Anatomy and Pathology of Leaf Spots on English Holly, Ilex Aquifolium (Tourn.) L

Home , Ilex aquifolium, Ilex opaca, Leaf spot, Phycomycetaceae

ANATOMY AND PATHOLOGY OF LEAF SPOTS ON ENGLISH HOLLY, ILEX AQUIFOLIUM (TOURN.) L.

ELIZABETH ANNE HERRIDGE

A THESIS submitted to

OREGON STATE COLLEGE

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

June 1960 APPROVED:

Professor of Botany

In Charge of Major

ti Chairman of Dep ment of otany and Plant Pathology

7 Chairman of School Graduate Committee

Dean of Graduate School

Date thesis is presented: May 13, 1960

Typed by Janice Cram ACKNOWLEDGMENTS

The author wishes to express her indebtedness to Dr. Roy

A. Young, Chairman of the Department of Botany and Plant Pathol-

ogy at Oregon State College, for suggesting this project.

Dr. Young is thanked for many helpful suggestions and his aid in

obtaining materials from various parts of Oregon.

Expressions of gratitude are inadequate to thank Dr. Frank

H. Smith for his limitless patience with the author and the many

problems of her research. His practical advice in microtech-

nique and in anatomical interpretation have proved invaluable.

By his constructive criticisms of the manuscript, Dr. Smith made

the writing of this thesis a less difficult task.

Many thanks are also extended to Dr. Lewis F. Roth for

his guidance in fungous identification. The use of his personal

library, together with his knowledge of appropriate literature,

saved many hours of work.

To Dr. Harry K. Phinney, the author wishes to extend most

sincere thanks for the precious time he spent instructing her in

photomicrography. The illustrations for this thesis were made

possible by the use of his equipment and by his encouraging help.

Thanks are also proffered to Mr. H. H. Millsap and Miss

. Janet Nelson for help in the preparation of figures and plates.

Finally the author wishes to thank those numerous friends

and collegues who have contributed to the preparation of this

thesis. Special thanks are given to Mr. Fred Merryfield and

_ J Mr. Ralph L. Carmichael for their encouragement and labors when clear thinking had reached a low ebb! TABLE OF CONTENTS

Page INTRODUCTION 1

LITERATURE 3

MATERIALS AND METHODS - 9

NORMAL ANATOMY 16

SPOTTING CAUSED BY FUNGI 24

Identification of Fungi 29

Morphology and Anatomy of the Inoculation Tests. . 36

Detached -Leaf Inoculations 39

SPOTTING CAUSED BY CHEMICAL SPRAYS 42

SPOTTING CAUSED BY MECHANICAL INJURY 48

SPOTTING CAUSED BY INSECT DAMAGE 53

DISCUSSION 56

SUMMARY 64

BIBLIOGRAPHY - 67

APPENDIX 71 ANATOMY AND PATHOLOGY OF LEAF SPOTS ON ENGLISH HOLLY,

ILEX AQUIFOLIUM (TOURN.) L.

INTRODUCTION

Cultivation of English holly in Oregon has developed in the last thirty years so that this plant has become a major specialty crop. Ten years ago the annual return from the 750 acres of commercial holly then under production was estimated to be $150,000 (37, p. 3). Since then, this income has increased approximately 33% and some 1300 acres are now under cultivation.1 A large number of varieties of Ilex aquifolium

('Turn.) L. are used in the production of Christmas holly, and as young ornamental planting stock. Silver and gold variegated and green varieties are shipped throughout the United

States. Over 150 varieties were described by Moore (24,25,26,

27) in England and since then many new ones have been selected for their leaf, berry and stem characteristics and are kept constant by propagation with cuttings, layering and grafting.

Some 'Dutch' forms have entire leaves with large berries while the 'French -English' is a blue -stemmed variety with green, spiny foliage. Many variegated forms are grown for their deco- rative leaves even though berries may not be produced.

Holly growers throughout Oregon have become concerned with the disfiguring spots on the foliage of Ilex aquifolium.

1Conklin, Melvin J. Asst. Ag. Economist G.S.C. Private com- munication of approximate statistics for 1957. 2

Several types of red or red -black discolorations and blister -like swellings occur on the abaxial and sometimes adaxial surfaces of the leaves. The condition increased during the 1950's so that considerable amounts of the crop were unsaleable on the Christmas markets.

Mechanical injury, insects, fungi and chemicals have been suggested as causes of the red spotting. The histological investigations re orted here have been conducted to determine the causes of different types of leaf spots and to observe changes in leaf anatomy resulting from certain external stimuli. 3

LITERATURE

Ilex aquifolium is produced extensively in the Pacific

Northwest and a second species, I. opaca Alton, occurs in the

Atlantic and New England states. These are the only two species of Ilex (Tourn.) L. grown commercially in the United States.

They belong to the dicotyledonous family Aquifoliaceae, which

consists of five genera of deciduous and evergreen trees and

shrubs bearing alternate leaves with or without minute stipules.

The genus Ilex is indigenous to North, Central and South America,

Asia, Africa, Australia and Europe (46, p. 340).

Ilex aquifolium, termed English holly, is native from

Great Britain to Iran and from Norway to Turkey (36, p. 307).

It was one of the first species described and is the best known.

English holly was a favorite for use during festivals in Roman

times. The early Greeks celled it Holly Agria, meaning wild

holly or holly of the fields. The Romans coined the word Agri -

folium and then Aquifolium from acutum, meaning sharp, and

folium, a leaf, hence the specific name. Holly is also an an-

cient name said by some to be a corruption of holy. This is

refuted by Hume (18, p. 5) who states that the connection is

purely one of sentiment with the Christmas festival, and the

words holy and holly originate from different roots.

In habit, I. aquifolium may form a bush of several stems

or a single - trunked tree of eighty or more feet in height. When 4

these evergreen trees grow alone, they are generally pyramidal in shape with branches arising from ground level. They are known

to live for 250 to 300 years and ancient writings of the Romans

suggest that some specimens have reached 800 to 1200 years. His-

torical writings, poetry, legends, superstitions and ancient medical practices have many references to I. aquifolium (11,

p. 36 -61).

Ilex opaca, another evergreen species, is known as Ameri-

can holly and is native to the United States. American holly

is very similar to I. aquifolium and is common in the South-

eastern part of the country where it is also used for holiday

decorations (33, p. 26). It has proved to be quite hardy in

that region. I. opaca has been known to withstand temperatures

of -30 °F. in Massachusetts. Also, in this locality holly was

under twenty feet of salt water during the hurricane of 1938

without sustaining injury. Nevertheless, it is considered bet-

ter to use local clones in the more rigorous Northern climate

although some of the New England varieties do well in North

Carolina, Kentucky and Tennessee (47, p. 68). I. opaca is, how-

ever, considered of inferior quality compared with I. aquifolium

and is rarely planted in the Pacific Northwest (37, p. 4).

Literature reviews of diseases that cause leaf spotting

on holly have produced little information to aid the present

study. In 1935, Grove published descriptions of six species of

Fungi Imperfecti which occur on the living leaves of Ilex 5 acuifoli.um in Great Britain. Asteroma ilicis Grove (15, p. 145) gives brown irregular spots; .Stazcncspera ilicis Grove (15, p. 347) occurs as hypcphylious black pycnidia; Phyllosticta aquifolina Grove (15, p. 23), Phoma ilicis Desm. (15, p. 87) and

Phoma ilicicola Saco. (15, p. 86) all produce grey -brown spots on leaves and twigs of English holly. The sixth species,

Ascochyta ilicis Grove (15, p. 304) appears to be a secondary invader occurring in the spots produced by leaf miners. Phomop- sis crustosa Tray., Ceuthospora phacidioides Grey. and Dendrophoma phyllogena Trail occur on dead or fallen leaves and twigs (15, p. 134, p. 192, p. 289).

Another species of Phoma, P. citricarpa McAlp., was reported to cause a leaf spot of I. aquifolium in the Kurrajong

Heights of New South Wales (32). Leaf pathogens reported on Eng- lish holly in Oregon by Shaw (41, p. 51; 42, p. 14, p. 49, p. 118) are Boydia insculpta (Oud) Grove, Diaporthe eres Nits. with the imperfect stage Phomopsis crustosa, and Nectria al- ligena Bres. with the imperfect stage Cylindrocarpon mali

(Allesch.) Wr. Subsequently, however, it has been shown that

Boydia insculpta is a secondary invader and only saprophytic upon the holly leaves (7). Fungi of the imperfect genus Cladosporium

Link ex. Fr. which are parasitic and saprophytic, and Fumago vagans Pers., a saprophyte, have also been described. Two other genera are reported on I. aquifolium in Washington and Califor- nia: wPhyllosticta Pers. ex. Desm. and Gleosporium aquifolii 6

Penz. & Sacc. are both imperfect fungi (45, p. 50). Phytophthora

ilicis Buddenhagen & Young, was diagnosed at Oregon State College

as the cause of a leaf spot and stem canker disease (7).

On I. opaca, dieback was reported by Bender (6) to be

caused by a Fusarium species. This species, besides producing

wilting of the leaves and current growth, also caused defoliation

of the trees but no report of leaf spots was given. Rhizoctonia

solani Kühn has also been the alleged cause of defoliation in

cuttings of I. opaca and the formation of zonate leaf spots on

the fallen leaves (10). Phacidium Curtisii (B. & Ray.) Luttrell

was considered to be the cause of Tar Spot of American holly (21).

This fungus, consisting of thin walled intracellular hyphae with

prominent nuclei was thought to have a single host, I. opaca.

Luttrell described the development of spots from tiny chlorotic

areas similar to insect damage, seen in May, through an increase

in size and coloration from red to brown until in the fall the

spots were shiny black cushions when subepidermal stromata had

formed. Cole (9) reported that great damage was caused by this

fungus attacking leaves and berries in years of excessive rain-

fall. Sanitation by pruning and removal of fallen leaves was

supplemented by spraying with Bordeau and then Phygon. Another pathogen of I. opaca, the uredospore stage of a rust, Crysomyxa ilicina Saville, was described from Tennessee and West Virginia

(40). 7

Plakidas (31) described a spot anthracnose of Chinese holly, I. cornuta Lindl. & Paxt. The symptoms were of two types, both confined to the upper surface of the leaf. Black spots occurred only in late fall, were generally numerous, and sometimes coalesced to form irregular patches. The second type, less common and consisting of much larger black lesions, was found in early summer. In both types, chlorosis was followed by brown spotting. The spots were caused by the intercellular hyphae of

Elsinoe ilicis Plakidas, which occurred mostly in the mesophyll but finally invaded the epidermis.

Ilex wightiana Wall. was noted in India by Ramakrishnan

(34) to have raised shiny black irregular spots on the adaxial surface of living leaves. They showed as discolored patches below. This was attributed to a new fungus species, Sclerotiopsis indica Ramakrishnan.

Weber and Roberts (44) described the invasion of I. crenata Thumb. by Rhizoctonia ramicola Weber & Roberts. Leaf lesions generally have necrotic centers and purple -brown margins and are brittle in texture. The hyphae penetrate the stomata and form intercellular growths which enzymatically break down parenchymatous tissue but leave vascular strands untouched.

Physalospora ilicis (Scheich. ex. Fr.) Saco. causes defoliation of I. crenata var. rotundifolia Hort. following greying and pycnidial development on the adaxial epidermis. These symptoms are rapidly succeeded by death of the whole plant (12). 8

Wyman (47, p. 70) reports that the holly leaf- miner,

working between the upper and lower epidermis at the larval

stage, is a severe pest on I. opaca. Control of this insect

by spraying has led to heavy infestations of red spider which

cause another type of spotting. However; a compound spray has been found which, with careful timing of application,

eliminates both pests. 9

MATERIALS METHODS

Holly branches were obtained from several locations throughout the state. To keep the leaves fresh, small twigs were packed in polyethylene bags and refrigerated as soon as possible after cutting. Notes were made of the external appearance of each sample and in some cases photographs were taken. The coloration, amount of swelling and distribution of the spots and their occurrence on both leaf surfaces were checked.

A sliding microtome was used to cut fresh sections of several samples of each collection after placing a small piece of tissue in a split carrot block. This method had the ad- vantage of giving a rapid answer concerning the state of the leaf tissue but the sections could not be cut less than 20

thick and details of structure were difficult to discern.

Material was collected for sectioning in paraffin by

taking approximately five typical lesions from each sample of holly received. These were killed and fixed for 24 hours in

Navashin's chrom- acetic- formalin solution. Formalin- acetic- alcohol was also used in parallel preparations at the outset

but when Navashin's proved successful the former was no longer employed. Rapidity of penetration of these solutions was in-

creased by using a vacuum pump. After fixation and thorough washing, the material was dehydrated and infiltrated by the 10 tertiary butyl alcohol -paraffin oil schedule described by

Johansen (19, p. 130) and embedded in 56 ° -58 °C. Tissuemat.

A single piece of material from each sample block was cut, shaped and mounted on a small wooden block. After expos- ing the cutting face of the material, it was soaked in water at room temperature for 5 -7 days to soften the lignified tissues. Sections were cut on a rotary microtome at 10 and ribbons of sections were mounted on new, grease -free slides using Haupt's adhesive (19, p. 20) and 4% formalin. The mounted ribbons were dried on a warming plate at a temperature of

44 °C. and then stored at room temperature for at least a week, to harden before staining.

A progressive technique employing iron -haematoxylin and safranin was used to stain the sectioned material. After removing the paraffin with two changes of xylol, the slides were rinsed thoroughly with 100% ethyl alcohol and transferred

through 95% and 50''.; alcohol to water, where they were thoroughly washed. The sections were first treated with a mordant of 2% ferric chloride for 5 -10 minutes. All the mordant not bound to the sections was removed by another thorough washing and the slides were rapidly dipped twice in a dilute solution of haematoxylin. This stain was prepared by adding approximately 70 drops of 10% haematoxylin in 100% alcohol to a Coplin jar of tap water. The slide was washed again and placed for 9 -12 hours in a very dilute solution of safranin, 15 -20 drops of 1% 11 aqueous safranin in a Coplin jar of tap water. The sections,

then overstained with safranin, were destained and dehydrated simultaneously, by changing in a series of ethyl alcohols from

50% to 95 until the material was the desired color. Final dehydration, by washing in three changes of 100% alcohol and clearing in xylol, left the slides ready for mounting with 60%

H.S.R. synthetic resin in toluene. Cross sections of the abnormal, spotted specimens were then compered with published descriptions and tha observed normal anatomy of holly leaves.

In connection with mechanical and insect damage, examination was made of a series of spines from holly leaves. One leaf of the present seasons growth and one of the previous year were taken as a sample of young and old leaves from each of fifteen trees on the Oregon State Experimental Farm at Corvallis.

Four spines were removed from each leaf and measurements were taken with a calibrated ocular micrometer of the breadth of the spines at their tips and the length of exposed fibers extending beyond the broken, necrotic epidermis. The average lengths and breadths at the tip for the two leaf groups were then calculated.

Applying Koch's rules, set up in 1382 for the proof of a parasitic disease (14, p. 3), isolations were made from some of the specimens to ascertain whether fungi were the cruse of the leaf spots. The fungi isolated were then used to inoculate the leaves of rooted holly cuttings in the greenhouse and the 12 morphology and anatomy of these le ves were checked to determine

the infectivity of the isolates. .eisolations from the infected

leaves were carried out and these cultures and those used as in-

oculum were identified as closely as possible. The isolations

were made by a modification of the technique described by Plaki-

das (30). Infected holly leaves were removed one at a time from

their twigs and immersed in a sterile petri plate containing 2%

Clorox for about two minutes. This treatment was followed by washing in two separate petri plates of sterile water to remove

excess Clorox. With a sharp scalpel, sterilized by flaming with alcohol, the epidermis was removed above one of the spots.

After reflaming the blade, a small piece of the exposed internal

tissue was removed and placed on sterile medium in another petri plate. All isolations, thus made, were incubated at room temperature and transfers were made from the original isolation in about seven days. To keep the cultures growing, transfers were made to fresh medium every fourteen days.

The culture medium used for these fungi was 2% potato dextrose agar (PDA) containing 20 gms. dextrose, 20 gms. agar and the juice of 200 gms. of boiled potatoes in one liter of water. This medium was autoclaved at 10 lbs. pressure for 20 minutes and 0.5 ml. of streptomycin nitrate was added per liter of medium to reduce bacterial growth. Plates were poured with approximately 20 ml. of medium per plate. 13

When the cultures were 7 -10 days old, they were used to inoculate young holly trees. One culture was inoculated in

September 1958, four different cultures were used in February

1959 and eight different cultures were used in July 1959. In- oculations were made by flame sterilizing a scalpel and, after allowing it to cool, removing a small portion of culture from the petri plate and spreading it over the under surfaces of the three or four uppermost leaves of the plant. Care was taken that the edge of the blade did not touch the epidermis to cause mechanical damage. In the first two tests, all the four -year- old rooted cuttings were immediately placed in a spray chamber with 100% humidity and were kept there for three weeks before placing in the greenhouse. The final test was carried out with young cuttings rooted for one year. The test was divided into three parts. Each fungus culture was used to inoculate three cuttings and the plants were placed in the moisture chamber for one week. At the end of this time, one cutting from each fungus group was removed and placed in the greenhouse at moderate temperature. The only moisture applied to these plants was the daily watering. Three weeks after inoculation a second

set of cuttings was removed and placed in the greenhouse. The r .

final set of inoculated hollies was left continuously in the

100;6 humidity and was only removed for examination. These three treatments will be designated the 'dry', 'moist' and 'wet' test groups. 14

After a period of seventeen weeks, one leaf was removed from each treatment inoculated with each fungus, a total of twenty -four leaves. The morphology was again recorded and samples were prepared for anatomical inspection and for isolation as previously described for the original material.

A pathogenicity test was also made in the laboratory.

Healthy leaves sterilized in 10 Clorox for 15 minutes were in- verted in sterile petri plates with a few milliliters of sterile rainwater. The lower epidermis of each leaf was scratched with a sterilized needle and a disc from each of eight streptomycin-

FDA cultures was placed, mycelium downward, on the damaged leaf surface. Four replicates were made for each fungus. A control was set up in three stages. A normal untouched leaf, a leaf with scratched lower epidermis and a scratched leaf with a sterile streptomycin -FDA disc on the wounded area were placed in petri plates in the same manner as the fungal test leaves. By

comparing the reactions of the stages of this control, the reaction of the final stage, the scratched leaf with the sterile agar disc, became the control for the fungal test material.

Identification of the cultures was made with isolates ob-

tained on agar plates. In some instances it was found necessary

to radiate the cultures with ultra -violet light to induce spore production. Barnett's key (4) was used to identify some of the

genera. 15

Specimens were acquired from the Astoria Station in

January, 1960 which showed small pustule -like outgrowths on the adaxial surface of the leaf. To induce sporulation in situ, single leaves were incubated in sterile petri plates, containing sterile water, for ten days. Later incubation was continued under a 12 -hour cycle of ultra -violet radiation for two more weeks.

For several years tri- basic -copper compounds have been used to combat the growth of lichens, which can mar the decor- ative quality of the foliage of I. aquifolium. These sprays have also been found useful in reducing the incidence of

Phytophthora leaf- and twig -blight. During 1956 and 1957, growers suggested that some of these compounds have been the cause of certain types of spotting. In response to their re- quests a new spray testing program was set up in the spring of

1958. Applications were made in April and June of that year with a three gallon hand spray. Concentrations of 2 lbs., 4 lbs. and 8 lbs. of tri- basic -copper, and 2 lbs. of copper sulfate per 100 gallons of water were applied tc young holly trees

3 -5 feet high, just to the point at which the liquid ran off the leaves. In all cases Triton spreader -sticker was added to the spray. The control was set up in two stages. Trees sprayed with spreader- sticker were compared with unsprayed trees and the former were then used as controls for the spreader- sticker- copper spray treatment. 16

NORMAL ANATOMY

A complete description of the normal anatomy of Ilex aquifolium cannot be found in any of the available literature.

It is therefore necessary to establish this criterion for comparison with the abnormalities associated with different types of spots. Several varieties of normal green and normal variegated English holly have been investigated for this purpose

(Fig. 1).

The cuticle of the leaf of I. aquifolium appears to be homogeneous when stained with safranin. The epidermal cells have thickened outer walls showing laminations of cutinized cellulose alternating with pectin. Fine radial lines occur in this laminated region and since these extend outward from the lumen of the cell it could reasonably be supposed that these are plasmadesmata which provide the passage for movement of fatty substances to the leaf surface as is suggested by Esau

(13, p. 50) for other genera. Metcalfe and Chalk (22, p. 3ë3) suggest that in some species, unspecified, the cuticle is composed of two distinct layers. Confusion may arise by mistak- ing the stratified epidermal wall for the internal layer of cuticle, since this region stains only very slightly with safranin and is ver, distinct from the red surface layer. The primary wall of the cells takes on the grey color of the haematoxylin. While the cells of the upper epidermis are of approximately regular depth and the external wall thickening is 17 uniform, the loner epidermal cells are frequently irregular in shape with uneven thickening of the walls. This leads to a smooth cuticle on the adaxial surface of the leaf and an undu- late appearance on the abaxial side. Stomata occur only on the abaxial surface and there are no special subsidiary cells. The guard cells are surrounded by an irregular arrangement of epidermal cells, a condition described by Metcalfe and Chalk as ranunculaceous (22, p. xv). An external stomatal chamber is formed beyond the opening of each stoma. This is produced by prolongation of the thickened cell wall and cuticle of the guard cells and forms a domed cavity which has an oval pore at its apex (Fig. 2).

A distinct hypodermal layer is found on the adaxial surface below the epidermis. This is in agreement with the observations of Holm (17) and :Metcalfe and Chalk (22, p. 383). The cells of this layer are larger than those of the epidermis, more regular in shape and have a uniformly thicker wall when comp red with the inner wall of the epidermal cells. There are numerous simple pits in the walls of the hypodermal cells

(Fig. 2).

The mesophyll is differentiated into the adaxial palisade and the abaxial spongy mesophyll as noted by Holm (17). The palisade region may consist of two or three layers of compact, elongated cells, the number of layers varying with the specimen of holly. The cells vary in size and shape from specimen to 18

specimen, some being approximately one and one half times as

long as wide, while the length of others is as much as five

times the width. The palisade thickness and cell shape may

be a varietal characteristic but no record of this information was kept and strict correlation cannot be made (Fig. 2).

Chloroplasts, packed closely together, line the walls

of the palisade cells and also occur in large numbers in the

spongy mesophyll. In the hypodermal cells, they are less num-

erous and are absent in the epidermis. Druse crystals of

calcium oxalate are found in cells situated primarily in the

central region of the leaf. However, they may be seen as close

to the adaxial surface as the outer layer of the palisade or

just on the abaxial side of the vascular traces. They do not appear in any regular arrangement and in some specimens seem

to be absent (Fig. 2).

Venation in holly leaves is of the reticulate form, typical of dicotyledons (Fig. 1). The mid vein runs throughout the leaf blade and into the terminal spine. From the mid vein, lateral veins extend out to the margin where they branch.

The spinal vein of each marginal spine is formed by the fusion of the two branches from adjacent lateral veins. One or two finer vascular strands from other parts of the reticulum also accompany the spinal vein.

The mid vein consists of a large, fan -shaped region of the vascular bundle with broad areas of strengthening tissue 19 above and below. The hypodermal layer, present in the lamina, is only one of several layers of thick- walled collenchymatous tissue. This was also observed by Pfitzer (29, p. 51) who related the origin of the collenchyma cells to the same initials that give rise to the hypodermis and spongy mesophyll. The epidermis did not divide periclinally in any of his observations of leaves of many ages and he concluded that the strengthening tissue could not arise from the epidermal initials. Closer to the xylem tissue the cell walls are thinner and small amounts of air space occur. This lacunar collenchyma has a few chloroplasts in most of the cells. The bundle is strengthened close to the xylem region by a group of thick -walled fibers. The thickness of these walls varies according to the age of the leaf.

The xylem tissue is arranged in rows radiating from a cambial region and the phloem is a less regular narrow band on the lower side of the cambium. Strengthening phloem fibers are reinforced by chlorenchymatous collenchyma which occurs between the border parenchyma and the lower epidermis. This lower region of col - lenchyma is wider than that below the upper epidermis and together with the large main vascular bundle forms the midrib which is prominent on the abaxial surface of the leaf. The lower epidermis and cuticle are very regular and there are no stomata in this midrib area (Fig. 3).

The lateral veins are centrally located in the tissues of the lamina. As in the midvein, the xylem and phloem are 20 separated by a cambial region but the types of strengthening tissue are restricted to a cap of primary phloem fibers and a small group of fibers adjacent to the xylem. Each vein is surrounded, in cross section, by border parenchyma which is a single layer of chlorenchymatous cells of a regular, rounded shape. These cells are elongated along the length of the vein.

The border parenchyma cells connect to the palisade mesophyll in the lower region (Fig. 2). There is no collenchyma development.

Small branches of the lateral veins contain no cambial cells and the only strengthening tissue, which may or may not be present, is the region of primary phloem fibers. The finest veins are surrounded by border parenchyma.

The spinal vein, composed of xylem, cambium and phloem, is strengthened by a cap of phloem fibers. The two branch veins in the spine region have phloem and phloem fibers accompanying the xylem but no cambial tissue is present. Several smaller veins, with and without fiber tissue, are also found and these are succeeded by mesophyll towards the spine tip.

The margin of all the holly leaves investigated is bounded by a strong band of fibers (Fig. 4). Like those associated with the vascualr bundles, these cells have few cell contents and, because of the thickened walls with simple pits, the cell lumina are much diminished. This sclerenchyma bundle was also noted by

Pfitzer (29, p. 51) and was described by him as modified 21 hypodermal tissue. The present study is in partial agreement with this. The fiber strand is bounded on all sides by chlorenchyma which at the leaf margin forms two to four layers of cells indistinguishable from the hypodermal layer which succeeds them to the outside. Chlorenchyma cells may also occur in the fiber strand. These facts suggest that the fibers are either

which have a common origin. modified - hypodermis or mesophyll

Toward the spine tips, the two fiber strands become orientated parallel to one another. At one margin the fiber strand joins with the phloem fibers of the spinal vein (Fig. 4). One of the branch veins anastamoses with the spinal vein and the second is ultimately succeeded by fiber cells. One fiber strand with

the main vascular bundle is separated from the other fiber strand by undifferentiated chlorenchyma and air space. Progres- sing toward the spine tip, this chlorenchyma is gradually re- placed by fibers until the vascular tissue is surrounded by

fibers (Fig. 5). At this stage the spine is almost circular in

cross section. The epidermis and one hypodermal layer of dead,

tannin -filled cells cover the fiber sheath. Further reduction

shows the vascular tissue to consist of about six or seven xylem vessels and one or two phloem elements (Fig. 6). These are approximately surrounded by parenchyma with living proto- plasta. This structural relationship continues until the 22 parenchyma is no longer present and the vascular elements diminish until only one xylem and one phloem element remain.

Finally, for a very short distance, the vessel alone is surrounded by fibers until at the tip, only fibers occur (Fig. 7).

Depending upon the age of the leaf, the tip of the spine may be covered by epidermis and hypodermis or, after one year, the fibers may be exposed for some distance from the tip.

A change in the palisade tissue is the main factor re- sponsible for the variegation found in some forms of English holly. In the variegated variety known as Ilex aquifolium var. marginata, horticultural variety Silvery, by Brownell, there is a decreasing concentration of green pigment and an increase in the yellow coloration from the center of the leaf toward the margin. The palisade cells in the central green area are the usual shape and in the same number of layers as are found in most green varieties (Fig. 8). These cells are densely packed with chloroplasts. However, the coloration of the outermost palisade layer below the hypodermal cells changes abruptly. The cells are shorter, only one and one half times the width, and there is a conspicuous absence of chloroplasts or, rarely, one or two plastids occur in a cell. This single row of small colorless cells continues for some distance until another abrupt change occurs in the row of palisade 23 below. Soon the third row changes and is followed rapidly by the loosely packed innermost layer of palisade. When the first abrupt change occurs in the palisade cells the spongy mesophyll cells close to the lower epidermis become slightly smaller and also lose most of their plastids. The sequence continues until this condition prevails throughout the chlorenchyma. The hypo - dermis in variegated leaves has never been found to contain chloroplasts. The diminution of these internal cells is evi- denced by a decrease in thickness of the leaf in this variegated region. In another variety of unknown origin, the decrease in cell size is not so marked and the number of chloroplasts is decreased but a few are always present. 24

SPOTTING CAUSED BY FUNGI

Some thirty varieties of Ilex aquifoiium were set out at the John Jacob Astor Branch Experiment Station in Astoria in 1951.

Since that time the trees have never been sprayed with any type of fungicide and by 1958 almost every tree in the orchard showed some signs of spotting.

In the spring, some trees are found with leaves bearing very small spots visible only on the abaxial surface (Fig. 9).

These are grouped in patches which under the binocular dissect- ing microscope appear as minute dark spots in the regions of the stomata. The anatomy of the leaf is only slightly disturbed.

Several adjacent stomata show penetration by fungal hyphae. The external chamber is usually filled with soiled mycelium and the guard cells close the stomatal opening with one hyphal strand passing between them into the leaf tissue (Fig. 10). With the advance of the invader, enlargement and proliferation of the spongy mesophyll cells occur. These cells pack together and block the air space. They are usually without chloroplasts or

these show degeneration. Portions of mycelium are found beyond

this area in the air space and between the lower layers of the palisade (Fig. 10). Where abnormality has reached two or three layers from the guard cells, one or two cells immediately adjacent to the stoma may show cytoplasmic degeneration with wound gum or resin deposition. 25

In the autumn, some leaves exhibit irregular areas composed of aggregates of small swellings visible on both leaf surfaces. These areas are translucent and chlorotic. Where the spots are more marked there are slight tinges of red (Fig.

11). Internal abnormalities show advanced hypertrophy and cell multiplication from inside the epidermis through to the palisade tissue. Few cells in these areas contain any chloroplasts but cytoplasm and nuclei are prominent. Hypertrophy is the main feature and the enlarged cells show an irregular arrangement (Fig. 12), particularly in the center of the tissue.

Necrosis inside the stomata remains slight, as in the earlier material, but a little occurs in the center of the tissue where dark -staining substances are sometimes present in cells adjacent to intercellular hyphae. The lower epidermis and some cells in the layer immediately adjacent are unaffected by the hypertrophy though there is sometimes degeneration and resin deposition.

The hypertrophy with absence of chloroplasts may extend to some cells of the outermost layer of the palisade tissue but most of these cells remain undisturbed in shape, size and content (Fig.

12).

Material obtained from another source also shows raised translucent blister spots with slight chlorotis. These, however, remain single and appear to have a pattern of formation following the veins. If visible on the adaxial surface, these spots appear as chlorotic discolorations without swelling (Fig. 13). 26

The proliferation is sometimes marked in the spongy mesophyll

by formation of palisade -like cells immediately within the sto-

mata and adjacent epidermal tissue. The hypertrophy and pro-

liferation in the central area of the leaf appears to have no

regular formation. Slight red tinges in some spots on the ab-

axial surface of the leaf may be partially due to necrosis of

some of tae guard cells in the swollen area. Chloroplasts are

missing in all enlarged and dividing cells. The number of

chloroplasts is also fewer in some cells adjacent to the air

space. All other tissues, the palisade mesophyll, the vascular

bundles, the hypodermis and the epidermis, are undisturbed.

The pathogen is not easily visible but does occur between the

tightly packed cells of the abnormal tissue (Fig. 14).

Still other material shows similar single translucent

blisters on the abaxial surface of the leaf, but without any

particular distribution pattern. The abnormalities are con-

fined to areas of spongy mesophyll around the stomata where en-

largement and division of the cells are irregular. The re-

sulting tissue is devoid of chlorophyll and slightly necrotic.

Where a vascular bundle traverses abnormal tissue, the border

parenchyma appears normal. Stomatal penetration by fungous

' hyphae is less frequently found but intercellular mycelium is

present as before (Fig. 15).

The most advanced symptoms occur throughout the year in

the two- and three -year -old leaves at the Astoria Experiment 27

Station. The external characteristics appear as large rough red -black spots of irregular margin and varying size. They are visible on both leaf surfaces with irregularly swollen contours and are scattered at random throughout the leaf tissue on the midrib, lateral veins and lamina (Fig. 16). Hyper- trophy and proliferation extend from the lower to the upper epidermis in the center of the spots. Cells of the palisade mesophyll, unaltered in shape, show absence of chloroplasts or a few degenerating plastids. The hypodermal layer is often necrotic and cells of the upper and lower epidermis have similar depositions of gums and resins. Complete breakdown of cells occurs in the spongy mesophyll and this degeneration continues through the hypodermis and epidermis in the center of some spots. However, the cuticle remains unbroken. The pathogen persists on the lower surface of the leaf and is found in some external stomatal chambers. Hyphae continue to be visible between the cells but do not occur in great quantity.

They are more marked in the degenerating tissues but may also be found in some parts of the air space existing between the proliferated areas (Fig. 17).

The largest aggregation of mycelium is present in material collected in March, 1959. It occurs within a few of the collapsed cells of the upper epidermis and in hypodermal cells in the center of the spot. Coils of mycelium fill the cells, which are without normal living contents, and some of their 28 walls are broken down. The outer wall of the epidermal cells and the cuticle of the leaf remain intact (Fig. 18).

Final samples for anatomical study were procured in

January, 1960. For the first time pustule -like extrusions were found on the adaxial surface of the leaf (Figs. 19,20,21).

Clusters of sterile hyphae issue from the ruptured epidermis and cuticle. They were produced from a dark stroma in the destroyed epidermal and hypodermal cells (Fig. 22), which cells show damage similar to that observed in the sample of March,

1959 (Fig. 18). White tufts extend from the pustules or acervuli (Fig. 23) after ultra -violet radiation. These are conidiospores developing on two types of conidiophores (Fig. 24).

In general, fungal spots are always swollen and rough, at least on the abaxial surface, and may be single or aggregated. Chlorosis and translucency are early symptoms which are often followed by a slightly red discoloration before the in- tense red -black color appears in the center of the spot. The internal structure of these spots always includes irregular hypertrophy and proliferation, at least in the spongy mesophyll around the stomata, but may spread into the palisade and hypodermal layers. Chloroplasts are always absent in the enlarged cells and necrosis may be found according to the state of development. Cell wall thickening and warts may also be present. Stomatal penetration by fungal hyphae is not always apparent but intercellular mycelium is a constant observable 29 feature which is often accompanied by deposition of pectin - aceous substances in adjacent host cell walls. In advanced stages, the hyphae may appear inside collapsed hypodermal and epidermal cells. This stage precedes the aggregation of dark mycelium which ruptures the outer epidermal wall and cuticle to produce an acervulus. From this extruded hyphal mass, conidiophores arise and abundant conidia production results

(Tables 3 & 4).

Identification of Fungi

Phomopsis and Boydia are the only two genera of fungi that have been identified in the present studies which were previously reported as occurring on Ilex aquifolium. Seven unreported genera were also found and these are identified as:

Gliocladium, Asteroma, Pullularia, Coniothyrium, Alternaria,

Stemphylium, and Epicoccum. For completeness, all nine species occurring on I. aquifolium are described in the succeed- ing paragraphs.

After radiation with ultra -violet -light, reproductive structures were produced in detached -leaf -cultures of leaves which were obtained from the Astoria Experiment Station since

January, 1960. From leaves of many other sources, isolates were cultured on streptomycin -PDA in 1958 and 1959. Of the nine cultures, five fungi were cultured on the agar medium only, two fungi were cultured on the agar and also on the 50 detached -leaf- cultures under ultra -violet radiation and two were identified on only detached -leaf -cultures that had been treated with ultra -violet radiation.

The first of these tests was set up in January, 1960 when the leaf material with the immature acervuli was obtained.

Fourteen days after treatment, conidiophores were produced which are hyaline and dimorphic. One form has biverticilliate symetrical penicilli (35, p. 46) and the other has a simple branching structure (Fig. 24). The conidia are ovoid -oblong, hyaline and cling together in gelatinous heads. When the leaves were four weeks old, dried out and brown, the small pustules were still visible and uncontaminated. By this time they were a distinct pink color when en masse. These factors have led to the diagnosis that the fungus is Gliocladium roseum

(Link) Bainier. It is commonly reported as parasitic or semi - parasitic on ornamentals and has been found both alone and associated with other species on Hibiscus L., Buxus L. and other hosts. Two other similar tests using ultra -violet light were set up in March, 1960 with further material from Astoria which also yielded Gliocladium roseum from separate leaf cultures.

A second leaf pathogen was first isolated and identified from sterile leaf spots on an unrecorded holly specimen in 1958.

In detached leaf cultures in early March, 1960, small, dark, globose pycnidia were produced. These were superficial, occurring in a mass of dark, radiating hyphae of a subiculum. The 31

unicellular spores were hyaline, ovoid -globose and were borne

on short, hyaline conidiophores. These characteristics are

typical of the genus Asteroma DC. ex. Fr., described by Barnett

(4, p. 166). The culture from the 1958 isolate produces, on

streptomycin -PDA, a yellow -brown colony which, after one week,

darkens and has a small amount of white surface mycelium. In

older cultures, the medium also becomes yellow with metabolic

by- products. The mycelium is septate and 'jointed', hyaline

when young and becoming yellow to brown with age. It is mod-

erately branched with some tendency to aggregate in strands of

several hyphae. At maturity the small, globose, dark pycnidia

enclosed by dark thick -walled, radiating hyphae are produced

without ultra- violet treatment. There is a very short beak

with a definite ostiole. The conidiophores are hyaline, bear-

ing conidia which are unicellular, ovoid -oblong, catenulate

and hyaline. They are extruded in a grey mass. This culture

(1A) of Asteroma was used for the inoculation experiments.

The literature concerning this genus and its association with holly includes two species. Both of the citations refer to

Asteroma ilicis. Roumeguére reports "Asteroma Ilicis Sp. nov.

Grog. in Herb. Reuni quelques fois au Depazea Ilicicola Fr. sur les feuilles de houx Nov. 1881. Forst de Fontainebleau."

(39, p. 218). Grove describes the symptoms and occurrence as follows, "Asteroma Ilicis Grove. Spots many on each leaf, indeterminate, irregular, confluent, olivaceous- brown; no 32

fibrils visible. On leaves of Ilex Aquifolium. Rissbury Camp,

Hereford. June." (15, p. 145). Since both descriptions are

nomena nuda there is no way to specify whether they are the

same or different fungi and no comparison can be drawn with the

present species of the same identified genus on Ilex aquifolium.

The dimorphism, characteristic of the genus Phomopsis, a plant parasite causing leaf spotting, was recognized in 1958.

This organism was cultured on streptomycin -PDA in plate culture

from holly leaf spots on sample 124 collected from Timmerman's orchard, Astoria, in July, 1958. The agar culture produced a grey -white spreading colony of a rough, fibrous nature with pycnidia containing dimorphic spores. This culture was used

to inoculate young holly trees and it reproduced the leaf spot-

ting. In detached -leaf- cultures, the innate, erumpent pycnidia were also dark colored. They were globose and ostiolate, each

fruiting body being topped by a coiled mass of spores varying

from white to beige. These spores were hyaline and showed dimorphism. The majority were obovoid but a few filiform, slightly curved cells were also present. Both spore types were unicellular.

One culture identified as Boydia insculpta was produced by the detached -leaf -culture under ultra- violet radiation.

This produced the typical dark, innate perithecium with the erumpent, curved, setaceous beak on the upper surface of the leaf. The asci were hyaline with a thickened wall at the 33 ostiole. Eight uniseptate ascospores were contained in the sporangium and these were also hyaline. The slender, dumb -bell shape of these spores is characteristic of the species. This pathogen was identified on only one occasion and was not cultured on the agar medium. The literature carries two reports in Washington and Oregon associating this organism with canker on holly. However, inoculation tests by Buddenhagen and Young

(7) indicate that Boydia insculpta is a secondary invader.

Culture 171, after three days of growth on streptomycin-

PDA shows a flesh -pink colony of a waxy nature. As the colony increases in size, grey -brown coloration appears, showing a regular banded formation of dart mycelium alternating with beige, which is also the color at the margin of the culture.

The dark coloration is due to the pigmentation of the old mycelium and the laterally -produced, one -celled, ovoid conidiospores. This culture corresponds to Pullularia pullulans (de

Bary) Berkhout, originally described as Dematium pullulans by de Bary (5, p. 293). This fungus is considered by many to be a saprophyte. However, a nine -fold higher incidence of the organism was found with spruce budworm infection in bud necrosis of Picea glauca (Moench) Voss, by Molnar and Silver (23).

Other investigators have found pathogenic reactions under certain conditions. This fungus is probably a weak facultative parasite; with which suggestion the present work is in agreement. 34

Culture 180, when grown for seven days on streptomycin -

PDA, shows a grey -white colony of woolly texture with a few moisture droplets. Yellow pigment is produced that diffuses into the agar at the point of inoculation. The colony becomes brown -khaki in color with aging. The mycelium is dry with a slightly irregular margin. Surface mycelium has a white appearance and the culture, as a whole, has a banded formation when seen from the lower surface of the plate. Pycnidia production on the agar culture is induced with ten days of ultra -violet lighting on a twelve hour cycle. Cn streptomycin -

PDA with only daylight, there was no pycnidial production.

Greater production is seen where the colony is disturbed before illuminating the plate. The disturbance was effected in the present work by scratching the colony to expose the mycelium on the surface of the agar. It was reported by Kunkel (in 28, p. 28 -29) that Alternaria responded in the same way to this

treatment. The pycnidia so formed in culture 180 are dark grey

to black and grow in loose aggregates. The conidiophores are very short, like sterigmata, on the basal cells of the pycnidial wall. Masses of ovoid, unicellular, dark conidiospores are produced. Using Barnett's key, this fungus was identified as a species of Coniothyrium Sacc.

Isolate 159 grows on streptomycin -PDA and produces a roughened, woolly colony with an irregular margin. From the center of the culture to the margin, the mycelium is several 35 shades of dark yellow to orange with drops of a dark orange liquid scattered over the surface. In one to two days, this water- soluble, pigmented fluid diffuses through the agar ahead of the mycelial growth. Sporulation did not occur on the streptomycin -PDA alone. After seven days under ultra- violet light, sporulation was induced in irregular clusters on the surface of the culture. The spores are black, echinulate and muriform. This isolate conforms with the species

Epicoccum nigrum Link and is considered a common saprophyte.

A dark brown colony is produced on streptomycin -PDA by culture 181. This becomes black with age but has a white surface mycelium. The conidiophores are dark due to brown pigment. They bear large, muriform conidiospores, the septa of which may be horizontal or horizontal and vertical. These spores are also pigmented and are borne laterally on the conidiophores. The catenulate spores are very varied in shape, subclavate to eliptical, with beaks absent or of varying length. The spore surface may be echinulate or smooth.

These features identify this culture with the genus Alternaria

Nees. It has been described as a facultative parasite on many

hosts and one isolate has been described that causes leaf spot - on apple.

Culture 172 is soot -like with fine grey -white threads on the surface. Moisture droplets occur at the margin which is regular in outline. There is abundant spore production which 36 gives a powdery texture to the colony. Like the mycelium!, the spores contain dark pigment. They are xnurif.orm and globose - oblong, borne terminally on short conidiophores. This isolate belongs to the genus Stemphylium Wallr.

In the summary of his book, Neergaard (28) describes

Alternaria tenuis and Stemphylium ilicis Teng. as "pronounced polyphagous facultative parasites with a presumably very wide host range." Later, in an additional note to the book, he in- dicates that the name S. ilicis is in fact a synonym for S. consortiale (Thun., 1876) Groves & Skolko (16), which is the older, and therefore correct, name to use.

The white-cotton-textured culture 194 on streptomycin -FDA spreads rapidly and thinly over the surface of the medium. The mycelium is partially septate and hyaline but fruiting bodies have not been found. Thus, other than classifying this as a

Phycomycete, no closer identification has been possible and discussion of its pathogenic nature is limited. Suffice to state that at least one species of this class, Phytophthora ilicis Buddenhagen & Young does produce leaf spots on I. a ui- folium in Oregon (7).

Morphology and Anatomy of the Inoculation Tests

The results of the inoculations on rooted holly cuttings show that spots are not produced with all treatments of the cultures used. However, where inoculated leaves do produce 37 spots, these are of the raised 'blister' form, translucent and often chlorotic. Intercellular mycelium is always present.

In the three treatments with Pullularia pullulans, isolate 171, hypertrophy, irregular proliferation and degeneration of chloroplasts are the main features of the pathological anatomy. Stomatal penetration and intercellular mycelium are visible. Some deposition of wall pectins is visible adjacent

to the path of the hyphae (Fig. 25).

With Phomopsis, isolate 124, the wet and moist tests show only small regions of abnormal tissue but stomatal pene-

tration and intercellular mycelium are present. In this dry

treatment, the infected area is larger (Fig. 26).

Spots were produced with specimen 194, the unidentified

Phycomycete, in wet and moist tests but none in the dry. The amount of abnormality produced here is approximately the same as with the previous two inocula but the amount of mycelium

found among the host cells is not so great. There is also more substomatal necrosis than in the previously mentioned materials.

Leaves inoculated with Alternaria, isolate 161, gave a little more reaction in the moist test than in the wet. The dry test produced very few, very small spots. Material from

the moist test shows hypertrophy and proliferation extending into the palisade layers; it also occurs around the vascular

bundles. Intercellular mycelium is present and degeneration 38 of the chloroplasts is complete in many cells. The same cell abnormalities occur in the wet and dry tests with this culture but they do not extend into the palisade tissues and the mycelium between the cells is much less conspicuous.

Considerable reaction occurs with the culture of

Coniothyrium, isolate 180, in the moist test but no spots are produced in the wet treatment and only a small affected area is found in the dry treatment. Mycelial strands in this material were difficult to distinguish.

In the tests with Epicoccum, isolate 159, very few spots occurred in all three groups. Stemphylium, isolate 172, showed a few spots in the wet, very slight reaction in the moist and none in the dry treatment. The dry and moist tests with Aster - oma, isolate 1rß, show very few spots and none are found in the wet treatment. In these three specimens giving a positive reaction, a small amount of hypertrophy and proliferation occurs around the stomata. Here, chlorosis is due to the breakdown of chloroplasts. Attempts at fungal penetration of the stomata are present but there are very few indications of intercellular hyphae.

By culturing from the inoculated trees in the greenhouse, four of the eight fungi originally used were recovered, each from one sample only of all the tests set up. Epicoccum and

Coniothyrium were returned from a wet test; Stemphylium was recovered from a leaf given moist treatment; Pullularia pullulans 39 was reisolated from a dry test leaf.

Detached -Leaf Inoculations

The results of the inoculations of leaves set up in sterile petri plates showed that the untouched leaves were still fresh and green eighteen days after starting this test.

Those which had been scratched with a sterile needle showed brown necrotic scratch marks and a small amount of chlorosis in one leaf. Similarly the scratched leaves with the sterile streptomycin -FDA discs showed red -brown necrotic lines and slight chlorosis in all cases (Fig. 27). In the fungus and agar portion of the test, the reactions were variable. These latter results are given in detail in the following paragraphs where they are compared with the scratched control with the agar disc.

Reactions to Epicoccum, Pullularia, Stemphylium,

Asteroma and the unidentified isolate 194, were identical with those of the agar disc controls and may therefore be considered as non organisms where wounded tissue is involved. At the same time the peripheral agar discs from the Epicoccum culture, which contained yellow by- products of the fungal metabolism but no mycelium, produced no apparent toxic reactions. 40

Table 1 Results of detached -leaf- inoculations

Treatment Results

Controls: Untouched leaf Green and fresh.

Scratched leaf Brown necrotic scratch lines. Slight chlorosis: 1 of 4 leaves.

Agar disc on Red -brown necrotic scratch lines. scratched leaf Slight chlorosis: 4 of 4 leaves. surface

Asteroma, lA Same as agar disc controls.

Pullularia, 171 Same as agar disc controls.

Stemphylium, 172 Same as agar disc controls.

Epicoccum, 159 Same as agar disc controls.

Peripheral agar Same as agar disc controls. of culture, 159

Unknown Same as agar disc controls. isolate, 194

Coniothyrium, 180 Black spot: 2 times diameter of inoculation disc, on both surfaces, covered with white mycelium, 4 of 4 leaves. First noticed after 10 days.

Alternaria, 181 Black spot: 2 times diameter of inoculation disc, on both surfaces, 3 of 4 leaves. First noticed after 10 days.

Phomopsis, 124 Black spot: Li times diameter of inoculation disc, on both surfaces, 2 of 4 leaves. 41

Coniothyrium, Alternaria and Phomopsis showed a positive reaction. In the case of Coniothyrium and Alternaria, black necrotic areas spread from the inoculation disc throughout the leaf, covering an area two to two and one half times the diameter of the disc. The necrosis showed on both the adaxial and abaxial surfaces. This necrosis was also apparent at 10 days after inoculation and, in the case of Coniothyrium, a white mycelium covered the whole area of infection. With the Phomop- sis culture, the same necrotic area was produced in eighteen days on both leaf surfaces of two samples, but the extent of the infected area was only one to one and one half times the diameter of the inoculation disc and occurred on two of the four inoculated leaves. Holly leaves in Figure 28 demonstrate these symptoms as they appeared three weeks after inoculation.

When the whole leaf was dead, the test with Phomopsis bore clusters of spore exudate from both the adaxial (Fig. 29) and

the abaxial surface of the leaves. Smears of these exudates showed the typical dimorphic conidia described for the culture

from which the inoculum was taken. These results are summari-

zed in Table 1. 42

SPOTTING CAUSED BY CHEMICAL SPRAYS

The experimental spray treatments with tri- basic- copper

and copper sulfate showed no deleterious effects throughout

the summer of 1958. The trees were checked at intervals but

no injury was observed until the winter. By March, 1959,

severe red -black spots were present on the abaxial surface of

the leaves which had been sprayed with high rates of copper.

These were slightly raised, circular spots with brown centers.

Some of the larger areas also showed as black discolorations

on the adaxial surface. A gradation of spotting was found,

the severity increasing with an increase in concentration of

the spray material (Fig. 30). Some leaves showed spots in

regions along the midrib and on the margin as if the spray had

run down the leaves and collected in these areas. This is in

agreement with Rohbaugh (38, p. 706) who found that oil spray

accumulated along the veins, midrib and leaf margins of treated

Citrus and then penetrated in those regions. Leaves sprayed

with both the tri- basic -copper and the copper sulfate developed

the same symptoms.

A few spots occurred on the check trees, both unsprayed

(Fig. 30, B) and those sprayed with spreader -sticker (Fig. 30,

C). These were discrete, blister -like spots similar to those

produced in other orchards over the state. Slight chlorosis

and dark discolorations occurred around the stomata. In such

L 43 spots, hyphae are found in many external stomatal chambers and there is penetration of the adjacent stomata. The characteristic intercellular mycelium with hypertrophy and proliferation of the spongy mesophyll, palisade like in the spreader -sticker check, occurs close to the stomata. Slight necrosis of a few cells and the absence of chloroplasts in many others are also recurring features.

Trees sprayed with the tri- basic -copper had no fungal material on the leaf surfaces examined and no penetration of the external stomatal chambers. In rare instances a small number of hyphae occurred on the abaxial surface of leaves which had the copper sulfate treatment (Fig. 30, F) but, again, no penetration occurred. It would appear, therefore, that the copper compounds are an effective protectant against fungal invasion since the check trees develop typical fungal spots.

The first indication of injury in leaves sprayed with the tri- basic -copper compound is the hypertrophy of spongy mesophyll cells which obliterates the substomatal cavities.

This reaction was also shown in experiments by Skoss (43, p. 65) who investigated the structure and permeability of the plant cuticle. He found that, when 2,4 -D was applied, hypertrophy and turgidity of cells were present 48 hours after spraying. The progressive development of spots in the present investigations shows that cells immediately around the substomatal cavities en- large towards the stoma. They form elongated cells which later 44

divide; the external ones become necrotic and develop wound -

gums. The outermost cells have a characteristic, unevenly

distributed thickening of their cell walls. Toward the sto-

matal aperture the structure is much thicker and laminated,

to the epider- . tapering off on the side walls perpendicular

mis (Fig. 31). This was also noted by Küster (20, p. 106) in

his discussion of hypertrophy. He referred to this as tylosis

formation in the substorn:ntal cavity but was uncertain whether

it was a reaction to injury.

In later stages, cell divisions below the stomata be-

come very regular (Fig. 32). These cells are small and in

older spots become necrotic and are filled with wound -gums.

Epidermal cells adjacent to the affected stoma become necrotic

as the phellogen develops below them. The regular cell de-

velopment proceeds more ra idly parallel to the lower epider-

mis than through the depth of the leaf. The younger cells of

the cork layer, adjacent to the phellogen often become uni-

formly thick walled with simple pits.

The amount of hypertrophy in the cells of the spongy

mesophyll below the phellogen depends upon the rapidity with

which the adjacent air space is obliterated. Thus cell size

in the abnormal areas of the more advanced stages of growth

is quite irregular (Fig. 33). Similar growth patterns were

described by Bachmann (2, p. 209 -216) in cork formation in

leaves of Ilex aquifolium. As expanding cells approach one 45 another, small pectinaceous protruberances develop on the outer surfaces of the walls. These warts vary in size and quantity in the abnormal tissues (Fig. 34) but have not been found in unin- jured holly leaves. Similar warts were described by Carlquist

(8) in Compositae where they occur in normal tissue on the walls of parenchyma cells which abut on intercellular spaces.

In the most advanced stage, the necrotic cells of the lower epidermis and several layers of cork cells show wound gums and broken cell walls. The border parenchyma of the vascular bundle is, in some regions, incorporated in the phellogen. As described by Bachmann (2, p. 209-216), the vascular tissue is sometimes surrounded by a multiple layered structure. The vascular tissue itself seems undisturbed but the hypertrophy extends around it into the palisade in some places (Fig. 35). In all instances, the cells showing hypertrophy in the center of the region of abnormal growth are completely devoid of plastids but nuclei persist. Those cells which are still in the process of extension into the air space sometimes contain a few chloroplasts.

The sequence of events is similar to that found by

slander (1, p. 1069) who determined the effects of sulfuric acid when used as a weed spray. After penetration of the stomata, and later a possible penetration of the epidermal cells since these show necrosis while cells beneath may remain unaffected, hypertrophy occurs. This is followed by a decomposition of chlorophyll causing the chlorosis, and a breakdown of 46

chloroplasts. Present results show necrosis and cell breakdown

to be a late stage of the disturbance. This is contrary to the report of slander who found no cell wall destruction.

Material acquired from Roseburg and Astoria is known to have been sprayed, but the history of other samples from the

Portland area is unknown. In all these specimens, slightly

swollen, red -black, round spots (Fig. 36) are shown to be the

result of hypertrophy and proliferation of the spongy mesophyll

with obliteration of the air space. Tannin deposits are fre-

quent and uneven laminated wall thickenings are constant in

cells with numerous simple pits are found only in a few samples.

As in the experimentally sprayed trees, chloroplasts are absent

or degenerating in cells showing enlargement or proliferation

but the nuclei are present (Fig. 3?). In some samples, numerous

druse crystals occur among the abnormal cells (Fig. 38), a con-

dition indicated by Luttrell (21) to be induced in Ilex opaca

by Phacidium Curtisii (B. & Ray.) Luttrell. Dark discolorations

on the adaxial surface of some samples (Fig. 39) are usually

associated with spots on the other side, but the internal organi-

zation reveals anthocyanin pigment in only the epidermal and

hypodermal tissues of these areas; the chlorenchyma shows no

color change. If anthocyanin is present in the abaxial tissue,

the coloration is masked by the wound gums present in the necrotic cells. 47

The general features of chemically induced leaf spots show discrete, smooth round spots that are rarely aggregated and only slightly raised. The spots are dark red on the lower surface and often a discoloration without swelling occurs on the upper surface. There is never any break in the cuticle.

The anatomical disturbance is always within stomata where the cells immediately adjacent to the substomatal cavity have unevenly thickened, laminated walls. A regular phellogen with cork formation is characteristic but slightly irregular hypertrophy and proliferation may fill the air space in the central regions of the leaf. Necrosis occurs only when damage is far advanced and cell breakdown takes place in the central region. Chloroplasts are always absent in the dividing cells but the cork region masks the chlorotic coloration. 48

SPOTTING CAUSED BY MECHANICAL INJURY

Mechanical damage frequently occurs when spines of one leaf scratch or penetrate the lamina ofanother leaf. The macroscopic symptoms are brown corky swellings ringing a scratch or hole, which partially or completely pierces the lamina (Fig. 40). Slight chlorosis and red -black coloration may also be found encircling the cork area.

The funnel- shaped puncture produced by a spine pene- trating the lower cuticle and epidermis is lined by broken cells which are necrotic and crushed (Fig. 41). Formation of phellogen occurs by periclinal divisions of the spongy mesophyll below the necrotic tissue. Some anticlinal divisions also occur particularly in the epidermis and mesophyll immediately adjacent. Cork development then takes place below the broken cells on the surface of the puncture. The innermost layer of the cork cells still have contents and the walls are unevenly thickened. The cells of the phellogen and phello- derm are without chloroplasts and a few of the spongy mesophyll cells within the cork cambial region have fewer plastids than normal chlorenchyma. Hypertrophy and proliferation are not apparent in the tissues of the center of the leaf and the air space and chlorenchyma are quite normal. The surface of this small lesion is only slightly raised at the periphery of the opening. i+9

Where two or more punctures occur close together (Fig.

43), the phellogen region extends across the area between as well as around the initial points of damage. The outermost layer of cells with nuclei show thick cellulose walls. When phellogen formation is complete, the production of cork causes a swelling on the abaxial surface of the leaf (Fig. 43). De- generation of chloroplasts occurs in cells around the phellogen.

With a shallow upper surface puncture of the leaf, the hypodermal layer forms the main phellogen tissue by periclinal division, although a few cells of the epidermis divide anticlinally where the phellogen approaches the surface. The spongy .mesophyll below the epidermis also becomes slightly irregular and some cells divide periclinally (Fig. 41). Again the outer layer of living cells show thickened walls.

When the wound is deep and penetrates from the upper almost through to the lower surface of the leaf (Fig. 44), the tissues which were in the path of the spine become necrotic to a depth of 5 -10 layers of cells surrounding the hole. Slight shrinkage of the cells and deposition of wound gums are characteristic of these necrotic regions. Inside this necrotic area which bounds the hole, hypertrophy and proliferation are marked.

This transforms the spongy mesophyll into a compact tissue of irregular cells which obliterates the normal air space. On the inner edge of this abnormal tissue, where the cells do abut onto the air space, pectinaceous warts are found. These are of 50

Table 2 Measurements of Leaf Spines of Holly

Leaf of Current Season Leaf of Previous Season

Tree Length in Tip breadth Length in Tip breadth i p Sample of fibers in p, at 1 p of fibers in p, at 1 p Number exposed exposed

1 33 11 80 18

2 16 13 96 broken

3 0 11 715 11

4 0 13 731 8

5 0 11 234 13

6 0 16 117 16

7 16 18 130 10

8 16 11 234 19

9 0 10 325 18

10 1 8 553 16

11 16 14 650 14

12 0 10 600 19

13 16 16 552 10

14 0 14 156 10

15 0 10 650 13

Total 114 186 5843 211

Average 7.5 17 388 14

These figures are the average of four spines for each tree sample. 51 the same type that result from copper injury. The regular formation of the palisade tissue also becomes lost. Through this proliferating region a typical phellogen is produced so that a narrow layer of cork cells separates the necrotic cells from the living leaf tissue. Where the necrotic tissue is adjacent to a vascular bundle, the cells of the border parenchyma show hypertrophy and proliferation before any phellogen develops.

Secondary invaders occur amongst the necrotic cells surrounding the penetration hole. These are primarily lichens but some fungous hyphae, a perithecium and a mite are known to be present. However, none of these seem to penetrate the phellogen and no pathogen occurs in the internal proliferating region.

In the mechanical injury of holly leaves, the characteristic wound is trough- or funnel -shaped. The damaged area has a cork border and is circled by dark red pigmentation which may also occur on the opposite surface of the leaf. Broken necrotic cells of the pierced epidermis and internal tissues cover the wound surface and an active phellogen produces cork tissue just beneath them. Hypertrophy and proliferation of the internal tissues may occur within the phellogen if the wound is deep.

Chloroplasts are absent in the phellogen and in areas of cell enlargement. Warts and other wall thickenings may occur.

Measurements taken from leaf spines showed two things in connection with mechanical damage. The length of exposed fibers due to loss of the epidermis is considerable in the two -year -old 52 leaves. The current seasons leaves show little or no fiber exposure. Since the breadth of the spine points shows an in- significant difference with leaf age, the presence or absence of epidermis has no effect on this. From Table 2 it may be seen that the average breadth of the spine tips is approximately 15r. 53

SPOTTING CAUSED BY INSECT DAMAGE

Most insect damage is believed to be caused by small sucking insects since the diameter of some cuticle damage does not exceed 50r. The breakage in the cuticle is small in many samples examined and only epidermal cells are pierced. The reaction to injury rarely extends deeper than the hypodermal layer in upper surface damage.

A fine passage, as if caused by a proboscis, often occurs in the necrotic epidermal cell (Fig. 45). Necrosis appears in this cell and sometimes in some of the adjacent cells but rarely in the layers of mesophyll tissue (Fig. 46).

The necrosis of epidermal cells adjoining the point of entry gives the appearance of a wider spot but neither the cuticle nor the cell walls of these other cells show any damage. Cell divisions producing phellogen tissue take place without hypertrophy, primarily in the hypodermic. These divisions are mainly periclinal but at the periphery of the abnormal area they may become anticlinal. Some epidermal cells may also show anticlinal division in this area (Fig. 47). In two instances, periclinal divisions also occur in the outer layer of palisade cells (Fig. 48). Chloroplast degeneration takes place in this disturbed tissue.

Insect damage without necrosis occurs in some material

(Fig. 49). Dark green 'water -lines' on the under surface of 54 the leaf are depressed rather than swollen in structure. This is caused by the broken and collapsed nature of the epidermal cells which are without protoplasmic contents. Within these broken cellulose walls, the first layer of living cells shows thickening of the walls to the outside but, within these, several layers of small narrow cells indicate the presence of cork cambial tissue. There is no necrotic tissue. Chloroplasts are absent in the phellogen tissue. Translucency is present due to the obliteration of the air space by the dividing phellogen.

Material with scale organisms still attached to the lower surface of the leaf show spots of a blister type. These are slightly yellow but not translucent and were found scattered over the surface. The cuticle is pierced in two places in the lower epidermis by introduction of the rostralis. These passages are narrow, approximately one tenth of the width of the cell wall below. The feeding organ passes through one cell of the epidermis. The mesophyll cell below is also damaged and both cells show necrosis. The passage is then between the cells of the mesophyll to the vascular bundle (Fig. 50). Several spongy mesophyll cells adjacent to the border parenchyma show considerable hypertrophy and, though nuclei are present, the cells are devoid of chloroplasts. Passing between the sheath cells and fibers of the bundle cap, the rostralis finally penetrates into the phloem tissue which shows no necrosis. This is 55

contrary to the work of Baranyovits (3) who describes scale on

Citrus and states they feed exclusively on parenchyma tissue.

A thick coating of pectinaceous material is deposited in the

cell walls along the path of the rostralis (Fig. 50). It is

presumably produced by the living host cells since it does not

occur where the organ passes through the cuticle or between

the fibers of the bundle cap. This deposit is particularly

thick in the region of the border parenchyma.

The main features of insect damage on holly show the

broken cuticle and a damaged epidermal cell or number of cells with little or no necrosis. There is development of a phello-

gen in the hypodermic or first layer of spongy mesophyll.

Hypertrophy is present only if the insect has penetrated beyond the epidermis. 56

DISCUSSION

A comparison of the lesions found on holly leaves shows

that there are certain features common to fungal, chemical,

mechanical and insect damage. Both morphological and anatomi-

cal similarities have been found. Red coloration occurs at

some stage of all the abnormal developments. This is a growth

reaction also seen in the tissue of normal leaves of Ilex.

The anthocyanin pigments causing the red coloration have long

been attributed to the presence of excess carbohydrates (20,

p. 58-60). Chlorosis appears at the outset of all types of

spotting which involves chlorenchyma. One of the first reactions

to stimulation is chlorophyll degeneration. In abnormal areas

showing hypertrophy, proliferation or phellogen formation,

chloroplasts are absent. A few plastids occur in cells develop-

ing abnormality at the periphery of these regions. ksctinaceous

warts are occasional on the outer walls of spongy mesophyll at

the periphery of abnormal areas in all types of lesions.

Separation of the four types of leaf damage may be made

on the basis of the characteristics of two groups and subse-

quently on individual factors. The fungal and chemical damage may be classified together because of certain common features.

Similarities between mechanical and insect injury place these

two together. 57

Fungal and chemical lesions develop as a result of stimulation through the stomata. Internal reaction, therefore, commences on the abaxial surface of the leaf. The resulting morphology shows raised swellings of various sizes. The main anatomical feature is hypertrophy. The irregular enlargement of cells, particularly in the spongy mesophyll, obliterates the air space in abnormal areas. This hypertrophy is generally followed by proliferation. These two processes cause the swollen spot which Küster called an intumescence (20, p. 83). He suggested that there is a connection between these intumes- cences and the distribution of stomata and postulated a correlation between swellings and the effects of "poisons, especially copper salts." The present investigations corroborate these theories. Translucency accompanies the hypertrophy in fungal and chemical damage. It appears in both types of lesions before necrosis occurs and is caused by the large liquid- filled vacuoles of the abnormal cells.

Details of morphology and anatomy separate fungal damage from chemical disturbances. Spots caused by fungal invaders produce an abaxial swelling of irregular, rough contours, frequently protruding on the adaxial surface. Hyper- trophy generally results in thin walled cells, but occasionally these may be evenly thickened with simple pits. In advanced damage, necrosis occurs in the center of the region and progresses toward the surface of the leaf. The presence of fungal 58 mycelium is diagnostic. Hyphae may occur in the external

stomatal chambers, in the substomatal cavities and as inter-

cellular or intracellular structures. Intercellular hyphae

are frequently associated with pectinaceous deposits in the

walls of associated mesophyll cells. Fungal lesions appear to

begin development at the time of stomatal blockage by hyphae.

Thus, it is possible that a hypha, whether or not it is able

to invade the leaf, may stimulate abnormal growth of the spongy

mesophyll in the vicinity of the stoma. If invasion occurs,

hypertrophy and proliferation become extensive. Following this

hypothesis, Epicoccum, 159. and Stemphylium, 172, produce small

or negligible spots because, though able to occlude the stomata,

e:;,ta li -hr :ent within the host can not be achieved. Epicoccem,

normally considered a saprophyte, and `'terphy!iuin, a very weak

facultative parasite, were isolated and reisolated since

material was taken from just within the epidermis of the host.

The possibility o poor technique must be considered and con-

taminants from the leaf surface may have produced the culture.

However, the Clorox, used to sterilize the leaves prior to iso-

lation, should have prevented this. There may be several

reasons why only two of the other five cultures could be reiso-

lated from considerable amounts of abnormal growth in the host.

Technical inaccuracies such ::s the use of a scalpel which was

too hot for removing the isolate may have rendered the pathogen

non -viable. Inadequate medium may also have prevented growth 59 in culture. If the invasion of the fungus had progressed beyond the region of tissue from which the isolation was made, the earlier growth in these regions was perhaps already dead and reisolation was impossible. The very definite intercellular growth of Phomopsis, 124, Pullularia, 171, Coniothyrium,

180, and the Phycomycete, 194, suggests two methods of host stimulation. One possible stimulus is that of contact between the fungus and leaf tissue. However, since the original invasion is from the abaxial surface and the hyphae are found in the air space of the spongy mesophyll, contact is not con- tinuous. The second stimulus is most probably a chemical produced by the fungus, since there is every indication of abnormality in areas where no fungal hyphae have been found.

In contrast to the fungal damage, the chemically produced spots are generally discrete, round, smooth structures, only slightly raised on the abaxial leaf surface and, if apparent, show only as discolorations on the adaxial surface. The hypertrophy of spongy mesophyll in the region of the substo- matai chamber is characteristic. The cells which fill the cavity have unilaterally thickened, laminated walls. These cells are generally the first to become necrotic and degeneration of the tissues progresses toward the center of the leaf.

Mechanical and insect damage may be distinguished from the preceeding group by the stimulation which takes place by the penetration through the cuticle and epidermis. This 60

results in initiation of lesions on both adaxial and abaxial

surfaces of the leaf. In this group, cell division occurs in

a regularly arranged pattern. The development of phellogen

occurs in the first or second layer of cells below the epider-

mis or within the first layer of living cells surrounding deep

wounds.

Consideration of the size and detailed nature of the

resulting damage by leaf spines and insects is used to dis-

tinguish these two types of injury. Spine tips causing

mechanical damage are approximately half the width of any epi-

dermal cell. The increase in breadth of the spines is rapid

from the tip towards the lamina. The injury they cause is

made by a rubbing action with the aid of wind. This injury is

normally many cells wide and will penetrate the mesophyll to

varying depths, even through the leaf. Lesions measured in

section across the cuticle vary from 113r, to 2181,, and in all

instances the hole is funnel shaped. Mechanical injury to cells

results in necrosis of the damaged cytoplasm in the immediate

vicinity of the wound.

Insect damage produces cuticle breakage from 10r. to 50r. wide. The hole is generally a fine, straight passage penetrat-

ing only the epidermal cell or the layer of cells just beneath. -

Removal of cytoplasm from the broken cells prevents necrosis and the chlorenchyma below these collapsed cells has a darker coloration than the undisturbed tissue. In some areas, the 61

Table 3 Morphology of Leaf Spots on Holly

Class of Type of Spot Coloration Position on Damage Leaf Surface

Fungal Raised blister, Chlorotic Lower surface rough surface Translucent Both surfaces in Single or Red becoming advanced stage Aggregated red -black Scattered

Chemical Slightly raised Red -black Lower surface smooth surface Both surfaces in Single, discrete advanced stage or coalescent, Scattered or irregular collected on midrib and margin

Mechanical Holes Red -black Upper and, or Scratches Cork centers lower surface irregular in Scattered shape

Insect Depressed lines Chlorotic Upper and, or Small blisters Chlorotic and lower surface red Scattered 62 TABLE 4 Anatomy of Leaf Spots on Holly

Class of Damage Cuticle and Upper Epidermis Hypodermal Layer Chlo re n chyma Vascular Bundle Lower Epidermis and Cuticle Fungal Unaffected Hyphae intercellular PALISADE AND SPONGY MESOPHYLL: Vascular elements: Unaffected Advanced stage: Advanced stages: Hypertrophy and proliferation Unaffected Stomatal penetration try Hyphae intracellular Hyphae intracellular irregular Border parenchyma: hyphae; external chamber Inner walls broken Cell walls broken Intercellular hyphae Hypertrophy and filled Pustule eruption: Pectinaceous deposits in cells proliferation Advanced stage: Outer walls ruptured Chloroplasts absent or few occasional Necrosis rare Cuticle ruptured Pectinaceous warts on spongy

Chemical Unaffected Unaffected PALISADE: Unaffected Unaffected Advanced stage: Advanced stage: General: Unaffected Advanced stage: Necrotic Necrotic Advanced stage: Hypertrophy Necrotic Cuticle unbroken and proliferation irregular SPONGY: General: Slight hypertrophy and proliferation irregular; pellogen formation Advanced stage: Necrosis and cell breakdown GENERAL: Chloroplasts absent Uneven laminated walls

Mechanical Abaxial penetration: Abaxial penetration: PALISADE: Penetration: Vascular elements: Abaxial penetration: Unaffected Unaffected Abaxial: Unaffected Unaffected Destroyed Adaxial penetration: Adaxial penetration: Adaxial: Phellogen Necrotic if pierced Phellogen formation by Destroyed Destroyed SPONGY: Penetration: Border parenchyma: anticlinal division Phellogen formation by Phellogen formation by Abaxial: Phellogen Hypertrophy and Adaxial penetration: anticlinal division anti- or periclinal Adaxial: Unaffected proliferation Unaffected Deep wound: division PALISADE AND SPONGY MESOPHYLL: below deep wound Deep wound: Necrotic or destroyed Deep wound: Deep wound: Necrotic or destroyed Necrotic or destroyed Phellogen formation Slight proliferation GENERAL: Chloroplasts absent

Insect Abaxial penetration: Abaxial penetration: PALISADE OR SPONGY MESOPHYLL: Vascular elements: Abaxial penetration: Unaffected Unaffected Hypertrophy and proliferation Unaffected Destroyed Adaxial penetration: Adaxial penetration: irregular and, or Border parenchyma: Adaxial penetration: Destroyed Destroyed Phellogen formation Unaffected Unaffected Phellogen formation by Chloroplasts absent Bundle penetration: anti- or periclinal Pectinaceous deposits Vascular necrosis division in cells Parenchyma necrosis Pectinaceous deposits in cells 63

color of the resulting lesions is white. This is due to the

thickening of cell walls in the living cells below the injured

tissue. Deep wounds caused by scale insects penetrate to the

vascular bundle where the rostralis is found in the phloem,

contrary to the report of Baranyovits (3). However, other

symptoms are similar to those caused by fungal damage. The

initially damaged epidermal cells become necrotic. The ros-

tralis then occupies an intercellular position. Along the

path of the feeding organ, hypertrophy and proliferation of the mesophyll occurs. Pectinaceous deposits are produced in the

walls of the leaf cells adpressed to the invader. These cell wall configurations were also reported by Baranyovits (3) who

suggests they are a reaction to insect saliva. They are similar to the reaction of fungal hyphae in contact with mesophyll.

This pectin deposition may also be related to the unevenly

thickened walls found in spongy mesophyll reacting to chemical

damage.

In conclusion, therefore, a distinct separation of fungal,

chemical, mechanical and insect damage to holly leaves may be made on the basis of a combination of both morphological and

detailed anatomical investigations (Tables 3 & 4). 64

SUMMARY

Four types of injury that result in the development of spots on holly leaves are recognized. These are fungal, chemical, mechanical and insect injury. The spots produced by these agents may be distinguished by their external appearance and internal anatomy.

Fungal entry occurs through the stomata and initial damage is near the abaxial surface of the leaf. The damage is characterized by intercellular mycelium which causes hypertrophy followed by proliferation, first of the spongy mesophyll and later of the palisade cells. This results in the development of raised spots on both leaf surfaces. Translucency is another morphological distinction due to the obliteration of intercellular spaces in the leaf.

Eight fungi were isolated and identified as Asteroma,

Phomopsis, Coniothyrium, Alternaria, Stemphylium, Pullularia,

Epicoccum and an unknown species of Phycomycetaceae. Tree inoculations were made and small blister spots were produced by all fungi. However, reisolations were successful only with

Coniothyrium, Alternaria, Pullularia and Epicoccum. Glio- cladium roseum, Boydia insculpta, Phomopsis and Asteroma were identified in detached -leaf-culture after the induction of spore production by ultra -violet radiation.

-_J 65

Chemical damage shows discrete, round, raised red -black spots on the abaxial leaf surface. These may or may not be visible on the adaxial surface as red spots that are not raised.

Hypertrophy and proliferation occur regularly in the spongy mesophyll but rarely in the palisade tissue. Characteristic unilaterally- thickened laminated cell walls are produced by the cells filling the substomatal chambers. These are the first cells to show necrosis.

Mechanical damage occurs on both leaf surfaces. The lesion produced is funnel -shaped and penetrates the mesophyll to varying depths or may pierce the leaf. A well developed phellogen produces cork which cuts off the damaged cells from healthy tissues adjacent to the injury. There are always necrotic cells on the outer surface of the cork.

insect damage usually results in lesions smaller than those caused by mechanical injury but they also occur on both surfaces of the leaf. They are light green or colorless but may show some red coloration with age. The feeding organ makes a distinct, narrow pathway through the cuticle and epidermal cell wall. Cell contents are removed and the collapsed cell walls give the colorless appearance of the lesion.

One or two adjacent cells may darken with the deposition of wound gums. A few cells of the hypodermal layer or first layer of spongy mesophyll show regular cell division similar to phellogen. Deep penetration by scale insects feeding in 66 vascular tissue produce some anatomical abnormalities similar to those found in fungal lesions.

All four types of leaf damage show red or red -black discolorations at some stage of their growth. Chlorosis is also a common morphological characteristic. The production of abnormal cells, by hypertrophy and proliferation or from phellogen, always occurs and chloroplast degeneration occurs in these cells. 67

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Figure 1. Normal holly; adaxial surface of (A) variegated and (B) green variety. X 1.

Figure 2. Cross section of normal green holly leaf. X 97.

Figure 3. Cross section of midrib. X 97.

Figure 4. Cross section at base of leaf spine. X 39.

Figure 5. Cross section at center of leaf spine. X 39.

Figure 6. Cross section towards tip of leaf spine. X 39.

Figure 7. Incomplete cross section of distil end of leaf spine. X 39.

Figure 8. Cross section of variegated holly leaf showing changes in chlorenchyma. X 39.

a - Spongy mesophyll b - External stomatal chamber ba - Branch vein c - Cuticle ca - Cambium co - Collenchyma d - Druse crystal e - Epidermis f - Fibers ff - Fiber strand h - Hypodermis k - Chlorenchyma 1 - Lateral vein m - Mid vein n - Necrosis p - Phloem pe - Phloem fibers q - Palisade mesophyll s - Spinal vein - Vascular tissue x - Xylem z - Substomatal chamber 73

A B 1 2

f ' t

ti t°10

5 6 - ..z i7

F Figure 9. Green holly from Experiment Station, Astoria; abaxial surface showing minute spotting of early fungus damage. X 1.

Figure 10. Cross section through abaxial surface of leaf shown in Figure 9. X 192.

Figure 11. Green holly from Experiment Station, Astoria; both leaf surfaces showing aggregated blister spots, chlorotic and red -tinged. X 1.

Figure 12. Cross section of leaf shown in Figure 11. X 96.

Figure 13. Green holly from Experiment station, Astoria; fungus spots; single translucent blisters on abaxial surface (A) and corresponding chlorosis on adaxial leaf surface (B). X 1.4.

Figure 14. Cross section of leaf from Figure 13. X 96.

g - Hypha n - Necrosis w - Hypertrophy and proliferation in spongy mesophyll y - Hypertrophy and proliferation in palisade mesophyll 9 10

'112

, .;.. .:t ,t' J. Figure 15. Cross section through abaxial surface of green holly leaf showing fungus penetration of stoma. X 181.

Figure 16. Green holly from Experiment Station, Astoria; extreme red -black fungus spots; A, abaxial surface, B, adaxial surface. X 9/10.

Figure 17. Cross section through leaf of Figure 16. X 90.

Figure 18. Cross section through adaxial surface of advanced fungus lesion. X 388.

c - Cuticle e - Epidermis g - Intercellular hypha g' - Intracellular hypha h - Hypodermis n - Necrosis w - Hypertrophy and proliferation in spongy mesophyll y - Hypertrophy and proliferation in palisade mesophyll 77

\ Figure 19. Adaxial surface of leaf with aggregated red -black spot showing acervuli with fungal hyphae. X 23.

Figure 20. Adaxial surface of leaf with one acervulus from Figure 19. X 126.

Figure 21. Lateral view of hyphae issuing from acervulus. X 126.

Figure 22. Section through acervulus and adaxial layers of leaf. X 444.

Figure 23. Green holly from Experiment Station, Astoria, after 14 days under ultra -violet light; adaxial surface showing sporulation from acervuli. X 9/10.

Figure 24. Mount of acervulus from Figure 23, showing simple and biverticilliately symmetrical conidiophores with conidiospores. X 168.

c - Cuticle, broken e - Epidermis g - Sterile erumpent hyphae g' - Intracellular hyphae h - Hypodermis s - Stroma of mycelium o - Conidiospores 79

J a. rr

20 r

141 C re

.1k

1', 23 . sn r 24 Figure 25. Cross section through adaxial region of leaf inoculated with Pullularia pullulans, culture 171.

X.eL 185.

Figure 26. Cross section through abaxial regiLn of leaf inoculated with Phomopsis, culture 124. X 185.

Figure 27. Control leaves of detached- leaf -inoculation -test. X 9/10. A - Scratched leaf with sterile agar P - Scratched leaf C - Untouched leaf

Figure 28. Test leaves of detached -leaf -inoculation -test after 3 weeks. X 9/10. D - Inoculated with Coniothyrium E - Inoculated with Alternaria F - Inoculated with Phomopsis G - Inoculated with Epicoccum

Figure 29. Tip of leaf inoculated with Phomopsis after 6 weeks; adaxial surface. X 5.5.

g - Intercellular hypha o - Spore exudate from pycnidia u - Deposition of pectin in cell wall w - Hypertrophy and proliferation in spongy mesophyll 81

. . 126

A. 27

i -'

i .

ßG 28

J SÌ

_29 Figure 30. Leaves from trees treated with varying concentrations of tri -basic- copper. X 1/2.

A - Normal holly B - Untreated leaf from tree next to check; small blister spots C - Spreader- sticker spray treatment; small blister spots D - 2 lbs. tri- basic -copper spray; typical discrete, red -black spots E - 2 lbs. tri- basic- copper spray; typical discrete, red -black spots F - 2 lbs. copper sulfate spray; typical discrete, red -black spots G - 4 lbs. tri- basic- copper spray; typical discrete, red -black spots H - 8 lbs. tri- basic -copper spray; typical discrete, red -black spots J - 8 lbs. tri- basic -copper spray; typical discrete, red -black spots

Figure 31. Cross section through abaxial surface of leaf sprayed with 2 lbs. copper sulfate showing unilaterally thickened laminated walls. X 187.

Figure 32. Cross section through abaxial surface of leaf sprayed with 2 lbs. tri -basic -copper showing pitted thickened walls. X 187.

Figure 33. Cross section through abaxial surface of leaf sprayed with 2 lbs. tri -basic - copper showing irregular hypertrophy and proliferation with initial necrosis. X 94.

Figure 34. Cells of spongy mesophyll at edge of abnormal region showing pectinaceous warts. X 403.

n - Necrosis q - Palisade unaffected u - Unevenly thickened, laminated walls ub - Evenly thickened, pitted walls qu - Pectinaceous warts w - Hypertrophy and proliferation of spongy mesophyll z - Substomatal chamber

-- 83

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4 Figure 35. Cross section of leaf sprayed with 8 lbs. tri- basic- copper showing advanced necrosis and cell breakdown. X 94.

Figure 36. Abaxial surface of green holly showing discrete round red -black spots of chemical damage. X 9/10.

Figure 37. Cross section through abaxial surface of leaf from tree having unknown spray treatments. X 187.

Figure 38. Cross section through leaf showing typical chemical damage. X 94.

Figure 39. Adaxial surface of leaf showing red discolorations corresponding to chemical type spotting on abaxial surface. X 9/10

d - Druse crystals k - Border parenchyma proliferation n - Necrosis u - Unevenly thickened laminated walls ub - Evenly thickened pitted walls w - Hypertrophy and proliferation of spongy mesophyll 85 1

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' 38 39 Figure 40. Green holly showing mechanical damage; A, abaxial surface, B, adaxial surface. X 9/10.

Figure 41. Cross section through abaxial surface of leaf with small mechanical lesion. X 187.

Figure 42. Cross section through abaxial surface of leaf with double mechanical lesion. X 94.

Figure 43. Cross section through abaxial surface of leaf showing necrosis and cork formation in mechanical lesion. X 187.

Figure 44. Cross section through leaf showing deep mechanical lesion initiated on the adaxial surface. X 94.

a - Spongy mesophyll, chlorotic c - Cuticle, broken ch - Cork k - Border parenchyma proliferation n - Necrosis ph - Phellogen s - Secondary invaders t - Unevenly thickened walls w - Slight hypertrophy and proliferation of spongy mesophyll 87 u

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j ;MP 4 41,4* t ! or, - Ili _ ! r PrZi Figure 45. Cross section through adaxial leaf surface showing insect damage of single epidermal cell. X 402.

Figure 46. Cross section through adaxial leaf surface showing entry path of insect proboscis and phellogen formation. X 402.

Figure 47. Cross section through adaxial leaf surface showing two points of insect penetration. X 402.

Figure 48. Cross section through adaxial leaf surface with epidermal penetration and hypodermal hypertrophy. X 402.

Figure 49. Cross section through abaxial leaf surface showing collapsed epidermal cells without contents. X 402.

Figure 50. Cross section through abaxial leaf surface showing rostralis of scale insect and hypertrophy of spongy mesophyll with pectinaceous deposits. X 402.

c - Cuticle, broken e - Epidermal proliferation ec - Collapsed epidermis h - Hypodermal proliferation k - Border parenchyma n - Necrosis p - Phloem pe - Phloem fibers - Rostralis u - Pectinaceous wall deposits w - Hypertrophy and proliferation, irregular, in spongy mesophyll y - Proliferation in palisade 89

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