem to be synchronous in their longitudinal growth." In Douglas fir, the witches' brooms

produced can be large and spectacular, often reaching 2 or 3 meters in diameter,

completely altering the normal growth pattern of the host (Figs. lB,D).

The anatomy of the endophytic or internal system within the secondary tissue of the host has received extensive treatment, including works by Solms-Laubach (1867-8) and Heinricher (1 92 1) on

8.

oxycedri, Thoday & Johnson (1 930a) on

A.

pusillum, Parke

(195 1) on

A.

dounlasii, Cohen (1954) on

A.

campvlopodum, Datta (1 954) and Bhandari & Nanda (1 970) on

A.

minutissimum, Gill (1 935), Kuijt (1 960), Srivastava & Esau (1961a, 1961b) and Calvin & Wilson (1 996) on several species. The literature on ultrastructure of the endophytic system is less extensive. Tainter (1971), Kuijt & Toth (1975), Alosi & Calvin (1984, 1985), Sadik (1986a, 1986b), and Calvin & Wilson (1996) have all dealt with the subject but only within the context of the host secondary tissues.

Hawksworth & Wiens (1 996) report that more than 6,400 papers have appeared since Johnson's statement (1888) "that so much has already been written on this genus (Arceuthobium).

. .

that many readers will no doubt be surprised that there should be anything new to be said on the subject." The endophytic system within the primary host tissue of the apical buds, however, has been little studied; in fact, only Kuijt (1960) and Thoday & Johnson (1 930a) make reference to it. Heinricher (1 92 1) states that he traced the endophyte of

A.

oxvcedri (on a small, lateral branch) to within 3 cm of the apical meristem of the host. Thoday & Johnson (1 930a) report that slender strands of an August collection of the

A.

pusillum endophyte have been found in the current year's growth reaching to within 2 mm of the apical meristem itself. This would suggest that the endophyte may occur even in the apical bud except that in their summary they state that 'the parasite

.

. .

by autumn has reached the base of the winter bud". Parke (1951) states that the terminal regions of the endophytic strands of

8.

dou~lasii are uniseriate and

extend to within a few centimeters of the twig apex; he W h e r asserts that the strands do not extend into the current years growth.

The first reference that specifically documents the presence of the parasite within apical buds appears to be by Kuijt (1960). He describes his observations of the endophyte

of

A.

americanum in the dormant apical buds of

P.

contorta where the uniseriate

filaments are observed to penetrate into the apical dome and even into a bract subtending a dwarf shoot. He also diagrammatically represents a possible pattern of growth of the endophytic system of _A. americanum in an apical bud of

2.

contorta (Fig. 3). Many subsequent authors, including Tainter (1971), Alosi & Calvin (1984, 1985) and Calvin & Wilson (1996) make reference to Kuijt's findings. Baranyay et al. (1971) repeats Kuijt's findings describing an isophasic infection as one in which the endophytic system is in the terminal bud and keeps pace with the apices of the host. In spite of these references no subsequent studies have reported any direct evidence of this important observation. Kuijt (1960) also theorizes a similar pattern of growth to that described f o r t . contorta /_A. americanum in the

P.

menziesii

/A.

dounlasii association but admits that his study of this association was limited, however the presence of the endophyte in the host bud was documented (his Fig. 39).

There also appear in the literature two references to the endophytic system penetrating even into the leaf tissue of the host. In commenting on the endophytic tissues of the isophasic system of Arceuthobium, Kuijt (1964) states that "even leaves may be invaded", but this statement is not substantiated. Hawksworth & Wiens (1996) in their discussion of _A. minutissimum, state that the species is unique in the genus because of the emergence of shoots from host needles. Their figure 16:124 indeed appears to show the condition described but it is very difficult to tell from the photograph if the mistletoe shoot does indeed emerge from the needle or if instead the emergence is from the base of the fascicle.

Kuij t's (1 960) discussion of the presence of the

A.

americanum endophyte in the apical buds of

P.

contorta is quite thorough but his similar discussion of the

A.

dourzlasii / P. menziesii association is incomplete and based on a very limited number of

-

observations.

The purpose of the present study is to confirm the presence of the endophytic filaments of

A.

americanum in the apical buds of

P.

contorta and the less well

documented

A.

douglasii /

P.

menziesii association and to chart the precise position of the A. douglasii endophyte within those buds. All dwarf mistletoe infections must begin with

-

the intrusive growth of the endophytic system into the host tissue; Kuijt's (1 960) observations suggest that once established in primary tissues, isophasic infections maintain a non-intrusive, synchronous pattern of growth in tune with the annual

periodicity of host apical bud development. This study will attempt to determine whether the parasite growth can be characterized as truly non-intrusive or if it should be more accurately described as combining elements of both intrusive and non-intrusive growth. It is hoped that a detailed anatomical study will lead to a more complete description of the endophyte anatomy and to a more thorough understanding of the growth pattern of isophasic infection. A limited ultrastructural examination will also be made of both parasite I host associations, with emphasis on intercellular plasmodesmatal connections within the parasite.

THE DOUGLAS FIR APICAL BUD

The vegetative apical bud including the helical arrangement of the primordial leaves of Douglas fir has been well described (Allen, 1947; Allen & Owens, 1972; Sterling, 1946; 1947). The arrangement of the leaves, observed in a dormant bud

(Figs. 4A,B) follow a phyllotaxy corresponding to an 8/13 fraction of the Fibonacci series, the bud scales being arranged in a 215 fraction series (Sterling, 1947). A cross section made at the crown (basal) region of a dormant bud reveals 13 vascular bundles (Fig. 5A), corresponding precisely with the leaf trace arrangement described by Sterling (1947). The organs of the entire following year's shoot, in a preformed state, lie within the bud scales of the dormant vegetative winter bud (Allen & Owens, 1972).

The stages in the development of the Douglas fir apical bud are fixed and

constant, following an annual periodicity of growth. This sequence of events is invariable although environmental factors may affect the duration and timing of each phase of growth. Sterling (1 946, 1947) describes this cycle for Marin County, California, and Allen & Owens (1972) describe it for coastal British Columbia. The area of collection of apical buds used in this study, in south-central British Columbia, has a cooler climate and shorter growing season than either of the study locales mentioned above yet the sequence described is the same. The description which follows reflects this shorter growing season and the correspondingly longer period of winter dormancy.

Growth begins in late spring with a gradual expansion of the shoot and preformed leaves within the apical bud which ultimately break through the bud scales in mid to late May, the bud scales being discarded. After emerging, the shoot and needles elongate and expand, rapidly pushing aloft the newly developing apical bud. As the apical bud expands a new series of bud scales rapidly develops in a tight helix resembling a rosette around the base of the bud. Each of these bud scales is supplied with a procambial strand

(Sterling, 1946). By mid July bud scale initiation is complete and the bud scales continue to expand in length and experience extremely active marginal growth (Sterling, 1947),

thus overarching and enclosing the upper portion of the new bud (Allen & Owens, 1972). During the period of bud scale initiation the apex slowly increases in size.

When bud scale initiation is complete the apex enlarges rapidly and a series of leaf primordia is initiated acropetally from the bud scales to the apex. Even before the leaf primordia are recognizable the procambial strands are evident (Sterling, 1947). By September leaf initiation is complete and the cells of the crown region differentiate, forming a shallow dome of tissue that extends to the base of the youngest bud scales. Above the crown region the vascular bundles formed from the procambial strands have few differentiated vascular elements (Sterling, 1946). Cells in the bud continue to enlarge and divide through late September and October but no new tissue types are initiated. By late November the bud is fully dormant (Owens & Allen, 1972) with the thick-walled crown tissue separating the newly formed bud from the rest of the shoot (Sterling, 1946). The vascular connections pass through the crown region and while they are largely undifferentiated above, they contain extensive tracts of differentiated xylem and phloem below the crown (Sterling, 1946). The interfascicular cambium between the 13 vascular bundles does not differentiate as a meristematic band of cells; the original bundles gradually enlarge and the subsequent bundle union forms a coherent, continuous cambial ring (Sterling, 1947).

Anatomically, the dormant bud of Douglas fir is composed of several zones (Fig. 4D) which may be described as follows. The apical meristem, located at the tip of the vegetative bud, is composed of a small cluster of apical initial cells that give rise to the protoderm through a series of periclinal divisions and to the central mother cell zone, immediately below the apical meristem, by a series of anticlinal divisions. This central

mother cell zone gives rise to the cortex and procambium, contributes tissue to the leaves, and below to the rib meristem, the source of the pith, a cylinder of tissue in the bud's centre (Allen & Owens, 1972). The cortex forms as a continuous sheath surrounding the pith. The procambium, which will ultimately give rise to the vascular cambium, develops in the innermost region of the cortex. The primordial or preformed leaves are helically arranged around the vegetative bud in the pattern described above. The crown region, composed of cells with thick primary walls impregnated with high levels of pectic substances, extends across the pith between the youngest bud scales forming an anatomical separation between last years growth and the current year's bud (Allen & Owens, 1972).

The organization of tissues in vegetative buds of

P.

menziesii, as described above, is somewhat different from that observed in Pinus spp. The Pinus bud contains two sets of bud scales; a small set located just below the very small apex, overarching and protecting it, and a larger set located at the base of the bud enclosing the apex as well as the young lateral buds and the young dwarf shoots (Fig. 3); additionally, each of the dwarf shoots is protected by its own set of bud scales. The well defined crown region described for Douglas fir is not present in Pinus (Mirov, 1967).

Materials

&

Methods

COLLECTIONS

Shoots from isophasic brooms of Arceuthobium dourrlasii infected Pseudotsuga menziesii (Douglas fir) were collected a total of 6 times in southern British Columbia. Collections were made in June and October 1999 from roadside trees a few kilometers

north of Olalla on Hwy 3a; the remaining 4 were made between July 2002 and November 2003 from a roadside area on Green Mountain Road approximately 3.6 kilometers north of the junction with Hwy 3a at an elevation of approximately 700 meters. This area was dominated by second growth Douglas fir heavily infested with

A.

douglasii. (See Table 2 for details of collections)

Shoots from isophasic brooms of

a.

americanum infected Pinus contorta (lodgepole pine) were collected a total of 7 times between July 1999 and November 2003. All were collected at a site with an elevation of approximately 1200 meters a few hundred meters south of the road to Lightning Lakes 1.5 kilometers south-east of the junction with Hwy 3 at Manning Lodge, 64 krn east of Hope, British Columbia. The

lodgepole pine in this area has since been decimated by a bark beetle infestation and several of the larger trees utilized for collections had succumbed along with the mistletoe by November 2003. (See Table 2 for details of collections)

Table 2. Details of field collections included in this study.

Collection Date 8-Jun-99 20-Oct-99 25-May-02 7-Jul-02 15-Feb-03 7-May-03 14-Jul-03 6-Nov-03 Sample ID MT E DFMT DFB Douglas fir 4 4 .\,

4

J 4 Lodgepole pine

4

4

4

4

.\I J .\I Collection notes GPS readings taken at both sites

apical buds and needles for PCR Needles collected 4 .\, J .! Fixative Glut. 4 4 4 d J .\, Nav. 4 d 4 d d

Specimens collected were immediately placed in plastic bags, wrapped in damp newspapers, placed on ice and transported to laboratory facilities at the Canadian Food Inspection Agency, Centre for Plant Health, Sidney, BC, or the University of Victoria, Department of Biology, Victoria, BC. All specimens were kept on ice until they were dissected and fixed approximately 18 to 24 hours after collection.

Morphological examinations of the shoot emergence patterns of the mistletoes were made within 48-72 hours of collection. Digital micrographs of most collections were made under a dissecting microscope with a Nikon Coolpix 885 camera. In a few instances a Sony Power HAD RGB camera coupled to an Olyrnpus SZX9 dissecting microscope were employed. The emergence patterns were compared with those ' diagrammed by Kuijt's Figs. 8 & 9 (1960), abridged in Figs. 2A & 2B.

SAMPLE PREPARATION

Apical buds of mistletoe infected Douglas fir and lodgepole pine were prepared for microscopical study by carefully removing bud scales, excising the buds below the crown region, and placing them in fixative and aspirating for 15-30 minutes. All dissections were made with the material immersed in a drop of fixative.

Some of the apical buds from the first five collections were fixed with Navashin's fixative (chromium, acetic acid and formalin) rinsed, dehydrated, infiltrated and

embedded in paraffin (Berlyn & Miksche, 1970; Ruzin, 1999). The balance of the study specimens was aspirated and fixed in glutaraldehyde (2% glutaraldehyde in 0.05 M

sodium cacodylate (pH 7.3) with 1 rnM CaC12 and 4% sucrose). Samples were rinsed 3 times with 0.05 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide for 1

buffer. Apical buds were infiltrated and embedded in Spurr's resin (Spurr, 1969) after dehydration with acetone or ethanol and propylene oxide, then polymerized overnight at 60•‹C. The collection made in November 2003 was en bloc stained overnight in 5% uranyl acetate in 50% EtOH before embedding.

The last four collections included mature needles from one and two year old flushes (See Table 2). Needles collected for paraffin embedding were fixed whole or halved; the cuticle was lightly abraded with 220 grit sandpaper to enhance infiltration of fixative and paraffin. Cross sectional slices of from 0.5 to 1 rnm thickness from basal, mid and apical portions of needles from several years flushes were fixed in

glutaraldehyde and embedded in Spurr's resin as described above.

Serial, longitudinal, semi-thin sections (0.5 ym) through portions of Spurr's resin embedded apical buds of infected Douglas fir and lodgepole pine were made utilizing knives of ultramicrotome glass cut with a Leica EMKMR2 knife maker. Two

ultramicrotomes were used for semi-thin sectioning; a Leica Reichert Ultracut E and a Leica Ultracut UCT. At approximately 10 ym intervals three or four sections were mounted on a glass slide and stained with Richardson's stain (Methylene Blue and Azure 11) (Richardson et al., 1960; Ruzin, 1999) or Toluidine Blue 0 (TBO) (Ruzin, 1999) for from 30 seconds to 2 minutes at 60•‹C, rinsed with distilled water, dried and cover-slipped with a drop of Perrnount SP 15-1 00 Histological Mounting Medium (Fisher Scientific). In all, portions of more than 40 apical buds were prepared in this manner. All of the buds examined appeared to contain mistletoe endophyte regardless of collection time or species.

A single representative Douglas fir apical bud, identified as E-22-2, was chosen from the collection made in October 1999 and the entire bud was longitudinally sectioned in 0.5 pm increments. Although this representative bud was too large to have been

optimally fixed for ultrastructural study, the relatively undamaged nature of the bud and the thorough resin infiltration and staining qualities made it a suitable choice for a detailed anatomical study. At approximately 10 pm intervals 3 or 4 sections were mounted and stained as described above and 129 successive digital micrographs were made.

Each micrograph was examined, the image cropped and the background eliminated. The host bud (without bud scales) was outlined, the individual cells of the parasite were identified and highlighted and the host tissue was masked leaving only the outlines of the host and of the mistletoe cells (Fig. 8). These individual graphic

representations generated from the micrographs could then be superimposed or stacked in series emphasizing and clarifying the pattern of endophyte growth (Figs. 8,9). Once all 129 of the serial graphics had been aligned, a crude animation was created utilizing

dob be@ ~ h o t o s h o ~ ~ CS Image Ready.

Several ~ h o t o s h o ~ @ tools were employed extensively in analyzing and

manipulating the graphic representations studied. Many of the sections examined were quite large and in order to obtain images with adequate detail it was often necessary to take overlapping images that were recombined using the photomerge function. The levels, curves and magic wand tools were used extensively as were layers. In order to accurately superimpose or stack the serial sections it was often necessary to use the free

transform tool to correct for the differential compression of the specimen caused by the glass knives used for sectioning.

These serial micrographs, produced from the apical bud described above and from portions of several others, were also used to approximate how many of each of the

preformed leaves within the dormant bud contained mistletoe endophyte. An attempt was

then made to quantify the number of infected leaf primordia present. Spreadsheets were used to plot infected and uninfected preformed leaves (See Appendix A for sample).

Prior to trimming specimen blocks for Transmission Electron Microscopy (TEM) study, 1 pm semi-thin sections were prepared as above but mounted on glass cover slips, stained with 5% uranyl acetate in 50% ethanol for one hour and 5% aqueous lead citrate for 1 hour, mounted on aluminum stubs and carbon coated. These samples were

examined at low magnification with a Hitachi S-3500N Scanning Electron Microscope (SEM) and TEM-like digital images were created (Figs. 5A,B). The specimen blocks were then trimmed in preparation for ultrathin sectioning.

Ultrathin (70-80 nm) sections for TEM study were cut with a 2.1 rnm Diatome diamond knife installed in either a Leica Reichert Ultracut E or a Leica Ultracut UCT ultrarnichrotome (as above), mounted on carbon-coated Formvar (1% Formvar in 1,2 - Dichloroethane) coated 50, or 200 mesh copper grids or slotted brass grids and stained with 5% uranyl acetate in 50% ethanol for 30 minutes and 5% aqueous lead citrate for 10 minutes.

SAMPLE IMAGING

Semi-thin serial sections were examined and photographed utilizing several different microscope and digital camera combinations. These included a Zeiss Universal

epi-fluorescence microscope coupled with a Nikon Coolpix 990 or a spotm model 7.0 camera with spotm version 4.0 imaging software (Diagnostics Instruments Inc.) and a Zeiss Axioskop microscope coupled to a DVC still digital camera with Northern Eclipse version 5.0 imaging software from Empix Imaging Inc. All digital micrographs were subsequently viewed and edited utilizing s do be@ ~ h o t o s h o ~ ~ CS, Educational Edition.

Grids were viewed with a Hitachi 7000 Transmission Electron Microscope (TEM) at 75 kv acceleration voltage. Images were captured on Kodak electron microscope film 4489 and scanned with a Polaroid Sprintscan 45 scanner with Polacolor Insight software. All subsequent digital editing was performed utilizing s do be@ ~ h o t o s h o ~ ~ CS software as above.

The Hitachi S-3500N SEM used for the low magnification imaging described above was also utilized for a simple morphological study (Figs. 4A,B) with unfixed, freshly harvested healthy Douglas fir apical buds. Buds were dissected as above, but without fixative, and mounted on an aluminum stub with a drop of silver paste. Samples were digitally imaged within an hour of collection under variable pressure at 15 kv accelerating voltage.

Results

&

Discussion

MORPHOLOGY OF SHOOT EMERGENCE

Little can be added to the description of shoot emergence patterns provided by Kuijt (1960) outlined above and represented diagrammatically in Figs. 2A & B, except in the case of staminate brooms observed in this study. In vigorous staminate brooms mistletoe shoots occasionally occurred up to 7 or 8 annual segments from the apex of

Douglas fir brooms infected with

A.

dounlasii. The fourth and fifth segments proximal to the apical bud were at times so uniformly and densely packed with mistletoe shoots that the branches supporting them were barely visible as evidenced in the photograph of the fourth segment of a staminate broom (Fig. 1C) collected in May of 2003.

ENDOPHYTE IDENTIFICATION

An essential early step in establishing the pattern of endophyte distribution within host apical buds was the identification of cellular features in which parasite and host differed. A list of five distinctive characteristics was developed from the scientific literature and direct observation. A11 characteristics described below were clearly visible in the 0.5 - 1.0 pm sections (Fig. 6) of glutaraldehyde fixed, resin infiltrated apical buds viewed with Kohler illumination at relatively low magnification (500 X).

1) Thickened external endophyte cell walls: Wherever the non-lignified cell walls of the endophyte contact host cells they are characterized by a pronounced and uniform thickening. These thick outer walls are clearly observed in the early illustrations of cross sections of small Arceuthobium strands in Solms-Laubach (1867-8, Figs. 6-10) and Cohen (1954, Figs. 7-9). The prominently thickened non-lignified external cell walls of the endophyte are usually two to three times as thick as those of the host parenchyma (Calvin & Wilson, 1996); (Kuijt & Toth, 1976). This thickening contrasts sharply with the much thinner internal transverse walls observed in uniseriate filaments and in transverse and longitudinal walls of the older tiered, multiseriate filaments (Kuijt, 1960). It should be noted that this thickening of the cell wall at the parasitelhost interface appears to be entirely limited to the parasite; by contrast the host cell walls, whether in contact with one another or with the

endophyte, appear remarkably consistent in being thinner than external endophyte walls.

2) Cell size and shape: Mistletoe cells in mature host branches are generally larger than the surrounding host cells, and in young uniseriate filaments are two to three times as long as they are wide (Thoday & Johnson, 1930a). This is consistent with

observations made of the endophyte within dormant host apical buds except that parasite cells were often four or more times as long as they are wide.

3) Nucleus: Plant nuclei can be divided into two basic types, chromocentric, characterized by the presence of dense chromatin masses and reticulate with chromatin evenly distributed throughout (Lafontaine, 1974). In interphase the chromocentric

A.

douglasii nuclei contain a fine meshwork of chromatin and conspicuous chromatin masses: those of

P.

menziesii have a more reticulate

distribution of chromatin and lack the masses (Alosi and Calvin, 1984, 1985). Both parasite and host cells may contain conspicuous nucleoli. Bhandari & Nanda (1970) note that the relative chromaticity of the nuclei of the host (P. excelsa) and parasite

(A.

minutissimum) is distinct. Kuijt (1960) in commenting on the difficulty of differentiating parasite fiom host relied on the conspicuous nucleus and the dark staining nucleoli of the cells of the endophyte. All of the studies mentioned (Lafontaine, 1974); (Alosi and Calvin, 1 984, 1 985); (Bhandari & Nanda, 1970); (Kuijt 1960) were made on Pinaceae; Sadik et al. (1986a), in their study of _A. oxycedri infecting Juniperus (Cupressaceae), note that nuclear contrasts were not sufficiently pronounced to differentiate host fiom parasite.

4) Presence of Lipids: The presence of lipids in the endophyte cells has been discussed by many researchers including Alosi & Calvin (1 984, 1985), Calvin & Wilson

(1996), Kuijt & Toth (1976), Sadik et al. (1986a, 1986b), Tainter (1971), and Thoday & Johnson (1930a).

A.

dounlasii parenchyma often contains large, regular, more or less spherical, dark staining lipids: in Douglas fir, lipid droplets, when present, were generally smaller and less abundant.

5) Plasmol~sis and Vacuolization: Kuijt (1 960) observes that the endophyte cytoplasm shows a more severe plasmolysis than that of its host, owing to fixation. In the material of this study a similar tendency was observed, but the distribution of

cytoplasm and presence of vacuole membranes indicated vacuolization in addition to plasmolysis.

A great deal of seasonal variation in all of the above characteristics was observed with the possible exception of the thickened endophyte cell walls. The size of the

endophyte cells, relative to those of the host show some seasonal variation. In late spring, as dormancy is replaced by active growth, host cells undergo a very rapid increase in size; the expanding bud breaks through the protective layer of bud scales and produces the current year's flush of growth. During the bud break stage the host cells appeared much closer in size to the mistletoe cells. Patterns of chromatin distribution also varied seasonally somewhat in both host and parasite but they remained sufficiently different to be an effective means of differentiation. During summer, when the host apical meristem is initiating the next season's growth, endophyte cells within the very small buds are heavily vacuolated and lipids are largely absent. While it was often difficult to apply all five characteristics listed above to any single cell (Fig. 6), a combination of 3 or 4

characteristics commonly sufficed for identification. There were instances in which the differentiation of the parasite cells from those of the host was difficult as in Fig. 11C; when this occurred any questionable cells were treated as belonging to the host.

PRELIMINARY FINDINGS

Upon observing the first 0.5 pm sections through the cortical region of Spurr's resin embedded dormant apical buds of Douglas fir infected with _A. dourrlasii, the

uniseriate strands of the endophyte were immediately recognizable. However, the amount of mistletoe tissue present and its complexity made it necessary to develop a method of visually separating the parasite from its host. Initially a single micrograph was examined, the mistletoe cells were identified and the host tissue masked to highlight the endophyte; if the host tissue was eliminated entirely the course of the ramifying strands of the mistletoe endophyte could be clarified (Fig. 7).

As more buds were examined two important facts emerged. First, it became apparent that the endophytic strands of the parasite were distributed in a fairly uniform, more or less predictable pattern within the host. Second, and of perhaps greater botanical interest, the filaments of the endophyte were present even in some of the preformed leaves or leaf primordia within the dormant buds (Figs. 10A,C).

The rather uniform, predictable pattern of endophyte growth led to the

development of the method described above. The entire bud identified as E-22-2 was serially sectioned and photographed. The micrographs were manipulated to create graphic representations of the endophyte's position within the host, and a crude animation created. Although this method could not be used to generate a three dimensional representation, this simple animation proved to be a useful tool for

visualizing and describing the pattern of endophyte distribution within the host apical bud.

LIPID DISTRIBUTION AND PLASMOLYSIS

It was noted that lipids were generally more abundant immediately above the crown region and in the basal region of the cortex; endophyte cells near the apex often contained no lipids at all, consistent with the assertion that meristematic cells have few lipid bodies (Alosi & Calvin, 1985). Sadik et al. (1 986a) in their descriptions of sinkers in host secondary tissue noted that lipids were abundant in sinkers but that more lipid

inclusions were present at the tips of sinkers. Both Thoday and Johnson (1930a), writing on

A.

pusillurn and Parke (1% 1) discussing

A.

douglasii theorized that sinker cells are generated from an intercalary meristem located at the host cambium. If this is true then the cells at the tips of the sinkers are developmentally older than those at the base; thus lipids are concentrated in the older endophyte cells, consistent with findings for the endophytic filaments in the host apical buds reported in this study.

It was also noted that plasmolysis and vacuolization increased as the abundance of lipids decreased; mistletoe cells near the apical meristems of host buds often had little cytoplasm present and vacuoles were the most prominent cellular feature.

ENDOPHYTE DISTRIBUTION

The endophyte in host apical buds is for the most part composed of a series of parenchymatous uniseriate strands that appear to grow in fairly regular helical patterns within the host cortex. Like the helix observed in the arrangement of the leaf primordia, the endophytic strands spiral in both clockwise and anticlockwise directions from crown

region to apex. Kuijt (1960) theorizes that the endophyte forms a loose wreath of

filaments just below the host crown region (Fig. 3). No evidence was found of this wreath

of filaments in the buds examined.

Kuijt diagrammatically represents, in his Fig. 7 (1 96O), his observation of the presence of endophytic filaments of _A. americanum in the pith of older stem tissue of

P.

contorta. In observations made in his Plate 38b (1 960), a micrograph taken of a

longitudinal section of the procambial region of a shoot apex from a broom of

P.

contorta, he theorizes that we may be witnessing an early stage of the displacement of a filament into the host pith region.

The current study does not extend below the crown region in Douglas fir buds or the corresponding area of the lowest bud scale insertion of Lodgepole pine apical buds. The occurrence of mistletoe endophyte was not observed in the pith of either species; it occurred exclusively in the cortex, except as noted below.

From observations made in this study I hypothesize that a series of uniseriate and multiseriate strands is located between the procambial strands and the epidermis

centrifugal to the host crown region. Above the crown region a complex network of largely uniseriate strands ramifies throughout the cortex centrifugal to the pith, extending into some of the bud scales and preformed leaves of the host. The transverse cell walls separating endophyte cells within these strands are generally uniformly thin and

plasmodesmata between cells are not uncommon (Figs. 12C,D; 13C,D; 14B). By contrast the outer cell walls of the mistletoe are much thicker where they interface with host cells (Figs. 5B, 6, 7C, 10, 1 1, 12A,B). What appear to be multiseriate strands are quite

common when viewed in longitudinal sections. These strands are of two very different forms.

In the first type of multiseriate strand the cells occur in longitudinal pairs, separated by the same thin wall described above (Fig. 11). This tiered arrangement of cells, indicative of their initially uniseriate structure, is consistent with descriptions of the cortical strands of the endophytic system as it occurs in the secondary tissues of the host (Heinricher, 192 1 ; Thoday & Johnson, 1930a; Parke, 195 1). Not unexpectedly, these multiseriate strands were more common near the base of the bud, especially in the crown region. Within the uniseriate and tiered multiseriate endophytic strands plasmodesmata were commonly observed (Figs. 12C,D; 13C,D; 14B), however, none were ever observed between parasite and host.

In the second type of multiseriate strand, the tiered arrangement of cells is absent; the walls between mistletoe cells are not uniformly thin but often appear for the most part more like the thick mistletoe-host interface. This non-tiered multiseriate appearance is in part due to the meandering course described by the uniseriate strands; as they make their convolute way through the host cortex they are often found to be immediately adjacent to one another. In many cases two or three individual strands will meet at a single point in the cortex and form an intertwining mass of tissues. Although difficult to interpret without careful study of the consecutive serial sections described above, what appear at first observation to be relatively irregular multiseriate strands are presumed to be a series of fused, intertwined uniseriate strands. In some of the places where these endophytic strands grow within close contact with one another, instead of the thick cell wall normally seen on the outside walls, there occur thin areas of cell wall alternating with

thick areas (Fig. 7C). It is possible that this represents reabsorption of the cell wall and a re-establishment of symplasty where two or more of the uniseriate strands contact one another within the cortex. In this study no plasmodesmata were observed in these areas of theorized reabsorption; extensive ultrastructural study of these regions would be required in order to determine whether interconnecting plasmodesmata form secondarily,

establishing a syrnplastic connection between individual strands of the endophyte.

Branching of the uniseriate strands of the endophyte was commonly observed and was consistent with the descriptions of

A.

pusillum by Thoday and Johnson (1930a),

A.

dounlasii by Parke (1 95 1) and _A. minutissimum by Bhandari and Nanda (1 970). Often a bulge arises from the side of a sub-terminal cell. Eventually an oblique or longitudinal division wall forms and the meristematic cell separated from the mother strand gives rise through a series of regular lateral divisions to a new strand, indistinguishable from that from which it has arisen. This branching was observed throughout the cortex of dormant host buds and at times even occurred in preformed leaves (Fig. 10C).

Early in this study the presence of the mistletoe endophyte was observed in the preformed leaves (Figs. 10A,C) and even the bud scales (Fig. 10B) of dormant buds. The endophyte was not present in every preformed leaf of an infected bud, however it did occur with consistency and regularity in every bud examined. Usually, but not exclusively, a single uniseriate strand of the endophyte, with its origin in the cortex, penetrates the preformed leaf of the host. Although at this stage of development the method employed did not clearly stain the provascular tissue within the primordial leaves, in each case the endophyte was observed toward the centre of the needle in the region that will ultimately give rise to the vascular tissue of the leaf. In many cases the

parasitic strand reached to within a few cells of the primordial leaf apex (Fig. 10A). In several instances sub-apical branching of the endophyte was observed even within the newly expanding leaf (Fig. 10C) and occasionally two individual uniseriate strands were observed within a single preformed leaf.

Kuijt (1 960) documents an interesting phenomenon occurring in the expanding material of an actively growing apical bud of Douglas fir; he reports the apical cell of a single filament of _A. dounlasii endophyte actually breaking through the epidermis of the host. His micrograph (Fig. 39) clearly shows this eruption of a filament in the axil of a bud scale subtending a primordial leaf. He further states that he observed several

instances of this phenomenon in material collected form southern British Columbia. The exact time of collection was not recorded, but the developmental state of the bud scale and leaf primordium in his micrograph are indicative of material collected in mid summer, possibly July or August. Even though several buds at a similar developmental stage were examined during this study no such emergences were observed, however the endophyte was observed within bud scales (Fig. 10B) and in close proximity to the epidermis immediately adaxial to the insertion point of the bud scale. In order to prepare apical buds for fixation it was necessary to excise the bud scales; it is probable that the emerging mistletoe filaments that Kuijt observed were present in our study material but were removed with the bud scales prior to fixation.

Endophyte filaments did not occur in every preformed leaf. An attempt was made to quantify the number of infected leaves as a percentage of the total leaves examined. The single serially sectioned apical bud, E22-2, described above, was examined at 10 pm intervals. Each leaf primordium was numbered and the endophyte presence documented.

In all, 37 of the 120 total leaf primordia present contained mistletoe tissue. Portions of four additional Douglas fir buds were longitudinally, serial sectioned and again the number of infected primordia recorded. In order to accurately calculate the number of preformed leaves infected when only a portion of the bud was studied by serial section it was necessary to adjust the number of leaves examined by subtracting fiom the total any of the needles that were only partially sectioned. This recognizes the fact that partially sectioned needles may contain the endophyte in the unsectioned and therefore

unexamined portion. In all of the apical buds examined there appeared to be no recognizable pattern of primordial leaf infection; infected needles appeared to be randomly and uniformly distributed in all regions of the bud.

While the sample size studied was too small for a detailed statistical analysis, the presence of the endophyte in every bud examined and the consistency of the results illustrated in Table 3 indicate that the mistletoe appears to infect approximately one quarter to one half of all the preformed leaves within a dormant bud.

Table 3. Quantitative estimate of the number of mistletoe infected needles expressed as a percentage of the total needles examined. The corrected data recognize that a needle that has only been partially sectioned may contain mistletoe in the unsectioned and therefore unstudied portion.

I Bud ID E22-1 E22-2 E l 7-2 E l 7-3 E l 7-7 Totals

1

Raw Data %

Total lnfected lnfected

58 27 46.6% 120 37 30.8% 67 24 35.8% 48 13 27.1% 81 26 32.1% 374 127 34.0% Corrected 'Yo

Total lnfected lnfected

50 27 54.0% 120 37 30.8% 55 24 43.6% 38 13 34.2% 75 26 34.7% 338 127 37.6%

The endophyte was present in some of the preformed leaves of every bud studied, both in dormant and actively growing buds. Because of this all sample collections made in 2003 included mature needles. The occurrence of the endophyte in primordial leaves immediately raised the question of the possible presence of the endophyte in the mature host leaves. An attempt was made to detect the mistletoe within the needles of the current year's flush. Although the needles were dissected from the stem within 1 mm of the insertion point of the petiole into the branch, no evidence was found of the mistletoe within the mature needles, with the one exception noted below. The preformed leaves within the dormant bud are generally less than 500 pm in length and in some cases the endophyte grows to within a few cells of the leaf primordium apex (Fig. 10A). The failure to detect the mistletoe in the emerged needle suggests that the growth of the endophyte may not keep pace with the rapidly elongating needle. Alternatively, the characteristics of the endophyte may change to such an extent that they cannot be recognized by the techniques employed in this study.

In a cross section from a single needle petiole, from the collection of needles from the current year's flush made in July 2003, an unusual group of cells was noted

immediately adjacent to the primary phloem of the host (Fig. 5D). Serial cross sections were made from the petiole toward the needle apex. When these sections were examined the unusual cells observed in the first section disappeared and the vascular bundle again appeared normal. The thick cell walls and unusual position suggests that the group of cells may have been associated with the tip of the endophytic strand. It is also possible that this unusual cell group was the early stage of astrosclereid formation, not unusual in the mesophyll of mature needles of Douglas fir (Al-Talib & Torrey, 1961; Apple et al.,

2002). This explanation seems unlikely as astrosclereids normally occur singly in more mature needles.

Even though it was the endophyte within the primary tissues of host apical buds that was the focus of this study, some observations of the crown region were also made. Kuijt (1960) illustrates a loose wreath of endophytic filaments girdling the base of the bud and repeated just subtending the apex of the bud of

P.

contorta infected with _A. americanum (Fig. 3). He further theorizes a similar pattern of endophyte distribution for

P_. menziesii infected with

8.

douglasii; with a wreath of endophytic filaments subtending the crown region. In this study no such wreaths of Douglas fir mistletoe cells were

observed; instead, numerous individual filaments were observed, always in the cortical region centrifugal to primary xylem of the vascular bundles. Many of the strands were uniseriate, but many multiseriate strands were also observed which displayed the tiered arrangement described above.

In the earlier discussion of endophyte identification, reference was made to the presence of an intercalary rneristem in the Arceuthobium endophyte located at the vascular cambium region of the host which generates sinker cells (Thoday & Johnson,

1930a; Parke, 1951) Other researchers agree with this interpretation (Gill, 1935; Kuijt, 1960; Srivastava & Esau, 1961a); however, all of these studies have been in the secondary tissues of the host. When a cross section through the crown region of an infected Douglas fir bud is examined, the uniseriate and multiseriate strands of the endophyte are clearly visible; these strands are always located in or near the primary

L

phloem area of the vascular traces or in the newly developing secondary phloem

sinker penetrating, or encased in, the primary xylem of the host is clearly visible (Figs. 5A,B). The wedge-shaped endophyte, in no case observed, penetrated into a host xylem cell, rather it appears to be pushing the files of primary xylem cells apart. This early ontogeny of sinker initiation supports Kuijt's (1960) argument that the radial

development of sinkers is not dependent on the endophytic contact with phloem rays as sinker initiation in this instance occurs before ray initiation.

The observations of the regular pattern of endophyte distribution in host apical buds made in this study, particularly the position within preformed leaves and bud scales, and in close proximity to the leaf a i l s where axillary buds will develop, suggest that the pattern of growth is truly isophasic, keeping pace with the cycles of seasonal growth experienced by the host. This distribution also makes Kuijt's observations of endophyte emergence in the a i l s of host bud scales likely and certainly relates to the pattern of endophyte emergence observed earlier in this discussion under the heading

-

Dwarf mistletoe shoot emergence in isophasic brooms.

ULTRASTRUCTURAL OBSERVATIONS

Although the main emphasis of this study was anatomical, specifically the organization of the

8.

dou~lasii endophyte within the apical buds of Pseudotsuna

menziesii, it was possible to make limited observations of the parasite and of the host I

parasite interface at an ultrastructural level. As noted in the introduction, all of the ultrastructural studies of the Arceuthobium endophyte have been only within the context of the host secondary tissues. In this study several hundred electron micrographs were made of the endophyte of

A.

dounlasii in Pseudotsuna menziesii and of

A.

americanum in

Pinus contorts, all in the primary tissue of the host apical buds, bud scales and leaf primordia.

In his writing on the ultrastructure of _A. pusillum, Tainter (1971) comments on the presence of chloroplasts in parenchyma cells of aerial shoots and endophyte. The presence of chloroplasts within the endophyte was also observed in

A.

oxvcedri by Heinricher (1921) and Sadik et al. (1986a); Solms-Laubach (1867-8) also states that he observed chloroplasts. In their ultrastructural studies, Alosi & Calvin (1984, 1985) and Calvin & Wilson (1 996) make no mention of the presence of chlorophyll within endophytic tissue of Arceuthobium although they document the presence of plastids.

In 1999 Marler et al. published a successful polymerase chain reaction (PCR) method developed to detect the presence of

A.

doualasii in the secondary tissues of young branches of isophasic brooms of Pseudotsuga menziesii. This test targets the large segment of the ribulose bisphophate carboxylase/oxygenase (rbcL) gene, located in the chloroplast genome. This effective PCR test relies on the presence of chloroplasts in both host and mistletoe, providing further evidence of the presence of chloroplasts in the endophytic cells of

A.

doualasii. Specific details of the PCR test are described in Appendix B.

This study found plastids to be abundant in the endophyte of

8.

doualasii and

A.

americanum (Figs. 12-14) but although chloroplasts were frequently observed in host cells (Fig. 12B), none were observed in the cells of the endophyte. Lipids were also common in both mistletoe species and were especially abundant in _A. americanum collected in late spring (Fig. 14A). In addition to numerous lipids, small dark staining

starch granules were also observed, most commonly in association with the cell wall of A. douglasii in dormant buds of

P.

menziesii (Figs. 12C,D).

All of the researchers mentioned above, except Tainter (1971), agree that no true plasmodesmata connect mistletoe endophyte cells with those of their respective hosts although Alosi & Calvin (1985) do note the presence of half-plasmodesmata extending fiom the protoplast of host cells to the middle lamella region separating host fiom parasite. Tainter (1971), in his examination of a spruce broom of unspecified age, states that he observed plasmodesmata in restricted areas between cells of the endophytic system of

A.

pusillum and host needle trace phloem parenchyma of Picea mariana, but his micrograph, figure 6b, fails to show complete plasmodesmata (Alosi & Calvin, 1985) and it seems possible that an error may have been made in cell identification (Kuijt & Toth, 1976). In this study no plasmodesmata were observed connecting host to parasite. Half-plasmodesmata similar to those observed by Alosi & Calvin (1985) were

documented (Figs. 13A,B) and plasmodesmata connecting endophyte cells within uniseriate strands were very common in both of the host 1 parasite associations studied (Figs. 12C,D; 13C,D; 14B). Transverse primary walls between mistletoe cells

occasionally contained massively thickened areas (Fig. 13D) referred to as peculiar beadlike thickenings by Kuijt (1960). These peculiar thickenings, with their clearly evident microfibril structure, resemble the cellulosic primary cell wall of the endophyte suggesting that they are not composed of callose, an amorphous carbohydrate associated with sieve areas and with wound response in parenchyma cells.

Within the apical buds studied very little physical evidence of crushing or tearing of host tissue was observed suggesting that little or no intrusive growth was occurring.

Indeed the cell walls which separate parasite from host are fused so completely that it was impossible to discern the precise location of the middle lamella. This implies that the uniseriate strands of the endophyte must be capable of a very rapid longitudinal growth in early spring when host shoot elongation occurs. The formation of transverse division walls was commonly observed in the cells composing uniseriate strands; these division walls were not confined to apical cells but were observed along the entire strand. In material collected in late spring it was not uncommon to observe these division walls in the earliest stage of development (Fig. 14D). This frequent division may explain in part the ability of the mistletoe to keep pace with host growth. The very thick cellulosic, primary wall of the mistletoe cell where it interfaces with the host may provide another, accommodating the stretching of the filament during the rapid elongation of the host branch.

SUGGESTIONS FOR FUTURE STUDIES

Fig. 9C demonstrates a thinning of the external cell wall where two or more uniseriate strands come in close contact with one another. No plasmodesmata have been observed in such contiguous filaments, but the possibility of such connections cannot be excluded. A detailed ultrastructural study could establish whether or not a symplastic connection of the plasmodesmata, so commonly observed between cells composing a single uniseriate strand, is in fact established.

The presence of the mistletoe endophyte in primordial leaf tissue and bud scales warrants further study. The fact that the mistletoe remained undetected in the emerged needles of Douglas fir suggests that the endophyte's growth within needles may not continue after bud break. Does the endophyte persist in the mature needles or does the