Citation
Evaluating the pathogenicity of Botryosphaeria ribis Gross. & Duggar on Melaleuca quinquenervia (Cav.) Blake in South Florida

Material Information

Title:
Evaluating the pathogenicity of Botryosphaeria ribis Gross. & Duggar on Melaleuca quinquenervia (Cav.) Blake in South Florida
Creator:
Rayachhetry, Min Bahadur, 1953-
Publication Date:
Language:
English
Physical Description:
xvii, 152 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Callus ( jstor )
Cells ( jstor )
Defoliation ( jstor )
Diseases ( jstor )
Fungi ( jstor )
Hyphae ( jstor )
Inoculation ( jstor )
Mycology ( jstor )
Pycnidia ( jstor )
Xylem ( jstor )
Canker (Plant disease) -- Florida ( lcsh )
Dissertations, Academic -- Forest Resources and Conservation -- UF
Forest Resources and Conservation thesis, Ph. D
Melaleuca quinquenervia -- Diseases and pests -- Florida ( lcsh )
Miami metropolitan area ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1995.
Bibliography:
Includes bibliographical references (leaves 143-151).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Min Bahadur Rayachhetry.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
021620274 ( ALEPH )
33410383 ( OCLC )
AKN3993 ( NOTIS )

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Full Text













EVALUATING THE PATHOGENICITY OF Botryosphaeria ribis
Gross. & Duggar ON Melaleuca quinquenervia (Cav.)
Blake IN SOUTH FLORIDA

















BY

MIN BAHADUR RAYACHHETRY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1995




EVALUATING THE PATHOGENICITY OF Botryosphaeria ribis
Gross. & Duggar ON Melaleuca quinquenervia (Cav.)
Blake IN SOUTH FLORIDA
BY
MIN BAHADUR RAYACHHETRY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995


I dedicate this dissertation to my parents, Kamal B.
Rayamazhi and Chhindra K. Rayamazhi, who always dreamed of their
children being educated. It is their love, encouragement, and
sacrifice that made their children's education possible.


ACKNOWLEDGEMENT
I am grateful to Dr. G.M. Blakeslee, the chairman of my
supervisory committee, for his willingness to take the
responsibility of guiding me through my graduate program following
Dr. R.S. Webb's relocation. His insight and advice in every step
of my graduate program were invaluable in accomplishing this goal.
I wish to extend my profound appreciation to him for his time,
patience, expertise, receptiveness, and response during
preparation of this dissertation.
I am also grateful to Dr. R.S. Webb for first accepting me
into this PhD program and guiding me as the chairman of my
supervisory committee until his relocation. I appreciate him for
allowing me to be involved in this Melaleuca biocontrol project,
providing me with a research assistantship, and being willing to
continue to serve on the supervisory committee.
My deep appreciation is extended to Dr. J.W. Kimbrough, a
member of my supervisory committee, who provided me with an
assistantship during a critical financial crisis in my graduate
program in 1993 and access to his laboratory as needed. His
expertise, time, and kindness were invaluable in improving the
quality of my graduate research.
I would like to extend my sincere thanks to Drs. R.
Charudattan, J. Gander, and T. Center, the members of my
supervisory committee, for invaluable input throughout my graduate
program and their willingness to dedicate time and expertise in
improving the quality of my research. I am grateful to every


member of my supervisory committee for reviewing and giving
creative suggestions during preparation of this dissertation.
I would like to extend my heartfelt thanks to Dr. L.
Arvanitis for his constant encouragement and help as an
administrator during the ups and downs of my life as a graduate
student in this school. Appreciation is also due to Dr. T. Miller
for reviewing and giving invaluable suggestions on Chapter V of
this dissertation. Thanks are also due to Jay Harrison and Jim
Allen for help and verification of the statistical analysis
performed on various experiments included in this manuscript.
I wish to thank Winnie Lante and Dr. Robert Berger for their
friendship, wisdom, and assistance in every possible way. I also
extend my thanks to Joel Smith, James DeValerio, Jeff English, Dan
Schultz, Tess Korhnak, Dave Nolletti, and Willy Wood for their
friendship as well as unfaltering assistance as needed.
Appreciation is also due to Drs. Mark Lesney, Jerry Benny, Mick
Popp, and Litzu Li for their wisdom and sincere friendship.
I would like to thank USDA-ARS, Fort Lauderdale, South
Florida Water Management District, West Palm Beach, Florida, and
SFRC, University of Florida, for funding this research and my
graduate program.
Last but not least, I extend my deepest love and gratitude
to my parents, wife Geeta, and sons Amit and Ajamber. It would
have been impossible for me to accomplish my PhD program without
their love, encouragement, and sacrifice.
IV


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES ix
LIST OF FIGURES xi
ABSTRACT xvi
CHAPTERS
I. GENERAL INTRODUCTION 1
Host Distribution 1
Problems Associated with Melaleuca
in South Florida 1
Pathology of Melaleuca 3
Research Rationale 4
General Hypotheses 6
General Objectives 7
Chapter Organization 7
II. MYCOLOGICAL CHARACTERISTICS OF THE ISOLATES OF
Botryosphaeria ribis 8
Introduction 8
Materials and Methods 11
Isolate Acquisition and
Identification 11
Mycelial Growth and Morphology 12
Vegetative Compatibility
Among Isolates 13
Pycnidial Production and Morphology 13
Conidial Characteristics 15
Germination of Macroconidia 15
Statistical Analyses 16
v


Results.
17
Fungal Identification 17
Mycelial Growth and Morphology 17
Vegetative Compatibility
Among Isolates 20
Pycnidial Production and
Morphology 22
Conidial Characteristics 24
Germination of Macroconidia 25
Discussion 27
Fungal Morphology and Taxonomy 27
Germination of Macroconidia 32
Conclusions 34
III. EVALUATION OF Botryosphaeria ribis ISOLATES FOR
PATHOGENICITY TO Melaleuca quinquenervia 41
Introduction 41
Materials and Methods 43
Tree Clone Production 43
Isolate Acquisition 44
Inocula Preparation 45
Leaf Inoculations 46
Influence of Hyphae vs Macroconidia
as Inocula 48
Pathogenicity of Botryosphaeria ribis
vs Pestalotia 49
Effects of Inoculation Positions
on Canker Development 50
Evaluation of Virulence of Isolates
Under Greenhouse Conditions 50
Evaluation of Virulence of Isolates
Under Field Conditions 52
Inocula 52
Site selection 53
Tree inoculation 53
Experimental design 53
Harvesting and canker evaluation 53
Effects of Season on Canker
Development 54
Simulated Storm-Damage and Inoculation
with Macroconidia 56
Statistical Analyses 58
Results 58
Leaf Inoculations 58
Influence of Hyphae vs Macroconidia
as Inocula 60
VI


60
Pathogenicity of Botryosphaeria ribis
vs Pestalotia
Effects of Inoculation Positions
on Canker Development 62
Evaluation of Virulence of Isolates
Under Greenhouse Conditions 62
Evaluation of Virulence of Isolates
Under Field Conditions 65
Effects of Season on Canker
Development 66
Simulated Storm-Damage and Inoculation
with Macroconidia 67
Discussion 69
Preliminary Screening of Macroconidial
Cultures 6 9
Evaluation of Isolates by Stem
Inoculations 70
Conclusions 76
IV. EFFECTS OF STRESS FACTORS ON CANKER DEVELOPMENT ON
STEMS OF Melaleuca quinquenervia INOCULATED WITH
Botryosphaeria ribis 87
Introduction 87
Low Moisture 8 8
Low Temperature 90
Defoliation 92
Objectives 93
Materials and Methods 93
Selection Criteria for Tree Clones
and Isolates 93
Low Moisture 94
Low Temperature 95
Defoliation 96
Statistical Analyses 97
Results 97
Low Moisture 97
Low Temperature 98
Defoliation 100
Discussion 100
Low Moisture 100
Low Temperature 103
Defoliation 105
Conclusions 106
vii


V. HISTOPATHOLOGY OF Botryosphaeria ribis IN Melaleuca
quinquenervia STEMS 112
Introduction 112
Materials and Methods 114
Preparation of Leaf Materials 114
Preparation of Stem Materials from
Unstressed Plants 115
Preparation of Stem Materials from
Stressed Plants 116
Results 117
Host Response 117
Leaves 117
Stems 117
Callused 118
Noncallused 120
Stressed 121
Fungal Invasion and Colonization 121
Leaf Tissues 121
Stem Tissues 122
Callused 122
Noncallused 123
Discussion 123
Leaf Invasion and Colonization 123
Stem Invasion and Colonization 124
Conclusions 129
VI. SUMMARY AND CONCLUSIONS 137
REFERENCES 143
BIBLIOGRAPHICAL SKETCH 152
viii


LIST OF TABLES
Table Page
2.1. Radial growth rates (mm/day) of the mycelia of
Botryosphaeria ribis isolates on potato dextrose agar
(PDA) ,cornmeal agar (CMA), mycological agar (MA) and
starch agar (SA) 18
2.2. Production of aerial hyphae, mycelial color, and
pycnidial stroma by isolates of B. ribis in four
artificial growth media at 21 days after inoculation. .. 19
2.3. Vegetative and reproductive behavior of the pairs of
the eight B. ribis isolates on and around the zone of
confrontation (the meeting point of two colonies
growing towards each other) on PDA 21
2.4. Range and average diameter of the pycnidial stroma
on 2- to 4-week-old culture of some B. ribis isolates. .. 24
2.5. Mean and range of dimensions (length and width) and
the number of nuclei in macroconidia of B. ribis 24
2.6. Effect of temperature on germination of macroconidia
by 4 h after incubation in WA at 100 percent ambient
humidity 26
3.1. Leaf bioassay experiments designed for preliminary
tests of virulence among 11 monoconidial cultures
within each of six isolates of B. ribis obtained from
Melaleuca quinquenervia from South Florida 59
3.2. Separation of means of the effects of form of
inocula on canker length (mm) on the stems of M.
quinquenervia in summer inoculations with isolate
BR-1 of B. ribis 61
3.3. Main-effect means and mean separations from analyses
of variance for comparison of B. ribis and Pestalotia
species, and determination of the effect of inoculation
point on disease development. Dependent variable was
total canker length (mm) measured at 8 weeks after
inoculation in spring 61
IX


3.4.
Percent callusing around the inoculation point and
percent reisolation of fungus from 5 mm beyond the
inoculation point on M. quinquenervia stems at 8
weeks after inoculation in spring 63
3.5. Effects of B. ribis isolates on canker development
on stems of M. quinquenervia clones at 8 weeks after
inoculation in spring 1994 64
3.6. Main-effect means and mean separations from analyses of
variance for stem-inoculation tests on total canker
length (mm) at 8 weeks after inoculation 65
3.7. Effects of simulated storm damage on the establishment
of BR-1 isolate of B. ribis on stems of M. quinquenervia
ramets inoculated with macroconidia 68
4.1. Analysis of variance of data of experiments designed to
assess the effects of stress factors on canker
development on stems inoculated with BR-2 and BR-5
of B. ribis at 8 weeks after inoculation 99
4.2. Main-effect means and mean separations from analyses
of variance for effects of stress factors on canker
length (mm) on the stems of M. quinquenervia inoculated
with isolates of B. ribis 99
x


LIST OF FIGURES
Figure Page
2.1. Aerial hyphae Botryosphaeria ribis imparting black
color and partially submerged pycnidia on PDA 37
2.2. Hyphae of B. ribis in and on the surface of media
imparting tan color and pycnidia immersed in CMA 37
2.3. Mycelial mat of B. ribis on PDA with hyphae of various
diameters 3 7
2.4. Blended mycelial mat of B. ribis grown in PDB under
continuous shaking conditions showing chains of
chlamydospore-like cells 37
2.5. Cross-section through a multilocular pycnidial stroma
of B. ribis produced on PDA showing numerous locules. .. 37
2.6. Enlarged pycnidia from a portion of multilocular
stroma of B. ribis showing macroconidia and an obscure
ostiole 37
2.7. Pycnidial stroma of B.ribis on Melaleuca quinquenervia
leaf, inoculated and maintained in a sterile moist
chamber. Evidence of solitary and botryose types of
pycnidia 3 7
2.8. Pycnidial stroma of B. ribis erupting from dead bark
of M. quinquenervia showing solitary and botryose
types of pycnidia 3 9
2.9. Section through a M .quinquenervia leaf showing a
solitary pycnidium of B. ribis erupting from epidermis.
Evidence of ostiole and pycnidial wall 3 9
2.10. Longitudinal section through a M. quinquenervia stem
showing a solitary pycnidium of B. ribis erupting
through bark. Evidence of ostiole and pycnidial wall
composed of textura angularis 39
2.11. Pycnidial stroma of B. ribis on M. quinquenervia leaf
oozing macroconidial cirrhi at room temperature 39
XI


2.12. Cross-section through a pycnidium of B. ribis showing
pycnidial wall composed of textura angularis, and
conidiophores 3 9
2.13. A portion of the inner wall of a 3-day-old pycnidium
of B. ribis. Evidence of conidiophores with bulbous
base bearing macroconidia at different stages of
deve 1 opment 3 9
2.14. Macro- and microconidia from freshly squashed 7-day-
old pycnidium of B. ribis showing the microconidia and
macroconidia 3 9
2.15. Macroconidia of B. ribis from PDA germinated in water.
Evidence of mixture of aseptate and septate conidia
bearing germ tubes 3 9
2.16. Germinating aseptate and septate macroconidia of B.
ribis showing one or more germ tubes 39
2.17. Germination of the spores of Isolate BR-1 of B. ribis
on water agar at 100 percent relative humidity 40
2.18. Germination of the spores of Isolate BR-7 of B. ribis
on water agar at 100 percent relative humidity 40
3.1. A portion of a declining M. quinquenervia stand in the
management area ACME-2 of the Loxahatchee National
Wildlife Refuge in South Florida. Crown thinning
among some trees and complete leaf-loss among others. .. 79
3.2. A M. quinquenervia tree with a basal canker showing
severe crown thinning above cankered area but still
sprouting from the root-collar 79
3.3. A typical canker on the stem of M. quinquenervia
revealing vertical depression surrounded by callus
ridges 79
3.4. A portion of a cankered stem of M. quinquenervia
split to reveal the discolored sapwood beneath the
canker 79
3.5. Mean necrotic leaf area of M. quinquenervia due to
treatments with isolates BR-1 through BR-6 of
B. ribis, F. moniliformis var subglutinans,
Lophotrichus sp., and wound only 8 0
3.6. A portion of a stem wounded but not inoculated with B.
ribis showing wound closed by callus ridges 82
3.7. A portion of a stem wounded and inoculated with
B. ribis showing canker extension above and below
the point of inoculation, and callus ridge around the
canker 82
xii


3.8.
A portion of a stem inoculated with B. ribis in the
fall, representing noncallused stem showing lack of
callus around the wound
82
3.9. A portion of a stem above the point of inoculation
of B. ribis showing pycnidia emerging from the bark
of M. quinquenervia stem 82
3.10. Pathogenic interaction between B. ribis and M.
quinquenervia showing means of canker length from
21 stems 83
3.11. Clonal susceptibility of M. quinquenervia towards B.
ribis shown by mean of canker length from 24 stems 83
3.12. Mean canker length at the proximal and distal parts
of the stem from the point of inoculation of M.
quinquenervia with B. ribis isolates 84
3.13. Mean necrotic tissues on M. quinquenervia stems at
5 mm above the point of B. ribis inoculations showing
mean percentage of necrosis of phloem and cambium 85
3.14. Mean canker length of M. quinquenervia ramets 8 weeks
after inoculation with BR-2 isolate of B. ribis 86
4.1 Xylem water potential of differentially moisture-
stressed stems of M. quinquenervia ramets inoculated
with B. ribis 107
4.2. Effects of low moisture on canker development on
the stems of M. quinquenervia ramets inoculated
with B. ribis 107
4.3. Four groups (gr.) of M. quinquenervia ramets inoculated
with B. ribis and watered every 24 (gr.l), 72 (gr.2),
168 (gr.3), and 288 (gr.4) h showing progressive crown
thinning (from gr.l to gr.4) due to leaf-drop 109
4.4. A stem showing a typical canker among M. quinquenervia
ramets wounded and inoculated with B. ribis and
watered every 24 h showing callus ridge surrounding a
canker 109
4.5. A stem showing a typical canker among M. quinquenervia
ramets wounded and inoculated with B. ribis and watered
every 288 h 109
4.6. Inner tissues in a typical stem canker among M.
quinquenervia ramets wounded and inoculated with
B. ribis and watered every 288 h revealing blackened
bark and sapwood 109
4.7. Effects of low temperature on canker development on
stems of M. quinquenervia ramets inoculated with
B. ribis 110
xiii


4.8. Effects of defoliation on canker development on stems
of M. quinquenervia ramets inoculated with B. ribis. ... Ill
4.9. Effects of defoliation on mortality of M. quinquenervia
ramets inoculated with B. ribis Ill
5.1. A cross-sectional view of a healthy M. quinquenervia
leaf stained with Pianeze's IIIB showing stoma,
epidermis, chlorenchyma cells, mesophyll cells,
and oil chamber 132
5.2. A wounded but noninoculated (with B. ribis) stem
showing callus closing wound 132
5.3. AM. quinquenervia stem wound-inoculated with B. ribis
showing canker surrounded by callus ridges 132
5.4. A cross-sectional view of a wounded M. quinquenervia
stem stained with Pianeze's IIIB showing callus
filling the wound, vascular cambium and xylem at the
time of wounding, and patches of ligno-suberized
cells in callus. Evidence of redifferentiated vascular
cambium and newly formed xylem in callus 132
5.5. A cross-section through unwounded M. quinquenervia
stem stained with phloroglucinol-HCl and Sudan IV
showing phelloderm and layers of suberized cells,
cortex, phloem, and xylem 132
5.6. A cross-sectional view through a portion of 4-wk-old
wound on stem stained with phloroglucinol-HCl and
Sudan IV showing suberized cell layers in callus ridges
extending to the ligno-suberized periderm enclosing
tanninoid and ligno-suberized parenchyma cells 132
5.7 A longitudinal section (stained with Pianeze's IIIB)
through the margin of callus on a B. ribis-inoculated M.
quinquenervia stem showing a gap at the interface of
xylem and callus, groups of ligno-suberized cells
enclosing tanninoid parenchymatous cells 132
5.8. A cross-section through a 8-wk-old, B. ribis
inoculated wound of a fresh M. quinquenervia stem
stained with phloroglucinol-HCl revealing xylem, and
suberized and ligno-suberized parenchyma cells in and
around the callus ridge 134
5.9. A longitudinal section through the canker margin of
a B. ribis-inoculated stem of M. quinquenervia showing
tyloses plugging a vessel in the xylem near disrupted
vascular cambium 134
xiv


5.10. A cross-section through a B. ribis-inoculated M.
quinquenervia leaf (stained with Pianeze's IIIB)
showing fungal hyphae penetrating epidermis through
stomata and colonizing chlorenchyma, mesophyll,
and oil chamber 134
5.11. A cross-section through a B. ribis-inoculated leaf M.
quinquenervia leaf (stained with Pianeze's IIIB)
showing a fungal hypha penetrating through stomata,
and a pycnidium emerging from the epidermis 134
5.12. A cross-section through a callus ridge of a stem
canker on M. quinquenervia caused by B. ribis showing
cells surrounded by hypha resulting in deterioration
of cellular integrity 134
5.13. A cross-section through a callus ridge of a stem canker
on M. quinquenervia caused by B. ribis revealing
intra- and intercellular hyphae in deteriorating areas
as shown in Fig. 5.12 134
5.14. A longitudinal section through xylem from the canker
margin of a B. ribis-inoculated stem of M.
quinquenervia showing hyphal growth along ray
parenchyma and tracheal cells 136
5.15. A longitudinal section through xylem from the canker
margin of a B. ribis-inoculated stem of M.
quinquenervia showing hyphae along inner wall clogging
tracheal cells, passing radially and producing hyphal
swellings 136
5.16. A longitudinal section through xylem from the canker
margin of a B. ribis-inoculated stem of M.
quinquenervia showing a hyphae branching in a tracheal
lumen 136
5.17. A cross-section through noncallused canker on M.
quinquenervia stem inoculated with B. ribis revealing
a pycnidium in phelloderm, hyphal concentration in
cambium and phloem, and hyphae in the lumen and
intercellular spaces of vessels and tracheids 136
xv


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EVALUATING THE PATHOGENICITY OF Botryosphaeria ribis
Gross. & Duggar ON Melaleuca quinquenervia (Cav.)
Blake IN SOUTH FLORIDA
By
Min Bahadur Rayachhetry
May, 1995
Chairman: Dr. G.M. Blakeslee
Major Department: School of Forest Resources and Conservation
Biology and virulence of the Fusicoccum anamorph of
Botryosphaeria ribis isolates obtained from the canker margins of
Melaleuca quinquenervia trees and gall tissues of Rhizophora
mangle from South Florida were evaluated under laboratory,
greenhouse, and field conditions. Morphologically, the isolates
were variable. Hyphal, pycnidial, and conidial attributes of the
isolates of B. ribis examined overlapped in the anamorph of B.
dothidea revealing their synonymy.
Spores and hyphae of B. ribis were equally effective in
inciting canker disease on M. quinquenervia stems. A weak
correlation existed between diameter of stem at the point of
inoculation and canker length. All eight tested isolates of B.
ribis were able to incite cankers under greenhouse and field
conditions but two isolates were more virulent than others.
Differential susceptibility of M. quinquenervia clones towards B.
ribis was evident.
XVI


Stem-wounds without B. ribis were closed in both spring and
fall inoculations. Cankers with or without callus were formed
following both spring and fall inoculations with B. ribis. Fall
inoculations had less callusing intensity and greater percentages
of wilting and dieback of ramets than those inoculated in spring
although the differences were small. Establishment of B. ribis in
stems of M. quinquenervia appeared to require wounds at the depth
of the sapwood or some kind of injury stressing the trees.
Low moisture in the xylem (<-1.5 Mpa) of B. ribis inoculated
stems favored rapid canker development and wilting of ramets. Low
temperature treatment of 6C for 3-day/wk was more effective than
6C for 6-day/wk and 0 (1)C for 16-h/wk in inciting longer
cankers on M. quinquenervia stems inoculated with B. ribis.
Partial defoliation followed by B. ribis inoculation did not cause
differential canker expansion. Complete defoliation plus B. ribis
inoculation caused 90 percent mortality of ramets within 4 weeks.
Callus tissues formed in response to wounding were
surrounded by suberized periderm, both in presence and absence of
inoculation with B. ribis. Tissues in the callus ridges were
tanninoid and contained groups of lignified cells. The fungus
gained entry into callus tissues through the ventral surface of
the wedges. Cells in the xylem, adjacent to the wound were also
intensely lignified. Despite these barriers, inner tissues in
sapwood and callus were invaded by fungal hyphae. Stems with
noncallused wounds were rapidly girdled by fungal hyphae.
Botryosphaeria ribis appeared to be able to persist in the
tissues of healthy trees but became more aggressive pathogen on M.
quinquenervia in presence of stress factors.
XVII


CHAPTER I
GENERAL INTRODUCTION
Host Distribution
Melaleuca quinquenervia (Cav.) Blake, the cajeput tree, is a
species in the Myrtaceae (Meskimen 1962, Bodle et al. 1994) It
is native to Australia and New Guinea (personal communication with
Dr. Center), and has become naturalized in the Philippines, India,
Madagascar, Zaire, Hawaii, the United States (Myers 1975), and
Indonesia (Holliday 1989) The genus Melaleuca includes more than
200 species in Australia alone (Holliday 1989).
Problems Associated with Melaleuca in South Florida
The introduction of M. quinquenervia to South Florida
occurred in the 1890s (Meskimen 1962) when seeds were imported.
The tree was considered useful for ornamental purposes and for a
possible commercial source of wood for forest industries (Myers
1975, Huffman 1980, Balciunas and Center 1991, Bodle et al. 1994).
Certain favorable biological characteristics, such as prolific
seed-producing ability and tolerance to brackish water, flooding,
complete submergence, and fire, have enabled this species to
aggressively invade portions of the Everglades National Park, and
the seasonally flooded areas of Lee, Collier, Martin, Palm Beach,
1


2
Broward, and Dade counties in South Florida (Meskimen 1962, Myers
1975, Huffman 1980, Hofstetter 1991). In 1976, M. quinquenervia
trees were estimated to cover an area of about 0.53 million ha
(Ewel et al. 1976). The trees reportedly produce allelopathic
chemicals (Di Stefano and Fisher 1983) which may enhance their
ability to displace local flora.
In 1983, this tree species was estimated to occupy 1.01
million ha, and the rate of invasion of new areas was estimated at
1012 ha/year (Di Stefano and Fisher 1983). Since 1980, the
invaded area has increased at least by 50 percent (Balciunas and
Center 1991). For example, the Florida peninsula south of Lake
Okeechobee has been estimated to have an infestation of 0.2 to
0.61 million ha out of the total acreage of 3.04 million ha (Bodle
et al. 1994). It has been estimated that most of the remaining
natural land south of Lake Okeechobee may be overtaken by
uncontrolled M. quinquenervia within 30 years (Bodle et al. 1994)
The aggressive colonization by M. quinquenervia of South
Florida wetland sites has created serious problems such as the
near elimination of native plant communities and freshwater
wildlife species (Crowder 1974), respiratory problems and allergic
reactions of people to pollen and vaporized oils (terpinine 4-ol)
(Morton 1962, 1969), increased fire hazard to urban developments,
and accelerated loss of ground water due to increased
evapotranspiration (Balciunas and Center 1991).


3
Pathology of Melaleuca
Spaulding (1961) listed a total of three fungal pathogens
(Fomes lignosus, causing root-rot in Uganda; Phyllosticta
leucadendri, causing leaf spot in Australia; and Puccinia
camargoi, causing leaf rust in Brazil) on cajeput (M.
leucadendron=M. quinquenervia) trees. According to Hepting
(1966), cajeput trees appear to be free of diseases in their
exotic as well as native habitats. Hepting (1966) mentioned that
the only disease record on cajeput trees in the United States is
the damping-off disease in the nurseries in Florida due to
Rhizoctonia species.
Farr et al. (1989) have recorded a few pathogens
(Botryosphaeria sp., Macrophoma sp., Phoma sp., and Phomopsis sp.
causing dieback; Pythium sp. and Fusarium sp. causing root rot;
Puccinia psidii causing leaf rust; and Al ternaria sp.,
Cylindrocladium pteridis, C. scoparium, Dothiorella sp., and
Phyllosticta sp. causing leaf spots) on M. quinquenervia from
South Florida.
Alfieri et al. (1994) have recorded several fungal pathogens
on M. quinquenervia from South Florida. These diseases are
related to damping-off (Pythium sp., and Rhizoctonia solani),
algal leaf spots (Cephaleuros virescens), fungal leaf spots
(Alternara sp., Cercospora sp Curvularia sp Cylindrocladium
pteridis, C. scoparium, Dothiorella sp., and Phyllosticta sp.),
foliar rust (Puccinia psidii), stem cankers and dieback
(Botryosphaeria sp., Macrophoma sp., Phoma sp., Phomopsis sp., and
Physalospora sp.), stem galls (Sphaeropsis sp.), and root rots


4
(Armillariella tabescens, Fomes sp., Fusarium sp., Gloeosporium
sp.( Pycnosporus sp., and Pythium sp.).
Thus far, no pathogenicity tests of any of these pathogens
on M. quinquenervia have been reported and the effects of these
fungi on the population of M. quinquenervia from South Florida are
unknown.
Research Rationale
Because of the increasing awareness of the negative impact
of M. quinquenervia in South Florida ecosystems, this species
recently has been listed officially as a federal noxious weed and
a Florida prohibited aquatic weed (Bodle et al. 1994). Various
research projects concerning the control of M. quinquenervia and
possible restoration of South Florida ecosystems are underway.
The cost of control ($1.70 per tree and 25,000-100,000 trees/ha)
of M. quinquenervia by herbicide use is very high (Bodle et al.
1994). Additionally, seed release is accelerated among trees
stressed by herbicides and new foci are created through natural
dispersal of these seeds (Laroche and Ferriter 1992). Such a
phenomenon of regeneration will require repeated herbicidal
treatment of these sites which compounds the pollution problems in
sensitive areas like the Everglades and other fresh water
reservoirs in South Florida. A problem of this magnitude warrants
the discovery and use of control agents that can perpetuate in the
tree stands, disperse naturally and destroy existing stands or
limit further invasion of new sites. For effective control of
this weed, we need agents that can attack one or more plant parts


5
such as roots, stems, leaves, flowers and fruits. Balciunas and
Center (1991) have discussed the suitability of M. quinquenervia
as a target for biological control.
With these strategies in mind, biological control of this
weed by suitable insect pests and/or fungal pathogens to ensure
ecological compatibility and economic viability (Balciunas and
Center 1991, Laroche and Ferriter 1992, Bodle et al. 1994) by
reducing the use of herbicides has received recent attention.
Weed control has been accomplished using insects and fungal
pathogens. Biological control of herbaceous weeds has been
successfully accomplished in the United States using an endemic
pathogen (e.g., TeBeest and Templeton 1985, Charudattan 1991).
The benefits of endemic pathogens as biological control agents has
been discussed by Charudattan (1988). In the past, such
activities have been successful against perennial woody weeds
(e.g., Wilson 1965, Griffith 1966, Adams 1988, Charudattan 1991,
TeBeest et al. 1992).
In August 1989, Dr. Mark D. Maffei, the Assistant Manager of
the Loxahatchee National Wildlife Refuge (Palm Beach County, FL)
discovered a patch of dead and dying M. quinquenervia trees in
Acme-2 of the Refuge. He contacted Dr. Ted D. Center, the Project
Leader of the USDA-ARS, Aquatic Plant Management laboratory at
Fort Lauderdale, FL. Dr. Center invited Dr. Roger S. Webb, then
Associate Professor in the School of Forest Resources and
Conservation at the University of Florida, to identify the cause
of tree mortality in the Refuge. A preliminary investigation by
Dr. Webb indicated that the decline and mortality were most likely
associated with the occurrence of stem vascular stain and cankers


6
located beneath the papery bark layers of the M. quinquenervia
trees. He consistently isolated a fungus from the margin of
diseased stem tissue that subsequently was identified by the
International Mycological Institute, Kew, Surrey, England, as the
Fusicoccum anamorph (order, Sphaeropsidales; family,
Sphaeropsidaceae) of Botryosphaeria ribis Gross. & Duggar.
Initial pathogenicity tests under greenhouse conditions
revealed B. ribis to be capable of inciting disease on M.
quinquenervia. Previously, isolates of B. ribis were reported to
possess some degree of host specificity towards their original
hosts (Hildebrand and Weber 1944, Witcher and Clayton 1963,
Schreiber 1964, Milholland 1972, Sutton and Boyne 1983, Latorre
and Toledo 1984, Pusey et al. 1986, Shearer et al. 1987). This
fungus is native to South Florida, so it should be safe to use as
a biological control agent, if a virulent and host specific
isolate could be identified.
General Hypotheses
1. Botryosphaeria ribis is capable of inciting disease on
M. quinquenervia,
2. certain isolates of B. ribis may be more virulent than
others on M. quinquenervia clones, and
3 stress factors may influence disease severity caused by
B. ribis on M. quinquenervia.


7
General Objectives
In general, the following objectives were considered in
testing the hypotheses mentioned above.
1. Establish mycological characteristics of B. ribis
isolates included in the experiments.
2. Evaluate B. ribis isolates for pathogenicity to M.
quinquenervia clones.
3. Test virulent isolate(s) for disease development on
stressed M. quinquenervia plants.
4. Analyze the histopathology of disease development on
inoculated stems.
Chapter Organization
This research work will be discussed in six chapters that
follow: "Mycological Characteristics of the Isolates of
Botryosphaeria ribis" (Chapter II); "Evaluation of Botryosphaeria
ribis Isolates for Pathogenicity to Melaleuca quinquenervia"
(Chapter III); "Effects of Stress Factors on Canker Development on
Melaleuca quinquenervia Inoculated with Botryosphaeria ribis"
(Chapter IV); "Histopathology of Botryosphaeria ribis in Melaleuca
quinquenervia Stems" (Chapter V); and "Summary and Conclusions"
(Chapter VI). Each chapter is intended to be a self-contained
unit, including the literature review pertinent to that section.


CHAPTER II
MYCOLOGICAL CHARACTERISTICS OF THE ISOLATES OF
Botryosphaeria ribis
Introduction
Botryosphaeria ribis Grossenb. & Dug. and B. dothidea (Moug.: Fr.)
Ces. & de Not. are often treated synonymously as established by
von Arx and Muller (1954) during reclassification of amerosporous
pyrenomycetes, and later supported by the work of Witcher and
Clayton (1963). However, as B. dothidea was described by de
Cesati and de Notaris in 1863 and B. ribis was described later by
Grossenbacher and Duggar in 1911, the former name has the priority
over the later (von Arx and Muller 1954, Witcher and Clayton 1963,
Maas and Uecker 1984). Since then the names, B. dothidea to B.
ribis, have been synonymously used by many authors (Witcher and
Clayton 1963, Crist and Schoeneweiss 1975, English et al. 1975,
Spiers 1977, Brown and Hendrix 1981, Latorre and Toledo 1984, Mass
and Uecker 1984, Pusey et al. 1986, Pusey 1989). However, others
consider B. ribis and B. dothidea as two distinct species (Smith
1934, Luttrell 1950, Punithalingam and Holliday 1973, Davison and
Tay 1983, Webb 1983, Rumbos 1987, Ramos et al. 1991).
Botryosphaeria ribis (order, Pleosporales; family,
Botryosphaeraceae) (Luttrell 1973, Barr 1987) was originally
described from currants (Ribes spp.) in New York (Grossenbacher
8


9
and Duggar 1911). Additionally, they reported two anamorphs
associated with B. ribis, a Macrophoma-form (pycnidial
conidiomata), and a Dothiorella-form (stromatic conidiomata). The
authors also recognized the existence of two different strains of
B. ribis, 1) chromogenic (purplish-pink coloration on starch
paste) and strongly parasitic, and 2) nonchromogenic and
saprophytic to currants. The strains reported by Grossenbacher
and Duggar (1911) were confirmed by Shear et al. (1924) and
additionally, they noted that the microconidia either remained
intermixed with the macroconidia or were produced in separate
locules. Stevens (1926) supported the existence of the
chromogenic strain of B. ribis and reported its occurrence in the
native hosts of Cuba and southeastern United States. However,
Witcher and Clayton (1963) considered B. ribis and B. dothidea to
be synonymous and mentioned that the latters' pathogenicity was
not correlated with chromogenesis in culture media. They grew two
blueberry-derived isolates in Czapek's solution at various pH
levels and produced four color groups, i,e., clear, yellow, and
purple pigments in pH 5.0-7.0, and red at pH 9.0.
So far, the taxonomy of the Fusicoccum anamorph of B. ribis
is in a confused state (Morgan-Jones and White 1987). The type
species F. aesculi Corda was described and illustrated originally
by Saccardo (1880, 1886). Petrak (1922) considered Saccardo1s F.
aesculi as an anamorph of B. dothidea so he established a new
binomial, Dothiorella aesculi Petrak. Despite many names for this
anamorph in the literature, it is accepted that the name
Fusicoccum, as an anamorph of at least some species of
Botryosphaeria Ces.& de Not., such as B. ribis and B. dothidea,


10
seems logical (Sutton 1980, Morgan-Jones and White 1987, Rumbos
1987, personal communication with Punithalingam 1990).
Morphologically, no clear differences have been established
for B. ribis and B. dothidea. Hyphal color in growth media has
been reported to change from white to dark-gray or greenish-black
(Weaver 1974, Mass and Uecker 1984, Rumbos 1987). Swollen cells
and chlamydospores have been reported in B. ribis (Ramos et al.
1991). Macroconidia are produced in uni- or multilocular
pycnidial stroma and the majority of them are hyaline and
aseptate, though 1-3 septate brown conidia were also reported in
both fungal species (Spiers 1977, Mass and Uecker 1984, Rumbos
1987). Conidia and ascospores are multinucleate but all nuclei in
the conidium and ascospore are homokaryotic, i.e., genetically
similar (Wolf and Wolf 1939).
The literature on B. ribis appears largely unsettled in
regard to its taxonomy, especially the anamorphic stage i.e.,
Fusicoccum vs Dothiorella, and morphological characteristics of
the fungus. Additionally, the teleomorph stages are very rare.
Many mycologists and pathologists refer to the teleomorphic stage
of this fungus i.e., B. ribis, to avoid uncertainty about its
anamorphic taxonomy.
Because of the uncertainties associated with the fungus, the
first objective of this research was to investigate morphological
and reproductive characteristics of the isolates of B. ribis
obtained from the canker margins on the stems of M. quinquenervia
(Cav.) Blake and stem galls on Rhizophora mangle L. from South
Florida.


11
Materials and Methods
Isolate Acquisition and Identification
During 1989, Dr. Roger S. Webb isolated and performed
preliminary pathogenicity tests of five isolates of B. ribis which
he had obtained from stem canker margins of Melaleuca
quinquenervia (Cav.) Blake trees from the Loxahatchee National
Wildlife Refuge. These isolates were tentatively identified as B.
ribis and later sent to International Mycological Institute (IMI),
Kew, Surrey, England.
In 1990, I obtained and identified an isolate of the same
fungus from the canker margin of a tree in the same area of
Loxahatchee National Wildlife Refuge. These six isolates of B.
ribis derived during 1989-1990 were subjected to monoconidial
cultures and 11 monoconidial cultures (MCs)/isolate were tested
for pathogenicity using a leaf bioassay method described later in
this chapter. The most virulent monoconidial culture/isolate was
selected (one MC for each of six isolates) and henceforth referred
to as isolates BR-1 through BR-6. I obtained two other isolates
of B. ribis during 1993 from the necrotic gall-tissues of red
mangrove trees (Rhizophora mangle L.) collected from the coastal
region of South Florida by Dr. Webb. These two isolates were
identified by Dr. J.W. Kimbrough and me in the Department of Plant
Pathology, at University of Florida. These two isolates were also
subjected to monoconidial culture, henceforth called isolates BR-7
and BR-8. These eight isolates were used to study mycological
characteristics.


12
Mycelial Growth and Morphology
Four artificial growth media, potato-dextrose-agar (PDA)
(Difeo), cornmeal-agar (CMA) (Difeo), mycological-agar (MA)
(Difco), and 24 gm of starch (Difco) supplemented with 15 gm of
agar (Difco)/liter (SA), were used to study the radial growth
rates of the eight isolates of B. ribis. Each isolate was
inoculated on four petri plates per growth medium. The inoculum
consisted of a 3-mm disk removed from the edge of 5-day-old colony
on acidic-PDA (3.3 ml of 50% lactic acid/liter of PDA). The
plates were sealed with Parafilm (American National Can,
Greenwich, CT) after inoculation, randomized with respect to
location on the laboratory bench and maintained at 30 C 1,
using a 12 h diurnal cycle of fluorescent light. The radial
extent of hyphal growth of each fungal colony was measured along 4
premarked radii at the time the first colony touched the edge of
its plate. The mean colony extension rates per plate, expressed
as mm/day (3-day basis for PDA, CMA, and MA, and 6-day basis for
SA), was used to compare isolates.
Culture plates used for growth rate assessment were further
incubated in the same condition for 21 days from the date of
treatment initiation. These plates were used to study other
reproductive and morphological characteristics such as,
sporulation behavior, mycelial color, and hyphal diameters of the
representative isolates.
Mycelial morphology of B. ribis isolates grown in potato
dextrose broth (PDB) was studied. A drop of macerated mycelial
suspension was inoculated into PDB and incubated 4 days at room


13
temperature under continuous shaking (100 rpm) conditions.
Macerated hyphae were stained with lactophenol cotton blue to
visualize dimensions for measurement of their diameter.
Photomicrographs of those hyphae were taken using a light
microscope (Olympus/BMH system).
Vegetative Compatibility Among Isolates
A modified form of half-diallel mating system (Zobel and
Tolbert 1984) was used. The isolates were given 1 chance to mate
with each other. The experiment was replicated 3 times.
Fungal isolates were grown on acidified potato dextrose agar
(APDA) (Difco) for 3 days, and agar blocks of 3-mm diameter were
cut from the periphery of the actively growing colonies using a
sterile cork borer. One block from each of the pair of isolates
to be mated was placed on opposite sides of APDA in 8.5 cm
diameter petri plates. The plates were sealed with parafilm and
incubated at 29(1)C under a 12 h cycle of fluorescent light.
Observations were made on days 3, 7, 14, and 35 following
inoculation to detect colony reaction at the confrontation zones
where the two colonies met. The confrontation zones were
evaluated for pigmentation, hyphal behavior, and production of
asexual and sexual structures.
Pycnidial Production and Morphology
Presence or absence of pycnidia in or on the culture medium
was recorded. The diameters of 30 solitary and botryose


14
(multilocular) pycnidial stroma of BR-4, BR-5, BR-7, and BR-8 were
measured. These four isolates were chosen because they sporulated
in all media and represented pycnidial morphology of the remaining
isolates.
Pycnidial stroma were fixed in formalin:propionic acid
(FPA), dehydrated through butyl alcohol series, and infiltrated
and embedded in paraffin (Johansen 1940) Sections of 8-10 fim
thickness were cut using a rotary microtome, dewaxed through a
xylene series, passed through 100 and 90 percent ethanol, and
stained with Pianeze's IIIB (Vaughan 1914). Numbers of locules
per stromata was counted and photomicrographs were taken using
light microscopy (Nikon/OPTIPHOT II or Olympus/BMH).
Fully expanded leaves of M. quinquenervia were wound-
inoculated with a hyphal suspension of each of eight isolates of
B. ribis, incubated in the sterile moist-chamber, and allowed to
produce pycnidial stroma. Stems of M. quinquenervia ramets were
wound-inoculated with isolates of B. ribis and maintained under
greenhouse conditions. Some of these inoculated stems produced
pycnidia on the bark. Segments of leaves and stems with pycnidial
stroma were processed for sectioning and staining by methods same
as used for pycnidial stroma from artificial growth media.
Pycnidial morphology from these leaves and stems were also noted
and photographed. These stromatic pycnidia from the leaves were
also used for the study of macroconidial characteristics.


15
Conidial Characteristics
Macroconidial length, width, and the number of nuclei were
studied using 14- to 18-day-old stromatic pycnidia from the M.
quinquenervia leaves inoculated with each of the eight isolates of
B. ribis. Pycnidia were placed on a glass slide, covered with a
drop of Haupt's adhesive, crushed with a cover slip, smeared, and
were allowed to dry for 24 h at room temperature. The
macroconidia were then stained with Pianeze's IIIB (Vaughan 1914),
and the number of nuclei in each of 100 macroconidia were recorded
for each isolate.
Germination of Macroconidia
Spore germination tests were performed using slight
modifications of the procedures of Brooks and Ferrin (1994) .
Isolate BR-1 and BR-7 were selected to represent isolates from M.
quinquenervia and red mangrove, respectively, both produce
superficial pycnidial stroma on PDA and hence macroconidia could
be extracted easily.
Isolates BR-1 and BR-7 were grown on PDA under a 12 h/day
fluorescent light period at 30(1)C for 2 wk. Mature,
superficial or partially submerged pycnidia were partially dried
under sterile conditions by temporarily removing lids from the
petri-plates. Within 5-10 minutes following this partial
desiccation treatment, masses of macroconidia were extruded from
the pycnidia onto the surface of PDA. Conidia were washed from
the medium with sterile distilled water and the resultant


16
macroconidial suspension was strained through 2 layers of sterile
cheesecloth. One drop of spore suspension was smeared on each of
3 premarked strips on water agar (WA) in petri plates. The petri
plates were then sealed with Parafilm. Seven culture plates of
each isolate were incubated at 5, 10, 15, 20, 25, 30, 35, 40, and
45C. One culture plate of each isolate per temperature treatment
was removed at 1, 2, 4, 8, 12, 16, and 24 h following incubation.
Following removal, the strips on WA containing germinating
macroconidia were immediately stained with cotton blue in
lactophenol and 100 macroconidia per strip, i.e., a total of 300
macroconidia per plate, were counted and the number of germinated
macroconidia were recorded for each plate. A spore was considered
germinated when the germ tube was equal or greater than the spore
length. Germination of macroconidia for each isolate per
temperature was expressed as a percentage. The experiment was
repeated twice using a different batch of WA and macroconidia.
Relevant photomicrographs of fungal materials were taken using
light microscopy (Olympus/BMH or Nikon/OPTIPHOT II).
Statistical Analyses
Wherever necessary, variances and means of main effects were
analyzed using GLM procedure in SAS (1985) Logistic procedures
in SAS (1985) were used to analyze maximum likelihood of the
effects of temperature and incubation periods on macroconidial
germination and their relationships were used to derive equations
to explain the combined effects of these two variables.


17
Correlation of temperature and incubation period with spore
germination was tested using Pearson's method.
Results
Fungal Identification
Identity of the fungal isolates collected during 1989 from
stem-canker margin of M. quinquenervia was confirmed by Dr.
Punithalingam from IMI, Kew, Surrey, England, as the Fusicoccum
anamorph of B. ribis. I had tentatively identified one isolate
derived from similar trees in 1990 and two other isolates derived
from cankered galls on red mangrove to be the Fusicoccum anamorph
of B. ribis. Those identifications were later confirmed by Dr.
J.W. Kimbrough of the Department of Plant Pathology, at University
of Florida.
Mycelial Growth and Morphology
The radial growth rates of the isolates of B. ribis in four
growth media are presented in the Table 2.1. In general, PDA and
MA supported the most rapid growth while SA produced the slowest
for most isolates. The growth rate of some isolates on a
particular medium was also significantly different.


18
Table 2.1. Radial growth rates (mm/day) of the mycelia of B.
ribis isolates in potato dextrose agar (PDA), cornmeal agar (CMA),
mycological agar (MA), and starch agar (SA).
Isolate no.
Growth rates
(mm/day) in
PDA
CMA
MA
SA
BR-1
10.9A,a
9.2A,b
12.5A,a
6.8A,b
BR-2
13.8A,a
10.1A,b
14.1A,a
6.3A,c
BR-3
13.8A,a
10.4A,b
14.3A,a
4.9A,C
BR-4
12.4A,a
10.1A,b
13.0A,a
4.9A,c
BR-5
10.5A,a
9.7A,ab
10.4B,a
6.8A,b
BR-6
13.3A,a
8.3A,b
14.2A,a
5.4A,b
BR-7
11.3A,a
7.0B,b
11.1A,a
5.7A,b
BR-8
12.7A,a
9.8A,b
14.3A, a
6.0A,c
Note: Since isolate-media interaction was significant(Pr>F=0.0001), Scheffe's multiple
comparison procedure was used to locate significant differences between least square means
indexed by both isolate and medium. Means followed by the same letters in upper case
(comparing columns) and lower case (comparing rows) are not significantly different
(minimum significant difference=3.35 mm at P=0.05).
Some of the isolates appeared to impart color on SA, PDA,
and MA during the incubation period. However, a close observation
with the aid of a light microscope revealed that the colors were
due to a dense network of smaller diameter hyphae submerged in the
given growth medium.
Mycelial color in the center of the colony of all except BR-
6 changed from white to tan within as early as 48 h following
incubation on PDA and MA. The mycelial color of all isolates
changed from white to light yellowish-green in SA within 72 h
following incubation. A pigment similar to that of mycelial color
was also secreted in SA surrounding the submerged mycelial
network. The mycelia of all isolates remained colorless in CMA
except BR-7 which turned light yellowish-green within 72 h
following incubation. The mycelial color variation among isolates


19
in four growth media was easily noticeable 21 days after
incubation (Table 2.2).
Texture of mycelia ranged from cottony in PDA to floccose in
MA. Aerial hyphae observed at 21 days after incubation were
relatively longer in PDA and MA than in CMA and SA (Figs. 2.1,
2.2). However, isolates BR-7 and BR-8, respectively, did not
produce any aerial hyphae in CMA and SA (Table 2.2). Hyphal
diameter of a 21-day-old fungal culture ranged from 1.8-10.2
(4.2), 1.2-10.2 (5.0), 2.8-10.2 (5.3), and 2.6-12.8 (5.3) /min
CMA, MA, PDA, and SA, respectively.
Table 2.2. Production of aerial hyphae, mycelial color, and
pycnidial stroma by isolates of B. ribis in four artificial growth
media at 21 days after inoculation.
Isolate number
BR-l BR-2 BR-3 BR-4 BR-5 BR-6 BR-7 BR-8
Mycelial color:
Potato dextrose agar
Corn meal agar
Mycological agar
Starch agar
Aerial hyphae:
Potato dextrose agar
Corn meal agar
Mycological agar
Starch agar
Pycnidial stroma:
Potato dextrose agar
Corn meal agar
Mycological agar
Starch agar
B
B
B
LB
LYB
LYB
W
LYB
LB
LB
LB
LB
YG
YG
LYG
YG
AM
AM
AM
AS
CS
AS
AS
BS
AS
AS
AS
AS
CS
CS
CS
DS
A
D
D
A
A
D
B
A
D
D
B
A
B
D
C
A
B
B
B
B
LYB
W
W
W
LB
T
B
B
YG
YG
YG
LYG
AM
AL
AM
AM
CS
CM
--
BS
AS
AS
AS
AS
BS
DS
DS
B
N
A
A
D
N
A
B
C
D
B
B
D
C
A
A
Note. Mycelial color: B=brown, LB=light brown, LYB=light yellowish-brown, YG=yellowish-
green, W=white, and T=tan, Aerial hyphae: Surface coverage of total media A=100%, B=50-
99%, C=26-49%, D=l-25; and upward mycelial growth L=up to petri dish cover, M=midway
between media surface and petri dish cover, --=not visible on media surface, and S= short
cottony on media surface. Pycnidial stroma: A= >100/plate, B=50-99/plate, C=26-49/plate,
D=l-25/plate and N=none.


20
Short-celled inflated hyphae (Fig. 2.3) were abundant across
the four media. Compared with the remaining three media, SA
contained a large number of hyphae with chains of chlamydospore-
like cells in the intercalary positions. The majority of cells in
these hyphae were guttulated. In contrast, mycelia in MA were
highly melanized compared with the mycelia in remaining three
media. The majority of 4-day-old hyphae grown in potato-dextrose-
broth under continuous shaking conditions consisted of chains of
chlamydospore-like cells of the diameter of up to 18.6 ¡im (Fig.
2.4) .
Vegetative Compatibility Among Isolates
The pairs of isolates can be divided into 3 groups based on
the pigmentation at the zone of confrontation of the two colonies
(Table 2.3): 1) no pigmentation by day 3, colonies diffused into
each other, and development of brown coloration similar to the
rest of the colony by day 7; 2) no pigmentation by day 3 but
mycelia not diffused into the opposite colonies and production of
dark brown coloration at the zone of confrontation; and 3) brown
pigmentation by day 3 but the mycelia not diffused into the
opposite colonies and zone of confrontation later intensely dark
brown. Microscopic observation of the agar blocks in groups 2 and
3 along with the mycelia from the zone of confrontation revealed
purplish brown pigmentation on the media around the mycelial mats.


21
Table 2.3. Vegetative and reproductive behavior of the pairs of
the eight isolates of B. ribis on and around the zone of
confrontation (the meeting point of two colonies growing towards
each other) on PDA.
Isolate
pairs
Zone Pi
Pigment
confrontation
Mycelial color
Sclerotia
Micro
conidia
Macro
conidia
Teleomor
ph stage
Groups
BR-lxBR-2
none
brown
(2 f)
(1)
(1)
none
1
BR-lxBR-3
none
brown
none
none
(1)
none
1
BR-lxBR-4
none
brown
none
(4)
(1,4)
none
1
BR-lxBR-5
none
brown
none
none
(1,5)
none
1
BR-lxBR-6
brown
dark brown
(6 f)
none
(1)
none
2
BR-lxBR-7
none
dark brown
none
none
(1,7)
none
2
BR-lxBR-8
none
dark brown
(8 f)
(8)
(1,8)
none
2
BR-2XBR-3
none
brown
(2 f)
none
(2,3)
none
1
BR-2XBR-4
none
brown
(2 f)
(4)
(2,4)
none
1
BR-2XBR-5
none
brown
none
(5)
(5)
none
1
BR-2xBR-6
brown
dark brown
(6 nf)
(6)
(6)
none
3
BR-2xBR-7
brown
dark brown
none
none
(7)
none
2
BR-2xBR-8
brown
dark brown
(8 f)
(8)
(8)
none
2
BR-3XBR-4
brown
dark brown
none
(4)
(3,4)
none
3
BR-3XBR-5
none
brown
none
none
(5)
none
1
BR-3XBR-6
brown
dark brown
none
none
none
none
3
BR-3XBR-7
none
brown
none
none
(7)
none
1
BR-3XBR-8
brown
dark brown
(8 f, 3
nf)
none
(8)
none
3
BR-4XBR-5
none
brown
none
(4)
(4,5)
none
1
BR-4XBR-6
brown
dark brown
(6 f)
(4)
(4,6)
none
3
BR-4XBR-7
brown
dark brown
none
(4)
(4,7)
none
3
BR-4XBR-8
brown
dark brown
(8 f)
(4)
(4,8)
none
3
BR-5XBR-6
brown
dark brown
(6 f)
(5,6)
(5,6)
none
3
BR-5XBR-7
none
dark brown
none
none
(5,7)
none
2
BR-5XBR-8
none
dark brown
(8 f)
none
(5,8)
none
2
BR-6XBR-7
brown
dark brown
(6 f)
(6)
(6,7)
none
3
BR-6XBR-8
brown
dark brown
(6 f, 8
f)
none
(6,8)
none
3
BR-7XBR-8
none
brown
(8 f)
none
(7,8)
none
1
Note: Numbers in the parenthesis under respective columns indicate the isolate numbers that produced sclerotia or
microconidia or macroconidia. Also, the 'f' and 'nf' in the parenthesis indicate presence and absence of
pycnidial sclerotia, respectively. Pigmentation and the mycelial colors at the zone of confrontation were noted
at day 3 and 7, respectively, following the date of inoculation onto PDA. The record of the presence of
sclerotia, microconidia, macroconidia, and teleomorph stages were based on the day 14 and 35, respectively,
following the date of inoculation onto PDA. Groups 1, and 2 and 3, represent vegetative compatibility and
noncompatibility, respectively.
In addition to pycnidia, isolates BR-2, BR-6, and BR-8
produced sclerotia on the surface of the media (Table 2.3). The
majority of those sclerotia contained pycnidial locules bearing
conidia. In one combination or another, the isolates BR-1, BR-4,


22
BR-5, BR-6, and BR-8 produced microconidia, whereas macroconidia
were produced by all the isolates (Table 2.3). Often, the
pycnidial concentration was intense at the zone of confrontation
of two colonies. However, as revealed by the observations of the
plates for 35 days following inoculation date, all pairs of
isolates failed to produce teleomorph stages (Table 2.3).
Pycnidial Production and Morphology
In general, production of pycnidial stroma was greater on
PDA followed by SA, CMA, and MA (Table 2.2). The best and the
least sporulation across four growth media at 12 h fluorescent
light cycle and 30C was achieved in isolates BR-4 and BR-6,
respectively (Table 2.2).
In SA and CMA, the pycnidial stroma were scattered and
submerged, whereas in PDA and MA, both superficial and submerged
pycnidia were produced (Figs. 2.1, 2.2). In general, the range
and average diameter of pycnidial stroma varied among growth media
(Table 2.4). Superficially produced pycnidial stroma were of
larger diameter than the submerged ones and were often seen
covered with appendage-like hyphae. Most of the submerged
pycnidia were solitary and unilocular, whereas most of the
superficial pycnidia were multilocular (botryose) and as much as
31 locules were observed in BR-8 (Figs. 2.5, 2.6). The diameter
of pycnidial cavities ranged from 57-200 (ave. 124) /xm. Across
isolates, the majority of 21-day-old pycnidial stroma in PDA, CMA,
and SA sporulated while a majority were pseudopycnidial in MA.


23
Pycnidial stroma in and on M. quinquenervia leaves were
produced within 3-14 days following inoculation. Both solitary
and botryose types of pycnidia were observed on host tissues
(Figs. 2.7, 2.8, 2.9, 2.10). Based on the mode of formation,
pycnidial stroma on leaves and bark of the host plant were of 2
kinds, 1) superficial on the surface and 2) erupting through the
epidermis (Figs. 2.9, 2.10). A majority of pycnidial stroma
produced on the surface were covered with short erect hyphae while
these structures were lacking in the erupting types. Both
solitary and botryose (Figs. 2.8, 2.10) pycnidia were produced on
the bark of the stems of M. quinquenervia. The majority of
pycnidia produced on the bark were immersed or submerged in the
layers of phellodermal tissues.
Immersed or submerged pycnidia were globose and the botryose
or eustromatic ones were lobulate and often had flat tops. Either
kind of pycnidia was ostiolate and the ostioles were often obscure
(Figs. 2.6, 2.8, 2.10). Superficial pycnidia produced
macroconidial cirri (Fig. 2.11) following partial drying by aerial
exposure for a few minutes. The pycnidial wall consisted of
heavily melanized cells of irregular textura-angularis becoming
gradually loose and hyaline towards the inner lining of the cavity
that produced conidiophores (Fig. 2.12). Conidiophores were short
and unbranched with bulbous bases produced from the lining of the
stromatal wall of the pycnidial cavities (Fig. 2.13). Mode of
conidial production was holoblastic and successional.
Microconidial production was very rare among most isolates. When
present, both macro- and microconidia (Fig. 2.14) were produced in
the same or separate pycnidial stroma.


24
Table 2.4. Range and average (parenthesis) diameter (mm) of the
pycnidial stroma on 2- to 4-week old culture of some B. ribis
isolates.
Isolate
Average diameter (mm) in
no.
PDA
CMA
SA
MA
BR-4
BR-5
BR-7
BR-8
0.1-1.0 (0.5)
0.1-1.3(0.6)
0.1-1.2 (0.5)
0.1-3.8(2.3)
0.1-0.5 (0.2)
0.1-1.0 (0.4)
0.1-0.5 (0.2)
0.3-1.2 (0.5)
0.1-0.5(0.2)
0.1-1.0 (0.4)
Table 2.5. Mean and range of dimensions (length and width) and
the number of nuclei in macroconidia.
Isolate
Dimensions (/x m)
Average Ranges
Number
of nuclei
Length
Width
Length
Width
Average
Range
BR-1
16.8c
6.2
5.7-23.0
4.1-8.9
6.0
1.0-12.0
BR-2
16.6c
6.2
8.9-23.0
4.3-8.9
5.0
1.0-11.0
BR-3
18.4a
6.2
14.0-23.2
3.3-8.9
5.0
1.0- 9.0
BR-4
17.9ab
6.1
8.9-26.8
4.1-7.9
5.0
1.0-11.0
BR-5
17.3bc
6.1
11.5-23.0
4.1-8.4
5.0
1.0-11.0
BR-6
18.Oab
6.2
7.7-23.0
3.1-8.9
4.0
1.0- 8.0
BR-7
16.7c
5.3
10.2-20.4
3.8-6.4
3.0
1.0- 8.0
BR-8
16.8c
5.5
9.7-21.0
3.9-6.5
4.0
1.0- 7.0
Note: B.
ribis isolates, i.e..
BR-l through BR-
6 from M.
quinquenervia, and BR-7 and BR-8
from R. mangle) from 21-day-old pycnidial stroma from inoculated M. quinquenervia-leaves.
For each isolate, N=100. Means with same letter(s) are not significantly different from
each other at P=0.05, according to Duncan's Multiple Range Test.
Conidial Characteristics
Macroconidia in younger (3- to 7-day-old) pycnidia across
isolates were fusoid, hyaline, and aseptate with truncate bases
(Fig. 2.14) when young and fresh, but some of them produced 1-4
septa in 2-week or older pycnidia (Fig. 2.15). Septation and


25
bluish-brown pigmentation was observed in some macroconidia
retained in cavities of pycnidia produced on host and growth
media. But, those extruded in the form of cirrhi remained hyaline
and aseptate even at 12 weeks of drying at room temperature. Mean
length of macroconidia were significantly different among isolates
(Pr>F=0.0001), and the differences in range of dimensions were
obvious (Table 2.5). When present, the microconidia (Figs. 2.14,
2.15) were allantoid to shortly elongated, hyaline, and
unicellular with a range of length and diameter of 2.7-5.1
(average 3.7) /im and 1.3-2.5 (average 1.6) /im, respectively.
Germination of Macroconidia
Most of the macroconidia germinating on WA were aseptate; a
few contained 1-3 septa. However, the majority of macroconidia
from the same batch developed 1-3 septa when germinated in water
under a coverslip (Figs. 2.15, 2.16). Regardless of the numbers
of septa, 1-4 germ tubes (majority with 2 germ tubes, 1 on each
end) were produced by germinating macroconidia within 16 h of
incubation (Fig. 2.16) at 30C. None of the microconidia, either
in water under a coverslip or on WA, germinated at 16 h after
incubation at 30C.
In WA, germination of macroconidia began as early as 1 h
after incubation at 30-35C (Figs. 2.17, 2.18). Within 4 h at 20-
35C germination had concluded and was over and below 90% in BR-1
and BR-7, respectively. However, after 24 h at 20-40C, spore
germination was 93-100% and 83-98%, in BR-1 and BR-7,
respectively. The correlation between germination and


26
temperature, and germination and incubation using 126 observations
for both isolates was weak (Pr>R=0.088), and strong (Pr>R=0.0001),
respectively. At a temperature range of 20-35C, a majority of
macroconidia in BR-1 and BR-7 germinated within 4 h of incubation
(Table 2.6 and Figs. 2.17, 2.18). Isolate BR-1 (isolated from
melaleuca canker) revealed a broad optimum temperature range (2 0-
30C) for germination of macroconidia opposed to a narrow range
(25C) for BR-7 (isolated from mangrove trees) (Table 2.6). In
both isolates, spore germination was insignificant at 5 and 45C.
At 45C, about 5% of the macroconidia in both isolates spilled
cellular contents onto the WA at 16 h after incubation.
Table 2.6. Effect of temperature on germination of macroconidia
by 4 h after incubation in WA at 100 percent humidity.
Percentage
of spore germination
at C
Isolate
5
10
15
20
25
30
35
40
45
BR-1
0.2
1.7de
0.0
0 Oe
3.7
11. Od
91.2
72.8ab
94.8
77.2ab
98.3
82.7a
90.3
72.0b
46.5
43.0c
0.0
O.Oe
BR-7
0.0
0 Of
0.0
0 Of
2.5
10. Oe
55.2
48.0c
89.8
71.5a
82.0
65.0b
64.3
53.4c
25.5
30.3d
0.0
O.Of
Note: Mean comparisons were performed for 4 h incubation period only since at the
temperature range of 20-35C, more than 50% spores germinated during this period. For each
isolate, numbers on the upper row represent actual percentages of spores germinated by 4 h.
Numberts in the second row for each isolate represent corresponding means from transforming
back to percentage the arcsin-square-root of X scale used in the analysis of data (Arauz
and Sutton 1989). Corrected percentages in the second row followed by the same letter are
not significantly different from each other at P=0.05 (minimum significant difference for
BR-1 and BR-2 was 10.65 and 5.67, respectively).
Inocula of isolates BR-1 and BR-2 appeared to vary in their
ability to survive in extremely low temperatures. Macroconidia of
BR-1 were viable after 3-month storage at -20C, whereas for
isolate BR-7 viability was lost after the same period of storage.


27
Initiation of the germination of macroconidia stored at -20C
appeared to be delayed by as much as 24 h.
The combined effects of the temperature and incubation
period on germination of macroconidia at 100 percent relative
humidity using data from 5-45C was described by the following
equation derived through logistic regression:
G(Isolate BR-l) = -12.856 + 0.255 INC+0.927 TEM-0.017 TEM2
G(Isolate BR-7) = -12.856+(0.255- 0.016 ) INC+(0.927-0.036 ) TEM
+(-0.017+0.006) TEM2
where G = logit (percent germination), INC = incubation
period, and TEM = temperature treatment (C) .
Discussion
Fungal Morphology and Taxonomy
A vast confusion exists in the literature regarding the
taxonomic identity of B. ribis and B. dothidea, and their
anamorph-teleomorph relationships. Since the description of B.
ribis by Grossenbacher and Duggar (1911), this fungus has been
reported to cause diseases on many hosts; frequently mycologists
and plant pathologists have stressed teleomorphic names to avoid
the taxonomic uncertainty of the anamorphic states (Shear et al.
1924, Smith 1934, Luttrell 1950, Wiehe 1952, Witcher and Clayton
1963, Schreiber 1964, Crist and Schoeneweiss 1975, English et al.
1975, Spiers 1977, Filer and Randall 1978, Shearer et al. 1987,
Brown and Hendrix 1981, Davison and Tay 1983, Webb 1983, Latorre


28
and Toledo 1984, Mass and Uecker 1984, Rumbos 1987, Ramos et al.
1991). However, in most of these studies the teleomorph stages
were not produced.
Some authors have referred to B. ribis as having an anamorph
belonging to Dothiorella (Wolf and Wolf 1939, Smith 1934, Anahosur
and Fazalnoor 1972, Smith and Fergus 1971, Webb 1983), while
others have referred to it having anamorphs in Fusicoccum
(Punithalingam and Holliday 1973, Morgan-Jones and White 1987).
Evidence from the results related to morphological aspects of the
vegetative and reproductive structures and the literature suggests
that the controversy in taxonomic position of B. ribis and B.
dothidea is due mainly to 1) variable mycelial and conidial
morphology in different growth conditions and stages, 2) rareness
of sexual stages, 3) pantropic distribution, and 4) ability to
cause diseases on many plant species.
In the present study, mycelial characteristics such as
growth pattern, color change over time, and dimensions in PDA
(Tables 2.1, 2.2) agrees with the mycelial characteristics
described for B. ribis (Morgan-Jones and White 1987) Most
descriptions of the anamorphic stages of B. ribis and B. dothidea
overlap. For example, the measurements (Table 2.5), and the
nature of pycnidia (Figs. 2.5, 2.6, 2.7, 2.8, 2.9, 210, 2.11) and
conidiophores (Fig. 2.12, 2.13), as well as fresh macroconidia
from my studies agree with Wolf and Wolf (1939), Punithalingam and
Holliday (1973), and Sivanesan's (1984) description for B. ribis.
The immersed or submersed pycnidia in four artificial media were
smaller than superficial pycnidia. Also, pycnidial stroma in PDA
were relatively larger than the pycnidia produced in CMA, MA, and


29
SA (Tables 2.1, 2.2). Based on these observations and the
evidence from literature (Grossenbacher and Duggar 1911, Shear et
al. 1924, Morgan-Jones and White 1987), I suggest that the
pycnidial dimension in the artificial media is influenced by the
quality of nutrient.
The possible pairing of all eight isolates of B. ribis did
not yield a teleomorph stage. Mass and Uecker (1984) had a
similar experience with B. dothidea that causes stem canker in
blackberry. According to the descriptions given by Punithalingam
and Holliday (1973), the ascal and ascospore dimensions of B.
ribis are 100-110 x 16-20 and 17-23 x 7-10 /xm, respectively. The
measurements given for asci and ascospores of B. dothidea are 65-
140 x 16-21 and 13-35 x 6-14 /xm, respectively (Pennycook and
Samuels 1985). These measurements reveal that the ascal and
ascospore dimension of B. ribis falls within the range of the
dimensions of these structures of B. dothidea. Therefore, I
suggest that the taxonomic identity of these so-called two
distinct species needs to be reviewed.
Based on the presence of macro- and microconidia, Morgan-
Jones and White (1987) considered their isolates obtained from the
leaf of Smilax to be the Fusicoccum anamorph of B. ribis. Though
rare, microconidia were produced by almost every isolate I have
studied. Microconidia in the present study were of larger
dimensions then those reported by Wolf and Wolf (1939),
Punithalingam and Holliday (1973), Sutton (1980), Pennycook and
Samuels (1985), and Sivanesan (1984), but were similar to those
described by Morgan-Jones and White (1987). These microconidial


30
characteristics are consistent with the previous descriptions of
B. ribis and its anamorph Fusicoccum.
The separation of the genera Dothiorella and Fusicoccum
seems to be based on the form of conidiomata, the mode of
conidiogenesis, and the nature of conidia. The criteria of
holoblastic mode of conidiogenesis yielding hyaline to brown
conidia with aseptate-to-biseptate conditions for Dothiorella, and
enteroblastic phialidic conidiogenesis producing hyaline to light
brown aseptate conidia for Fusicoccum, has been used (Barr 1987)
to differentiate these two form-genera. Sutton (1980) examined
the illustrations of the type specimen of Saccardo (1886) and
found holoblastic conidiogenesis for F. aesculi. Though Pennycook
and Samuels (1985) agree with Sutton (1980), they noted that
Saccardo's specimen was immature. It has been demonstrated that
the older conidiogenous cells are enteroblastic phialidic forming
2-3 irregularly shaped annelations (Mass and Uecker 1984,
Pennycook and Samuels 1985).
The upper range of the dimensions, and color and aseptate
condition of macroconidia from the younger pycnidia in my studies
(Table 2.5) closely match the ranges of the dimension of
macroconidia reported by Weaver (1974) for B. dothidea. Septate
and pigmented conidia have been reported in B. ribis (Wiehe 1952,
Taylor 1958, Rumbos 1987) as well as in B. dothidea (English et
al. 1975, Spiers 1977). However, Pennycook and Samuel (1985)
mentioned that the conidia of B. dothidea do not become light
brown or septate with age or at the time of germination.
Septation of macroconidia when germinated under water (Figs.
2.15, 2.16) and those retained in dry pycnidia were similar to


31
those described by Mass and Uecker (1984) for B. dothidea from the
cankers on thornless blackberry. Also, the uni- as well as bi
polar and sometimes lateral germ-tube production (Fig. 2.16) fits
to the description of English et al. (1975) for B. dothidea.
However, the majority of macroconidia germinated on water agar at
100% relative humidity were aseptate. The septation of some of
the older and/or germinating conidia in my studies create doubt
since reportedly the macroconidia of Fusicoccum are not septate
(Sutton 1980). I suggest that the septation of macroconidia of
the anamorphs of B. ribis and B. dothidea may be related to the
level of oxygen/carbondioxide or combinations of some other
factors in their microenvironment which are yet to be discovered.
Thus, it appears that the morphological characteristics of
B. ribis and B. dothidea, especially of their anamorphs, overlap
and become difficult to be separated from one another. For
example, Michailides (1991) isolated a pycnidial fungus from
panicle and shoot blight of pistachio from California and
identified as B. dothidea, but he reported that the isolates fit
the description of the pycnidial stage of B. ribis. As discussed
by Morgan-Jones and White (1987), small variations in conidial
dimensions occur in different circumstances.
The separation of these form genera Dothiorella and
Fusicoccum may have been based on their morphological characters
observed at a certain stage of development at a given
microenvironment. If hyphal and conidial characteristics of some
of the anamorphic stages of these two fungi are studied side by
side at similar conditions and followed through different stages
of development, it may be difficult to separate the taxonomic


32
identity of these two fungal species. My opinion is further
supported by the view of some other authors while discussing the
anamorph-teleomorph connections of these two teleomorph stages
(Pennycook and Samuel 1985, Morgan-Jones and White 1987,
Michailides 1991).
In light of my results and the evidence from literature, the
isolates of fungi obtained from M. quinquenervia and R. mangle are
closer to the Fusicoccum complex. However, the systematic
position of the form genera Dothiorella and Fusicoccum, and their
teleomorphs {B. ribis and B. dothidea) need to be reviewed.
Germination of Macroconidia
The studies of representative isolates, BR-1 and BR-7 from
M. quinquenervia and R. mangle, respectively, revealed that the
macroconidia of both isolate are capable of germinating at 5-40C
with a range of 25-35C being optimum for germination (Fig. 2.17,
2.18). Similar range of optimum temperature range for germination
of macroconidia has been reported for B. dothidea from California
(Brooks and Ferrin 1994). However, the isolates, BR-1 and BR-7
differ in the speed of the germination of their macroconidia.
Macroconidia of isolate BR-1 had >90% germination within 4 h at
25-35C, while macroconidia of BR-7 needed 8 h to achieved 90% at
the same range of temperature (Table 2.6 and Figs. 2.17, 2.18)
The isolates, BR-1 and BR-7 had a similar threshold for
survival at high temperature (45C) but differed at a low
temperature threshold. It was interesting to note that
macroconidial suspensions (in sterile distilled water) and aerial


33
hyphae (in sealed petri plates) of BR-1 remained viable for a
period of 3 months at -20C, whereas similar inocula of BR-7 lost
their viability in comparable period and storage conditions. The
isolate BR-1 came from inland trees growing in a fresh water swamp
and the BR-7 came from trees in coastal swamps of saline water.
Such a differential tolerance of these two isolates to low
temperature may be related to adaptation in their respective
environment, though actual temperature differentials may vary only
moderately between these areas. Based on the results from the
observations of variables such as temperature and incubation
period, no conclusion can be drawn regarding the variable
adaptability of this fungal species. Arauz and Sutton (1989) have
reported natural variability between isolates of B. obtusa
collected from western and central North Carolina. The isolate
coming from drier region required high temperature and low
relative humidity to germinate than the one that came from
relatively wet area.
In general, it appears that isolate BR-1 has better chance
of survival than BR-7 among a population of susceptible trees
where freezing temperature is frequent in inland ecosystems. In
general, it is assumed that the high temperature along with yet to
be discovered factors may have been limiting this fungus from
building its population to an epidemic level among trees of
susceptible species though it is endemic to South Florida.


34
Conclusions
Eight tentatively identified isolates of B. ribis: six from
M. quinquenervia, and two from R. mangle were obtained from South
Florida. Morphological characteristics of these isolates were
studied using artificial growth media (CMA, MA, PDA, and SA), and
excised leaves and potted ramets of M. quinquenervia.
Mycelial morphology and growth rate of isolates in
artificial media varied, and the overall best growth-rate was
achieved on MA and PDA. Sporulation intensity and dimension of
conidiomata varied by isolates and growth media and except BR-6,
the majority of isolates sporulated best on PDA, SA, and excised
leaves incubated in moist chamber in sterile condition.
Conidiophores were short and hyaline with a bulbous base arising
from the lining of the stromatal locules, and conidiogenesis was
holoblastic among younger conidiomata. Macroconidia from younger
conidiomata were fusiform, truncate at the base, obtuse at the
apex, hyaline, aseptate, but some of them developed 1-3 septa when
retained in dry conidiomata or germinated in water under
coverslips. Regardless of the number of septa, 1-5 germ tubes
were observed to be produced from polar as well as lateral sides
of conidia. Microconidia were rare but were produced by most
isolates, but none of them was observed to germinate.
In isolates BR-1 and BR-7, the percentage of macroconidia
germinating within 4 to 8 h was greater at 25-35C than outside
that temperature range. In both isolates, macroconidial
germination was at the least or absent at 5C, and viability was
lost at 45C.


35
The teleomorph stage was not observed at any growth media
and conditions. Morphological characteristics revealed the
isolates to be closer to the Fusicoccum anamorph of B. ribis.
From variation in characteristics of mycelia, conidiomata, and
conidia of the isolate(s) among different growth media and
microenvironment examined, I conclude that the so-called
teleomorph(s) B. ribis or B. dothidea and their anamorph(s)
Fusicoccum and Dothiorella complexes are highly variable. The
variation in their morphological characteristics may have root in
their adaptive ability in a given host or microenvironment.
Therefore, their taxonomic relationships need to be further
clarified. I suggest that these two teleomorphs are synonymous
and so are their two anamorphs.


Figs. 2.1-2
Figure 2.1.
Figure 2.2.
Figure 2.3.
Figure 2.4.
Figure 2.5.
Figure 2.6.
.7 Mycelial and pycnidial characteristics of the
Fusicoccum anamorph of B. ribis on PDA, CMA, PDB,
and M. quinquenervia leaf.
Aerial hyphae imparting black color and partially
submerged pycnidia (arrows) on PDA.
Hyphae in and on the surface of media imparting
tan color and pycnidia (arrow) immersed in CMA.
Mycelial mat on PDA with hyphae of various
diameters. Bar=50 /xm.
Blended mycelial mat grown in PDB under
continuous shaking conditions (100 rpm). Note
hyphae containing chains of chlamydospore-like
cells (arrows) Bar=50 ¡xm.
Cross-section through a multilocular pycnidial
stroma produced on PDA. Note numerous locules
(lo) Bar=500 /xm.
Enlarged pycnidia from a portion of multilocular
stroma. Note macroconidia (arrow) and an obscure
ostiole (os) Bar=100 /xm.
Pycnidial stroma on the leaf inoculated and
maintained in a sterile moist chamber. Note
solitary (arrows) and botryose (arrow heads)
types of pycnidia.
Figure 2.7.


37


Figs. 2.8-2.16. Pycnidia and macroconidia of B. ribis associated
with artificial growth media and M. quinquenervia
leaves and stems.
Figure 2.8.
Pycnidial stroma erupting from dead bark. Note
solitary (arrow) and botryose (arrow heads) types
of pycnidia.
Figure 2.9.
Section through leaf showing a solitary pycnidium
erupting from epidermis. Note leaf epidermis
(arrow), ostiole (os), and pycnidial wall (arrow
head) Bar=50 /xm.
Figure 2.10.
Longitudinal section through stem showing a
solitary pycnidium erupting through bark. Note
an obscure ostiole (os), and pycnidial wall (pw)
composed of textura angularis. Bar=50 /xm.
Figure 2.11.
Pycnidial stroma on leaf oozing macroconidial
cirrhi(arrows) on drying at room temperature.
Figure 2.12.
A portion from the cross-section through a
pycnidium showing pycnidial wall (pw) composed of
textura angularis, and conidiophores (arrows).
Bar=25 /xm.
Figure 2.13.
A portion of the inner wall of pycnidium squashed
from a 3-day-old pycnidium. Note conidiophores
with bulbous base (arrow) bearing macroconidia
(arrow head) at different stages of development.
Bar=25 /xm.
Figure 2.14.
Macro- (arrow) and microconidia (arrow heads)
from freshly squashed 7-day-old pycnidium. Note
the microconidia are allantoid-slightly elongated
and the macroconidia are truncate base and obtuse
apices. Bar=25 /xm.
Figure 2.15.
Macroconidia (from 3-day-old pycnidium) from PDA
germinated in water. Note a mixture of aseptate
and septate conidia some of them bearing germ
tubes (arrows) Bar=50 /xm.
Figure 2.16.
Germinating aseptate (arrow) and septate (arrow
heads) macroconidia showing one or more germ
tubes. Bar=50 /xm.


39


40
Figure 2.17. Germination of the macroconidia of isolate
BR-1 of Botryosphaeria ribis on water agar (WA) at 100 percent
relative humidity. Refer to Table 2.6 for significant diferences
in percentage of germination by 4 h after incubation at the
temperature ranges shown in this figure.
4-1
O
tn
nJ
-U
G
Q)
u
u
cu
Figure 2.18. Germination of the macroconidia of isolate
BR-7 of Botryosphaeria ribis on water agar (WA) at 100 percent
relative humidity. Refer to Table 2.6 for significant differences
in percentage of germination by 4 h after incubation at the
temperature ranges shown in this figure.


CHAPTER III
EVALUATION OF Botryosphaeria ribis ISOLATES FOR
PATHOGENICITY TO Melaleuca quinquenervia
Botryosphaeria dothidea (Moug ex Fr.) Ces. et de Not. has
been considered synonymous to B. ribis Grossenb. & Dug. by some
authors (Shear et al. 1924, Wiehe 1952, Witcher and Clayton 1963,
Crist and Schoeneweiss 1975, English et al. 1975, Spiers 1977,
Brown and Hendrix 1981, Latorre and Toledo 1984, Mass and Uecker
1984). However, others consider B. ribis and B. dothidea as two
distinct species (Smith 1934, Luttrell 1950, Punithalingam and
Holliday 1973, Davison and Tay 1983, Webb 1983, Rumbos 1987, Ramos
et al. 1991). Evidently, canker diseases on various hosts
formerly reported to be caused by B. ribis are often ascribed to
B. dothidea due to their synonymy (Weaver 1974).
Botryosphaeria ribis has been reported to be pathogenic on
numerous tree species (Rumbos 1987). Dieback of branches and
twigs has been reported in Prunus parsica L. (Weaver 1974), P.
amygdalus Batsch 'Nonpareil' (English et al. 1975), Malus pumila
Miller (Brown and Hendrix 1981, Latorre and Toledo 1984),
Liquidamber styraciflua L. (Vannacci and Lorenzini 1981),
Eucalyptus marginata Donn ex Sm. (Davison and Tay 1983), E.
camaldulensis Dehnh. (Webb 1983), Rubus spp. (Mass and Uecker
1984), and Juglans regia L. (Rumbos 1987). In an attempt to
41


42
explore potential biological control agents of a pest tree
species, Myrica faya Ait., in Hawaii, Gardner and Hodges (1990)
reported the association of B. ribis with twig dieback of M. faya.
Recently, this fungus has been found to be associated with tip
dieback of mango (Mangifera indica L.) in South Florida (Ramos et
al. 1991) .
Cankers caused by B. ribis on E. marginata Donn. ex Sm. were
often unnoticeable from the stem surface and became evident only
after removal of outer bark (Davison and Tay 1983). Generally,
the cankered xylem and phloem tissues were characterized by brown
discoloration (Rumbos 1987) and were often associated with insect
damage that ranged from oviposition on small twigs to cerambycid,
buprestid, and scolytid galleries in branches and trunks (Davison
and Tay 1983).
Botryosphaeria ribis has been described frequently as a
wound parasite (Rumbos 1987) which can cause severe damage to
stressed trees (Crist and Schoeneweiss 1975). A test on blueberry
stems indicated variability in virulence between two isolates of
B. ribis (Witcher and Clayton 1963). A similar view has been
presented where the degree of susceptibility of the host plant has
been attributed to the cultivar (Hildebrand and Weber 1944),
isolate, and inoculation position on the trunk (Latorre and Toledo
1984). Schreiber (1964) reported that B. ribis isolates from
apple (Malus sp.), American holly (Ilex sp.), and Rhododendron
varied in their pathogenicity on Rhododendron. In his experiment,
the isolate originally obtained from Rhododendron was most
virulent, and the apple isolate was the least. In another
experiment using four Eucalyptus species [E. radiata Sieb. ex DC.,


43
E. calophylla R. Br., E. marginata, and E. cladocalyx ), B. ribis
isolates were found to be relatively virulent on only one species
(E. radiata) (Shearer et al. 1987). Similar phenomenon of host-
selective virulence of Hypoxylon maimatum (Wahl.) Mill, has been
reported in clones of Populus tremuloides (French et al. 1969).
To date, the pathogenicity and relative virulence of B.
ribis towards M. quinquenervia (Cav.) Blake has not been studied.
Identification of a virulent isolate of this fungus could be an
important first step toward biological control of M. quinquenervia
in South Florida. Therefore, the specific objectives of the
experiments presented in this chapter were to: 1) perform
pathogenicity tests of B. ribis on M. quinquenervia, 2) test
monoconidial cultures from each of eight isolates of B. ribis for
variation in virulence, and 3) to quantify virulence of one
selected monoconidial culture from each of the eight isolates of
B. ribis on stems of M. quinquenervia clones.
Materials and Methods
Tree Clone Production
As described in Chapter I, a 0.8-hectare stand of declining
trees of M. quinquenervia was discovered (Fig. 3.1) in a
management unit (Acme-2) of the Loxahatchee National Wildlife
refuge in South Florida (see introduction in Chapter I, II).
These trees had active cankers (Fig. 3.2) on the stem which were
marked by a vertical depression or callus (Fig. 3.3) and sapwood


44
discoloration (Fig. 3.4). The main stems above the cankers were
dead (Fig. 3.2). Observations revealed that some of the trees in
the stand were affected more than others by the cankers.
Therefore, this site was selected for collection of tree clones
for testing pathogenicity of B. ribis isolates and variable
susceptibility of the M. quinquenervia clones.
Seven M. quinquenervia trees (henceforth referred to as tree
clones MQ-1 through MQ-7) located at least 50 m apart (to avoid
possible clonal similarity) at the Acme-2 site on the Loxahatchee
National Wildlife Refuge were selected and approximately 20 stem
cuttings were obtained from each tree. The cuttings were
transported to Gainesville and rooted by placing them in water in
plastic buckets. Following rooting, the ramets were transferred
to 5-gallon plastic containers containing 1:1 mixture of peat and
sand. These clonal materials were then maintained as stock trees
in a greenhouse at the University of Florida, School of Forest
Resources and Conservation plant growth facility. Rooted cuttings
(ramets) obtained from the stock trees were used for various
laboratory and greenhouse experiments involving B. ribis
inoculations.
Isolate Acquisition
During 1989, Dr. Roger S. Webb, then Associate Professor in
the School of Forest Resources and Conservation, isolated and
performed preliminary pathogenicity tests with five isolates of B.
ribis which he had obtained from the margin of stem cankers on
declining M. quinquenervia trees from the Loxahatchee National


45
Wildlife Refuge. These isolates were tentatively identified as B.
ribis and later sent to the International Mycological Institute,
Kew, Surrey, England. When I began my doctoral program in 1990, I
obtained and identified another isolate of the same fungus from a
canker margin of a declining tree in the same area of the
Loxahatchee National Wildlife Refuge. From each of these six
isolates, 11 monoconidial cultures (MCC) were produced. These MCC
were tested for pathogenicity using a leaf bioassay method
described later in this chapter. The most virulent monoconidial
culture/isolate was selected from each set of 11 MCC, and are
henceforth referred to as isolates BR-1 through BR-6.
During 1993, I obtained two additional isolates of B. ribis
from the necrotic gall-tissues of red mangrove trees (Rhizophora
mangle L.) collected form the coastal region of South Florida by
Dr. Webb. These two isolates were also single-spored and
identified by Dr. J.W. Kimbrough and myself in the Department of
Plant Pathology, at the University of Florida, and henceforth are
called isolates BR-7 and BR-8. All of these eight isolates, BR-1
through BR-6 and BR-7 and BR-8 were used to study mycological
characteristics (Chapter II) and in the pathogenicity tests
described in this chapter.
Inocula Preparation
The MCC of B. ribis were grown on potato dextrose agar (PDA)
for 3 days and disks (5 mm diameter) were removed from the margin
of the colonies. The disks were placed in a sterile test tube
containing 3 mm glass beads and deionized water. After closing,


46
the tubes were shaken vigorously until the disks were completely
macerated. Three drops of the resulting suspension of MCC were
added to potato dextrose broth (PDB) (Difco) in 500 ml Erlenmeyer
flasks and maintained under continuous shaking conditions (100
rpm) using a rotary shaker. Mycelia were aseptically harvested in
the log phase of growth (4-day-old) and were filtered through
sterile cheese cloth. The mycelial mass retained on the cheese
cloth was then weighed and blended for 15 seconds under aseptic
conditions after which sterile deionized water was added to
prepare a 1% (dry weight/volume) mycelial suspensions. The fresh
inocula were then refrigerated and used in pathogenicity testing.
Leaf Inoculations
The ability of MCC to cause leaf necrosis will reflect their
ability to cause stem diseases. Accordingly, a series of initial
pathogenicity tests were developed. The tests utilized detached
leaves as a bioassay technique to detect variation in relative
virulence, if any, among 11 MCC (only eight MCC in case of isolate
3, since three of the MCC were not used because of contamination)
within each of six B. ribis isolates originally obtained from M.
quinquenervia during 1989-90. Four M. quinquenervia clones were
used in the test. Three check treatments involving inoculation of
1) Fusarium moniliforme var. subglutinans (FM), a pathogenic
deuteromycetous fungus affecting Pinus species; 2) Lophotrichus
sp., a saprophytic ascomycetous fungus; and 3) sterile deionized
water were also included in each experiment.


47
Fully expanded leaves of M. quinquenervia, i.e., 4th, 5th,
and 6th, from stem apex were obtained from the potted ramets,
dipped in 15% (of 5.25% stock) NaOCl for 15 minutes, and rinsed
six times (each 5 minutes) with sterile deionized water (DW).
Three sterile leaves from each M. quinquenervia clone were placed
on pre-moistened sterile filter paper contained in a sterile petri
dish. A small (about a 0.5 sq. mm) scratch was made on the dorsal
surface of each sterile leaf and 10 ¡jl! of the 1% mycelial
suspension was placed on each wound. Following inoculation, the
petri dishes were sealed with Parafilm and incubated at 25C under
10 h light/day for 120 h. In the checks, the leaves were
similarly inoculated with FM, Lophotrichus spp., and sterile
deionized water and maintained under same conditions. The
experiment was repeated twice and with each repetition treated as
block for analysis. Data were analyzed using ANOVA for a
randomized block design.
The area of necrotic leaf was measured using transparent
graph paper at 120 h after inoculation. Mean necrotic areas of
three leaves/MCC/M. quinquenervia clones were calculated.
Variance in virulence among 11-MCC per isolate, overall variance
in susceptibility among four M. quinquenervia clones, and
interaction between MCC and M. quinquenervia clones were analyzed
considering isolates and tree clones as random effects. The MCC
were ranked for their relative virulence using Waller-Duncan
Multiple Range Test. The checks were excluded from F-test since
they exhibited negligible amount of leaf necrosis compared to the
magnitude of necrosis caused by MCC from isolates of B. ribis.
The most virulent MCC from each isolate (hereafter referred to as


48
"isolate") was used in the experiments involving mycological
characteristics (Chapter II), and the stem-inoculation described
later in this Chapter.
Influence of Hyphae vs Macroconidia as Inocula
In all stem-inoculation experiments, I had to rely on hyphal
inocula since many of the B. ribis isolates did not sporulate,
thus failing to provide adequate macroconidial inocula (see
Chapter II). Therefore, it was necessary to evaluate the form of
inoculum (spores vs hyphal fragments) for its ability to cause
diseases on the stems of M. quinquenervia.
Effect of the form of inocula, i.e., macerated hyphae vs
macroconidia, on disease development was evaluated in seven M.
quinquenervia clones (MQ-1 through MQ-7). Isolate BR-1 was chosen
to represent B. ribis isolates for this experiment because it
produced abundant superficial pycnidial stroma and extraction of
macroconidia was more easily accomplished. Hyphal inocula of this
isolate were prepared as described under "Inocula Preparation" in
this chapter. Macroconidia were obtained by incubating the fungal
cultures on PDA under 12 h fluorescent light cycle at 30(1)C for
7 days. Spore suspensions were obtained by partially drying
pycnidia under aseptic conditions to promote conidial extrusion,
followed by washing the plates with sterile deionized water. The
spore concentration was adjusted to llxlO4 macroconidia/ml.
Presence of a few hyphal fragments in the suspension was
disregarded.


49
Each of the two form of inocula (macroconidia and hyphal
fragments) were inoculated on seven M. quinquenervia tree clones.
Each treatment contained three replications, i.e., three
ramets/treatment/tree clone. Inoculation techniques, experimental
conditions, and incubation periods were as described in the
section "Evaluation of Isolate Virulence Under Greenhouse
Conditions" later in this chapter. Fungal isolates and tree
clones were evaluated as fixed and random effects, respectively.
Pathogenicity of Botryosphaeria ribis vs Pestalotia
Attempts at isolating a causal organism from canker margins
on naturally infected M. quinquenervia trees in the field often
yielded Pestalotia sp. along with B. ribis. Therefore, it was
necessary to test the ability of Pestalotia sp. to cause canker
diseases on M. quinquenervia.
Three treatments comprised of the inoculation of 1)
composite inocula of B. ribis isolates, i.e., mixture of the equal
proportion of 1% mycelial (wt/vol) suspension of BR-1 through BR-
6, 2) 1:1 mixture of the composite inocula of B. ribis and
Pestalotia species, and 3) 1% mycelial suspension of Pestalotia.
This experiment used ramets of MQ-4 with the stem diameter of 4.5-
7.0 mm at the point of inoculation. Each treatment contained four
replications, i.e., four ramets/treatment. Fungal inocula,
inoculation methods, incubation period, other experimental
conditions, and analytical methods were similar to those described
for "Evaluation of Isolate Virulence Under Greenhouse Conditions,"


50
as discussed in this chapter. In this experiment, the treatments
were evaluated as fixed effects.
Effects of Inoculation Positions on Canker Development
Preliminary observations and evidence from the literature
(Latorre and Toledo 1984) suggested possible differences in
susceptibility of M. quinquenervia stems toward B. ribis in
relation to point of inoculation (root-collar, mid-section, and
near stem apex). Therefore, this experiment was designed to test
this hypothesis.
This experiment, using MQ-4 and a composite inocula of B.
ribis isolates, utilized points of inoculation as treatments: 1)
at the root-collar region, 2) at the approximate mid-point on the
main stem, and 3) at a point approximately 15 cm behind the apex
of the main stem. Each treatment was replicated five times.
Fungal inocula production, inoculation methods, incubation period,
experimental conditions, and analytical methods were same as those
described in this chapter for "Evaluation of Isolate Virulence
Under Greenhouse Conditions." Fungal isolates and inoculation
positions were evaluated as fixed effects.
Evaluation of Virulence of Isolate Under Greenhouse Conditions
In March 1993, an experiment was initiated to evaluate
variation in 1) virulence among eight B. ribis isolates, and 2)
susceptibility among seven M. quinquenervia clones under
greenhouse conditions. Ramets of M. quinquenervia with stem


51
diameters of 4.5 to 10.0 mm, at least at 10 cm above the root-
collar, were used in the experiment. Each of eight B. ribis
isolates were wound-inoculated on each of seven M. quinquenervia
clones. Two check treatments: 1) wounded and inoculated with
sterile deionized-water, and 2) wounded and inoculated with FM, on
seven M. quinquenervia clones, were also included in this
experiment. Thus, there were a total of 10 treatments (eight B.
ribis isolates and two checks) for each of seven tree clones. The
experiment was repeated three times and each repetition was
considered a block for analysis.
Ramets were wounded by making 1.5 mm x 2.0 mm drill-holes at
a height where the stem was at least 4.5 mm in diameter. The
drill holes were filled with freshly prepared mycelial inocula of
B. ribis. The same kind of drill holes were made for two check
treatments and were inoculated with diluted PDB or 1% hyphal
inocula of FM. Following inoculation, the wounds were immediately
wrapped with Parafilm (American National Can, Greenwich, CT
06830) .
Botryosphaeria ribis-treated ramets were randomized within
each block. Three blocks were maintained separately in the
greenhouse. Ramets in each of the two check treatments were
grouped and placed separately in the greenhouse. All treatments
were maintained at ca 25C under ambient day/night cycle and daily
watering schedule.
The ramets were evaluated every week following inoculation
for wilting, dieback, and mortality. Plants showing these
symptoms were destructively harvested during the day of
observation; the remaining ramets were harvested 8 weeks after


52
inoculation. The harvested ramets were evaluated for symptoms of
pathogenesis. The evaluation included 1) visual estimation of
presence or absence of callus at the inoculation point, 2)
measurement of distal and proximal extension of canker (tissue
discoloration) beneath bark from the point of inoculation, 3)
visual estimation (percent) of sapwood discoloration in cross
section and cambium discoloration both at 5 mm distal to the point
of inoculation, and 4) reisolation of fungus from cankered or
stained tissue beyond 5 mm from the point of inoculation. A 5 mm
long segment of stem beyond 5 mm from the point of inoculation
from each of the B. ribis and FM treated ramets was recovered from
the stem, surface sterilized by quick-dip in 95% ethyl alcohol and
followed by brief flaming. The surface-sterilized segments were
placed on acidified potato-dextrose-agar (APDA), incubated for 14
days (at 25C and ca 10 h fluorescent light cycle), and evaluated
for B. ribis colony development. Fungal isolates and tree clones
were evaluated as random effects. Data were analyzed using ANOVA
for a randomized block design.
Evaluation of Virulence of Isolate Under Field Conditions
Inocula. Hyphal inocula of each of the eight isolates were
prepared as described under "Inocula Preparation" in this chapter.
To facilitate field inoculations, 10 ml of aqueous hyphal inocula
(1% wt/vol.) were containerized into plastic capsules by ARBOR,
Tree Technology Systems, Inc., 1014 Rein Road, Cheektowga, NY
14225 according to arrangements made with this company by Dr.
Webb. The plastic capsules were designed to deliver the mycelial


53
contents into drill-holes made in the tree stems. The capsules
were to remain attached to the trees to prevent wound
contamination as needed. Viability of the inocula in the capsule
was tested the week before field application.
Site selection. A young stand of M. quinquenervia located
on the Strazulla tract of the South Florida Water Management
District in West Palm Beach at Palm Beach County was selected for
the field experiment on stem-inoculation with B. ribis. The stand
was selected for uniform tree sizes (about 2-10 cm diameter at
breast height) and accessibility for installing the experiment.
Tree inoculation. In March 1994, trees were wounded at
breast height by drilling holes 5 mm diameter and 10 mm deep into
the stem. A capsule containing the appropriate inocula was
inserted into the hole to deliver the inocula to the sapwood
region of the stem. The capsules were left attached to the trees
for 12 weeks. As a check treatment, 25 trees were similarly
drilled but injected with sterile distilled water only.
Experimental design. Each of the eight isolates were
inoculated into a set of 25 trees, five of which were scheduled to
be harvested at 3-month intervals to determine the rate of canker
development by B. ribis isolates under field conditions. Because
of time constraints, data was only collected from the first subset
of five trees/isolate; this information is presented in this
chapter.
Harvesting and canker evaluation. Five trees from each of
nine treatment (eight B. ribis isolates and one check) were
harvested after 3 months. Stem segments of about 40 cm long with
the inoculation point at the middle were removed and sealed in


54
plastic bags. These were then placed under refrigerated storage,
and transported to the Forest Pathology laboratory of the School
of Forest Resources and Conservation at the University of Florida.
There they were stored at 5C for three days in a cold room. The
stems were split longitudinally through the inoculation point and
the length of the discoloration of sapwood at the proximal and
distal segments from the inoculation point was measured. Fungal
isolates were evaluated as random effects.
Tissue samples about 5x5 mm square were removed from an area
located ca 10 cm distally from the point of inoculation. These
samples were surface sterilized by quickly dipping them into 95%
ethanol and then flaming them momentarily. The flamed samples
were cultured on APDA for 14 days at 25C and a 10 h photoperiod.
The plates were then evaluated for the presence of B. ribis
colonies.
Effect of Season on Canker Development
In September 1992, a preliminary pathogenicity test was
conducted with a composite sample of B. ribis isolates. This
measured the response of M. quinquenervia to inoculations during
the fall. A general comparison of these results with spring
inoculations (see "Evaluation of Isolate Virulence Under
Greenhouse Conditions") revealed seasonal variation in canker
development. Therefore, it was necessary to test the seasonal
effects on disease development.
The seven M. quinquenervia tree clones, used in the
"Evaluation of Isolate Virulence Under Greenhouse Conditions"


55
experiment, were also used in this experiment. However, only
isolate BR-2 of B. ribis was utilized since its canker-causing
ability had been determined to be greater than those of other
isolates.
Data on canker length, mortality, and callus formation
obtained by inoculating BR-2 in spring (March 1994) on stems of
all seven M. quinquenervia clones (see "Isolate Virulence Under
Greenhouse Conditions") were compared with the data obtained by
inoculating the same isolate on the same seven tree clones in the
fall (October 1994) as described. Ramets of M. quinquenervia with
stem diameter of 5.1 to 10.1 mm, at 10 cm above root-collar were
used. Three ramets from each of seven tree clones were wounded
and inoculated with inocula of BR-2 using the same methodology
described for the experiment, "Evaluation of Isolate Virulence
Under Greenhouse Conditions." Ramets inoculated with BR-2 were
randomized on a greenhouse bench. As a check treatment, three
ramets of similar diameter were wounded using the same method and
treated with sterile distilled water, randomized according to
location, and maintained on a greenhouse bench as a separate group
to avoid possible contamination. Both treatments (BR-2 inoculated
and checks) maintained under a daily watering schedule without
temperature control, were evaluated every week for mortality, and
harvested 8 weeks after inoculation. The canker evaluation
techniques and criteria were similar to those described for
"Evaluation of Isolate Virulence Under Greenhouse Conditions."
Seasonal (spring and fall inoculations) effects and the tree
clones were evaluated as fixed effects.


56
Simulated Storm Damage and Inoculation with Macroconidia
Plants growing under field conditions are injured by various
natural factors such as storms and hurricanes, and provide
infection courts for pathogens. If B. ribis is to be developed as
a successful mycoherbicide, a desirable attribute would be the
ability to be established on the host when inoculated in mass.
This fungus appears to be a wound pathogen. Therefore, it was
necessary to test whether it is capable of establishing itself
into leaf and stem tissues that are wounded by natural or induced
damage to the leaves or on the stem due to friction between
adjacent trees. This experiment tested the ability of B. ribis to
establish on the injuries created by a simulated storm.
Potted M. quinquenervia ramets of ca 1.0 m height, and
macroconidial inocula of BR-1 of B. ribis (based on the
availability of adequate spores) were used. The canopy and the
sides of all ramets were exposed to a blast of wind with a force
of 180 lb/sq inch for five minutes. This resulted in breakage of
a few small twigs and detachment or tearing of some leaves.
Additionally, the upper-halves of the main stem of each member of
a pair of adjacent ramets were deliberately rubbed against each
other only once to simulate injury that may occur during a storm.
Using these injured ramets, four treatment groups (Gr.) were
identified. They were Gr. 1 (not inoculated with fungal spores),
Gr.2 (inoculated with 4.0xl04 spores/ml), Gr.3 (5.7xl04 spores/ml),
and Gr.4 (11.6xl04 spores/ml). The ramets in a fifth treatment
group (Gr.5) were not injured but were inoculated with


57
macroconidial inocula at 11.6xl04 spores/ml). Each treatment
group contained five ramets.
The inocula were produced using the methods described for
the experiment "Germination of Macroconidia" in Chapter II.
Macroconidia were suspended in sterile distilled water, diluted to
the appropriate concentrations, and applied on the plants with an
atomizer until the plant were completely wet. All the ramets in
Grs. 2, 3, 4, and 5 were placed together in a dew chamber at 100%
RH and 27C for 72 h. Ramets in Gr.l were placed separately in
one corner of the same dew chamber. After incubation for 72 h,
surfaces of the leaves and small twigs were examined for spore
germination. Then the ramets in Grs. 2-5 were transferred to a
greenhouse and placed together on a bench in a randomized manner.
Ramets in Gr.l were also transferred to the greenhouse but were
placed on a separate bench.
Following transfer to the greenhouse bench, the plants were
watered daily, and evaluated weekly for leaf necrosis and stem
canker. The ramets were harvested eight weeks after incubation
and evaluated for canker development and the presence of fungus on
the unwounded and the wounded parts of the stem. A segment of
bark and sapwood (5 mm long) from the margins of cankers initiated
on wounded, unwounded, and leaf scars of a stem of each ramet in
all five treatment groups were surface sterilized by quickly
dipping them in 95% ethyl alcohol followed by a brief flaming.
The surface sterilized segments were placed on APDA, incubated for
7 days (at 25C and ca 10 h fluorescent light cycle) and
evaluated for B. ribis colony development.


58
Statistical Analyses
Data from laboratory, greenhouse, and field experiments were
analyzed using similar statistical procedures. Total canker
length (proximal + distal canker length from the point of
inoculation) was used as the response variable for all stem-
inoculation experiments. Analysis of variance (ANOVA), linear
contrasts among B. ribis isolates vs checks, and mean separations
among variables were performed using GLM procedures in SAS (1985).
Pearson's correlation coefficient was used to test the
relationship between stem diameter at inoculation point and total
canker length.
Results
Leaf Inoculations
Leaf bioassays revealed that six B. ribis isolates obtained
from M. quinquenervia are capable of causing a greater amount of
leaf necrosis when compared to the wound alone, wound plus FM, and
wound plus Lophotrichus species (Fig. 3.5). The F-values from
ANOVA for leaf inoculation experiments are presented in Table 3.1.
Difference in virulence of MCC within isolate and interaction
between MCC and M. quinquenervia clones were insignificant. These
findings indicate that MCC within an isolate were equally
pathogenic on a given M. quinquenervia.


59
Table 3.1. Leaf bioassay experiments (necrotic leaf area in
square mm) designed for preliminary tests of virulence among 11
monoconidial cultures within each of six isolates of B. ribis
obtained from M. quinquenervia from South Florida.
Source
Analyses
of variance
df
MS
F values
Pr > F
Isolate 1
MCC
10
2978.9
2.60
0.0146
MQ clones
3
10716.4
9.35
0.0001
Blocks
1
130.1
0.11
0.7379
MCC x MQ clones
30
1105.9
0.96
0.5344
Error
43
1146.6
Isolate 2
MCC
10
966.9
0.59
0.8118
MQ clones
3
4656.9
2.85
0.0485
Blocks
1
34060.7
20.84
0.0001
MCC x MQ clones
30
834.2
0.51
0.9720
Error
43
1634.7
Isolate 3
MCC
7
1755.2
1.11
0.3844
MQ clones
3
3310.1
2.08
0.1225
Blocks
1
50740.3
31.95
0.0001
MCC x MQ clones
21
2346.3
1.48
0.1581
Error
31
1588.0
MCC
10
2416.8
1.15
0.3492
MQ clones
3
17639.3
8.40
0.0002
Blocks
1
71535.0
34.07
0.0001
MCC x MQ clones
30
1578.6
0.75
0.7921
Error
43
2099.8
Isolate 5
MCC
10
508.0
0.43
0.9256
MQ clones
3
18667.5
15.68
0.0001
Blocks
1
17220.0
14.46
0.0004
MCC x MQ clones
30
747.7
0.63
0.9082
Error
43
1190.9
Isolate 6
MCC
10
1544.3
0.87
0.5704
MQ clones
3
13639.5
7.65
0.0003
Blocks
1
8920.4
5.00
0.0305
MCC x MQ clones
30
1307.7
0.73
0.8117
Error
43
1782.3
Note: MCC=monoconidial culture; MQ^Melaleuca quinquenervia.
The differences in susceptibility among M. quinquenervia
clones towards cumulative effects of individual isolates (based on
average of cumulative necrosis caused by 11 MCC within isolate)
were significant (P=0.05); this result has indicated that


60
combination of some isolate-M. quinquenervia clones may cause more
disease development than some other combinations. Treatment
blocks, which represent experiments performed at two different
times, were also significant (P=0.5) among all the isolates
revealing the effect of leaf age on disease development.
Influence of Hyphae vs Macroconidia as Inocula
The overall effect of the type of inocula, i.e.,
macroconidia vs hyphae, on canker length (mm) on stems of the
seven clones of M. quinquenervia was not significant (P=0.05, Pr
>F=0.7. Therefore, the type of inocula did not influence ultimate
canker length (32.8 and 31.3 mm for macroconidial and hyphal
inocula, respectively). The interaction between type of inocula
and tree clones was also not significant (P=0.05, Pr>F=0.5).
Therefore, both hyphal and macroconidial inocula appeared to have
similar effect on stem canker development on a given clone. The
mean canker length by inocula and isolate are presented in Table
3.2. Callusing was observed among ramets from both macroconidial
and hyphal inoculations.
Pathogenicity of Botryosphaeria ribis vs Pestalotia
Attempts to isolate causal organisms from stem cankers on M.
quinquenervia from Acme-2 of the Loxahatchee National wildlife
refuge yielded B. ribis and also Pestalotia species. Therefore,
pathogenicity of both fungal species was tested. Botryosphaeria
ribis caused longer canker than Pestalotia alone (Table 3.3).


61
Canker development resulting from the mixture of these two fungal
species was some what smaller, though not statistically different
(Table 3.3).
Table 3.2. Separation of means of the effects of form of inocula
on canker length (mm) on the stems of M. quinquenervia (MQ) in
summer inoculations
with
isolate
BR-1 of
B. ribis.
Tree clones
Inocula
MO-1
MO-2
MO-3
MO-4
MO-5
MO-6
MO-7
Macroconidia
Hyphae
26 Oa
19.7a
19.3a
23.3a
21.3a
17.3a
53.3a
43 Oa
27.8a
44 Oa
36 Oa
43 Oa
35.7a
44 Oa
Note: Means followed by the same letter in the rows are not significantly different
according to Scheffe's mean separation test at P=0.05.
Table 3.3. Main-effect means and mean separations from analyses
of variance for comparison of B. ribis and Pestalotia species, and
determination of the effect of inoculation point on disease
development. Dependent variable was total canker length (mm)
measured at 8 weeks after inoculation in spring.
Experiments Treatments Mean canker length
Botryosphaeria ribis vs Pestalotia
B. ribis only
111.Oa
B. ribis + Pestalotia
85 Oa
Pestalotia only
5.5b
Inoculation positions vs canker development
Root-collar
26.8b
Middle
129.8a
Top
63.3ab
Note: Means followed by the same letter(s) are not significantly different according to
Scheffe's mean separation test at P=0.05.


62
Effects of Inoculation Positions on Canker Development
The location of the inoculation points influenced the
development of stem cankers on M. quinquenervia clones
(Pr>F=0.008). Largest cankers were developed by inoculating at
the middle-, and smallest by inoculating at the root-collar
section of the stems (Table 3.3). Callusing was observed at all
inoculation positions among ramets of all clones inoculated with
B. ribis.
Evaluation of Virulence of Isolates Under Greenhouse Conditions
Under greenhouse conditions no mortality of B. ribis
inoculated M. quinquenervia ramets was recorded during the 8-week
incubation period. Wilting and dieback above the point of
inoculation on stems was noted in about 20 and 10 percent of the
ramets inoculated with BR-2 and BR-6, respectively. Except in the
checks, cankers of various dimensions were observed among ramets
inoculated with B. ribis. In these spring-inoculated stems of all
M. quinquenervia clones, callus formation occurred in all
treatments, i.e., checks (wound, wound plus FM), and wound plus B.
ribis, except those that developed wilting and eventual dieback.
However, the nature of the callus development was different among
treatments.
Callus development, among ramets in checks as well as B.
ribis inoculations, was initiated within two weeks after
inoculations. Within 8 weeks, 90-to-100 percent of the inoculated
wounds callused to various degrees (Table 3.4). All of the wounds


63
in checks (wound only and wound + FM) were closed (Fig. 3.6) and
no tissue discoloration was observed beyond 5 mm from the
inoculation point. In contrast, B. ribis-inoculated wounds were
still unclosed and were laterally surrounded by callus tissues
(Fig. 3.7). These cankers were elliptical or evidenced long
fissures (Fig. 3.7). Discoloration of the bark, cambium, and
sapwood occurred up to 100 mm beyond the callus margin on some
stems. These cankers were similar to those observed in the field
(Figs. 3.2, 3.3). The cankers among non-callused stem wounds
(Fig. 3.8) inoculated with B. ribis were marked by depressions on
proximally and distally to the point of inoculation. Often, B.
ribis produced fertile pycnidial stroma on the bark (Fig. 3.9) of
proximal and distal segments of the stem from the inoculation
points. Botryosphaeria ribis was reisolated from 5 mm above or
below the inoculation point in 86-to-100 percent of the inoculated
stems (Table 3.4).
Table 3.4. Percent callusing around the inoculation point and
percent reisolation of fungus from 5 mm beyond the inoculation
point on M. quinquenervia stems at 8 weeks after inoculation in
spring.
BR-1
BR-2
BR-3
BR-4
BR-5
BR-6
BR-7
BR-8
FM
Wound
Callusing
90
90
100
86
100
90
95
95
100
100
Reisolation
90
95
95
95
86
100
86
86
33*
14**
Note: 'FM reisolated from inoculation point, but none from 5 mm above the inoculation
point; "Botryosphaeria, Pestalotia, and Fusarium, were reisolated from the inoculation
point, but none from 5 mm above the inoculation point.
The linear contrasts between check treatments (wound alone
and wound plus FM) and wound-inoculation with B. ribis isolates
were significant (Table 3.5). This revealed that the canker


64
causing ability of the wound alone or wound plus FM was
insignificant. Therefore, the checks were not included in further
tests of main effects (without checks in Table 3.5) and mean
separations (Table 3.6) for isolates and clones. With or without
checks, the variances among isolates and clones were highly
significant, whereas interactions among isolates and clones were
insignificant (Table 3.5). Linear contrasts revealed the effects
of B. ribis isolates to be significantly higher from individual as
well as overall checks (Table 3.5).
Table 3.5. Effects of B. ribis isolates on canker development on
stems of M. quinquenervia clones at 8 weeks after inoculation in
spring 1994.
Sources
df
Analysis of variance
MS F value Pr > F
With checks
BR Isolates
9
12811.1
14.13
0.0001
MQ clones
6
4236.2
4.67
0.0007
BR Isolates x MQ clones
54
906.8
1.08
0.3613
Error
140
843.2
Linear contrasts
FM and wound vs BR isolates


0.0001
FM vs BR isolates


0.0001
Wound vs BR isolates


0.0001
FM vs wound


0.8087
Without checks
BR Isolates
7
6073.3
5.77
0.0001
MQ clones
6
4986.0
4.74
0.0003
BR Isolates x MQ clones
42
1051.9
1.00
0.4797
Error
112
1048.7
Note: BR=B. ribis, FM=F. moniliformis var subglutinans, and MQ=M. quinquenervia.


65
Table 3.6. Main-effect means and mean separations from analyses
of variance for stem-inoculation tests on total canker length (mm)
at 8 weeks after inoculation.
Isolate
number
Mean canker
Greenhouse
length
Field
Clone
number
Mean canker
lenath
BR-1
39.91ab
52.4ab
MQ -1
37.00b
BR-2
81.17a
78.2ab
MQ-2
34.85b
BR-3
50.76ab
70.4ab
MQ 3
38.33ab
BR-4
66.33 ab
91.6a
MQ-4
71.08a
BR-5
31.86b
45.2ab
MQ- 5
43.58ab
BR- 6
43.7ab
61.8ab
MQ 6
63,54ab
BR-7
32.2b
65.6ab
MQ- 7
56.80ab
BR- 8
48.57ab
64.2ab


Note: Means followed by the same letter(s) are not significantly different according to
Scheffe's test at P=0.05. Observations for each of B. ribis (BR) isolates and M.
quinquenervia (MQ) clones were 21 and 24, respectively.
Stem-inoculation experiments revealed that BR-2 and BR-5
were the most and least virulent B. ribis isolates, respectively
(Fig. 3.10). On the other hand, MQ-4 and MQ-2 were the most and
least susceptible clones (Fig. 3.11). Canker progress from the
inoculation point was greater distally than proximally in the
stems (Fig. 3.12). Cambium and phloem discoloration was maximum
(44 percent) in BR-2 and minimum in BR-7 (Fig. 3.13). The
correlation between stem diameter at point of inoculation and
total canker length was very weak (Pr>R=0.01).
Evaluation of Virulence of Isolates Under Field Conditions
After inoculation with B. ribis, no dieback or mortality of
M. quinquenervia trees occurred in the field within the 3 months
observation period. However, dissection of the inoculated stems
revealed evidence of host colonization, and significant


66
differences in canker length among B. ribis isolates were detected
(Table 3.5). Slight changes in pathogenicity ranking among
isolates from greenhouse to field inoculations were noted. For
example, BR-4 ranked first in this field inoculation compared to
BR-2 which ranked first in the greenhouse inoculations. However,
in both experiments these two isolates were at the top two
positions in ranking based on mean canker length of the stem.
Among check-trees, tissue discoloration was limited to a maximum
of about 12 mm (an average of 5.8 mm proximal and distal to the
center of the point of inoculation). As with the spring
inoculations under greenhouse conditions, all wounds stimulated
callus production.
Reisolation of B. ribis from ca 10 mm distal to the
inoculation point on stem ranged from 60 to 100 percent, and no
fungi were isolated from the check plants. However, bacterial
contamination was present among the majority of the reisolations
from checks (from point of inoculation) as well as the B. ribis
treated stems.
Effects of Season on Canker Development
Mortality did not occur among M. quinquenervia ramets
inoculated on stems with B. ribis isolate BR-2 in both spring
(March) and fall (October). However, wilting and dieback of
ramets above the inoculation point was 10 and 20 percent in spring
and fall inoculations, respectively. Callusing did not occur in
10 and 33 percent of the total stems inoculated in spring and
fall. Among checks, the wounds were completely closed in both


67
spring and fall inoculations, respectively. Characteristics of
cankers and fungal sporulation on bark among callused and
noncallused wounds on the stems were the same as described for
spring inoculations (Figs. 3.6-3.8).
Overall canker length (mm) (81a and 93a for spring and fall
inoculations, respectively) among ramets in seven tree clones was
not significantly different (Pr>F=0.2871 at P=0.05) between spring
and fall inoculations. Though statistically not significant in
all but two M. quinquenervia clone, a majority of clones produced
larger stem cankers in fall-inoculations than those inoculated in
spring (Fig. 3.14). The circumference of necrotic tissues on
sapwood surface was also larger (data not presented) in fall-
inoculated ramets than those inoculated in spring. Botryosphaeria
ribis was reisolated from 98 percent of the cankers.
Simulated Storm-Damage and Inoculation with Macroconidia
During the 8-week incubation period, mortality of M.
quinquenervia ramets was not observed in any of the five
treatments tested in this experiment. Germination of macroconidia
and mycelial proliferation on the surface of leaves and stems was
prevalent within the 72-h incubation period. During this period,
some hyphal tips were observed to gain entry into the leaves
through stomata of uninjured leaves. However, following transfer
from the moist chamber to the greenhouse bench, further necrosis
of uninjured as well as injured leaves stopped. Other effects of
the simulated storm on disease development on M. quinquenervia
stems are presented in Table 3.7.


68
Regardless of the concentration of macroconidia used, all
the wounds made by friction were still unclosed and surrounded by
thin strips of callus ridges (Table 3.7). In contrast, the wounds
on uninoculated stems were closed by callus ridges. Small bark-
limited cankers (2-15 mm long) developed on the leaf scars and
unwounded upper parts of some wind-blasted stems (Table 3.7) where
the bark was green at the time of treatment. Also, branch tip
dieback was observed among some wind-blasted ramets inoculated
with higher inoculum concentrations (Table 3.7). Percentage of
cankered and uncankered part of the stems from which B. ribis was
reisolated has been presented in Table 3.7. In check (Gr.l), 20
and 40% of the wounds yielded B. ribis and Pestalotia sp.,
respectively.
Table 3.7. Effects of simulated storm damage on the establishment
of BR-1 isolate of B. ribis on stems of M. quinquenervia ramets
inoculated with macroconidia.
Treatment groups
1
2
3
4
5
A.
Percentaae of ramets
forming cankers on
Wounds
Closed
Unclosed
Unclosed
Unclosed
None
Unwounded part
of stems
None
40
60
60
None
Leaf scars
None
40
40
60
None
B.
Percentage of dieback
of stem tips
None
None
20
60
None
C.
Percentage of
B. ribis-reisolation
from
Wound-cankers
20
100
100
100
None
Bark-limited cankers
None
80
100
80
None
Uncankered stems
0
60
80
20
29
Note: Treatment groups (Gr.) were : Gr.l (injured, not inoculated with fungal spores),
Gr.2 (injured, inoculated with 4.0x10* spores/ml), Gr.3 (injured, inoculated with 5.7x10*
spores/ml), Gr.4 (injured, inoculated with 11.6x10* spores/ml, and Gr.5 (ramets not injured
but inoculated with 11.6x10* spores/ml). The numbers in "A," "B," and "C" represent the
percentage of ramets developing stem cankers, tip dieback of branches, and uncankered or
cankered stems that yielded B. ribis, respectively.


69
DisCUSSign
An extensive literature search on the pathology of M.
quinquenervia indicated that very little is known about disease
affecting this species. Farr et al. (1989) and Alfieri et al.
(1994) recorded several pathogenic fungi on M. quinquenervia in
South Florida. The first reported testing of pathogenicity of B.
ribis on M. quinquenervia is presented in this work. In this
work, the isolates of B. ribis originally obtained from the canker
margins of M. quinquenervia trees and necrotic galls of red
mangrove trees from South Florida, were evaluated by inoculation
of foliage and stem tissues.
Preliminary Screening of Monoconidial Cultures
The observed variations in B. ribis isolates (Chapter II), made it
necessary to test for variation in pathogenicity of monoconidial
cultures (MCC) within each isolate. Sutton and Boyne (1983) have
proven that isolates of B. dothidea obtained from one part (fruit)
of an apple tree were found to be equally pathogenic on another
part (stems). Based on this evidence, a leaf bioassay procedure
was developed to provide a relatively rapid screening of numerous
MCC for their relative pathogenesis to M. quinquenervia although
the isolates were originally derived from stem cankers.
The leaf bioassay procedures (Fig. 3.5, Table 3.1) revealed
three important points: 1) the isolates of B. ribis were
pathogenic to M. quinquenervia clones, 2) despite differences in
conidial dimensions, MCC within each isolate were not different in


70
their ability to cause tissue discoloration, and 3) differential
susceptibility occurs among M. quinquenervia clones towards B.
ribis isolates, i.e., some isolates were more virulent on one M.
quinquenervia clone than others.
Evaluation of Isolates by Stem-Inoculations
Cankers among M. quinquenervia trees from which B. ribis
isolates were isolated during 1989-1990 were often unnoticeable on
the surface of the affected stems. On these trees, cankers of the
shape of elliptical fissures became visible only after removal of
several layers of the papery bark. Lateral margins of those
cankers contained ridges of callus tissues (Fig. 3.2, 3.3). In
advanced cankers, callus tissues were discolored, imparting a
brown to black coloration from the margin of the diseased and
healthy tissues to the advanced stages of rot in the center.
Under greenhouse conditions, similar kinds of cankers and tissue
discolorations were observed on the stems inoculated with B. ribis
isolates (Fig. 3.7). Necrotic cortex, phloem, and cambium under
the bark among noncallused cankers were darker compared with
progressive darkening of tissues at the margin of the callus and
healthy tissues.
Some species of Pestalotia have been described as weak plant
pathogens. This fungus was isolated along with some isolates of
B. dothidea causing gummosis disease on peach trees (Weaver 1974).
Ramos et al. (1991) reported Pestalotia from the tissues killed by
the dieback diseases caused by B. ribis. The present study
revealed canker-inciting ability of Pestalotia species on M.


71
quinquenervia to be insignificant compared with B. ribis (Table
3.2). It is assumed that Pestalotia species is a secondary-
colonizer of the tissues infected and killed by B. ribis.
However, the ability of B. ribis to cause stem cankers on M.
quinquenervia was retarded by Pestalotia when inoculated in
mixture. The ability of Pestalotia species to inhibit development
of B. ribis was further supported by an observation where B. ribis
hyphal growth was completely suppressed on PDA by the more rapidly
developing hyphae of Pestalotia when an equal proportion of hyphal
fragments of these fungi were inoculated onto the media. It seems
reasonable that the presence of Pestalotia species in M.
quinquenervia-induced cankers may slow down further canker
development.
The relationship between total canker length and the
diameter of the stem at the inoculation point was not strong.
These results follow the findings of Shearer et al. (1987) who
found E. radiata stems of all ages and diameters to be equally
susceptible to invasion by B. ribis. However, this does not mean
that stems of different diameters inoculated with B. ribis will be
killed in the same amount of time. Mortality of infected trees
depends on the ability of fungus to girdle the stem, which in
turn, depends on the ability of fungus to grow tangentially
through xylem, cambium, and cortex.
Results of the present study revealed rapid stem canker
development at the middle segment compared with the top and the
root-collar regions (Table 3.2). These results agree with Bagga
and Smalley (1969) who found that wound sites midway on the stem
of P. tremuloides were most susceptible to canker development when


72
inoculated with H. pruinatum. When northern red oaks (Quercus
rubra L.) were inoculated with Phytopthora cinnamomi Rands, the
root-collar appeared less susceptible (to canker formation) then
the upper portion of trunks (Robin et al. (1992). However, the
results of the present research are not in agreement with Latorre
and Toledo (1984) who reported that one of their isolates of B.
dothidea produced larger canker lesions on apple trees when
inoculated at the lower portion of the trunk.
The time of year during which B. ribis inoculations were
made did not appear to have an effect on the canker length on the
stems of M. quinquenervia clones. However, on B. ribis-inoculated
stems, wound callusing was more frequent and pronounced in spring
than in fall inoculations. Additionally, the decline and wilt of
ramets was relatively greater in fall inoculations.
Callus formation on stem around a wound is viewed as an
attempt of a host to contain the fungus and to subsequently close
the wound. Among noncallused stems, canker development beneath
the bark was marked by a depression visible from the surface.
This finding agrees with the work of Filer and Randall (1978) who
inoculated B. ribis on 21 sweetgum families in open-pollinated
progeny tests and found maximum infection of trees when
inoculations were made in September and least infection among
trees inoculated in May. However, the results of my study do not
completely agree with the work of Davison and Tay (1983) who
reported B. ribis as an extensive invader of the phloem of E.
marginata seedlings when inoculations were made in winter, spring,
and summer. Inoculation of B. dothidea on southern California


73
chaparral vegetation during September was found to result in rapid
disease development (Brooks and Ferrin 1994).
English et al. (1975) reported B. ribis to be highly
virulent on almond (Primus amygdalus) in spring, summer and fall.
Therefore, it appears that susceptibility of tree species to B.
ribis may be influenced by the season of the year and it varies
among species. Under greenhouse conditions, fall inoculation of
M. quinquenervia with B. ribis appeared to be more effective in
inhibiting callus formation around wounds on stems than the spring
and summer inoculations. Although, the overall effect (across
clones) of season on canker length on B. ribis inoculated stems
was not statistically significant, certain clones appeared to be
more susceptible to canker development in fall than in spring
(Fig. 3.14) and vice versa.
The present study revealed significant differences in
virulence of B. ribis isolates on M. quinquenervia clones. Under
greenhouse conditions, isolate BR-2 and BR-4 were ranked first and
second, respectively, in virulence towards B. ribis clones (Table
3.6). However, in field inoculations this ranking was reversed.
Unlike greenhouse inoculations, I did not have control over tree
clones in the field inoculations. Therefore, it is possible that
trees inoculated with isolate BR-4 may be relatively more
susceptible to this isolate or the conditions around those trees
may be more conducive to disease development than the trees
inoculated with isolate BR-2. The remaining six isolates
including two from red mangrove also incited canker disease on M.
quinquenervia (Table 3.6). Similar variation in virulence among
isolates of B. ribis have been reported on currants (Ribes


74
species) (Hildebrand and Weber 1944) and on blueberry (Witcher and
Clayton 1963). Isolates of B. dothidea have shown variation in
pathogenicity on apple cultivars in Chile (Latorre and Toledo
1984). Based on the results from the inoculation on apple
cultivars, Latorre and Toledo (1984) suggested the existence of
strains of B. dothidea. Significant variation in pathogenicity
among isolates of B. dothidea has also been reported on apple
trees (Sutton and Boyne 1983).
Cultivar dependent susceptibility towards isolates of B.
ribis and B. dothidea has been reported on currant (Hildebrand and
Weber 1944) and apple (Latorre and Toledo 1984), respectively.
Milholland (1972) tested 11 isolates of B. dothidea on 18
blueberry cultivars and found that six isolates were virulent on
10 cultivars, and five isolates were virulent on the remaining
eight cultivars. These findings agree with present observations
under greenhouse inoculations in which variable susceptibility
among clones of M. quinquenervia was evident (Fig. 3.9). Similar
phenomenon of host selective virulence and variable clonal
susceptibility has been reported also for the host-parasite
relationship of P. tremuloides and H. mammatum (French et al.
1969) .
Tissue discoloration among B. ribis inoculated stems of M.
quinquenervia was more rapid in distal than in proximal segments
with respect to the point of inoculation (Fig. 3.10). It is
assumed that upward movement of sap through vessels and tracheids
may interfere with the downward growth of hyphae and hence slowed
discoloration of stem tissues at the proximal segments. In
contrast, tissues above the inoculation point may have facilitated


75
quick establishment and growth of the fungus due to suction of
inocula further into the stem through vessels and tracheids by-
transpiration pull from above. These factors may have contributed
to relatively rapid tissue discoloration in distal segments of M.
quinquenervia stems.
In the present study, two isolates obtained from the galls
of red mangrove were as virulent as some of the isolates which
were originally derived from M. quinquenervia (Table 3.6, Fig.
3.8). However, both of the isolates (BR-2 and BR-4) that
performed best in greenhouse and field conditions were originally
derived from M. quinquenervia. Schreiber (1964) reported
Rhododendron-derived isolates of B. ribis to be more virulent on
that same host than were isolates obtained from non-Rhododendron
hosts. Similar views have been expressed for the isolates of B.
ribis on Eucalyptus species in Australia (Shearer et al. 1987).
Pusey et al. (1986) reported a new biotype of B. dothidea causing
gummosis disease on peach trees. Many researchers agree on the
existence of moderate host specificity (Schreiber 1964, Shearer
1987), possibility of development of new strain or biotype
(Latorre and Toledo 1984, Pusey et al. 1986), and differences in
virulence (Hildebrand and Weber 1944, Witcher and Clayton 1963,
Milholland 1972, Sutton and Boyne 1983) among isolates of B. ribis
and B. dothidea complex.
In the present study, uninjured ramets inoculated with
macroconidial inocula of B. ribis neither produced cankers nor
induced any decline symptoms (Table 3.7). On the other hand, some
of the wind blasted ramets inoculated with B. ribis produced bark-
limited cankers on the uninjured part of the stem and resulted in


76
cankers typical of wound inoculation (Table 3.7). The bark
limited cankers in the uninjured stem segments of wind blasted
ramets were similar to those described by Pusey et al. (1986) for
infection of peach stems through lenticels and development of
sunken necrotic lesions. The observations in the present studies
revealed that establishment of B. ribis in M. quinquenervia
requires either a wound exposing the sapwood or injury-stresses
such as loss or damage of leaves or branch breakages. These
results agree with Milholland (1972), Crist and Schoeneweiss
(1975), and Rumbos (1987), who found that B. dothidea or B. ribis
requires wounds, and/or some other form of injury and stress to
establish on host stems and cause canker diseases leading to tree
decline and dieback.
Conclusions
Eight isolates of B. ribis, six from M. quinquenervia
(collected during 1989-90) and two from red mangrove (collected
during 1993) were evaluated for pathogenicity on M. quinquenervia
under laboratory, greenhouse, and field conditions.
Preliminary evaluation of 11 monoconidial cultures from each
of six isolates (collected during 1989-90), using a leaf bioassay
(on leaves from four M. quinquenervia clones) revealed: 1)
nonsignificant difference in the ability of monoconidial cultures
within isolates to cause necrosis of M. quinquenervia leaves, and
2) significantly variable susceptibility among M. quinquenervia
clones towards the isolates of B. ribis.


77
Stem-inoculations under greenhouse conditions using eight
isolates of B. ribis on ramets from seven M. quinquenervia clones
and field inoculations of the same isolates revealed 1) elliptical
and/or long fissured cankers on ramets which were similar to those
observed in the field during 1989-90, 2) a correlation between the
inoculation position on the stem and the canker length to be
significant, i.e., middle segments of main stems of trees were
more susceptible to canker development than the root-collar
regions and top segments, 3) a weak correlation between the
diameter of the stem at the inoculation point and canker length,
4) the disease-inciting ability of hyphal and macroconidial
inocula not to be significantly different, 5) that callusing of
wounds among ramets across clones of M. quinquenervia inoculated
in spring was more frequent and pronounced compared with those
inoculated in fall, 6) that all eight isolates were able to incite
cankers on stems across clones, but isolates BR-2 and BR-4 were
the most virulent, 7) some M. quinquenervia clones to be more
susceptible to some isolates than others, and 8) that
establishment of this fungus in the stem tissues requires a wound
or some form of injury that stresses the trees.


Figs. 3.1-3
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
.4. Declining M. quinquenervia trees in a stand in
management area ACME-2 of the Loxahatchee
National Wildlife Refuge in South Florida,
observed during 1989-1990, from where six B.
ribis isolates were obtained.
A portion of a declining M. quinquenervia stand
in the management area ACME-2 of the Loxahatchee
National Wildlife Refuge in South Florida. Note
crown thinning among some trees (arrows) and
complete leaf-loss among others (arrow heads).
A M. quinquenervia tree with a basal canker
(arrow) showing severe crown thinning above
cankered area but still sprouting from the root-
collar (arrow head).
A typical canker on the stem revealing vertical
depression (arrow head) surrounded by callus
ridges (arrow).
A portion of a cankered stem split to reveal
inner tissues. Note discolored sapwood (arrows)
beneath the canker.


79


80
Figure 3.5. Mean necrotic leaf area of M. quinquenervia due to
various treatments. Treatments 1-6, 7, 8, and 9 represent the six
B. ribis isolates from 1989-90 collections, F. moniliformis var
subglutinans, Lophotrichus sp., and wound alone, respectively.


Figs. 3.6-3.9. Stems of M. quinquenervia ramets with wound alone
and wound inoculated with B. ribis, and
maintained under greenhouse conditions for 8
weeks.
Figure 3.6.
Figure 3.7.
Figure 3.8.
Figure 3.9.
A portion of a stem wounded but not inoculated
with fungus. Note the wound has been closed by
callus ridges (arrow).
A portion of a stem wounded and inoculated with
B. ribis. Note canker extension above and below
the point of inoculation (arrow head) and callus
ridge (arrow) around the canker.
A portion of a stem inoculated with B. ribis in
the fall, representing non-callused stem. Note
lack of callus around the wound (arrow).
A portion of a stem above the point of
inoculation of B. ribis (in fall) showing
pycnidia (arrows) emerging from the bark.


82


Full Text
EVALUATING THE PATHOGENICITY OF Botryosphaeria ribis
Gross. & Duggar ON Melaleuca quinquenervia (Cav.)
Blake IN SOUTH FLORIDA
BY
MIN BAHADUR RAYACHHETRY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1995

I dedicate this dissertation to my parents, Kamal B.
Rayamazhi and Chhindra K. Rayamazhi, who always dreamed of their
children being educated. It is their love, encouragement, and
sacrifice that made their children's education possible.

ACKNOWLEDGEMENT
I am grateful to Dr. G.M. Blakeslee, the chairman of my
supervisory committee, for his willingness to take the
responsibility of guiding me through my graduate program following
Dr. R.S. Webb's relocation. His insight and advice in every step
of my graduate program were invaluable in accomplishing this goal.
I wish to extend my profound appreciation to him for his time,
patience, expertise, receptiveness, and response during
preparation of this dissertation.
I am also grateful to Dr. R.S. Webb for first accepting me
into this PhD program and guiding me as the chairman of my
supervisory committee until his relocation. I appreciate him for
allowing me to be involved in this Melaleuca biocontrol project,
providing me with a research assistantship, and being willing to
continue to serve on the supervisory committee.
My deep appreciation is extended to Dr. J.W. Kimbrough, a
member of my supervisory committee, who provided me with an
assistantship during a critical financial crisis in my graduate
program in 1993 and access to his laboratory as needed. His
expertise, time, and kindness were invaluable in improving the
quality of my graduate research.
I would like to extend my sincere thanks to Drs. R.
Charudattan, J. Gander, and T. Center, the members of my
supervisory committee, for invaluable input throughout my graduate
program and their willingness to dedicate time and expertise in
improving the quality of my research. I am grateful to every

member of my supervisory committee for reviewing and giving
creative suggestions during preparation of this dissertation.
I would like to extend my heartfelt thanks to Dr. L.
Arvanitis for his constant encouragement and help as an
administrator during the ups and downs of my life as a graduate
student in this school. Appreciation is also due to Dr. T. Miller
for reviewing and giving invaluable suggestions on Chapter V of
this dissertation. Thanks are also due to Jay Harrison and Jim
Allen for help and verification of the statistical analysis
performed on various experiments included in this manuscript.
I wish to thank Winnie Lante and Dr. Robert Berger for their
friendship, wisdom, and assistance in every possible way. I also
extend my thanks to Joel Smith, James DeValerio, Jeff English, Dan
Schultz, Tess Korhnak, Dave Nolletti, and Willy Wood for their
friendship as well as unfaltering assistance as needed.
Appreciation is also due to Drs. Mark Lesney, Jerry Benny, Mick
Popp, and Litzu Li for their wisdom and sincere friendship.
I would like to thank USDA-ARS, Fort Lauderdale, South
Florida Water Management District, West Palm Beach, Florida, and
SFRC, University of Florida, for funding this research and my
graduate program.
Last but not least, I extend my deepest love and gratitude
to my parents, wife Geeta, and sons Amit and Ajamber. It would
have been impossible for me to accomplish my PhD program without
their love, encouragement, and sacrifice.
IV

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES ix
LIST OF FIGURES xi
ABSTRACT xvi
CHAPTERS
I. GENERAL INTRODUCTION 1
Host Distribution 1
Problems Associated with Melaleuca
in South Florida 1
Pathology of Melaleuca 3
Research Rationale 4
General Hypotheses 6
General Objectives 7
Chapter Organization 7
II. MYCOLOGICAL CHARACTERISTICS OF THE ISOLATES OF
Botryosphaeria ribis 8
Introduction 8
Materials and Methods 11
Isolate Acquisition and
Identification 11
Mycelial Growth and Morphology 12
Vegetative Compatibility
Among Isolates 13
Pycnidial Production and Morphology 13
Conidial Characteristics 15
Germination of Macroconidia 15
Statistical Analyses 16
v

Results.
17
Fungal Identification 17
Mycelial Growth and Morphology 17
Vegetative Compatibility
Among Isolates 20
Pycnidial Production and
Morphology 22
Conidial Characteristics 24
Germination of Macroconidia 25
Discussion 27
Fungal Morphology and Taxonomy 27
Germination of Macroconidia 32
Conclusions 34
III. EVALUATION OF Botryosphaeria ribis ISOLATES FOR
PATHOGENICITY TO Melaleuca quinquenervia 41
Introduction 41
Materials and Methods 43
Tree Clone Production 43
Isolate Acquisition 44
Inocula Preparation 45
Leaf Inoculations 46
Influence of Hyphae vs Macroconidia
as Inocula 48
Pathogenicity of Botryosphaeria ribis
vs Pestalotia 49
Effects of Inoculation Positions
on Canker Development 50
Evaluation of Virulence of Isolates
Under Greenhouse Conditions 50
Evaluation of Virulence of Isolates
Under Field Conditions 52
Inocula 52
Site selection 53
Tree inoculation 53
Experimental design 53
Harvesting and canker evaluation 53
Effects of Season on Canker
Development 54
Simulated Storm-Damage and Inoculation
with Macroconidia 56
Statistical Analyses 58
Results 58
Leaf Inoculations 58
Influence of Hyphae vs Macroconidia
as Inocula 60
VI

60
Pathogenicity of Botryosphaeria ribis
vs Pestalotia
Effects of Inoculation Positions
on Canker Development 62
Evaluation of Virulence of Isolates
Under Greenhouse Conditions 62
Evaluation of Virulence of Isolates
Under Field Conditions 65
Effects of Season on Canker
Development 66
Simulated Storm-Damage and Inoculation
with Macroconidia 67
Discussion 69
Preliminary Screening of Macroconidial
Cultures 6 9
Evaluation of Isolates by Stem
Inoculations 70
Conclusions 76
IV. EFFECTS OF STRESS FACTORS ON CANKER DEVELOPMENT ON
STEMS OF Melaleuca quinquenervia INOCULATED WITH
Botryosphaeria ribis 87
Introduction 87
Low Moisture 8 8
Low Temperature 90
Defoliation 92
Objectives 93
Materials and Methods 93
Selection Criteria for Tree Clones
and Isolates 93
Low Moisture 94
Low Temperature 95
Defoliation 96
Statistical Analyses 97
Results 97
Low Moisture 97
Low Temperature 98
Defoliation 100
Discussion 100
Low Moisture 100
Low Temperature 103
Defoliation 105
Conclusions 106
vii

V. HISTOPATHOLOGY OF Botryosphaeria ribis IN Melaleuca
quinquenervia STEMS 112
Introduction 112
Materials and Methods 114
Preparation of Leaf Materials 114
Preparation of Stem Materials from
Unstressed Plants 115
Preparation of Stem Materials from
Stressed Plants 116
Results 117
Host Response 117
Leaves 117
Stems 117
Callused 118
Noncallused 120
Stressed 121
Fungal Invasion and Colonization 121
Leaf Tissues 121
Stem Tissues 122
Callused 122
Noncallused 123
Discussion 123
Leaf Invasion and Colonization 123
Stem Invasion and Colonization 124
Conclusions 129
VI. SUMMARY AND CONCLUSIONS 137
REFERENCES 143
BIBLIOGRAPHICAL SKETCH 152
viii

LIST OF TABLES
Table Page
2.1. Radial growth rates (mm/day) of the mycelia of
Botryosphaeria ribis isolates on potato dextrose agar
(PDA) ,cornmeal agar (CMA), mycological agar (MA) , and
starch agar (SA) 18
2.2. Production of aerial hyphae, mycelial color, and
pycnidial stroma by isolates of B. ribis in four
artificial growth media at 21 days after inoculation. .. 19
2.3. Vegetative and reproductive behavior of the pairs of
the eight B. ribis isolates on and around the zone of
confrontation (the meeting point of two colonies
growing towards each other) on PDA 21
2.4. Range and average diameter of the pycnidial stroma
on 2- to 4-week-old culture of some B. ribis isolates. .. 24
2.5. Mean and range of dimensions (length and width) and
the number of nuclei in macroconidia of B. ribis 24
2.6. Effect of temperature on germination of macroconidia
by 4 h after incubation in WA at 100 percent ambient
humidity 26
3.1. Leaf bioassay experiments designed for preliminary
tests of virulence among 11 monoconidial cultures
within each of six isolates of B. ribis obtained from
Melaleuca quinquenervia from South Florida 59
3.2. Separation of means of the effects of form of
inocula on canker length (mm) on the stems of M.
quinquenervia in summer inoculations with isolate
BR-1 of B. ribis 61
3.3. Main-effect means and mean separations from analyses
of variance for comparison of B. ribis and Pestalotia
species, and determination of the effect of inoculation
point on disease development. Dependent variable was
total canker length (mm) measured at 8 weeks after
inoculation in spring 61
IX

3.4.
Percent callusing around the inoculation point and
percent reisolation of fungus from 5 mm beyond the
inoculation point on M. quinquenervia stems at 8
weeks after inoculation in spring 63
3.5. Effects of B. ribis isolates on canker development
on stems of M. quinquenervia clones at 8 weeks after
inoculation in spring 1994 64
3.6. Main-effect means and mean separations from analyses of
variance for stem-inoculation tests on total canker
length (mm) at 8 weeks after inoculation 65
3.7. Effects of simulated storm damage on the establishment
of BR-1 isolate of B. ribis on stems of M. quinquenervia
ramets inoculated with macroconidia 68
4.1. Analysis of variance of data of experiments designed to
assess the effects of stress factors on canker
development on stems inoculated with BR-2 and BR-5
of B. ribis at 8 weeks after inoculation 99
4.2. Main-effect means and mean separations from analyses
of variance for effects of stress factors on canker
length (mm) on the stems of M. quinquenervia inoculated
with isolates of B. ribis 99
x

LIST OF FIGURES
Figure Page
2.1. Aerial hyphae Botryosphaeria ribis imparting black
color and partially submerged pycnidia on PDA 37
2.2. Hyphae of B. ribis in and on the surface of media
imparting tan color and pycnidia immersed in CMA 37
2.3. Mycelial mat of B. ribis on PDA with hyphae of various
diameters 3 7
2.4. Blended mycelial mat of B. ribis grown in PDB under
continuous shaking conditions showing chains of
chlamydospore-like cells 37
2.5. Cross-section through a multilocular pycnidial stroma
of B. ribis produced on PDA showing numerous locules. .. 37
2.6. Enlarged pycnidia from a portion of multilocular
stroma of B. ribis showing macroconidia and an obscure
ostiole 37
2.7. Pycnidial stroma of B.ribis on Melaleuca quinquenervia
leaf, inoculated and maintained in a sterile moist
chamber. Evidence of solitary and botryose types of
pycnidia 3 7
2.8. Pycnidial stroma of B. ribis erupting from dead bark
of M. quinquenervia showing solitary and botryose
types of pycnidia 3 9
2.9. Section through a M .quinquenervia leaf showing a
solitary pycnidium of B. ribis erupting from epidermis.
Evidence of ostiole and pycnidial wall 3 9
2.10. Longitudinal section through a M. quinquenervia stem
showing a solitary pycnidium of B. ribis erupting
through bark. Evidence of ostiole and pycnidial wall
composed of textura angularis 39
2.11. Pycnidial stroma of B. ribis on M. quinquenervia leaf
oozing macroconidial cirrhi at room temperature 39
XI

2.12. Cross-section through a pycnidium of B. ribis showing
pycnidial wall composed of textura angularis, and
conidiophores 3 9
2.13. A portion of the inner wall of a 3-day-old pycnidium
of B. ribis. Evidence of conidiophores with bulbous
base bearing macroconidia at different stages of
deve 1 opment 3 9
2.14. Macro- and microconidia from freshly squashed 7-day-
old pycnidium of B. ribis showing the microconidia and
macroconidia 3 9
2.15. Macroconidia of B. ribis from PDA germinated in water.
Evidence of mixture of aseptate and septate conidia
bearing germ tubes 3 9
2.16. Germinating aseptate and septate macroconidia of B.
ribis showing one or more germ tubes 39
2.17. Germination of the spores of Isolate BR-1 of B. ribis
on water agar at 100 percent relative humidity 40
2.18. Germination of the spores of Isolate BR-7 of B. ribis
on water agar at 100 percent relative humidity 40
3.1. A portion of a declining M. quinquenervia stand in the
management area ACME-2 of the Loxahatchee National
Wildlife Refuge in South Florida. Crown thinning
among some trees and complete leaf-loss among others. .. 79
3.2. A M. quinquenervia tree with a basal canker showing
severe crown thinning above cankered area but still
sprouting from the root-collar 79
3.3. A typical canker on the stem of M. quinquenervia
revealing vertical depression surrounded by callus
ridges 79
3.4. A portion of a cankered stem of M. quinquenervia
split to reveal the discolored sapwood beneath the
canker 79
3.5. Mean necrotic leaf area of M. quinquenervia due to
treatments with isolates BR-1 through BR-6 of
B. ribis, F. moniliformis var subglutinans,
Lophotrichus sp., and wound only 8 0
3.6. A portion of a stem wounded but not inoculated with B.
ribis showing wound closed by callus ridges 82
3.7. A portion of a stem wounded and inoculated with
B. ribis showing canker extension above and below
the point of inoculation, and callus ridge around the
canker 82
xii

3.8. A portion of a stem inoculated with B. ribis in the
fall, representing noncallused stem showing lack of
callus around the wound 82
3.9. A portion of a stem above the point of inoculation
of B. ribis showing pycnidia emerging from the bark
of M. quinquenervia stem 82
3.10. Pathogenic interaction between B. ribis and M.
quinquenervia showing means of canker length from
21 stems 83
3.11. Clonal susceptibility of M. quinquenervia towards B.
ribis shown by mean of canker length from 24 stems 83
3.12. Mean canker length at the proximal and distal parts
of the stem from the point of inoculation of M.
quinquenervia with B. ribis isolates 84
3.13. Mean necrotic tissues on M. quinquenervia stems at
5 mm above the point of B. ribis inoculations showing
mean percentage of necrosis of phloem and cambium 85
3.14. Mean canker length of M. quinquenervia ramets 8 weeks
after inoculation with BR-2 isolate of B. ribis 86
4.1 Xylem water potential of differentially moisture-
stressed stems of M. quinquenervia ramets inoculated
with B. ribis 107
4.2. Effects of low moisture on canker development on
the stems of M. quinquenervia ramets inoculated
with B. ribis 107
4.3. Four groups (gr.) of M. quinquenervia ramets inoculated
with B. ribis and watered every 24 (gr.l), 72 (gr.2),
168 (gr.3), and 288 (gr.4) h showing progressive crown
thinning (from gr.l to gr.4) due to leaf-drop 109
4.4. A stem showing a typical canker among M. quinquenervia
ramets wounded and inoculated with B. ribis and
watered every 24 h showing callus ridge surrounding a
canker 109
4.5. A stem showing a typical canker among M. quinquenervia
ramets wounded and inoculated with B. ribis and watered
every 288 h 109
4.6. Inner tissues in a typical stem canker among M.
quinquenervia ramets wounded and inoculated with
B. ribis and watered every 288 h revealing blackened
bark and sapwood 109
4.7. Effects of low temperature on canker development on
stems of M. quinquenervia ramets inoculated with
B. ribis
xiii
110

4.8. Effects of defoliation on canker development on stems
of M. quinquenervia ramets inoculated with B. ribis. ... Ill
4.9. Effects of defoliation on mortality of M. quinquenervia
ramets inoculated with B. ribis Ill
5.1. A cross-sectional view of a healthy M. quinquenervia
leaf stained with Pianeze's IIIB showing stoma,
epidermis, chlorenchyma cells, mesophyll cells,
and oil chamber 132
5.2. A wounded but noninoculated (with B. ribis) stem
showing callus closing wound 132
5.3. AM. quinquenervia stem wound-inoculated with B. ribis
showing canker surrounded by callus ridges 132
5.4. A cross-sectional view of a wounded M. quinquenervia
stem stained with Pianeze's IIIB showing callus
filling the wound, vascular cambium and xylem at the
time of wounding, and patches of ligno-suberized
cells in callus. Evidence of redifferentiated vascular
cambium and newly formed xylem in callus 132
5.5. A cross-section through unwounded M. quinquenervia
stem stained with phloroglucinol-HCl and Sudan IV
showing phelloderm and layers of suberized cells,
cortex, phloem, and xylem 132
5.6. A cross-sectional view through a portion of 4-wk-old
wound on stem stained with phloroglucinol-HCl and
Sudan IV showing suberized cell layers in callus ridges
extending to the ligno-suberized periderm enclosing
tanninoid and ligno-suberized parenchyma cells 132
5.7 A longitudinal section (stained with Pianeze's IIIB)
through the margin of callus on a B. ribis-inoculated M.
quinquenervia stem showing a gap at the interface of
xylem and callus, groups of ligno-suberized cells
enclosing tanninoid parenchymatous cells 132
5.8. A cross-section through a 8-wk-old, B. ribis
inoculated wound of a fresh M. quinquenervia stem
stained with phloroglucinol-HCl revealing xylem, and
suberized and ligno-suberized parenchyma cells in and
around the callus ridge 134
5.9. A longitudinal section through the canker margin of
a B. ribis-inoculated stem of M. quinquenervia showing
tyloses plugging a vessel in the xylem near disrupted
vascular cambium 134
xiv

5.10. A cross-section through a B. ribis-inoculated M.
quinquenervia leaf (stained with Pianeze's IIIB)
showing fungal hyphae penetrating epidermis through
stomata and colonizing chlorenchyma, mesophyll,
and oil chamber 134
5.11. A cross-section through a B. ribis-inoculated leaf M.
quinquenervia leaf (stained with Pianeze's IIIB)
showing a fungal hypha penetrating through stomata,
and a pycnidium emerging from the epidermis 134
5.12. A cross-section through a callus ridge of a stem
canker on M. quinquenervia caused by B. ribis showing
cells surrounded by hypha resulting in deterioration
of cellular integrity 134
5.13. A cross-section through a callus ridge of a stem canker
on M. quinquenervia caused by B. ribis revealing
intra- and intercellular hyphae in deteriorating areas
as shown in Fig. 5.12 134
5.14. A longitudinal section through xylem from the canker
margin of a B. ribis-inoculated stem of M.
quinquenervia showing hyphal growth along ray
parenchyma and tracheal cells 136
5.15. A longitudinal section through xylem from the canker
margin of a B. ribis-inoculated stem of M.
quinquenervia showing hyphae along inner wall clogging
tracheal cells, passing radially and producing hyphal
swellings 136
5.16. A longitudinal section through xylem from the canker
margin of a B. ribis-inoculated stem of M.
quinquenervia showing a hyphae branching in a tracheal
lumen 136
5.17. A cross-section through noncallused canker on M.
quinquenervia stem inoculated with B. ribis revealing
a pycnidium in phelloderm, hyphal concentration in
cambium and phloem, and hyphae in the lumen and
intercellular spaces of vessels and tracheids 136
xv

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EVALUATING THE PATHOGENICITY OF Botryosphaeria ribis
Gross. & Duggar ON Melaleuca quinquenervia (Cav.)
Blake IN SOUTH FLORIDA
By
Min Bahadur Rayachhetry
May, 1995
Chairman: Dr. G.M. Blakeslee
Major Department: School of Forest Resources and Conservation
Biology and virulence of the Fusicoccum anamorph of
Botryosphaeria ribis isolates obtained from the canker margins of
Melaleuca quinquenervia trees and gall tissues of Rhizophora
mangle from South Florida were evaluated under laboratory,
greenhouse, and field conditions. Morphologically, the isolates
were variable. Hyphal, pycnidial, and conidial attributes of the
isolates of B. ribis examined overlapped in the anamorph of B.
dothidea revealing their synonymy.
Spores and hyphae of B. ribis were equally effective in
inciting canker disease on M. quinquenervia stems. A weak
correlation existed between diameter of stem at the point of
inoculation and canker length. All eight tested isolates of B.
ribis were able to incite cankers under greenhouse and field
conditions but two isolates were more virulent than others.
Differential susceptibility of M. quinquenervia clones towards B.
ribis was evident.
XVI

Stem-wounds without B. ribis were closed in both spring and
fall inoculations. Cankers with or without callus were formed
following both spring and fall inoculations with B. ribis. Fall
inoculations had less callusing intensity and greater percentages
of wilting and dieback of ramets than those inoculated in spring
although the differences were small. Establishment of B. ribis in
stems of M. quinquenervia appeared to require wounds at the depth
of the sapwood or some kind of injury stressing the trees.
Low moisture in the xylem (<-1.5 Mpa) of B. ribis inoculated
stems favored rapid canker development and wilting of ramets. Low
temperature treatment of 6°C for 3-day/wk was more effective than
6°C for 6-day/wk and 0 (±1)°C for 16-h/wk in inciting longer
cankers on M. quinquenervia stems inoculated with B. ribis.
Partial defoliation followed by B. ribis inoculation did not cause
differential canker expansion. Complete defoliation plus B. ribis
inoculation caused 90 percent mortality of ramets within 4 weeks.
Callus tissues formed in response to wounding were
surrounded by suberized periderm, both in presence and absence of
inoculation with B. ribis. Tissues in the callus ridges were
tanninoid and contained groups of lignified cells. The fungus
gained entry into callus tissues through the ventral surface of
the wedges. Cells in the xylem, adjacent to the wound were also
intensely lignified. Despite these barriers, inner tissues in
sapwood and callus were invaded by fungal hyphae. Stems with
noncallused wounds were rapidly girdled by fungal hyphae.
Botryosphaeria ribis appeared to be able to persist in the
tissues of healthy trees but became more aggressive pathogen on M.
quinquenervia in presence of stress factors.
XVII

CHAPTER I
GENERAL INTRODUCTION
Host Distribution
Melaleuca quinquenervia (Cav.) Blake, the cajeput tree, is a
species in the Myrtaceae (Meskimen 1962, Bodle et al. 1994) . It
is native to Australia and New Guinea (personal communication with
Dr. Center), and has become naturalized in the Philippines, India,
Madagascar, Zaire, Hawaii, the United States (Myers 1975), and
Indonesia (Holliday 1989) . The genus Melaleuca includes more than
200 species in Australia alone (Holliday 1989).
Problems Associated with Melaleuca in South Florida
The introduction of M. quinquenervia to South Florida
occurred in the 1890s (Meskimen 1962) when seeds were imported.
The tree was considered useful for ornamental purposes and for a
possible commercial source of wood for forest industries (Myers
1975, Huffman 1980, Balciunas and Center 1991, Bodle et al. 1994).
Certain favorable biological characteristics, such as prolific
seed-producing ability and tolerance to brackish water, flooding,
complete submergence, and fire, have enabled this species to
aggressively invade portions of the Everglades National Park, and
the seasonally flooded areas of Lee, Collier, Martin, Palm Beach,
1

2
Broward, and Dade counties in South Florida (Meskimen 1962, Myers
1975, Huffman 1980, Hofstetter 1991). In 1976, M. quinquenervia
trees were estimated to cover an area of about 0.53 million ha
(Ewel et al. 1976). The trees reportedly produce allelopathic
chemicals (Di Stefano and Fisher 1983) which may enhance their
ability to displace local flora.
In 1983, this tree species was estimated to occupy 1.01
million ha, and the rate of invasion of new areas was estimated at
1012 ha/year (Di Stefano and Fisher 1983) . Since 1980, the
invaded area has increased at least by 50 percent (Balciunas and
Center 1991). For example, the Florida peninsula south of Lake
Okeechobee has been estimated to have an infestation of 0.2 to
0.61 million ha out of the total acreage of 3.04 million ha (Bodle
et al. 1994). It has been estimated that most of the remaining
natural land south of Lake Okeechobee may be overtaken by
uncontrolled M. quinquenervia within 30 years (Bodle et al. 1994)
The aggressive colonization by M. quinquenervia of South
Florida wetland sites has created serious problems such as the
near elimination of native plant communities and freshwater
wildlife species (Crowder 1974), respiratory problems and allergic
reactions of people to pollen and vaporized oils (terpinine 4-ol)
(Morton 1962, 1969), increased fire hazard to urban developments,
and accelerated loss of ground water due to increased
evapotranspiration (Balciunas and Center 1991) .

3
Pathology of Melaleuca
Spaulding (1961) listed a total of three fungal pathogens
(Fomes lignosus, causing root-rot in Uganda; Phyllosticta
leucadendri, causing leaf spot in Australia; and Puccinia
camargoi, causing leaf rust in Brazil) on cajeput (M.
leucadendron=M. quinquenervia) trees. According to Hepting
(1966), cajeput trees appear to be free of diseases in their
exotic as well as native habitats. Hepting (1966) mentioned that
the only disease record on cajeput trees in the United States is
the damping-off disease in the nurseries in Florida due to
Rhizoctonia species.
Farr et al. (1989) have recorded a few pathogens
(Botryosphaeria sp., Macrophoma sp., Phoma sp., and Phomopsis sp.
causing dieback; Pythium sp. and Fusarium sp. causing root rot;
Puccinia psidii causing leaf rust; and Al ternaria sp.,
Cylindrocladium pteridis, C. scoparium, Dothiorella sp., and
Phyllosticta sp. causing leaf spots) on M. quinquenervia from
South Florida.
Alfieri et al. (1994) have recorded several fungal pathogens
on M. quinquenervia from South Florida. These diseases are
related to damping-off (Pythium sp., and Rhizoctonia solani),
algal leaf spots (Cephaleuros virescens), fungal leaf spots
(Alternaría sp., Cercospora sp. , Curvularia sp . , Cylindrocladium
pteridis, C. scoparium, Dothiorella sp., and Phyllosticta sp.),
foliar rust (Puccinia psidii), stem cankers and dieback
(Botryosphaeria sp., Macrophoma sp., Phoma sp., Phomopsis sp., and
Physalospora sp.), stem galls (Sphaeropsis sp.), and root rots

4
(Armillariella tabescens, Fomes sp., Fusarium sp., Gloeosporium
sp.( Pycnosporus sp., and Pythium sp.).
Thus far, no pathogenicity tests of any of these pathogens
on M. quinquenervia have been reported and the effects of these
fungi on the population of M. quinquenervia from South Florida are
unknown.
Research Rationale
Because of the increasing awareness of the negative impact
of M. quinquenervia in South Florida ecosystems, this species
recently has been listed officially as a federal noxious weed and
a Florida prohibited aquatic weed (Bodle et al. 1994). Various
research projects concerning the control of M. quinquenervia and
possible restoration of South Florida ecosystems are underway.
The cost of control ($1.70 per tree and 25,000-100,000 trees/ha)
of M. quinquenervia by herbicide use is very high (Bodle et al.
1994). Additionally, seed release is accelerated among trees
stressed by herbicides and new foci are created through natural
dispersal of these seeds (Laroche and Ferriter 1992). Such a
phenomenon of regeneration will require repeated herbicidal
treatment of these sites which compounds the pollution problems in
sensitive areas like the Everglades and other fresh water
reservoirs in South Florida. A problem of this magnitude warrants
the discovery and use of control agents that can perpetuate in the
tree stands, disperse naturally and destroy existing stands or
limit further invasion of new sites. For effective control of
this weed, we need agents that can attack one or more plant parts

5
such as roots, stems, leaves, flowers and fruits. Balciunas and
Center (1991) have discussed the suitability of M. quinquenervia
as a target for biological control.
With these strategies in mind, biological control of this
weed by suitable insect pests and/or fungal pathogens to ensure
ecological compatibility and economic viability (Balciunas and
Center 1991, Laroche and Ferriter 1992, Bodle et al. 1994) by
reducing the use of herbicides has received recent attention.
Weed control has been accomplished using insects and fungal
pathogens. Biological control of herbaceous weeds has been
successfully accomplished in the United States using an endemic
pathogen (e.g., TeBeest and Templeton 1985, Charudattan 1991).
The benefits of endemic pathogens as biological control agents has
been discussed by Charudattan (1988). In the past, such
activities have been successful against perennial woody weeds
(e.g., Wilson 1965, Griffith 1966, Adams 1988, Charudattan 1991,
TeBeest et al. 1992).
In August 1989, Dr. Mark D. Maffei, the Assistant Manager of
the Loxahatchee National Wildlife Refuge (Palm Beach County, FL)
discovered a patch of dead and dying M. quinquenervia trees in
Acme-2 of the Refuge. He contacted Dr. Ted D. Center, the Project
Leader of the USDA-ARS, Aquatic Plant Management laboratory at
Fort Lauderdale, FL. Dr. Center invited Dr. Roger S. Webb, then
Associate Professor in the School of Forest Resources and
Conservation at the University of Florida, to identify the cause
of tree mortality in the Refuge. A preliminary investigation by
Dr. Webb indicated that the decline and mortality were most likely
associated with the occurrence of stem vascular stain and cankers

6
located beneath the papery bark layers of the M. quinquenervia
trees. He consistently isolated a fungus from the margin of
diseased stem tissue that subsequently was identified by the
International Mycological Institute, Kew, Surrey, England, as the
Fusicoccum anamorph (order, Sphaeropsidales; family,
Sphaeropsidaceae) of Botryosphaeria ribis Gross. & Duggar.
Initial pathogenicity tests under greenhouse conditions
revealed B. ribis to be capable of inciting disease on M.
quinquenervia. Previously, isolates of B. ribis were reported to
possess some degree of host specificity towards their original
hosts (Hildebrand and Weber 1944, Witcher and Clayton 1963,
Schreiber 1964, Milholland 1972, Sutton and Boyne 1983, Latorre
and Toledo 1984, Pusey et al. 1986, Shearer et al. 1987). This
fungus is native to South Florida, so it should be safe to use as
a biological control agent, if a virulent and host specific
isolate could be identified.
General Hypotheses
1. Botryosphaeria ribis is capable of inciting disease on
M. quinquenervia,
2. certain isolates of B. ribis may be more virulent than
others on M. quinquenervia clones, and
3 . stress factors may influence disease severity caused by
B. ribis on M. quinquenervia.

7
General Objectives
In general, the following objectives were considered in
testing the hypotheses mentioned above.
1. Establish mycological characteristics of B. ribis
isolates included in the experiments.
2. Evaluate B. ribis isolates for pathogenicity to M.
quinquenervia clones.
3. Test virulent isolate(s) for disease development on
stressed M. quinquenervia plants.
4. Analyze the histopathology of disease development on
inoculated stems.
Chapter Organization
This research work will be discussed in six chapters that
follow: "Mycological Characteristics of the Isolates of
Botryosphaeria ribis" (Chapter II); "Evaluation of Botryosphaeria
ribis Isolates for Pathogenicity to Melaleuca quinquenervia"
(Chapter III); "Effects of Stress Factors on Canker Development on
Melaleuca quinquenervia Inoculated with Botryosphaeria ribis"
(Chapter IV); "Histopathology of Botryosphaeria ribis in Melaleuca
quinquenervia Stems" (Chapter V); and "Summary and Conclusions"
(Chapter VI). Each chapter is intended to be a self-contained
unit, including the literature review pertinent to that section.

CHAPTER II
MYCOLOGICAL CHARACTERISTICS OF THE ISOLATES OF
Botryosphaeria ribis
Introduction
Botryosphaeria ribis Grossenb. & Dug. and B. dothidea (Moug.: Fr.)
Ces. & de Not. are often treated synonymously as established by
von Arx and Muller (1954) during reclassification of amerosporous
pyrenomycetes, and later supported by the work of Witcher and
Clayton (1963). However, as B. dothidea was described by de
Cesati and de Notaris in 1863 and B. ribis was described later by
Grossenbacher and Duggar in 1911, the former name has the priority
over the later (von Arx and Muller 1954, Witcher and Clayton 1963,
Maas and Uecker 1984). Since then the names, B. dothidea to B.
ribis, have been synonymously used by many authors (Witcher and
Clayton 1963, Crist and Schoeneweiss 1975, English et al. 1975,
Spiers 1977, Brown and Hendrix 1981, Latorre and Toledo 1984, Mass
and Uecker 1984, Pusey et al. 1986, Pusey 1989). However, others
consider B. ribis and B. dothidea as two distinct species (Smith
1934, Luttrell 1950, Punithalingam and Holliday 1973, Davison and
Tay 1983, Webb 1983, Rumbos 1987, Ramos et al. 1991).
Botryosphaeria ribis (order, Pleosporales; family,
Botryosphaeraceae) (Luttrell 1973, Barr 1987) was originally
described from currants (Ribes spp.) in New York (Grossenbacher
8

9
and Duggar 1911). Additionally, they reported two anamorphs
associated with B. ribis, a Macrophoma-form (pycnidial
conidiomata), and a Dothiorella-form (stromatic conidiomata). The
authors also recognized the existence of two different strains of
B. ribis, 1) chromogenic (purplish-pink coloration on starch
paste) and strongly parasitic, and 2) nonchromogenic and
saprophytic to currants. The strains reported by Grossenbacher
and Duggar (1911) were confirmed by Shear et al. (1924) and
additionally, they noted that the microconidia either remained
intermixed with the macroconidia or were produced in separate
locules. Stevens (1926) supported the existence of the
chromogenic strain of B. ribis and reported its occurrence in the
native hosts of Cuba and southeastern United States. However,
Witcher and Clayton (1963) considered B. ribis and B. dothidea to
be synonymous and mentioned that the latters' pathogenicity was
not correlated with chromogenesis in culture media. They grew two
blueberry-derived isolates in Czapek's solution at various pH
levels and produced four color groups, i,e., clear, yellow, and
purple pigments in pH 5.0-7.0, and red at pH 9.0.
So far, the taxonomy of the Fusicoccum anamorph of B. ribis
is in a confused state (Morgan-Jones and White 1987). The type
species F. aesculi Corda was described and illustrated originally
by Saccardo (1880, 1886). Petrak (1922) considered Saccardo1s F.
aesculi as an anamorph of B. dothidea so he established a new
binomial, Dothiorella aesculi Petrak. Despite many names for this
anamorph in the literature, it is accepted that the name
Fusicoccum, as an anamorph of at least some species of
Botryosphaeria Ces.& de Not., such as B. ribis and B. dothidea,

10
seems logical (Sutton 1980, Morgan-Jones and White 1987, Rumbos
1987, personal communication with Punithalingam 1990).
Morphologically, no clear differences have been established
for B. ribis and B. dothidea. Hyphal color in growth media has
been reported to change from white to dark-gray or greenish-black
(Weaver 1974, Mass and Uecker 1984, Rumbos 1987). Swollen cells
and chlamydospores have been reported in B. ribis (Ramos et al.
1991). Macroconidia are produced in uni- or multilocular
pycnidial stroma and the majority of them are hyaline and
aseptate, though 1-3 septate brown conidia were also reported in
both fungal species (Spiers 1977, Mass and Uecker 1984, Rumbos
1987). Conidia and ascospores are multinucleate but all nuclei in
the conidium and ascospore are homokaryotic, i.e., genetically
similar (Wolf and Wolf 1939).
The literature on B. ribis appears largely unsettled in
regard to its taxonomy, especially the anamorphic stage i.e.,
Fusicoccum vs Dothiorella, and morphological characteristics of
the fungus. Additionally, the teleomorph stages are very rare.
Many mycologists and pathologists refer to the teleomorphic stage
of this fungus i.e., B. ribis, to avoid uncertainty about its
anamorphic taxonomy.
Because of the uncertainties associated with the fungus, the
first objective of this research was to investigate morphological
and reproductive characteristics of the isolates of B. ribis
obtained from the canker margins on the stems of M. quinquenervia
(Cav.) Blake and stem galls on Rhizophora mangle L. from South
Florida.

11
Materials and Methods
Isolate Acquisition and Identification
During 1989, Dr. Roger S. Webb isolated and performed
preliminary pathogenicity tests of five isolates of B. ribis which
he had obtained from stem canker margins of Melaleuca
quinquenervia (Cav.) Blake trees from the Loxahatchee National
Wildlife Refuge. These isolates were tentatively identified as B.
ribis and later sent to International Mycological Institute (IMI),
Kew, Surrey, England.
In 1990, I obtained and identified an isolate of the same
fungus from the canker margin of a tree in the same area of
Loxahatchee National Wildlife Refuge. These six isolates of B.
ribis derived during 1989-1990 were subjected to monoconidial
cultures and 11 monoconidial cultures (MCs)/isolate were tested
for pathogenicity using a leaf bioassay method described later in
this chapter. The most virulent monoconidial culture/isolate was
selected (one MC for each of six isolates) and henceforth referred
to as isolates BR-1 through BR-6. I obtained two other isolates
of B. ribis during 1993 from the necrotic gall-tissues of red
mangrove trees (Rhizophora mangle L.) collected from the coastal
region of South Florida by Dr. Webb. These two isolates were
identified by Dr. J.W. Kimbrough and me in the Department of Plant
Pathology, at University of Florida. These two isolates were also
subjected to monoconidial culture, henceforth called isolates BR-7
and BR-8. These eight isolates were used to study mycological
characteristics.

12
Mycelial Growth and Morphology
Four artificial growth media, potato-dextrose-agar (PDA)
(Difeo), cornmeal-agar (CMA) (Difeo), mycological-agar (MA)
(Difco), and 24 gm of starch (Difco) supplemented with 15 gm of
agar (Difco)/liter (SA), were used to study the radial growth
rates of the eight isolates of B. ribis. Each isolate was
inoculated on four petri plates per growth medium. The inoculum
consisted of a 3-mm disk removed from the edge of 5-day-old colony
on acidic-PDA (3.3 ml of 50% lactic acid/liter of PDA). The
plates were sealed with Parafilm (American National Canâ„¢,
Greenwich, CT) after inoculation, randomized with respect to
location on the laboratory bench and maintained at 30° C ± 1,
using a 12 h diurnal cycle of fluorescent light. The radial
extent of hyphal growth of each fungal colony was measured along 4
premarked radii at the time the first colony touched the edge of
its plate. The mean colony extension rates per plate, expressed
as mm/day (3-day basis for PDA, CMA, and MA, and 6-day basis for
SA), was used to compare isolates.
Culture plates used for growth rate assessment were further
incubated in the same condition for 21 days from the date of
treatment initiation. These plates were used to study other
reproductive and morphological characteristics such as,
sporulation behavior, mycelial color, and hyphal diameters of the
representative isolates.
Mycelial morphology of B. ribis isolates grown in potato
dextrose broth (PDB) was studied. A drop of macerated mycelial
suspension was inoculated into PDB and incubated 4 days at room

13
temperature under continuous shaking (100 rpm) conditions.
Macerated hyphae were stained with lactophenol cotton blue to
visualize dimensions for measurement of their diameter.
Photomicrographs of those hyphae were taken using a light
microscope (Olympus/BMH system).
Vegetative Compatibility Among Isolates
A modified form of half-diallel mating system (Zobel and
Tolbert 1984) was used. The isolates were given 1 chance to mate
with each other. The experiment was replicated 3 times.
Fungal isolates were grown on acidified potato dextrose agar
(APDA) (Difco) for 3 days, and agar blocks of 3-mm diameter were
cut from the periphery of the actively growing colonies using a
sterile cork borer. One block from each of the pair of isolates
to be mated was placed on opposite sides of APDA in 8.5 cm
diameter petri plates. The plates were sealed with parafilm and
incubated at 29(±1)°C under a 12 h cycle of fluorescent light.
Observations were made on days 3, 7, 14, and 35 following
inoculation to detect colony reaction at the confrontation zones
where the two colonies met. The confrontation zones were
evaluated for pigmentation, hyphal behavior, and production of
asexual and sexual structures.
Pycnidial Production and Morphology
Presence or absence of pycnidia in or on the culture medium
was recorded. The diameters of 30 solitary and botryose

14
(multilocular) pycnidial stroma of BR-4, BR-5, BR-7, and BR-8 were
measured. These four isolates were chosen because they sporulated
in all media and represented pycnidial morphology of the remaining
isolates.
Pycnidial stroma were fixed in formalin:propionic acid
(FPA), dehydrated through butyl alcohol series, and infiltrated
and embedded in paraffin (Johansen 1940) . Sections of 8-10 /¿m
thickness were cut using a rotary microtome, dewaxed through a
xylene series, passed through 100 and 90 percent ethanol, and
stained with Pianeze's IIIB (Vaughan 1914). Numbers of locules
per stromata was counted and photomicrographs were taken using
light microscopy (Nikon/OPTIPHOT II or Olympus/BMH).
Fully expanded leaves of M. quinquenervia were wound-
inoculated with a hyphal suspension of each of eight isolates of
B. ribis, incubated in the sterile moist-chamber, and allowed to
produce pycnidial stroma. Stems of M. quinquenervia ramets were
wound-inoculated with isolates of B. ribis and maintained under
greenhouse conditions. Some of these inoculated stems produced
pycnidia on the bark. Segments of leaves and stems with pycnidial
stroma were processed for sectioning and staining by methods same
as used for pycnidial stroma from artificial growth media.
Pycnidial morphology from these leaves and stems were also noted
and photographed. These stromatic pycnidia from the leaves were
also used for the study of macroconidial characteristics.

15
Conidial Characteristics
Macroconidial length, width, and the number of nuclei were
studied using 14- to 18-day-old stromatic pycnidia from the M.
quinquenervia leaves inoculated with each of the eight isolates of
B. ribis. Pycnidia were placed on a glass slide, covered with a
drop of Haupt's adhesive, crushed with a cover slip, smeared, and
were allowed to dry for 24 h at room temperature. The
macroconidia were then stained with Pianeze's IIIB (Vaughan 1914),
and the number of nuclei in each of 100 macroconidia were recorded
for each isolate.
Germination of Macroconidia
Spore germination tests were performed using slight
modifications of the procedures of Brooks and Ferrin (1994) .
Isolate BR-1 and BR-7 were selected to represent isolates from M.
quinquenervia and red mangrove, respectively, both produce
superficial pycnidial stroma on PDA and hence macroconidia could
be extracted easily.
Isolates BR-1 and BR-7 were grown on PDA under a 12 h/day
fluorescent light period at 30(±1)°C for 2 wk. Mature,
superficial or partially submerged pycnidia were partially dried
under sterile conditions by temporarily removing lids from the
petri-plates. Within 5-10 minutes following this partial
desiccation treatment, masses of macroconidia were extruded from
the pycnidia onto the surface of PDA. Conidia were washed from
the medium with sterile distilled water and the resultant

16
macroconidial suspension was strained through 2 layers of sterile
cheesecloth. One drop of spore suspension was smeared on each of
3 premarked strips on water agar (WA) in petri plates. The petri
plates were then sealed with Parafilm. Seven culture plates of
each isolate were incubated at 5, 10, 15, 20, 25, 30, 35, 40, and
45°C. One culture plate of each isolate per temperature treatment
was removed at 1, 2, 4, 8, 12, 16, and 24 h following incubation.
Following removal, the strips on WA containing germinating
macroconidia were immediately stained with cotton blue in
lactophenol and 100 macroconidia per strip, i.e., a total of 300
macroconidia per plate, were counted and the number of germinated
macroconidia were recorded for each plate. A spore was considered
germinated when the germ tube was equal or greater than the spore
length. Germination of macroconidia for each isolate per
temperature was expressed as a percentage. The experiment was
repeated twice using a different batch of WA and macroconidia.
Relevant photomicrographs of fungal materials were taken using
light microscopy (Olympus/BMH or Nikon/OPTIPHOT II).
Statistical Analyses
Wherever necessary, variances and means of main effects were
analyzed using GLM procedure in SAS (1985) . Logistic procedures
in SAS (1985) were used to analyze maximum likelihood of the
effects of temperature and incubation periods on macroconidial
germination and their relationships were used to derive equations
to explain the combined effects of these two variables.

17
Correlation of temperature and incubation period with spore
germination was tested using Pearson's method.
Results
Fungal Identification
Identity of the fungal isolates collected during 1989 from
stem-canker margin of M. quinquenervia was confirmed by Dr.
Punithalingam from IMI, Kew, Surrey, England, as the Fusicoccum
anamorph of B. ribis. I had tentatively identified one isolate
derived from similar trees in 1990 and two other isolates derived
from cankered galls on red mangrove to be the Fusicoccum anamorph
of B. ribis. Those identifications were later confirmed by Dr.
J.W. Kimbrough of the Department of Plant Pathology, at University
of Florida.
Mycelial Growth and Morphology
The radial growth rates of the isolates of B. ribis in four
growth media are presented in the Table 2.1. In general, PDA and
MA supported the most rapid growth while SA produced the slowest
for most isolates. The growth rate of some isolates on a
particular medium was also significantly different.

18
Table 2.1. Radial growth rates (mm/day) of the mycelia of B.
ribis isolates in potato dextrose agar (PDA), cornmeal agar (CMA),
mycological agar (MA), and starch agar (SA).
Isolate no.
Growth rates
(mm/day) in
PDA
CMA
MA
SA
BR-1
10.9A,a
9.2A,b
12.5A,a
6.8A,b
BR-2
13.8A,a
10.1A,b
14.1A,a
6.3A,c
BR-3
13.8A,a
10.4A,b
14.3A,a
4.9A,C
BR-4
12.4A,a
10.1A,b
13.0A,a
4.9A,c
BR-5
10.5A,a
9.7A,ab
10.4B,a
6.8A,b
BR-6
13.3A,a
8.3A,b
14.2A,a
5.4A,b
BR-7
11.3A,a
7.0B,b
11.1A,a
5.7A,b
BR-8
12.7A,a
9.8A,b
14.3A, a
6.0A,c
Note: Since isolate-media interaction was significant(Pr>F=0.0001), Scheffe's multiple
comparison procedure was used to locate significant differences between least square means
indexed by both isolate and medium. Means followed by the same letters in upper case
(comparing columns) and lower case (comparing rows) are not significantly different
(minimum significant difference=3.35 mm at P=0.05).
Some of the isolates appeared to impart color on SA, PDA,
and MA during the incubation period. However, a close observation
with the aid of a light microscope revealed that the colors were
due to a dense network of smaller diameter hyphae submerged in the
given growth medium.
Mycelial color in the center of the colony of all except BR-
6 changed from white to tan within as early as 48 h following
incubation on PDA and MA. The mycelial color of all isolates
changed from white to light yellowish-green in SA within 72 h
following incubation. A pigment similar to that of mycelial color
was also secreted in SA surrounding the submerged mycelial
network. The mycelia of all isolates remained colorless in CMA
except BR-7 which turned light yellowish-green within 72 h
following incubation. The mycelial color variation among isolates

19
in four growth media was easily noticeable 21 days after
incubation (Table 2.2).
Texture of mycelia ranged from cottony in PDA to floccose in
MA. Aerial hyphae observed at 21 days after incubation were
relatively longer in PDA and MA than in CMA and SA (Figs. 2.1,
2.2). However, isolates BR-7 and BR-8, respectively, did not
produce any aerial hyphae in CMA and SA (Table 2.2). Hyphal
diameter of a 21-day-old fungal culture ranged from 1.8-10.2
(4.2), 1.2-10.2 (5.0), 2.8-10.2 (5.3), and 2.6-12.8 (5.3) ¿/min
CMA, MA, PDA, and SA, respectively.
Table 2.2. Production of aerial hyphae, mycelial color, and
pycnidial stroma by isolates of B. ribis in four artificial growth
media at 21 days after inoculation.
Isolate number
BR-l BR-2 BR-3 BR-4 BR-5 BR-6 BR-7 BR-8
Mycelial color:
Potato dextrose agar
Corn meal agar
Mycological agar
Starch agar
Aerial hyphae:
Potato dextrose agar
Corn meal agar
Mycological agar
Starch agar
Pycnidial stroma:
Potato dextrose agar
Corn meal agar
Mycological agar
Starch agar
B
B
B
LB
LYB
LYB
W
LYB
LB
LB
LB
LB
YG
YG
LYG
YG
AM
AM
AM
AS
CS
AS
AS
BS
AS
AS
AS
AS
CS
CS
CS
DS
A
D
D
A
A
D
B
A
D
D
B
A
B
D
C
A
B
B
B
B
LYB
W
W
W
LB
T
B
B
YG
YG
YG
LYG
AM
AL
AM
AM
CS
CM
--
BS
AS
AS
AS
AS
BS
DS
DS
B
N
A
A
D
N
A
B
C
D
B
B
D
C
A
A
Note. Mycelial color: B=brown, LB=light brown, LYB=light yellowish-brown, YG=yellowish-
green, W=white, and T=tan, Aerial hyphae: Surface coverage of total media A=100%, B=50-
99%, C=26-49%, D=l-25; and upward mycelial growth L=up to petri dish cover, M=midway
between media surface and petri dish cover, --=not visible on media surface, and S= short
cottony on media surface. Pycnidial stroma: A= >100/plate, B=50-99/plate, C=26-49/plate,
D=l-25/plate and N=none.

20
Short-celled inflated hyphae (Fig. 2.3) were abundant across
the four media. Compared with the remaining three media, SA
contained a large number of hyphae with chains of chlamydospore-
like cells in the intercalary positions. The majority of cells in
these hyphae were guttulated. In contrast, mycelia in MA were
highly melanized compared with the mycelia in remaining three
media. The majority of 4-day-old hyphae grown in potato-dextrose-
broth under continuous shaking conditions consisted of chains of
chlamydospore-like cells of the diameter of up to 18.6 ¡im (Fig.
2.4) .
Vegetative Compatibility Among Isolates
The pairs of isolates can be divided into 3 groups based on
the pigmentation at the zone of confrontation of the two colonies
(Table 2.3): 1) no pigmentation by day 3, colonies diffused into
each other, and development of brown coloration similar to the
rest of the colony by day 7; 2) no pigmentation by day 3 but
mycelia not diffused into the opposite colonies and production of
dark brown coloration at the zone of confrontation; and 3) brown
pigmentation by day 3 but the mycelia not diffused into the
opposite colonies and zone of confrontation later intensely dark
brown. Microscopic observation of the agar blocks in groups 2 and
3 along with the mycelia from the zone of confrontation revealed
purplish brown pigmentation on the media around the mycelial mats.

21
Table 2.3. Vegetative and reproductive behavior of the pairs of
the eight isolates of B. ribis on and around the zone of
confrontation (the meeting point of two colonies growing towards
each other) on PDA.
Isolate
pairs
Zone Pi
Pigment
confrontation
Mycelial color
Sclerotia
Micro¬
conidia
Macro¬
conidia
Teleomor¬
ph stage
Groups
BR-lxBR-2
none
brown
(2 f)
(1)
(1)
none
1
BR-lxBR-3
none
brown
none
none
(1)
none
1
BR-lxBR-4
none
brown
none
(4)
(1,4)
none
1
BR-lxBR-5
none
brown
none
none
(1,5)
none
1
BR-lxBR-6
brown
dark brown
(6 f)
none
(1)
none
2
BR-lxBR-7
none
dark brown
none
none
(1,7)
none
2
BR-lxBR-8
none
dark brown
(8 f)
(8)
(1,8)
none
2
BR-2XBR-3
none
brown
(2 f)
none
(2,3)
none
1
BR-2XBR-4
none
brown
(2 f)
(4)
(2,4)
none
1
BR-2XBR-5
none
brown
none
(5)
(5)
none
1
BR-2xBR-6
brown
dark brown
(6 nf)
(6)
(6)
none
3
BR-2xBR-7
brown
dark brown
none
none
(7)
none
2
BR-2XBR-8
brown
dark brown
(8 f)
(8)
(8)
none
2
BR-3XBR-4
brown
dark brown
none
(4)
(3,4)
none
3
BR-3XBR-5
none
brown
none
none
(5)
none
1
BR-3XBR-6
brown
dark brown
none
none
none
none
3
BR-3XBR-7
none
brown
none
none
(7)
none
1
BR-3XBR-8
brown
dark brown
(8 f, 3
nf)
none
(8)
none
3
BR-4XBR-5
none
brown
none
(4)
(4,5)
none
1
BR-4XBR-6
brown
dark brown
(6 f)
(4)
(4,6)
none
3
BR-4XBR-7
brown
dark brown
none
(4)
(4,7)
none
3
BR-4XBR-8
brown
dark brown
(8 f)
(4)
(4,8)
none
3
BR-5XBR-6
brown
dark brown
(6 f)
(5,6)
(5,6)
none
3
BR-5XBR-7
none
dark brown
none
none
(5,7)
none
2
BR-5XBR-8
none
dark brown
(8 f)
none
(5,8)
none
2
BR-6XBR-7
brown
dark brown
(6 f)
(6)
(6,7)
none
3
BR-6XBR-8
brown
dark brown
(6 f, 8
f)
none
(6,8)
none
3
BR-7XBR-8
none
brown
(8 f)
none
(7,8)
none
1
Note: Numbers in the parenthesis under respective columns indicate the isolate numbers that produced sclerotia or
microconidia or macroconidia. Also, the 'f' and 'nf' in the parenthesis indicate presence and absence of
pycnidial sclerotia, respectively. Pigmentation and the mycelial colors at the zone of confrontation were noted
at day 3 and 7, respectively, following the date of inoculation onto PDA. The record of the presence of
sclerotia, microconidia, macroconidia, and teleomorph stages were based on the day 14 and 35, respectively,
following the date of inoculation onto PDA. Groups 1, and 2 and 3, represent vegetative compatibility and
noncompatibility, respectively.
In addition to pycnidia, isolates BR-2, BR-6, and BR-8
produced sclerotia on the surface of the media (Table 2.3). The
majority of those sclerotia contained pycnidial locules bearing
conidia. In one combination or another, the isolates BR-1, BR-4,

22
BR-5, BR-6, and BR-8 produced microconidia, whereas macroconidia
were produced by all the isolates (Table 2.3). Often, the
pycnidial concentration was intense at the zone of confrontation
of two colonies. However, as revealed by the observations of the
plates for 35 days following inoculation date, all pairs of
isolates failed to produce teleomorph stages (Table 2.3).
Pycnidial Production and Morphology
In general, production of pycnidial stroma was greater on
PDA followed by SA, CMA, and MA (Table 2.2). The best and the
least sporulation across four growth media at 12 h fluorescent
light cycle and 30°C was achieved in isolates BR-4 and BR-6,
respectively (Table 2.2).
In SA and CMA, the pycnidial stroma were scattered and
submerged, whereas in PDA and MA, both superficial and submerged
pycnidia were produced (Figs. 2.1, 2.2). In general, the range
and average diameter of pycnidial stroma varied among growth media
(Table 2.4). Superficially produced pycnidial stroma were of
larger diameter than the submerged ones and were often seen
covered with appendage-like hyphae. Most of the submerged
pycnidia were solitary and unilocular, whereas most of the
superficial pycnidia were multilocular (botryose) and as much as
31 locules were observed in BR-8 (Figs. 2.5, 2.6). The diameter
of pycnidial cavities ranged from 57-200 (ave. 124) /xm. Across
isolates, the majority of 21-day-old pycnidial stroma in PDA, CMA,
and SA sporulated while a majority were pseudopycnidial in MA.

23
Pycnidial stroma in and on M. quinquenervia leaves were
produced within 3-14 days following inoculation. Both solitary
and botryose types of pycnidia were observed on host tissues
(Figs. 2.7, 2.8, 2.9, 2.10). Based on the mode of formation,
pycnidial stroma on leaves and bark of the host plant were of 2
kinds, 1) superficial on the surface and 2) erupting through the
epidermis (Figs. 2.9, 2.10). A majority of pycnidial stroma
produced on the surface were covered with short erect hyphae while
these structures were lacking in the erupting types. Both
solitary and botryose (Figs. 2.8, 2.10) pycnidia were produced on
the bark of the stems of M. quinquenervia. The majority of
pycnidia produced on the bark were immersed or submerged in the
layers of phellodermal tissues.
Immersed or submerged pycnidia were globose and the botryose
or eustromatic ones were lobulate and often had flat tops. Either
kind of pycnidia was ostiolate and the ostioles were often obscure
(Figs. 2.6, 2.8, 2.10). Superficial pycnidia produced
macroconidial cirri (Fig. 2.11) following partial drying by aerial
exposure for a few minutes. The pycnidial wall consisted of
heavily melanized cells of irregular textura-angularis becoming
gradually loose and hyaline towards the inner lining of the cavity
that produced conidiophores (Fig. 2.12). Conidiophores were short
and unbranched with bulbous bases produced from the lining of the
stromatal wall of the pycnidial cavities (Fig. 2.13). Mode of
conidial production was holoblastic and successional.
Microconidial production was very rare among most isolates. When
present, both macro- and microconidia (Fig. 2.14) were produced in
the same or separate pycnidial stroma.

24
Table 2.4. Range and average (parenthesis) diameter (mm) of the
pycnidial stroma on 2- to 4-week old culture of some B. ribis
isolates.
Isolate
Average diameter (mm) in
no.
PDA
CMA
SA
MA
BR-4
BR-5
BR-7
BR-8
0.1-1.0 (0.5)
0.1-1.3(0.6)
0.1-1.2 (0.5)
0.1-3.8(2.3)
0.1-0.5 (0.2)
0.1-1.0 (0.4)
0.1-0.5 (0.2)
0.3-1.2 (0.5)
0.1-0.5(0.2)
0.1-1.0 (0.4)
Table 2.5. Mean and range of dimensions (length and width) and
the number of nuclei in macroconidia.
Isolate
Dimensions (/x m)
Average Ranges
Number
of nuclei
Length
Width
Length
Width
Average
Range
BR-1
16.8c
6.2
5.7-23.0
4.1-8.9
6.0
1.0-12.0
BR-2
16.6c
6.2
8.9-23.0
4.3-8.9
5.0
1.0-11.0
BR-3
18.4a
6.2
14.0-23.2
3.3-8.9
5.0
1.0- 9.0
BR-4
17.9ab
6.1
8.9-26.8
4.1-7.9
5.0
1.0-11.0
BR-5
17.3bc
6.1
11.5-23.0
4.1-8.4
5.0
1.0-11.0
BR-6
18.Oab
6.2
7.7-23.0
3.1-8.9
4.0
1.0- 8.0
BR-7
16.7c
5.3
10.2-20.4
3.8-6.4
3.0
1.0- 8.0
BR-8
16.8c
5.5
9.7-21.0
3.9-6.5
4.0
1.0- 7.0
Note: B.
ribis isolates, i.e..
BR-l through BR-
â– 6 from M.
quinquenervia, and BR-7 and BR-8
from R. mangle) from 21-day-old pycnidial stroma from inoculated M. guinguenervia-leaves.
For each isolate, N=100. Means with same letter(s) are not significantly different from
each other at P=0.05, according to Duncan's Multiple Range Test.
Conidial Characteristics
Macroconidia in younger (3- to 7-day-old) pycnidia across
isolates were fusoid, hyaline, and aseptate with truncate bases
(Fig. 2.14) when young and fresh, but some of them produced 1-4
septa in 2-week or older pycnidia (Fig. 2.15). Septation and

25
bluish-brown pigmentation was observed in some macroconidia
retained in cavities of pycnidia produced on host and growth
media. But, those extruded in the form of cirrhi remained hyaline
and aseptate even at 12 weeks of drying at room temperature. Mean
length of macroconidia were significantly different among isolates
(Pr>F=0.0001), and the differences in range of dimensions were
obvious (Table 2.5). When present, the microconidia (Figs. 2.14,
2.15) were allantoid to shortly elongated, hyaline, and
unicellular with a range of length and diameter of 2.7-5.1
(average 3.7) /im and 1.3-2.5 (average 1.6) /im, respectively.
Germination of Macroconidia
Most of the macroconidia germinating on WA were aseptate; a
few contained 1-3 septa. However, the majority of macroconidia
from the same batch developed 1-3 septa when germinated in water
under a coverslip (Figs. 2.15, 2.16). Regardless of the numbers
of septa, 1-4 germ tubes (majority with 2 germ tubes, 1 on each
end) were produced by germinating macroconidia within 16 h of
incubation (Fig. 2.16) at 30°C. None of the microconidia, either
in water under a coverslip or on WA, germinated at 16 h after
incubation at 30°C.
In WA, germination of macroconidia began as early as 1 h
after incubation at 30-35°C (Figs. 2.17, 2.18). Within 4 h at 20-
35°C germination had concluded and was over and below 90% in BR-1
and BR-7, respectively. However, after 24 h at 20-40°C, spore
germination was 93-100% and 83-98%, in BR-1 and BR-7,
respectively. The correlation between germination and

26
temperature, and germination and incubation using 126 observations
for both isolates was weak (Pr>R=0.088), and strong (Pr>R=0.0001),
respectively. At a temperature range of 20-35°C, a majority of
macroconidia in BR-1 and BR-7 germinated within 4 h of incubation
(Table 2.6 and Figs. 2.17, 2.18). Isolate BR-1 (isolated from
melaleuca canker) revealed a broad optimum temperature range (2 0-
30°C) for germination of macroconidia opposed to a narrow range
(25°C) for BR-7 (isolated from mangrove trees) (Table 2.6). In
both isolates, spore germination was insignificant at 5 and 45°C.
At 45°C, about 5% of the macroconidia in both isolates spilled
cellular contents onto the WA at 16 h after incubation.
Table 2.6. Effect of temperature on germination of macroconidia
by 4 h after incubation in WA at 100 percent humidity.
Percentage
of spore germination
at °C
Isolate
5
10
15
20
25
30
35
40
45
BR-1
0.2
1.7de
0.0
0 . Oe
3.7
11. Od
91.2
72.8ab
94.8
77.2ab
98.3
82.7a
90.3
72.0b
46.5
43.0c
0.0
O.Oe
BR-7
0.0
0 . Of
0.0
0 . Of
2.5
10. Oe
55.2
48.0c
89.8
71.5a
82.0
65.0b
64.3
53.4c
25.5
30.3d
0.0
O.Of
Note: Mean comparisons were performed for 4 h incubation period only since at the
temperature range of 20-35°C, more than 50% spores germinated during this period. For each
isolate, numbers on the upper row represent actual percentages of spores germinated by 4 h.
Numberts in the second row for each isolate represent corresponding means from transforming
back to percentage the arcsin-square-root of X scale used in the analysis of data (Arauz
and Sutton 1989). Corrected percentages in the second row followed by the same letter are
not significantly different from each other at P=0.05 (minimum significant difference for
BR-1 and BR-2 was 10.65 and 5.67, respectively).
Inocula of isolates BR-1 and BR-2 appeared to vary in their
ability to survive in extremely low temperatures. Macroconidia of
BR-1 were viable after 3-month storage at -20°C, whereas for
isolate BR-7 viability was lost after the same period of storage.

27
Initiation of the germination of macroconidia stored at -20°C
appeared to be delayed by as much as 24 h.
The combined effects of the temperature and incubation
period on germination of macroconidia at 100 percent relative
humidity using data from 5-45°C was described by the following
equation derived through logistic regression:
G(Isolate BR-l) = -12.856 + 0.255 INC+0.927 TEM-0.017 TEM2
G(Isolate BR-7) = -12.856+(0.255- 0.016 ) INC+ (0.927-0.036 ) TEM
+(-0.017+0.006) TEM2
where G = logit (percent germination), INC = incubation
period, and TEM = temperature treatment (°C) .
Discussion
Fungal Morphology and Taxonomy
A vast confusion exists in the literature regarding the
taxonomic identity of B. ribis and B. dothidea, and their
anamorph-teleomorph relationships. Since the description of B.
ribis by Grossenbacher and Duggar (1911), this fungus has been
reported to cause diseases on many hosts; frequently mycologists
and plant pathologists have stressed teleomorphic names to avoid
the taxonomic uncertainty of the anamorphic states (Shear et al.
1924, Smith 1934, Luttrell 1950, Wiehe 1952, Witcher and Clayton
1963, Schreiber 1964, Crist and Schoeneweiss 1975, English et al.
1975, Spiers 1977, Filer and Randall 1978, Shearer et al. 1987,
Brown and Hendrix 1981, Davison and Tay 1983, Webb 1983, Latorre

28
and Toledo 1984, Mass and Uecker 1984, Rumbos 1987, Ramos et al.
1991). However, in most of these studies the teleomorph stages
were not produced.
Some authors have referred to B. ribis as having an anamorph
belonging to Dothiorella (Wolf and Wolf 1939, Smith 1934, Anahosur
and Fazalnoor 1972, Smith and Fergus 1971, Webb 1983), while
others have referred to it having anamorphs in Fusicoccum
(Punithalingam and Holliday 1973, Morgan-Jones and White 1987).
Evidence from the results related to morphological aspects of the
vegetative and reproductive structures and the literature suggests
that the controversy in taxonomic position of B. ribis and B.
dothidea is due mainly to 1) variable mycelial and conidial
morphology in different growth conditions and stages, 2) rareness
of sexual stages, 3) pantropic distribution, and 4) ability to
cause diseases on many plant species.
In the present study, mycelial characteristics such as
growth pattern, color change over time, and dimensions in PDA
(Tables 2.1, 2.2) agrees with the mycelial characteristics
described for B. ribis (Morgan-Jones and White 1987) . Most
descriptions of the anamorphic stages of B. ribis and B. dothidea
overlap. For example, the measurements (Table 2.5), and the
nature of pycnidia (Figs. 2.5, 2.6, 2.7, 2.8, 2.9, 210, 2.11) and
conidiophores (Fig. 2.12, 2.13), as well as fresh macroconidia
from my studies agree with Wolf and Wolf (1939), Punithalingam and
Holliday (1973), and Sivanesan's (1984) description for B. ribis.
The immersed or submersed pycnidia in four artificial media were
smaller than superficial pycnidia. Also, pycnidial stroma in PDA
were relatively larger than the pycnidia produced in CMA, MA, and

29
SA (Tables 2.1, 2.2). Based on these observations and the
evidence from literature (Grossenbacher and Duggar 1911, Shear et
al. 1924, Morgan-Jones and White 1987), I suggest that the
pycnidial dimension in the artificial media is influenced by the
quality of nutrient.
The possible pairing of all eight isolates of B. ribis did
not yield a teleomorph stage. Mass and Uecker (1984) had a
similar experience with B. dothidea that causes stem canker in
blackberry. According to the descriptions given by Punithalingam
and Holliday (1973), the ascal and ascospore dimensions of B.
ribis are 100-110 x 16-20 and 17-23 x 7-10 /xm, respectively. The
measurements given for asci and ascospores of B. dothidea are 65-
140 x 16-21 and 13-35 x 6-14 /xm, respectively (Pennycook and
Samuels 1985). These measurements reveal that the ascal and
ascospore dimension of B. ribis falls within the range of the
dimensions of these structures of B. dothidea. Therefore, I
suggest that the taxonomic identity of these so-called two
distinct species needs to be reviewed.
Based on the presence of macro- and microconidia, Morgan-
Jones and White (1987) considered their isolates obtained from the
leaf of Smilax to be the Fusicoccum anamorph of B. ribis. Though
rare, microconidia were produced by almost every isolate I have
studied. Microconidia in the present study were of larger
dimensions then those reported by Wolf and Wolf (1939),
Punithalingam and Holliday (1973), Sutton (1980), Pennycook and
Samuels (1985), and Sivanesan (1984), but were similar to those
described by Morgan-Jones and White (1987). These microconidial

30
characteristics are consistent with the previous descriptions of
B. ribis and its anamorph Fusicoccum.
The separation of the genera Dothiorella and Fusicoccum
seems to be based on the form of conidiomata, the mode of
conidiogenesis, and the nature of conidia. The criteria of
holoblastic mode of conidiogenesis yielding hyaline to brown
conidia with aseptate-to-biseptate conditions for Dothiorella, and
enteroblastic phialidic conidiogenesis producing hyaline to light
brown aseptate conidia for Fusicoccum, has been used (Barr 1987)
to differentiate these two form-genera. Sutton (1980) examined
the illustrations of the type specimen of Saccardo (1886) and
found holoblastic conidiogenesis for F. aesculi. Though Pennycook
and Samuels (1985) agree with Sutton (1980), they noted that
Saccardo's specimen was immature. It has been demonstrated that
the older conidiogenous cells are enteroblastic phialidic forming
2-3 irregularly shaped annelations (Mass and Uecker 1984,
Pennycook and Samuels 1985).
The upper range of the dimensions, and color and aseptate
condition of macroconidia from the younger pycnidia in my studies
(Table 2.5) closely match the ranges of the dimension of
macroconidia reported by Weaver (1974) for B. dothidea. Septate
and pigmented conidia have been reported in B. ribis (Wiehe 1952,
Taylor 1958, Rumbos 1987) as well as in B. dothidea (English et
al. 1975, Spiers 1977). However, Pennycook and Samuel (1985)
mentioned that the conidia of B. dothidea do not become light
brown or septate with age or at the time of germination.
Septation of macroconidia when germinated under water (Figs.
2.15, 2.16) and those retained in dry pycnidia were similar to

31
those described by Mass and Uecker (1984) for B. dothidea from the
cankers on thornless blackberry. Also, the uni- as well as bi¬
polar and sometimes lateral germ-tube production (Fig. 2.16) fits
to the description of English et al. (1975) for B. dothidea.
However, the majority of macroconidia germinated on water agar at
100% relative humidity were aseptate. The septation of some of
the older and/or germinating conidia in my studies create doubt
since reportedly the macroconidia of Fusicoccum are not septate
(Sutton 1980). I suggest that the septation of macroconidia of
the anamorphs of B. ribis and B. dothidea may be related to the
level of oxygen/carbondioxide or combinations of some other
factors in their microenvironment which are yet to be discovered.
Thus, it appears that the morphological characteristics of
B. ribis and B. dothidea, especially of their anamorphs, overlap
and become difficult to be separated from one another. For
example, Michailides (1991) isolated a pycnidial fungus from
panicle and shoot blight of pistachio from California and
identified as B. dothidea, but he reported that the isolates fit
the description of the pycnidial stage of B. ribis. As discussed
by Morgan-Jones and White (1987), small variations in conidial
dimensions occur in different circumstances.
The separation of these form genera Dothiorella and
Fusicoccum may have been based on their morphological characters
observed at a certain stage of development at a given
microenvironment. If hyphal and conidial characteristics of some
of the anamorphic stages of these two fungi are studied side by
side at similar conditions and followed through different stages
of development, it may be difficult to separate the taxonomic

32
identity of these two fungal species. My opinion is further
supported by the view of some other authors while discussing the
anamorph-teleomorph connections of these two teleomorph stages
(Pennycook and Samuel 1985, Morgan-Jones and White 1987,
Michailides 1991).
In light of my results and the evidence from literature, the
isolates of fungi obtained from M. quinquenervia and R. mangle are
closer to the Fusicoccum complex. However, the systematic
position of the form genera Dothiorella and Fusicoccum, and their
teleomorphs (B. ribis and B. dothidea) need to be reviewed.
Germination of Macroconidia
The studies of representative isolates, BR-1 and BR-7 from
M. quinquenervia and R. mangle, respectively, revealed that the
macroconidia of both isolate are capable of germinating at 5-40°C
with a range of 25-35°C being optimum for germination (Fig. 2.17,
2.18). Similar range of optimum temperature range for germination
of macroconidia has been reported for B. dothidea from California
(Brooks and Ferrin 1994). However, the isolates, BR-1 and BR-7
differ in the speed of the germination of their macroconidia.
Macroconidia of isolate BR-1 had >90% germination within 4 h at
25-35°C, while macroconidia of BR-7 needed 8 h to achieved 90% at
the same range of temperature (Table 2.6 and Figs. 2.17, 2.18)
The isolates, BR-1 and BR-7 had a similar threshold for
survival at high temperature (45°C) , but differed at a low
temperature threshold. It was interesting to note that
macroconidial suspensions (in sterile distilled water) and aerial

33
hyphae (in sealed petri plates) of BR-1 remained viable for a
period of 3 months at -20°C, whereas similar inocula of BR-7 lost
their viability in comparable period and storage conditions. The
isolate BR-1 came from inland trees growing in a fresh water swamp
and the BR-7 came from trees in coastal swamps of saline water.
Such a differential tolerance of these two isolates to low
temperature may be related to adaptation in their respective
environment, though actual temperature differentials may vary only
moderately between these areas. Based on the results from the
observations of variables such as temperature and incubation
period, no conclusion can be drawn regarding the variable
adaptability of this fungal species. Arauz and Sutton (1989) have
reported natural variability between isolates of B. obtusa
collected from western and central North Carolina. The isolate
coming from drier region required high temperature and low
relative humidity to germinate than the one that came from
relatively wet area.
In general, it appears that isolate BR-1 has better chance
of survival than BR-7 among a population of susceptible trees
where freezing temperature is frequent in inland ecosystems. In
general, it is assumed that the high temperature along with yet to
be discovered factors may have been limiting this fungus from
building its population to an epidemic level among trees of
susceptible species though it is endemic to South Florida.

34
Conclusions
Eight tentatively identified isolates of B. ribis: six from
M. quinquenervia, and two from R. mangle were obtained from South
Florida. Morphological characteristics of these isolates were
studied using artificial growth media (CMA, MA, PDA, and SA), and
excised leaves and potted ramets of M. quinquenervia.
Mycelial morphology and growth rate of isolates in
artificial media varied, and the overall best growth-rate was
achieved on MA and PDA. Sporulation intensity and dimension of
conidiomata varied by isolates and growth media and except BR-6,
the majority of isolates sporulated best on PDA, SA, and excised
leaves incubated in moist chamber in sterile condition.
Conidiophores were short and hyaline with a bulbous base arising
from the lining of the stromatal locules, and conidiogenesis was
holoblastic among younger conidiomata. Macroconidia from younger
conidiomata were fusiform, truncate at the base, obtuse at the
apex, hyaline, aseptate, but some of them developed 1-3 septa when
retained in dry conidiomata or germinated in water under
coverslips. Regardless of the number of septa, 1-5 germ tubes
were observed to be produced from polar as well as lateral sides
of conidia. Microconidia were rare but were produced by most
isolates, but none of them was observed to germinate.
In isolates BR-1 and BR-7, the percentage of macroconidia
germinating within 4 to 8 h was greater at 25-35°C than outside
that temperature range. In both isolates, macroconidial
germination was at the least or absent at 5°C, and viability was
lost at 45°C.

35
The teleomorph stage was not observed at any growth media
and conditions. Morphological characteristics revealed the
isolates to be closer to the Fusicoccum anamorph of B. ribis.
From variation in characteristics of mycelia, conidiomata, and
conidia of the isolate(s) among different growth media and
microenvironment examined, I conclude that the so-called
teleomorph(s) B. ribis or B. dothidea and their anamorph(s)
Fusicoccum and Dothiorella complexes are highly variable. The
variation in their morphological characteristics may have root in
their adaptive ability in a given host or microenvironment.
Therefore, their taxonomic relationships need to be further
clarified. I suggest that these two teleomorphs are synonymous
and so are their two anamorphs.

Figs. 2.1-2
Figure 2.1.
Figure 2.2.
Figure 2.3.
Figure 2.4.
Figure 2.5.
Figure 2.6.
.7 Mycelial and pycnidial characteristics of the
Fusicoccum anamorph of B. ribis on PDA, CMA, PDB,
and M. quinquenervia leaf.
Aerial hyphae imparting black color and partially
submerged pycnidia (arrows) on PDA.
Hyphae in and on the surface of media imparting
tan color and pycnidia (arrow) immersed in CMA.
Mycelial mat on PDA with hyphae of various
diameters. Bar=50 /xm.
Blended mycelial mat grown in PDB under
continuous shaking conditions (100 rpm). Note
hyphae containing chains of chlamydospore-like
cells (arrows) . Bar=50 ¡xm.
Cross-section through a multilocular pycnidial
stroma produced on PDA. Note numerous locules
(lo) . Bar=500 /xm.
Enlarged pycnidia from a portion of multilocular
stroma. Note macroconidia (arrow) and an obscure
ostiole (os) . Bar=100 /xm.
Pycnidial stroma on the leaf inoculated and
maintained in a sterile moist chamber. Note
solitary (arrows) and botryose (arrow heads)
types of pycnidia.
Figure 2.7.

37

Figs. 2.8-2.16. Pycnidia and macroconidia of B. ribis associated
with artificial growth media and M. quinquenervia
leaves and stems.
Figure 2.8.
Pycnidial stroma erupting from dead bark. Note
solitary (arrow) and botryose (arrow heads) types
of pycnidia.
Figure 2.9.
Section through leaf showing a solitary pycnidium
erupting from epidermis. Note leaf epidermis
(arrow), ostiole (os), and pycnidial wall (arrow
head) . Bar=50 /xm.
Figure 2.10.
Longitudinal section through stem showing a
solitary pycnidium erupting through bark. Note
an obscure ostiole (os), and pycnidial wall (pw)
composed of textura angularis. Bar=50 /xm.
Figure 2.11.
Pycnidial stroma on leaf oozing macroconidial
cirrhi(arrows) on drying at room temperature.
Figure 2.12.
A portion from the cross-section through a
pycnidium showing pycnidial wall (pw) composed of
textura angularis, and conidiophores (arrows).
Bar=25 /xm.
Figure 2.13.
A portion of the inner wall of pycnidium squashed
from a 3-day-old pycnidium. Note conidiophores
with bulbous base (arrow) bearing macroconidia
(arrow head) at different stages of development.
Bar=25 /xm.
Figure 2.14.
Macro- (arrow) and microconidia (arrow heads)
from freshly squashed 7-day-old pycnidium. Note
the microconidia are allantoid-slightly elongated
and the macroconidia are truncate base and obtuse
apices. Bar=25 /xm.
Figure 2.15.
Macroconidia (from 3-day-old pycnidium) from PDA
germinated in water. Note a mixture of aseptate
and septate conidia some of them bearing germ
tubes (arrows) . Bar=50 /xm.
Figure 2.16.
Germinating aseptate (arrow) and septate (arrow
heads) macroconidia showing one or more germ
tubes. Bar=50 /xm.

39

40
Figure 2.17. Germination of the macroconidia of isolate
BR-1 of Botryosphaeria ribis on water agar (WA) at 100 percent
relative humidity. Refer to Table 2.6 for significant diferences
in percentage of germination by 4 h after incubation at the
temperature ranges shown in this figure.
Figure 2.18. Germination of the macroconidia of isolate
BR-7 of Botryosphaeria ribis on water agar (WA) at 100 percent
relative humidity. Refer to Table 2.6 for significant differences
in percentage of germination by 4 h after incubation at the
temperature ranges shown in this figure.

CHAPTER III
EVALUATION OF Botryosphaeria ribis ISOLATES FOR
PATHOGENICITY TO Melaleuca quinquenervia
Introduction
Botryosphaeria dothidea (Moug ex Fr.) Ces. et de Not. has
been considered synonymous to B. ribis Grossenb. & Dug. by some
authors (Shear et al. 1924, Wiehe 1952, Witcher and Clayton 1963,
Crist and Schoeneweiss 1975, English et al. 1975, Spiers 1977,
Brown and Hendrix 1981, Latorre and Toledo 1984, Mass and Uecker
1984). However, others consider B. ribis and B. dothidea as two
distinct species (Smith 1934, Luttrell 1950, Punithalingam and
Holliday 1973, Davison and Tay 1983, Webb 1983, Rumbos 1987, Ramos
et al. 1991). Evidently, canker diseases on various hosts
formerly reported to be caused by B. ribis are often ascribed to
B. dothidea due to their synonymy (Weaver 1974) .
Botryosphaeria ribis has been reported to be pathogenic on
numerous tree species (Rumbos 1987). Dieback of branches and
twigs has been reported in Prunus parsica L. (Weaver 1974), P.
amygdalus Batsch 'Nonpareil' (English et al. 1975), Malus pumila
Miller (Brown and Hendrix 1981, Latorre and Toledo 1984),
Liquidamber styraciflua L. (Vannacci and Lorenzini 1981),
Eucalyptus marginata Donn ex Sm. (Davison and Tay 1983), E.
camaldulensis Dehnh. (Webb 1983), Rubus spp. (Mass and Uecker
1984), and Juglans regia L. (Rumbos 1987). In an attempt to
41

42
explore potential biological control agents of a pest tree
species, Myrica faya Ait., in Hawaii, Gardner and Hodges (1990)
reported the association of B. ribis with twig dieback of M. faya.
Recently, this fungus has been found to be associated with tip
dieback of mango (Mangifera indica L.) in South Florida (Ramos et
al. 1991) .
Cankers caused by B. ribis on E. marginata Donn. ex Sm. were
often unnoticeable from the stem surface and became evident only
after removal of outer bark (Davison and Tay 1983). Generally,
the cankered xylem and phloem tissues were characterized by brown
discoloration (Rumbos 1987) and were often associated with insect
damage that ranged from oviposition on small twigs to cerambycid,
buprestid, and scolytid galleries in branches and trunks (Davison
and Tay 1983).
Botryosphaeria ribis has been described frequently as a
wound parasite (Rumbos 1987) which can cause severe damage to
stressed trees (Crist and Schoeneweiss 1975). A test on blueberry
stems indicated variability in virulence between two isolates of
B. ribis (Witcher and Clayton 1963). A similar view has been
presented where the degree of susceptibility of the host plant has
been attributed to the cultivar (Hildebrand and Weber 1944),
isolate, and inoculation position on the trunk (Latorre and Toledo
1984). Schreiber (1964) reported that B. ribis isolates from
apple (Malus sp.), American holly (Ilex sp.), and Rhododendron
varied in their pathogenicity on Rhododendron. In his experiment,
the isolate originally obtained from Rhododendron was most
virulent, and the apple isolate was the least. In another
experiment using four Eucalyptus species [E. radiata Sieb. ex DC.,

43
E. calophylla R. Br., E. marginata, and E. cladocalyx ), B. ribis
isolates were found to be relatively virulent on only one species
(E. radiata) (Shearer et al. 1987). Similar phenomenon of host-
selective virulence of Hypoxylon maimatum (Wahl.) Mill, has been
reported in clones of Populus tremuloides (French et al. 1969).
To date, the pathogenicity and relative virulence of B.
ribis towards M. quinquenervia (Cav.) Blake has not been studied.
Identification of a virulent isolate of this fungus could be an
important first step toward biological control of M. quinquenervia
in South Florida. Therefore, the specific objectives of the
experiments presented in this chapter were to: 1) perform
pathogenicity tests of B. ribis on M. quinquenervia, 2) test
monoconidial cultures from each of eight isolates of B. ribis for
variation in virulence, and 3) to quantify virulence of one
selected monoconidial culture from each of the eight isolates of
B. ribis on stems of M. quinquenervia clones.
Materials and Methods
Tree Clone Production
As described in Chapter I, a 0.8-hectare stand of declining
trees of M. quinquenervia was discovered (Fig. 3.1) in a
management unit (Acme-2) of the Loxahatchee National Wildlife
refuge in South Florida (see introduction in Chapter I, II).
These trees had active cankers (Fig. 3.2) on the stem which were
marked by a vertical depression or callus (Fig. 3.3) and sapwood

44
discoloration (Fig. 3.4). The main stems above the cankers were
dead (Fig. 3.2). Observations revealed that some of the trees in
the stand were affected more than others by the cankers.
Therefore, this site was selected for collection of tree clones
for testing pathogenicity of B. ribis isolates and variable
susceptibility of the M. quinquenervia clones.
Seven M. quinquenervia trees (henceforth referred to as tree
clones MQ-1 through MQ-7) located at least 50 m apart (to avoid
possible clonal similarity) at the Acme-2 site on the Loxahatchee
National Wildlife Refuge were selected and approximately 20 stem
cuttings were obtained from each tree. The cuttings were
transported to Gainesville and rooted by placing them in water in
plastic buckets. Following rooting, the ramets were transferred
to 5-gallon plastic containers containing 1:1 mixture of peat and
sand. These clonal materials were then maintained as stock trees
in a greenhouse at the University of Florida, School of Forest
Resources and Conservation plant growth facility. Rooted cuttings
(ramets) obtained from the stock trees were used for various
laboratory and greenhouse experiments involving B. ribis
inoculations.
Isolate Acquisition
During 1989, Dr. Roger S. Webb, then Associate Professor in
the School of Forest Resources and Conservation, isolated and
performed preliminary pathogenicity tests with five isolates of B.
ribis which he had obtained from the margin of stem cankers on
declining M. quinquenervia trees from the Loxahatchee National

45
Wildlife Refuge. These isolates were tentatively identified as B.
ribis and later sent to the International Mycological Institute,
Kew, Surrey, England. When I began my doctoral program in 1990, I
obtained and identified another isolate of the same fungus from a
canker margin of a declining tree in the same area of the
Loxahatchee National Wildlife Refuge. From each of these six
isolates, 11 monoconidial cultures (MCC) were produced. These MCC
were tested for pathogenicity using a leaf bioassay method
described later in this chapter. The most virulent monoconidial
culture/isolate was selected from each set of 11 MCC, and are
henceforth referred to as isolates BR-1 through BR-6.
During 1993, I obtained two additional isolates of B. ribis
from the necrotic gall-tissues of red mangrove trees (Rhizophora
mangle L.) collected form the coastal region of South Florida by
Dr. Webb. These two isolates were also single-spored and
identified by Dr. J.W. Kimbrough and myself in the Department of
Plant Pathology, at the University of Florida, and henceforth are
called isolates BR-7 and BR-8. All of these eight isolates, BR-1
through BR-6 and BR-7 and BR-8 were used to study mycological
characteristics (Chapter II) and in the pathogenicity tests
described in this chapter.
Inocula Preparation
The MCC of B. ribis were grown on potato dextrose agar (PDA)
for 3 days and disks (5 mm diameter) were removed from the margin
of the colonies. The disks were placed in a sterile test tube
containing 3 mm glass beads and deionized water. After closing,

46
the tubes were shaken vigorously until the disks were completely
macerated. Three drops of the resulting suspension of MCC were
added to potato dextrose broth (PDB) (Difco) in 500 ml Erlenmeyer
flasks and maintained under continuous shaking conditions (100
rpm) using a rotary shaker. Mycelia were aseptically harvested in
the log phase of growth (4-day-old) and were filtered through
sterile cheese cloth. The mycelial mass retained on the cheese
cloth was then weighed and blended for 15 seconds under aseptic
conditions after which sterile deionized water was added to
prepare a 1% (dry weight/volume) mycelial suspensions. The fresh
inocula were then refrigerated and used in pathogenicity testing.
Leaf Inoculations
The ability of MCC to cause leaf necrosis will reflect their
ability to cause stem diseases. Accordingly, a series of initial
pathogenicity tests were developed. The tests utilized detached
leaves as a bioassay technique to detect variation in relative
virulence, if any, among 11 MCC (only eight MCC in case of isolate
3, since three of the MCC were not used because of contamination)
within each of six B. ribis isolates originally obtained from M.
quinquenervia during 1989-90. Four M. quinquenervia clones were
used in the test. Three check treatments involving inoculation of
1) Fusarium moniliforme var. subglutinans (FM), a pathogenic
deuteromycetous fungus affecting Pinus species; 2) Lophotrichus
sp., a saprophytic ascomycetous fungus; and 3) sterile deionized
water were also included in each experiment.

47
Fully expanded leaves of M. quinquenervia, i.e., 4th, 5th,
and 6th, from stem apex were obtained from the potted ramets,
dipped in 15% (of 5.25% stock) NaOCl for 15 minutes, and rinsed
six times (each 5 minutes) with sterile deionized water (DW).
Three sterile leaves from each M. quinquenervia clone were placed
on pre-moistened sterile filter paper contained in a sterile petri
dish. A small (about a 0.5 sq. mm) scratch was made on the dorsal
surface of each sterile leaf and 10 ¡jlI of the 1% mycelial
suspension was placed on each wound. Following inoculation, the
petri dishes were sealed with Parafilm and incubated at 25°C under
10 h light/day for 120 h. In the checks, the leaves were
similarly inoculated with FM, Lophotrichus spp., and sterile
deionized water and maintained under same conditions. The
experiment was repeated twice and with each repetition treated as
block for analysis. Data were analyzed using ANOVA for a
randomized block design.
The area of necrotic leaf was measured using transparent
graph paper at 120 h after inoculation. Mean necrotic areas of
three leaves/MCC/M. quinquenervia clones were calculated.
Variance in virulence among 11-MCC per isolate, overall variance
in susceptibility among four M. quinquenervia clones, and
interaction between MCC and M. quinquenervia clones were analyzed
considering isolates and tree clones as random effects. The MCC
were ranked for their relative virulence using Waller-Duncan
Multiple Range Test. The checks were excluded from F-test since
they exhibited negligible amount of leaf necrosis compared to the
magnitude of necrosis caused by MCC from isolates of B. ribis.
The most virulent MCC from each isolate (hereafter referred to as

48
"isolate") was used in the experiments involving mycological
characteristics (Chapter II), and the stem-inoculation described
later in this Chapter.
Influence of Hyphae vs Macroconidia as Inocula
In all stem-inoculation experiments, I had to rely on hyphal
inocula since many of the B. ribis isolates did not sporulate,
thus failing to provide adequate macroconidial inocula (see
Chapter II). Therefore, it was necessary to evaluate the form of
inoculum (spores vs hyphal fragments) for its ability to cause
diseases on the stems of M. quinquenervia.
Effect of the form of inocula, i.e., macerated hyphae vs
macroconidia, on disease development was evaluated in seven M.
quinquenervia clones (MQ-1 through MQ-7). Isolate BR-1 was chosen
to represent B. ribis isolates for this experiment because it
produced abundant superficial pycnidial stroma and extraction of
macroconidia was more easily accomplished. Hyphal inocula of this
isolate were prepared as described under "Inocula Preparation" in
this chapter. Macroconidia were obtained by incubating the fungal
cultures on PDA under 12 h fluorescent light cycle at 30(±1)°C for
7 days. Spore suspensions were obtained by partially drying
pycnidia under aseptic conditions to promote conidial extrusion,
followed by washing the plates with sterile deionized water. The
spore concentration was adjusted to llxlO4 macroconidia/ml.
Presence of a few hyphal fragments in the suspension was
disregarded.

49
Each of the two form of inocula (macroconidia and hyphal
fragments) were inoculated on seven M. quinquenervia tree clones.
Each treatment contained three replications, i.e., three
ramets/treatment/tree clone. Inoculation techniques, experimental
conditions, and incubation periods were as described in the
section "Evaluation of Isolate Virulence Under Greenhouse
Conditions" later in this chapter. Fungal isolates and tree
clones were evaluated as fixed and random effects, respectively.
Pathogenicity of Botryosphaeria ribis vs Pestalotia
Attempts at isolating a causal organism from canker margins
on naturally infected M. quinquenervia trees in the field often
yielded Pestalotia sp. along with B. ribis. Therefore, it was
necessary to test the ability of Pestalotia sp. to cause canker
diseases on M. quinquenervia.
Three treatments comprised of the inoculation of 1)
composite inocula of B. ribis isolates, i.e., mixture of the equal
proportion of 1% mycelial (wt/vol) suspension of BR-1 through BR-
6, 2) 1:1 mixture of the composite inocula of B. ribis and
Pestalotia species, and 3) 1% mycelial suspension of Pestalotia.
This experiment used ramets of MQ-4 with the stem diameter of 4.5-
7.0 mm at the point of inoculation. Each treatment contained four
replications, i.e., four ramets/treatment. Fungal inocula,
inoculation methods, incubation period, other experimental
conditions, and analytical methods were similar to those described
for "Evaluation of Isolate Virulence Under Greenhouse Conditions,"

50
as discussed in this chapter. In this experiment, the treatments
were evaluated as fixed effects.
Effects of Inoculation Positions on Canker Development
Preliminary observations and evidence from the literature
(Latorre and Toledo 1984) suggested possible differences in
susceptibility of M. quinquenervia stems toward B. ribis in
relation to point of inoculation (root-collar, mid-section, and
near stem apex). Therefore, this experiment was designed to test
this hypothesis.
This experiment, using MQ-4 and a composite inocula of B.
ribis isolates, utilized points of inoculation as treatments: 1)
at the root-collar region, 2) at the approximate mid-point on the
main stem, and 3) at a point approximately 15 cm behind the apex
of the main stem. Each treatment was replicated five times.
Fungal inocula production, inoculation methods, incubation period,
experimental conditions, and analytical methods were same as those
described in this chapter for "Evaluation of Isolate Virulence
Under Greenhouse Conditions." Fungal isolates and inoculation
positions were evaluated as fixed effects.
Evaluation of Virulence of Isolate Under Greenhouse Conditions
In March 1993, an experiment was initiated to evaluate
variation in 1) virulence among eight B. ribis isolates, and 2)
susceptibility among seven M. quinquenervia clones under
greenhouse conditions. Ramets of M. quinquenervia with stem

51
diameters of 4.5 to 10.0 mm, at least at 10 cm above the root-
collar, were used in the experiment. Each of eight B. ribis
isolates were wound-inoculated on each of seven M. quinquenervia
clones. Two check treatments: 1) wounded and inoculated with
sterile deionized-water, and 2) wounded and inoculated with FM, on
seven M. quinquenervia clones, were also included in this
experiment. Thus, there were a total of 10 treatments (eight B.
ribis isolates and two checks) for each of seven tree clones. The
experiment was repeated three times and each repetition was
considered a block for analysis.
Ramets were wounded by making 1.5 mm x 2.0 mm drill-holes at
a height where the stem was at least 4.5 mm in diameter. The
drill holes were filled with freshly prepared mycelial inocula of
B. ribis. The same kind of drill holes were made for two check
treatments and were inoculated with diluted PDB or 1% hyphal
inocula of FM. Following inoculation, the wounds were immediately
wrapped with Parafilm (American National Canâ„¢, Greenwich, CT
06830) .
Botryosphaeria ribis-treated ramets were randomized within
each block. Three blocks were maintained separately in the
greenhouse. Ramets in each of the two check treatments were
grouped and placed separately in the greenhouse. All treatments
were maintained at ca 25°C under ambient day/night cycle and daily
watering schedule.
The ramets were evaluated every week following inoculation
for wilting, dieback, and mortality. Plants showing these
symptoms were destructively harvested during the day of
observation; the remaining ramets were harvested 8 weeks after

52
inoculation. The harvested ramets were evaluated for symptoms of
pathogenesis. The evaluation included 1) visual estimation of
presence or absence of callus at the inoculation point, 2)
measurement of distal and proximal extension of canker (tissue
discoloration) beneath bark from the point of inoculation, 3)
visual estimation (percent) of sapwood discoloration in cross
section and cambium discoloration both at 5 mm distal to the point
of inoculation, and 4) reisolation of fungus from cankered or
stained tissue beyond 5 mm from the point of inoculation. A 5 mm
long segment of stem beyond 5 mm from the point of inoculation
from each of the B. ribis and FM treated ramets was recovered from
the stem, surface sterilized by quick-dip in 95% ethyl alcohol and
followed by brief flaming. The surface-sterilized segments were
placed on acidified potato-dextrose-agar (APDA), incubated for 14
days (at 25°C and ca 10 h fluorescent light cycle), and evaluated
for B. ribis colony development. Fungal isolates and tree clones
were evaluated as random effects. Data were analyzed using ANOVA
for a randomized block design.
Evaluation of Virulence of Isolate Under Field Conditions
Inocula. Hyphal inocula of each of the eight isolates were
prepared as described under "Inocula Preparation" in this chapter.
To facilitate field inoculations, 10 ml of aqueous hyphal inocula
(1% wt/vol.) were containerized into plastic capsules by ARBORâ„¢,
Tree Technology Systems, Inc., 1014 Rein Road, Cheektowga, NY
14225 according to arrangements made with this company by Dr.
Webb. The plastic capsules were designed to deliver the mycelial

53
contents into drill-holes made in the tree stems. The capsules
were to remain attached to the trees to prevent wound
contamination as needed. Viability of the inocula in the capsule
was tested the week before field application.
Site selection. A young stand of M. quinquenervia located
on the Strazulla tract of the South Florida Water Management
District in West Palm Beach at Palm Beach County was selected for
the field experiment on stem-inoculation with B. ribis. The stand
was selected for uniform tree sizes (about 2-10 cm diameter at
breast height) and accessibility for installing the experiment.
Tree inoculation. In March 1994, trees were wounded at
breast height by drilling holes 5 mm diameter and 10 mm deep into
the stem. A capsule containing the appropriate inocula was
inserted into the hole to deliver the inocula to the sapwood
region of the stem. The capsules were left attached to the trees
for 12 weeks. As a check treatment, 25 trees were similarly
drilled but injected with sterile distilled water only.
Experimental design. Each of the eight isolates were
inoculated into a set of 25 trees, five of which were scheduled to
be harvested at 3-month intervals to determine the rate of canker
development by B. ribis isolates under field conditions. Because
of time constraints, data was only collected from the first subset
of five trees/isolate; this information is presented in this
chapter.
Harvesting and canker evaluation. Five trees from each of
nine treatment (eight B. ribis isolates and one check) were
harvested after 3 months. Stem segments of about 40 cm long with
the inoculation point at the middle were removed and sealed in

54
plastic bags. These were then placed under refrigerated storage,
and transported to the Forest Pathology laboratory of the School
of Forest Resources and Conservation at the University of Florida.
There they were stored at 5°C for three days in a cold room. The
stems were split longitudinally through the inoculation point and
the length of the discoloration of sapwood at the proximal and
distal segments from the inoculation point was measured. Fungal
isolates were evaluated as random effects.
Tissue samples about 5x5 mm square were removed from an area
located ca 10 cm distally from the point of inoculation. These
samples were surface sterilized by quickly dipping them into 95%
ethanol and then flaming them momentarily. The flamed samples
were cultured on APDA for 14 days at 25°C and a 10 h photoperiod.
The plates were then evaluated for the presence of B. ribis
colonies.
Effect of Season on Canker Development
In September 1992, a preliminary pathogenicity test was
conducted with a composite sample of B. ribis isolates. This
measured the response of M. quinquenervia to inoculations during
the fall. A general comparison of these results with spring
inoculations (see "Evaluation of Isolate Virulence Under
Greenhouse Conditions") revealed seasonal variation in canker
development. Therefore, it was necessary to test the seasonal
effects on disease development.
The seven M. quinquenervia tree clones, used in the
"Evaluation of Isolate Virulence Under Greenhouse Conditions"

55
experiment, were also used in this experiment. However, only
isolate BR-2 of B. ribis was utilized since its canker-causing
ability had been determined to be greater than those of other
isolates.
Data on canker length, mortality, and callus formation
obtained by inoculating BR-2 in spring (March 1994) on stems of
all seven M. quinquenervia clones (see "Isolate Virulence Under
Greenhouse Conditions") were compared with the data obtained by
inoculating the same isolate on the same seven tree clones in the
fall (October 1994) as described. Ramets of M. quinquenervia with
stem diameter of 5.1 to 10.1 mm, at 10 cm above root-collar were
used. Three ramets from each of seven tree clones were wounded
and inoculated with inocula of BR-2 using the same methodology
described for the experiment, "Evaluation of Isolate Virulence
Under Greenhouse Conditions." Ramets inoculated with BR-2 were
randomized on a greenhouse bench. As a check treatment, three
ramets of similar diameter were wounded using the same method and
treated with sterile distilled water, randomized according to
location, and maintained on a greenhouse bench as a separate group
to avoid possible contamination. Both treatments (BR-2 inoculated
and checks) maintained under a daily watering schedule without
temperature control, were evaluated every week for mortality, and
harvested 8 weeks after inoculation. The canker evaluation
techniques and criteria were similar to those described for
"Evaluation of Isolate Virulence Under Greenhouse Conditions."
Seasonal (spring and fall inoculations) effects and the tree
clones were evaluated as fixed effects.

56
Simulated Storm Damage and Inoculation with Macroconidia
Plants growing under field conditions are injured by various
natural factors such as storms and hurricanes, and provide
infection courts for pathogens. If B. ribis is to be developed as
a successful mycoherbicide, a desirable attribute would be the
ability to be established on the host when inoculated in mass.
This fungus appears to be a wound pathogen. Therefore, it was
necessary to test whether it is capable of establishing itself
into leaf and stem tissues that are wounded by natural or induced
damage to the leaves or on the stem due to friction between
adjacent trees. This experiment tested the ability of B. ribis to
establish on the injuries created by a simulated storm.
Potted M. quinquenervia ramets of ca 1.0 m height, and
macroconidial inocula of BR-1 of B. ribis (based on the
availability of adequate spores) were used. The canopy and the
sides of all ramets were exposed to a blast of wind with a force
of 180 lb/sq inch for five minutes. This resulted in breakage of
a few small twigs and detachment or tearing of some leaves.
Additionally, the upper-halves of the main stem of each member of
a pair of adjacent ramets were deliberately rubbed against each
other only once to simulate injury that may occur during a storm.
Using these injured ramets, four treatment groups (Gr.) were
identified. They were Gr. 1 (not inoculated with fungal spores),
Gr.2 (inoculated with 4.0xl04 spores/ml), Gr.3 (5.7xl04 spores/ml),
and Gr.4 (11.6xl04 spores/ml). The ramets in a fifth treatment
group (Gr.5) were not injured but were inoculated with

57
macroconidial inocula at 11.6xl04 spores/ml). Each treatment
group contained five ramets.
The inocula were produced using the methods described for
the experiment "Germination of Macroconidia" in Chapter II.
Macroconidia were suspended in sterile distilled water, diluted to
the appropriate concentrations, and applied on the plants with an
atomizer until the plant were completely wet. All the ramets in
Grs. 2, 3, 4, and 5 were placed together in a dew chamber at 100%
RH and 27°C for 72 h. Ramets in Gr.l were placed separately in
one corner of the same dew chamber. After incubation for 72 h,
surfaces of the leaves and small twigs were examined for spore
germination. Then the ramets in Grs. 2-5 were transferred to a
greenhouse and placed together on a bench in a randomized manner.
Ramets in Gr.l were also transferred to the greenhouse but were
placed on a separate bench.
Following transfer to the greenhouse bench, the plants were
watered daily, and evaluated weekly for leaf necrosis and stem
canker. The ramets were harvested eight weeks after incubation
and evaluated for canker development and the presence of fungus on
the unwounded and the wounded parts of the stem. A segment of
bark and sapwood (5 mm long) from the margins of cankers initiated
on wounded, unwounded, and leaf scars of a stem of each ramet in
all five treatment groups were surface sterilized by quickly
dipping them in 95% ethyl alcohol followed by a brief flaming.
The surface sterilized segments were placed on APDA, incubated for
7 days (at 25°C and ca 10 h fluorescent light cycle) , and
evaluated for B. ribis colony development.

58
Statistical Analyses
Data from laboratory, greenhouse, and field experiments were
analyzed using similar statistical procedures. Total canker
length (proximal + distal canker length from the point of
inoculation) was used as the response variable for all stem-
inoculation experiments. Analysis of variance (ANOVA), linear
contrasts among B. ribis isolates vs checks, and mean separations
among variables were performed using GLM procedures in SAS (1985).
Pearson's correlation coefficient was used to test the
relationship between stem diameter at inoculation point and total
canker length.
Results
Leaf Inoculations
Leaf bioassays revealed that six B. ribis isolates obtained
from M. quinquenervia are capable of causing a greater amount of
leaf necrosis when compared to the wound alone, wound plus FM, and
wound plus Lophotrichus species (Fig. 3.5). The F-values from
ANOVA for leaf inoculation experiments are presented in Table 3.1.
Difference in virulence of MCC within isolate and interaction
between MCC and M. quinquenervia clones were insignificant. These
findings indicate that MCC within an isolate were equally
pathogenic on a given M. quinquenervia.

59
Table 3.1. Leaf bioassay experiments (necrotic leaf area in
square mm) designed for preliminary tests of virulence among 11
monoconidial cultures within each of six isolates of B. ribis
obtained from M. quinquenervia from South Florida.
Source
Analyses
of variance
df
MS
F values
Pr > F
Isolate 1
MCC
10
2978.9
2.60
0.0146
MQ clones
3
10716.4
9.35
0.0001
Blocks
1
130.1
0.11
0.7379
MCC x MQ clones
30
1105.9
0.96
0.5344
Error
43
1146.6
Isolate 2
MCC
10
966.9
0.59
0.8118
MQ clones
3
4656.9
2.85
0.0485
Blocks
1
34060.7
20.84
0.0001
MCC x MQ clones
30
834.2
0.51
0.9720
Error
43
1634.7
Isolate 3
MCC
7
1755.2
1.11
0.3844
MQ clones
3
3310.1
2.08
0.1225
Blocks
1
50740.3
31.95
0.0001
MCC x MQ clones
21
2346.3
1.48
0.1581
Error
31
1588.0
MCC
10
2416.8
1.15
0.3492
MQ clones
3
17639.3
8.40
0.0002
Blocks
1
71535.0
34.07
0.0001
MCC x MQ clones
30
1578.6
0.75
0.7921
Error
43
2099.8
Isolate 5
MCC
10
508.0
0.43
0.9256
MQ clones
3
18667.5
15.68
0.0001
Blocks
1
17220.0
14.46
0.0004
MCC x MQ clones
30
747.7
0.63
0.9082
Error
43
1190.9
Isolate 6
MCC
10
1544.3
0.87
0.5704
MQ clones
3
13639.5
7.65
0.0003
Blocks
1
8920.4
5.00
0.0305
MCC x MQ clones
30
1307.7
0.73
0.8117
Error
43
1782.3
Note: MCC=monoconidial culture; MQ^Melaleuca quinquenervia.
The differences in susceptibility among M. quinquenervia
clones towards cumulative effects of individual isolates (based on
average of cumulative necrosis caused by 11 MCC within isolate)
were significant (P=0.05); this result has indicated that

60
combination of some isolate-M. quinquenervia clones may cause more
disease development than some other combinations. Treatment
blocks, which represent experiments performed at two different
times, were also significant (P=0.5) among all the isolates
revealing the effect of leaf age on disease development.
Influence of Hyphae vs Macroconidia as Inocula
The overall effect of the type of inocula, i.e.,
macroconidia vs hyphae, on canker length (mm) on stems of the
seven clones of M. quinquenervia was not significant (P=0.05, Pr
>F=0.7. Therefore, the type of inocula did not influence ultimate
canker length (32.8 and 31.3 mm for macroconidial and hyphal
inocula, respectively). The interaction between type of inocula
and tree clones was also not significant (P=0.05, Pr>F=0.5).
Therefore, both hyphal and macroconidial inocula appeared to have
similar effect on stem canker development on a given clone. The
mean canker length by inocula and isolate are presented in Table
3.2. Callusing was observed among ramets from both macroconidial
and hyphal inoculations.
Pathogenicity of Botryosphaeria ribis vs Pestalotia
Attempts to isolate causal organisms from stem cankers on M.
quinquenervia from Acme-2 of the Loxahatchee National wildlife
refuge yielded B. ribis and also Pestalotia species. Therefore,
pathogenicity of both fungal species was tested. Botryosphaeria
ribis caused longer canker than Pestalotia alone (Table 3.3).

61
Canker development resulting from the mixture of these two fungal
species was some what smaller, though not statistically different
(Table 3.3).
Table 3.2. Separation of means of the effects of form of inocula
on canker length (mm) on the stems of M. quinquenervia (MQ) in
summer inoculations
with
isolate
BR-1 of
B. ribis.
Tree clones
Inocula MO-1
MO-2
MO-3
MO-4
MO-5
MO-6
MO-7
Macroconidia 26.0a
Hyphae 19.7a
19.3a
23.3a
21.3a
17.3a
53.3a
43.0a
27.8a
44.0a
36.0a
43.0a
35.7a
44.0a
Note: Means followed by the same letter in the rows are not significantly different
according to Scheffe's mean separation test at P=0.05.
Table 3.3. Main-effect means and mean separations from analyses
of variance for comparison of B. ribis and Pestalotia species, and
determination of the effect of inoculation point on disease
development. Dependent variable was total canker length (mm)
measured at 8 weeks after inoculation in spring.
Experiments Treatments Mean canker length
Botryosphaeria ribis vs Pestalotia
B. ribis only
111.0a
B. ribis + Pestalotia
85.0a
Pestalotia only
5.5b
Inoculation positions vs canker development
Root-collar
26.8b
Middle
129.8a
Top
63.3ab
Note: Means followed by the same letter(s) are not significantly different according to
Scheffe's mean separation test at P=0.05.

62
Effects of Inoculation Positions on Canker Development
The location of the inoculation points influenced the
development of stem cankers on M. quinquenervia clones
(Pr>F=0.008). Largest cankers were developed by inoculating at
the middle-, and smallest by inoculating at the root-collar
section of the stems (Table 3.3). Callusing was observed at all
inoculation positions among ramets of all clones inoculated with
B. ribis.
Evaluation of Virulence of Isolates Under Greenhouse Conditions
Under greenhouse conditions no mortality of B. ribis
inoculated M. quinquenervia ramets was recorded during the 8-week
incubation period. Wilting and dieback above the point of
inoculation on stems was noted in about 20 and 10 percent of the
ramets inoculated with BR-2 and BR-6, respectively. Except in the
checks, cankers of various dimensions were observed among ramets
inoculated with B. ribis. In these spring-inoculated stems of all
M. quinquenervia clones, callus formation occurred in all
treatments, i.e., checks (wound, wound plus FM), and wound plus B.
ribis, except those that developed wilting and eventual dieback.
However, the nature of the callus development was different among
treatments.
Callus development, among ramets in checks as well as B.
ribis inoculations, was initiated within two weeks after
inoculations. Within 8 weeks, 90-to-100 percent of the inoculated
wounds callused to various degrees (Table 3.4). All of the wounds

63
in checks (wound only and wound + FM) were closed (Fig. 3.6) and
no tissue discoloration was observed beyond 5 mm from the
inoculation point. In contrast, B. ribis-inoculated wounds were
still unclosed and were laterally surrounded by callus tissues
(Fig. 3.7). These cankers were elliptical or evidenced long
fissures (Fig. 3.7). Discoloration of the bark, cambium, and
sapwood occurred up to 100 mm beyond the callus margin on some
stems. These cankers were similar to those observed in the field
(Figs. 3.2, 3.3). The cankers among non-callused stem wounds
(Fig. 3.8) inoculated with B. ribis were marked by depressions on
proximally and distally to the point of inoculation. Often, B.
ribis produced fertile pycnidial stroma on the bark (Fig. 3.9) of
proximal and distal segments of the stem from the inoculation
points. Botryosphaeria ribis was reisolated from 5 mm above or
below the inoculation point in 86-to-100 percent of the inoculated
stems (Table 3.4).
Table 3.4. Percent callusing around the inoculation point and
percent reisolation of fungus from 5 mm beyond the inoculation
point on M. quinquenervia stems at 8 weeks after inoculation in
spring.
BR-1
BR-2
BR-3
BR-4
BR-5
BR-6
BR-7
BR-8
FM
Wound
Callusing
90
90
100
86
100
90
95
95
100
100
Reisolation
90
95
95
95
86
100
86
86
33*
14**
Note: 'FM reisolated from inoculation point, but none from 5 mm above the inoculation
point; "Botryosphaeria, Pestalotia, and Fusarium, were reisolated from the inoculation
point, but none from 5 mm above the inoculation point.
The linear contrasts between check treatments (wound alone
and wound plus FM) and wound-inoculation with B. ribis isolates
were significant (Table 3.5). This revealed that the canker

64
causing ability of the wound alone or wound plus FM was
insignificant. Therefore, the checks were not included in further
tests of main effects (without checks in Table 3.5) and mean
separations (Table 3.6) for isolates and clones. With or without
checks, the variances among isolates and clones were highly
significant, whereas interactions among isolates and clones were
insignificant (Table 3.5). Linear contrasts revealed the effects
of B. ribis isolates to be significantly higher from individual as
well as overall checks (Table 3.5).
Table 3.5. Effects of B. ribis isolates on canker development on
stems of M. quinquenervia clones at 8 weeks after inoculation in
spring 1994.
Sources
df
Analysis of variance
MS F value Pr > F
With checks
BR Isolates
9
12811.1
14.13
0.0001
MQ clones
6
4236.2
4.67
0.0007
BR Isolates x MQ clones
54
906.8
1.08
0.3613
Error
140
843.2
Linear contrasts
FM and wound vs BR isolates
—
—
0.0001
FM vs BR isolates
—
—
0.0001
Wound vs BR isolates
—
—
0.0001
FM vs wound
—
—
0.8087
Without checks
BR Isolates
7
6073.3
5.77
0.0001
MQ clones
6
4986.0
4.74
0.0003
BR Isolates x MQ clones
42
1051.9
1.00
0.4797
Error
112
1048.7
Note: BR=B. ribis, FM=F. moniliformis var subglutinans, and MQ=M. quinquenervia.

65
Table 3.6. Main-effect means and mean separations from analyses
of variance for stem-inoculation tests on total canker length (mm)
at 8 weeks after inoculation.
Isolate
number
Mean canker
Greenhouse
length
Field
Clone
number
Mean canker
lenath
BR-1
39.91ab
52.4ab
MQ -1
37.00b
BR-2
81.17a
78.2ab
MQ-2
34.85b
BR-3
50.76ab
70.4ab
MQ - 3
38.33ab
BR-4
66.33 ab
91.6a
MQ-4
71.08a
BR-5
31.86b
45.2ab
MQ- 5
43.58ab
BR- 6
43.7ab
61.8ab
MQ- 6
63,54ab
BR-7
32.2b
65.6ab
MQ- 7
56.80ab
BR- 8
48.57ab
64.2ab
—
—
Note: Means followed by the same letter(s) are not significantly different according to
Scheffe's test at P=0.05. Observations for each of B. ribis (BR) isolates and M.
quinquenervia (MQ) clones were 21 and 24, respectively.
Stem-inoculation experiments revealed that BR-2 and BR-5
were the most and least virulent B. ribis isolates, respectively
(Fig. 3.10). On the other hand, MQ-4 and MQ-2 were the most and
least susceptible clones (Fig. 3.11). Canker progress from the
inoculation point was greater distally than proximally in the
stems (Fig. 3.12). Cambium and phloem discoloration was maximum
(44 percent) in BR-2 and minimum in BR-7 (Fig. 3.13). The
correlation between stem diameter at point of inoculation and
total canker length was very weak (Pr>R=0.01).
Evaluation of Virulence of Isolates Under Field Conditions
After inoculation with B. ribis, no dieback or mortality of
M. quinquenervia trees occurred in the field within the 3 months
observation period. However, dissection of the inoculated stems
revealed evidence of host colonization, and significant

66
differences in canker length among B. ribis isolates were detected
(Table 3.5). Slight changes in pathogenicity ranking among
isolates from greenhouse to field inoculations were noted. For
example, BR-4 ranked first in this field inoculation compared to
BR-2 which ranked first in the greenhouse inoculations. However,
in both experiments these two isolates were at the top two
positions in ranking based on mean canker length of the stem.
Among check-trees, tissue discoloration was limited to a maximum
of about 12 mm (an average of 5.8 mm proximal and distal to the
center of the point of inoculation). As with the spring
inoculations under greenhouse conditions, all wounds stimulated
callus production.
Reisolation of B. ribis from ca 10 mm distal to the
inoculation point on stem ranged from 60 to 100 percent, and no
fungi were isolated from the check plants. However, bacterial
contamination was present among the majority of the reisolations
from checks (from point of inoculation) as well as the B. ribis
treated stems.
Effects of Season on Canker Development
Mortality did not occur among M. quinquenervia ramets
inoculated on stems with B. ribis isolate BR-2 in both spring
(March) and fall (October). However, wilting and dieback of
ramets above the inoculation point was 10 and 20 percent in spring
and fall inoculations, respectively. Callusing did not occur in
10 and 33 percent of the total stems inoculated in spring and
fall. Among checks, the wounds were completely closed in both

67
spring and fall inoculations, respectively. Characteristics of
cankers and fungal sporulation on bark among callused and
noncallused wounds on the stems were the same as described for
spring inoculations (Figs. 3.6-3.8).
Overall canker length (mm) (81a and 93a for spring and fall
inoculations, respectively) among ramets in seven tree clones was
not significantly different (Pr>F=0.2871 at P=0.05) between spring
and fall inoculations. Though statistically not significant in
all but two M. quinquenervia clone, a majority of clones produced
larger stem cankers in fall-inoculations than those inoculated in
spring (Fig. 3.14). The circumference of necrotic tissues on
sapwood surface was also larger (data not presented) in fall-
inoculated ramets than those inoculated in spring. Botryosphaeria
ribis was reisolated from 98 percent of the cankers.
Simulated Storm-Damage and Inoculation with Macroconidia
During the 8-week incubation period, mortality of M.
quinquenervia ramets was not observed in any of the five
treatments tested in this experiment. Germination of macroconidia
and mycelial proliferation on the surface of leaves and stems was
prevalent within the 72-h incubation period. During this period,
some hyphal tips were observed to gain entry into the leaves
through stomata of uninjured leaves. However, following transfer
from the moist chamber to the greenhouse bench, further necrosis
of uninjured as well as injured leaves stopped. Other effects of
the simulated storm on disease development on M. quinquenervia
stems are presented in Table 3.7.

68
Regardless of the concentration of macroconidia used, all
the wounds made by friction were still unclosed and surrounded by
thin strips of callus ridges (Table 3.7). In contrast, the wounds
on uninoculated stems were closed by callus ridges. Small bark-
limited cankers (2-15 mm long) developed on the leaf scars and
unwounded upper parts of some wind-blasted stems (Table 3.7) where
the bark was green at the time of treatment. Also, branch tip
dieback was observed among some wind-blasted ramets inoculated
with higher inoculum concentrations (Table 3.7). Percentage of
cankered and uncankered part of the stems from which B. ribis was
reisolated has been presented in Table 3.7. In check (Gr.l), 20
and 40% of the wounds yielded B. ribis and Pestalotia sp.,
respectively.
Table 3.7. Effects of simulated storm damage on the establishment
of BR-1 isolate of B. ribis on stems of M. quinquenervia ramets
inoculated with macroconidia.
Treatment groups
1
2
3
4
5
A.
Percentaae of ramets
forming cankers on
Wounds
Closed
Unclosed
Unclosed
Unclosed
None
Unwounded part
of stems
None
40
60
60
None
Leaf scars
None
40
40
60
None
B.
Percentage of dieback
of stem tips
None
None
20
60
None
C.
Percentage of
B. ribis-reisolation
from
Wound-cankers
20
100
100
100
None
Bark-limited cankers
None
80
100
80
None
Uncankered stems
0
60
80
20
29
Note: Treatment groups (Gr.) were : Gr.l (injured, not inoculated with fungal spores),
Gr.2 (injured, inoculated with 4.0x10* spores/ml), Gr.3 (injured, inoculated with 5.7x10*
spores/ml), Gr.4 (injured, inoculated with 11.6x10* spores/ml, and Gr.5 (ramets not injured
but inoculated with 11.6x10* spores/ml). The numbers in "A," "B," and "C" represent the
percentage of ramets developing stem cankers, tip dieback of branches, and uncankered or
cankered stems that yielded B. ribis, respectively.

69
DisCUSSign
An extensive literature search on the pathology of M.
quinquenervia indicated that very little is known about disease
affecting this species. Farr et al. (1989) and Alfieri et al.
(1994) recorded several pathogenic fungi on M. quinquenervia in
South Florida. The first reported testing of pathogenicity of B.
ribis on M. quinquenervia is presented in this work. In this
work, the isolates of B. ribis originally obtained from the canker
margins of M. quinquenervia trees and necrotic galls of red
mangrove trees from South Florida, were evaluated by inoculation
of foliage and stem tissues.
Preliminary Screening of Monoconidial Cultures
The observed variations in B. ribis isolates (Chapter II), made it
necessary to test for variation in pathogenicity of monoconidial
cultures (MCC) within each isolate. Sutton and Boyne (1983) have
proven that isolates of B. dothidea obtained from one part (fruit)
of an apple tree were found to be equally pathogenic on another
part (stems). Based on this evidence, a leaf bioassay procedure
was developed to provide a relatively rapid screening of numerous
MCC for their relative pathogenesis to M. quinquenervia although
the isolates were originally derived from stem cankers.
The leaf bioassay procedures (Fig. 3.5, Table 3.1) revealed
three important points: 1) the isolates of B. ribis were
pathogenic to M. quinquenervia clones, 2) despite differences in
conidial dimensions, MCC within each isolate were not different in

70
their ability to cause tissue discoloration, and 3) differential
susceptibility occurs among M. quinquenervia clones towards B.
ribis isolates, i.e., some isolates were more virulent on one M.
quinquenervia clone than others.
Evaluation of Isolates by Stem-Inoculations
Cankers among M. quinquenervia trees from which B. ribis
isolates were isolated during 1989-1990 were often unnoticeable on
the surface of the affected stems. On these trees, cankers of the
shape of elliptical fissures became visible only after removal of
several layers of the papery bark. Lateral margins of those
cankers contained ridges of callus tissues (Fig. 3.2, 3.3). In
advanced cankers, callus tissues were discolored, imparting a
brown to black coloration from the margin of the diseased and
healthy tissues to the advanced stages of rot in the center.
Under greenhouse conditions, similar kinds of cankers and tissue
discolorations were observed on the stems inoculated with B. ribis
isolates (Fig. 3.7). Necrotic cortex, phloem, and cambium under
the bark among noncallused cankers were darker compared with
progressive darkening of tissues at the margin of the callus and
healthy tissues.
Some species of Pestalotia have been described as weak plant
pathogens. This fungus was isolated along with some isolates of
B. dothidea causing gummosis disease on peach trees (Weaver 1974).
Ramos et al. (1991) reported Pestalotia from the tissues killed by
the dieback diseases caused by B. ribis. The present study
revealed canker-inciting ability of Pestalotia species on M.

71
quinquenervia to be insignificant compared with B. ribis (Table
3.2). It is assumed that Pestalotia species is a secondary-
colonizer of the tissues infected and killed by B. ribis.
However, the ability of B. ribis to cause stem cankers on M.
quinquenervia was retarded by Pestalotia when inoculated in
mixture. The ability of Pestalotia species to inhibit development
of B. ribis was further supported by an observation where B. ribis
hyphal growth was completely suppressed on PDA by the more rapidly
developing hyphae of Pestalotia when an equal proportion of hyphal
fragments of these fungi were inoculated onto the media. It seems
reasonable that the presence of Pestalotia species in M.
quinquenervia-induced cankers may slow down further canker
development.
The relationship between total canker length and the
diameter of the stem at the inoculation point was not strong.
These results follow the findings of Shearer et al. (1987) who
found E. radiata stems of all ages and diameters to be equally
susceptible to invasion by B. ribis. However, this does not mean
that stems of different diameters inoculated with B. ribis will be
killed in the same amount of time. Mortality of infected trees
depends on the ability of fungus to girdle the stem, which in
turn, depends on the ability of fungus to grow tangentially
through xylem, cambium, and cortex.
Results of the present study revealed rapid stem canker
development at the middle segment compared with the top and the
root-collar regions (Table 3.2). These results agree with Bagga
and Smalley (1969) who found that wound sites midway on the stem
of P. tremuloides were most susceptible to canker development when

72
inoculated with H. pruinatum. When northern red oaks (Quercus
rubra L.) were inoculated with Phytopthora cinnamomi Rands, the
root-collar appeared less susceptible (to canker formation) then
the upper portion of trunks (Robin et al. (1992). However, the
results of the present research are not in agreement with Latorre
and Toledo (1984) who reported that one of their isolates of B.
dothidea produced larger canker lesions on apple trees when
inoculated at the lower portion of the trunk.
The time of year during which B. ribis inoculations were
made did not appear to have an effect on the canker length on the
stems of M. quinquenervia clones. However, on B. ribis-inoculated
stems, wound callusing was more frequent and pronounced in spring
than in fall inoculations. Additionally, the decline and wilt of
ramets was relatively greater in fall inoculations.
Callus formation on stem around a wound is viewed as an
attempt of a host to contain the fungus and to subsequently close
the wound. Among noncallused stems, canker development beneath
the bark was marked by a depression visible from the surface.
This finding agrees with the work of Filer and Randall (1978) who
inoculated B. ribis on 21 sweetgum families in open-pollinated
progeny tests and found maximum infection of trees when
inoculations were made in September and least infection among
trees inoculated in May. However, the results of my study do not
completely agree with the work of Davison and Tay (1983) who
reported B. ribis as an extensive invader of the phloem of E.
marginata seedlings when inoculations were made in winter, spring,
and summer. Inoculation of B. dothidea on southern California

73
chaparral vegetation during September was found to result in rapid
disease development (Brooks and Ferrin 1994).
English et al. (1975) reported B. ribis to be highly
virulent on almond (Primus amygdalus) in spring, summer and fall.
Therefore, it appears that susceptibility of tree species to B.
ribis may be influenced by the season of the year and it varies
among species. Under greenhouse conditions, fall inoculation of
M. quinquenervia with B. ribis appeared to be more effective in
inhibiting callus formation around wounds on stems than the spring
and summer inoculations. Although, the overall effect (across
clones) of season on canker length on B. ribis inoculated stems
was not statistically significant, certain clones appeared to be
more susceptible to canker development in fall than in spring
(Fig. 3.14) and vice versa.
The present study revealed significant differences in
virulence of B. ribis isolates on M. quinquenervia clones. Under
greenhouse conditions, isolate BR-2 and BR-4 were ranked first and
second, respectively, in virulence towards B. ribis clones (Table
3.6). However, in field inoculations this ranking was reversed.
Unlike greenhouse inoculations, I did not have control over tree
clones in the field inoculations. Therefore, it is possible that
trees inoculated with isolate BR-4 may be relatively more
susceptible to this isolate or the conditions around those trees
may be more conducive to disease development than the trees
inoculated with isolate BR-2. The remaining six isolates
including two from red mangrove also incited canker disease on M.
quinquenervia (Table 3.6). Similar variation in virulence among
isolates of B. ribis have been reported on currants (Ribes

74
species) (Hildebrand and Weber 1944) and on blueberry (Witcher and
Clayton 1963). Isolates of B. dothidea have shown variation in
pathogenicity on apple cultivars in Chile (Latorre and Toledo
1984). Based on the results from the inoculation on apple
cultivars, Latorre and Toledo (1984) suggested the existence of
strains of B. dothidea. Significant variation in pathogenicity
among isolates of B. dothidea has also been reported on apple
trees (Sutton and Boyne 1983).
Cultivar dependent susceptibility towards isolates of B.
ribis and B. dothidea has been reported on currant (Hildebrand and
Weber 1944) and apple (Latorre and Toledo 1984), respectively.
Milholland (1972) tested 11 isolates of B. dothidea on 18
blueberry cultivars and found that six isolates were virulent on
10 cultivars, and five isolates were virulent on the remaining
eight cultivars. These findings agree with present observations
under greenhouse inoculations in which variable susceptibility
among clones of M. quinquenervia was evident (Fig. 3.9). Similar
phenomenon of host selective virulence and variable clonal
susceptibility has been reported also for the host-parasite
relationship of P. tremuloides and H. mammatum (French et al.
1969) .
Tissue discoloration among B. ribis inoculated stems of M.
quinquenervia was more rapid in distal than in proximal segments
with respect to the point of inoculation (Fig. 3.10). It is
assumed that upward movement of sap through vessels and tracheids
may interfere with the downward growth of hyphae and hence slowed
discoloration of stem tissues at the proximal segments. In
contrast, tissues above the inoculation point may have facilitated

75
quick establishment and growth of the fungus due to suction of
inocula further into the stem through vessels and tracheids by-
transpiration pull from above. These factors may have contributed
to relatively rapid tissue discoloration in distal segments of M.
quinquenervia stems.
In the present study, two isolates obtained from the galls
of red mangrove were as virulent as some of the isolates which
were originally derived from M. quinquenervia (Table 3.6, Fig.
3.8). However, both of the isolates (BR-2 and BR-4) that
performed best in greenhouse and field conditions were originally
derived from M. quinquenervia. Schreiber (1964) reported
Rhododendron-derived isolates of B. ribis to be more virulent on
that same host than were isolates obtained from non-Rhododendron
hosts. Similar views have been expressed for the isolates of B.
ribis on Eucalyptus species in Australia (Shearer et al. 1987).
Pusey et al. (1986) reported a new biotype of B. dothidea causing
gummosis disease on peach trees. Many researchers agree on the
existence of moderate host specificity (Schreiber 1964, Shearer
1987), possibility of development of new strain or biotype
(Latorre and Toledo 1984, Pusey et al. 1986), and differences in
virulence (Hildebrand and Weber 1944, Witcher and Clayton 1963,
Milholland 1972, Sutton and Boyne 1983) among isolates of B. ribis
and B. dothidea complex.
In the present study, uninjured ramets inoculated with
macroconidial inocula of B. ribis neither produced cankers nor
induced any decline symptoms (Table 3.7). On the other hand, some
of the wind blasted ramets inoculated with B. ribis produced bark-
limited cankers on the uninjured part of the stem and resulted in

76
cankers typical of wound inoculation (Table 3.7). The bark
limited cankers in the uninjured stem segments of wind blasted
ramets were similar to those described by Pusey et al. (1986) for
infection of peach stems through lenticels and development of
sunken necrotic lesions. The observations in the present studies
revealed that establishment of B. ribis in M. quinquenervia
requires either a wound exposing the sapwood or injury-stresses
such as loss or damage of leaves or branch breakages. These
results agree with Milholland (1972), Crist and Schoeneweiss
(1975), and Rumbos (1987), who found that B. dothidea or B. ribis
requires wounds, and/or some other form of injury and stress to
establish on host stems and cause canker diseases leading to tree
decline and dieback.
Conclusions
Eight isolates of B. ribis, six from M. quinquenervia
(collected during 1989-90) and two from red mangrove (collected
during 1993) were evaluated for pathogenicity on M. quinquenervia
under laboratory, greenhouse, and field conditions.
Preliminary evaluation of 11 monoconidial cultures from each
of six isolates (collected during 1989-90), using a leaf bioassay
(on leaves from four M. quinquenervia clones) revealed: 1)
nonsignificant difference in the ability of monoconidial cultures
within isolates to cause necrosis of M. quinquenervia leaves, and
2) significantly variable susceptibility among M. quinquenervia
clones towards the isolates of B. ribis.

77
Stem-inoculations under greenhouse conditions using eight
isolates of B. ribis on ramets from seven M. quinquenervia clones
and field inoculations of the same isolates revealed 1) elliptical
and/or long fissured cankers on ramets which were similar to those
observed in the field during 1989-90, 2) a correlation between the
inoculation position on the stem and the canker length to be
significant, i.e., middle segments of main stems of trees were
more susceptible to canker development than the root-collar
regions and top segments, 3) a weak correlation between the
diameter of the stem at the inoculation point and canker length,
4) the disease-inciting ability of hyphal and macroconidial
inocula not to be significantly different, 5) that callusing of
wounds among ramets across clones of M. quinquenervia inoculated
in spring was more frequent and pronounced compared with those
inoculated in fall, 6) that all eight isolates were able to incite
cankers on stems across clones, but isolates BR-2 and BR-4 were
the most virulent, 7) some M. quinquenervia clones to be more
susceptible to some isolates than others, and 8) that
establishment of this fungus in the stem tissues requires a wound
or some form of injury that stresses the trees.

Figs. 3.1-3
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
.4. Declining M. quinquenervia trees in a stand in
management area ACME-2 of the Loxahatchee
National Wildlife Refuge in South Florida,
observed during 1989-1990, from where six B.
ribis isolates were obtained.
A portion of a declining M. quinquenervia stand
in the management area ACME-2 of the Loxahatchee
National Wildlife Refuge in South Florida. Note
crown thinning among some trees (arrows) and
complete leaf-loss among others (arrow heads).
A M. quinquenervia tree with a basal canker
(arrow) showing severe crown thinning above
cankered area but still sprouting from the root-
collar (arrow head).
A typical canker on the stem revealing vertical
depression (arrow head) surrounded by callus
ridges (arrow).
A portion of a cankered stem split to reveal
inner tissues. Note discolored sapwood (arrows)
beneath the canker.

79

80
Figure 3.5. Mean necrotic leaf area of M. quinquenervia due to
various treatments. Treatments 1-6, 7, 8, and 9 represent the six
B. ribis isolates from 1989-90 collections, F. moniliformis var
subglutinans, Lophotrichus sp., and wound alone, respectively.

Figs. 3.6-3.9. Stems of M. quinquenervia ramets with wound alone
and wound inoculated with B. ribis, and
maintained under greenhouse conditions for 8
weeks.
Figure 3.6.
Figure 3.7.
Figure 3.8.
Figure 3.9.
A portion of a stem wounded but not inoculated
with fungus. Note the wound has been closed by
callus ridges (arrow).
A portion of a stem wounded and inoculated with
B. ribis. Note canker extension above and below
the point of inoculation (arrow head) and callus
ridge (arrow) around the canker.
A portion of a stem inoculated with B. ribis in
the fall, representing non-callused stem. Note
lack of callus around the wound (arrow).
A portion of a stem above the point of
inoculation of B. ribis (in fall) showing
pycnidia (arrows) emerging from the bark.

82

83
Figure 3.10. Pathogenic interaction between B. ribis and M.
quinquenervia. Each bar represents the mean of canker length
of 21 stems.
Figure 3.11. Clonal susceptibility of M. quinquenervia towards
B. ribis. Each bar represents the mean of canker length of
24 stems.

84
70
Figure 3.12. Mean canker length at the proximal and distal parts
of the stem from the point of inoculation of M. quinquenervia
with B. ribis isolates. Each bar represents a mean of 21
stems.

85
Isolate number
Figure 3.13. Mean necrotic tissue on M. quinquenervia stems
at 5 mm above the point of B. ribis inoculations. Each bar
represents mean percentage of necrosis of phloem and cambium
along the circumference and cross-sectional areas of the phloem
and sapwood, respectively, from 21 stems.

86
Figure 3.14. Mean canker length of the ramets of M. quinquenervia
clones (MQ) 8 weeks after inoculation with BR-2 isolate of B. ribis.
Bars with the same letter(s) between spring and fall-inoculations
in each clone are not significantly different at P=0.05 (T-test).

CHAPTER IV
EFFECTS OF STRESS FACTORS ON CANKER DEVELOPMENT ON STEMS
OF Melaleuca quinquenervia INOCULATED
WITH Botryosphaeria ribis
Introduction
Canker diseases and dieback of Melaleuca quinquenervia
(Cav.) Blake have been observed at an isolated location in the
naturalized range of this plant in South Florida (personal
communication with Dr. Ted Center). We (Webb and Rayachhetry)
isolated Botryosphaeria ribis Grossenb. & Dug., a canker causing
pathogen, from symptomatic M. quinquenervia trees at this
location. The sporadic occurrence of canker disease caused by B.
ribis in natural populations of M. quinquenervia may be due to 1)
the predispositional effect of one or more environmental factors,
2) a change in pathogenicity in the fungal population, or 3) a
combination of these two elements. Identification of the
predispositional factors influencing disease occurrence and
development may be important, especially in the light of an
integrated approach (use of herbivorous insects and pathogenic
fungi) to controlling M. quinquenervia. Use of insects for
defoliation, combined with pathogens, might be a viable, long-term
solution for control of the M. quinquenervia population in South
Florida.
87

88
Stress factors often play an important role in predisposing
plant communities to disease and the occurrence of epiphytotics.
Factors such as 1) water stress (Towers and Stambaugh 1968, Bagga
and Smalley 1969, Crist and Schoeneweiss 1975, Old et al. 1990),
2) freezing temperature, and 3) repeated defoliation (indirectly
due to environmental factors or by herbivores) have been reported
as important to disease progress in woody plants (Schoeneweiss
1981, Old et al. 1990).
Low Moisture
Variation in precipitation and moisture availability is a
frequent event throughout the world, except in areas supported by
artificial irrigation (Schoeneweiss 1975). In the United States,
tree disease syndromes such as ash dieback, maple decline,
sweetgum blight, birch dieback, oak decline, and pitch streak of
slash pine, have been attributed to below normal precipitation for
an extended period in 1930s (Hepting 1963). However, the
association and the role of pathogens in many of these disease
syndromes were not clearly stated, but host predisposition to
various pathogens was one of the major factors involved in disease
development (Schoeneweiss 1975).
Some experimental evidence of host predisposition to
pathogen invasion is available. Bagga and Smalley (1969, 1974)
inoculated differentially watered (daily, every third-, and fifth-
day) Populus tremuloides Michx. seedlings with Hypoxylon pruinatum
(Klotz.) Cke. and established that the water stress was needed for
stem canker development. Similarly, Crist and Schoeneweiss (1975)

89
created differential xylem water potential in European white ash
(Betula alba L.) and inoculated with B. dothidea (Moug. ex Fr.)
Ces. & de Not. They reported that canker formation and
colonization of the wood and bark increased with decreased water
potential (< -12 bar). Appel and Stipes (1984) exposed 5- to-7-
year-old pin oak (Quercus palustris Muenchh.) trees to episodes of
drought in greenhouse conditions and wound inoculated these trees
with mycelial plugs of Endothia gyrosa (Schw.) Fries. They
discovered that only moisture-stressed pin oaks were colonized by
the fungus and that regular watering consistently inhibited
colonization. They also reported that increased water stress
decreased the ability of the host to resist E. gyrosa invasion.
Old et al. (1990) studied the effect of drought on the
development of E. gyrosa and B. ribis cankers on Eucalyptus
delegatensis R.T. Bak. and E. maculosa R.T. Bak., and reported
that cankers developed only in the water-stressed seedings and
saplings. However, resumption of regular watering resulted in
callus growth that covered the cankers after 8 weeks (Old et al.
1990). Brier (1961) linked low moisture content in bark tissues
with the promotion of invasion of living tissues by some bark-
inhabiting facultative fungi. This view was further supported by
Ross (1964) who found that the low moisture content of bark
tissues in white ash (Fraxinus americana L.) favored canker
formation by Fusicoccum species.

90
Low Temperature
Low-temperature effects can injure plant tissues and
predispose them to various pathogenic organisms (Schoeneweiss
1975, Schoeneweiss and Wene 1977, Kable 1967, Helton 1961 and
1962). Low temperature effects on plants can be viewed as
chilling stress and freezing stress (Levitt 1972) . Chilling
stress is generally elastic, occurring when plants are exposed to
temperatures above the freezing point, whereas plastic stress
occurs when plants are exposed to temperature below the freezing
point, and in this situation, the plants are severely stressed
(Schoeneweiss 1975) due to frost crack development in the roots
and stems. With respect to disease susceptibility, Schoeneweiss
(1975) sees the effect of low temperature on the host as being
reversible, as long as the plant tissues are not directly killed
by freezing.
Helton (1961, 1962) studied the effect of low temperature
injuries on the incidence of Cytospora canker on plum (Prunus
domestica L.) trees. He showed that the epiphytotics of Cytospora
canker on plum tree resulted from wounds created by low
temperatures during winter. Similarly, Kable et al. (1967)
reported that the fungus, C. leucostoma, causing cankers on sweet
cherry (P. avium L.) stems gained access through winter-injured
tissues, and formed perennial cankers. These findings are
supported by the work of Schoeneweiss and Wene (1977) who showed
that stem cankers caused by Nectria cinnaberina Todd ex Fr. on
Euonymus alatus (Thunb.) Sieb. required a freezing temperature
below -22.5°C to allow development of canker diseases.

91
Schoeneweiss (1974) has experimentally proven an increase in
disease susceptibility of Tallhedge (Rhamnus frángula L.) towards
Tubercularia ulmea Carter when temperature was gradually reduced
to -30°C. In another experiment, Wene and Schoeneweiss (1980)
were able to predispose 2-year-old stems of European mountain ash
(Sorbus aucuparia L.) to a nonaggressive stem canker isolate of B.
dothidea by differential exposure to low temperature (up to
-30°C) . Similarly, frozen flowers and twigs of peach trees from
northern Canada were found to provide entry points for Leuscostoma
cincta (Fr.) Hohn which causes nodal cankers on the peach stems
(Dhanvantari 1979). However, the susceptibility of the host
tissues within individual trees often varies with the season of
the year (Quamme et al. 1972).
Low temperature not only enhances colonization and stem
canker formation by some pathogens, but it can also encourage
invasion of the root system by root-invading pathogens. For
instance, low temperature treatment (10- to-12 C) in association
with drought was found to encourage the penetration of Pinus taeda
L. roots by Fomes annosus (Fr.) Cke. in field as well as in
greenhouse conditions (Towers and Stambaugh 1968).
Epiphytotics in trees caused by relatively nonaggressive
pathogens appear to reflect an alteration of the physical and
chemical ability of the host to respond to invasion. Freezing
resulted in wounds which facilitate the establishment of the
pathogen (Schoeneweiss 1974 and 1975, Kable 1967) which then can
gradually grow beyond the limit of the wound due to the decreased
ability of the host to produce chemical barriers, such as lignin
and suberin. Following freezing, almond trees, P. dulcís (Mill.)

92
Webb, became susceptible to the bark canker fungus, Phytophthora
syringae (Kleb.) Kleb due to its reduced ability to produce lignin
and suberin (Doster and Bostock 1988). Similarly, wound periderm
formation in apple trees (Doster and Bostock 1988) and wound
response of the bark of three conifer species (Mullick and Jenson
1976) have been reported to be much slower in the winter than in
the summer.
DafQ.lia.tiQn
Repeated defoliation of trees during the growing season can
weaken and predispose trees to invasion by various pathogenic
organisms (Schoeneweiss 1981 and 1967, Wargo et al. 1973), and
subsequently favor canker development. Staley (1965) attributed
declining and dying Q. rubra L. and Q. coccinia Muenchh. to insect
defoliation along with other environmental factors. Defoliated E.
delegatensis and E. regnans F. Muell. seedlings developed
susceptibility to invasion and canker development by B. ribis,
during which most stems were girdled (Old et al. 1990) .
Similarly, the studies on relationship between canker incidence
and length of exposure to defoliation of European white birch have
indicated that B. dothidea can result in girdling of completely
defoliated (for 8 weeks) seedlings in 4 weeks after inoculation
(Crist and Schoeneweiss 1975). The events of starch depletion
have been also reported for defoliated shoots of B. papyrifera
Marsh. (Gross and Larsen 1971).
Defoliation of sugar maple trees, induced artificially or by
insect feeding, resulted in depletion of overall starch levels and

93
increased levels of fructose, glucose and certain amino acids
(threonine, cysteine, tyrosine, histidine, and proline) in the
outer wood of the root system (Wargo et al. 1973, Wargo 1972) .
The amendment of growth media with an extract from outer wood of
defoliated trees has greatly enhanced the hyphal growth of
Armillaria mellea (Vahl. ex Fr.) Quel, an indication that the
fungus is probably favored by the increased level of these
chemical changes in the root tissue. Old et al. (1990) have
reported similar decreases in the starch level of the defoliated
eucalyptus stems. They have attributed the increased
susceptibility of seedlings following defoliation to the reduced
level of soluble and stored carbohydrates in the plant tissues.
Objectives
The objectives of the present research were to evaluate the
effects of low moisture, low temperature, and defoliation stresses
on the occurrence and severity of canker disease caused by B.
ribis on M. quinquenervia under greenhouse conditions.
Materials and Methods
Selection Criteria for Tree Clones and Isolates
The tree clones used in the following experiments were
selected according to the availability of an adequate number of
ramets maintained in greenhouse conditions. Fungal isolates BR-2

94
and BR-5 used in the following three experiments were chosen based
on their differential (most and least) virulence identified
through greenhouse experiments (see Chapter III).
Low Moisture
Tree clone MQ-6 of M. quinquenervia and isolates BR-2 and
BR-5 of B. ribis were used in this experiment. Four treatment
groups (Gr.) were selected to create different levels of xylem
water potential. These four groups were composed of ramets
watered every 24 (Gr.l), 72 (Gr.2), 168 (Gr.3), and 288 (Gr.4) h.
Five ramets per isolate in each treatment group were left in
greenhouse for the remainder of the 8-week experimental period.
The groups of ramets were exposed to their respective
watering cycle one time and were then inoculated with the
appropriate fungal isolate. Inoculation was performed at the mid¬
point of the stem by filling 1.5 mm diameter and 2.0 mm deep holes
with 5-day-old, 1% mycelial suspension (wt/vol) grown on PDB under
continuous shaking conditions and macerated by blending for 25
seconds. The inoculation points were wrapped with Parafilm
immediately after inoculation. The ramets were then randomized
according to location and maintained on a greenhouse bench at
30(+5)°C.
Thereafter, the ramets were exposed to their respective
watering cycle throughout an 8-week experimental period. Xylem
water potential of three twigs from each of the four groups of
plants was measured using a pressure bomb (Plant Moisture Stress
Inc., Corvallis, Oregon, USA) at 7:30-8:30 AM before and after

95
(next morning) watering. During measurement of xylem water
potential, the ambient temperature in the greenhouse was ca 25°C.
Ramets were evaluated every week for wilting, wilted ramets
were harvested, and the canker length was measured after splitting
stems longitudinally through the point of inoculation. After 8
weeks, the remaining ramets were harvested, the stems split
through the point of inoculation, and the proximal and distal
canker extension (from the point of inoculations) were measured.
Low Temperature
Effects of low temperature on disease development among M.
quinquenervia ramets were determined using tree clone MQ-9 and
isolates BR-2 and BR-5 of B. ribis. Treatment groups (Gr.) were
exposed to 3 0(±5)°C on the greenhouse bench for 7 days a week
(Gr.l), 6°C for 3-day/wk (Gr.2), 6°C for 6-day/wk (Gr.3), and
0(±1)°C for 16 h/wk (Gr.4). Each of the treatment groups were
represented by five ramets per isolate.
Groups of ramets were exposed one time to their respective
temperature before inoculating with B. ribis isolates according to
the procedures described under "Low Moisture" treatment experiment
in this chapter. Groups 2, 3, and 4 (assigned to low temperature
treatments) were also maintained together as a group on the
greenhouse bench at 30(+5)°C for the remainder of time in a week.
When the ramets were on the greenhouse bench, they were watered
daily and evaluated for wilt symptoms. Incubation period and
canker measurement methods were the same as described under "Low
Moisture" treatment experiment in this chapter.

96
Defoliation
Effects of defoliation on canker disease development among
M. quinquenervia ramets were determined using tree clone MQ-9 and
isolates BR-2 and BR-5 of B. ribis. Treatment groups (Gr.) were
composed of 0 (Gr.l), 50 (Gr.2), and 100 (Gr.3) percent
defoliation of the total leaf area. Each group per isolate was
represented by five ramets. Additionally, check plants (wounded
but not inoculated with fungus), composed of five ramets/isolate,
were maintained for Gr.3 treatments.
Ramets in groups 2 and 3 were defoliated by removing the
appropriate proportion of the area of new leaves every week for
two weeks. After two weeks, ramets were inoculated in the mid¬
point of the main stem using same inocula and inoculation
techniques as described under "Low Moisture" treatment experiment
in this chapter. The ramets of each group were maintained
separately on a greenhouse bench at 30(±5)°C under a regular
watering schedule.
Thereafter, the level of defoliation among groups was
maintained by removing an appropriate area of new leaves every
week for the 4-week experimental period. Ramets in all three
groups were harvested 4 weeks after inoculation since the majority
of the ramets in group 3 treated with fungus stopped producing new
leaves and appeared to be dead. Canker lengths were measured for
the ramets in the groups 1 and 2. However, percentage of
mortality was recorded for checks and fungus-inoculated ramets in
group 3 because canker length could not be recorded due to
advanced tissue deterioration.

97
Statistical Analysis
Total canker length (proximal + distal) was used as a
response variable for all the experiments used to evaluate the
effect of stress factors on canker development. Analysis of
variance and mean separations among variables were performed using
GLM procedures in SAS (1985). In all three experiments, host
clones and inocula were analyzed as fixed effects.
Results
Low Moisture
During the experimental period, the xylem water potential
among groups of ramets fluctuated markedly among treatment groups
(Fig. 4.1). Before as well as 24 h after watering, xylem water
potential among ramets in groups 3 and 4 was more negative than in
the ramets in groups 1 and 2 (Fig. 4.1). The more negative xylem
water potential may render ramets in groups 3 and 4 more
susceptible (to B. ribis) than those in groups 1 and 2.
The water stress treatments had a significant effect on stem
canker development (Table 4.1). The 7-day interval of watering
(Gr.3) had greater influence on canker development than the daily,
5-day, and 12-day intervals (Fig. 4.2). The differences in
virulence between fungal isolates, BR-2 and BR-5 were also
significant (Table 4.2). After about 4 weeks, shedding of older
leaves was pronounced among the most stressed ramets (groups 3 and

98
4) (Fig. 4.3) and 50 percent of the plants wilted in both groups
within 4 weeks after inoculation. Callus ridges around cankers
were prominent among least stressed ramets (groups 1 and 2) (Fig.
4.4) but was absent or nearly so in the most stressed ramets
(groups 3 and 4) (Fig. 4.5). The removal of bark from noncallused
stems revealed the development of black necrotic phloem, cambium,
and sapwood beyond the points of inoculations (Fig. 4.6).
Low Temperature
In the low temperature treatments, both isolates and
treatment levels were significantly different; the interaction
between isolates and temperature was insignificant (Table 4.1).
Mean canker length for isolate BR-2 was significantly greater than
for isolate BR-5 (Table 4.2). Canker development was greater
among stressed ramets in group 2 (treated with 3-day alternate
exposure to 6°C and 30°C (±5) than in non-stressed ramets in group
1 (Fig. 4.7). The stressed ramets in groups 3 and 4 developed
lesser amount of canker than the ramets in group 2. Also, sapwood
discoloration among ramets was extensive in group 2. Wound¬
callusing was rudimentary among most stressed ramets (groups 2, 3,
and 4) (as shown in Figure 4.5) compared to the nonstressed ramets
(group 1) (as shown in Figure 4.4) where cankers were surrounded
by pronounced ridges of calluses.

99
Table 4.1. Analysis of variance of data of experiments designed
to assess the effects of stress factors on canker development on
stems inoculated
inoculation.
with BR-2 and BR-5 of
B. ribis
at 8 weeks
after
Experiments
Source of
variation
Degree of
freedom
F-values
Pr>F
Water stress
Isolate
1
14.79
0.0002
Trtlevels
3
9.08
0.0001
Isolate * Trtlevels
3
0.89
0.1216
Error
32
Low temperature
Isolate
1
10.32
0.0030
Trtlevels
3
3.58
0.0244
Isolate * Trtlevels
3
0.55
0.6491
Error
32
Defoliation
Isolate
1
15.61
0.0011
Trtlevels
1
0.73
0.4060
Isolate * Trtlevels
1
1.22
0.2850
Error
16
Note: Trtlevels = Treatment levels. Significance was determined
at P=0.05, and
n=5. Under
"Defoliation,” only treatment levels 1 (group 1, 0%
defoliation)
and 2 (group 2,
50%
defoliation) has been
included in this analysis.
Table 4.2. Main effect means and mean separations from analyses
of variance for effects of stress factors on canker length (mm) on
the stems of M. quinquenervia inoculated with isolates of B.
ribis.
Experiments
Fungal
Mean canker
isolates
length
Water stress
BR-2
68.60a
BR-5
45.70b
Low temperature
BR-2
72.42a
BR-5
43.90b
Defoliation
BR-2
60.67a
BR-5
52.33b
Note: Means followed by the same letter(s) are not significantly different according to
Scheffe's test at P=0.05. For each isolate in water stress and low temperature experiments,
n=20; and for defoliation experiment, n=15.

100
Defoliation
The effects of 0 and 50 percent defoliation of the ramets in
groups 1 and 2, respectively, on canker development was
insignificant; the differences in virulence between isolates BR-2
and BR-5 was significant (Tables 4.1 and 4.2, Fig. 4.8). In these
two groups, no ramet mortality occurred during the 4-week
experimental period. Ramets in 0 and 50 percent defoliation
responded to the wound inoculation of fungus by developing a
substantial callus around the wound, whereas ramets defoliated 100
percent failed to respond with callusing around the wound
inoculated with fungus. Removal of the bark around the point of
inoculation revealed blackened necrotic phloem similar to those
depicted in Figure 4.6. The average mortality among ramets with
100 percent defoliation was 90 percent within 4 weeks after
inoculation in contrast to 14 percent among check plants that were
100 percent defoliated but not inoculated with fungus (Fig. 4.9).
Discussion
Low Moisture
The xylem water potential in the stem reflects the free
water content in the xylem. The more negative the xylem water
content in the plant, the less water is available to plants for
various chemical reactions including those related to chemical-
defense mechanism against pathogenic organisms. Therefore, in the

101
low moisture experiment, xylem water potential of the ramets was
used as indicator of the level of moisture stress.
The effect of drought on disease development in a variety of
host-pathogen systems is a well documented phenomenon (Hepting
1963, Bagga and Smalley 1969 and 1974, Crist and Schoeneweiss
1975, Bertrend et al. 1976, Appel and Stipes 1984, Old et al.
1990). In this study, the xylem water potential influenced canker
development on the stems of M. quinquenervia. Disease level
increased with the decreased xylem water potential (Figs. 4.1,
4.2). Disease level increased significantly among groups when
xylem water potential reached a maximum of -1.5 MPa before or -0.6
MPa after watering (Figs. 4.1, 4.2). Similar effect of drought
has been reported for B. dothidea on European white birch (Crist
and Schoeneweiss 1975) and H. pruinatum on P. tremuloides (Bagga
and Smalley 1969).
After reaching a threshold of about -1.7 MPa, further
decrease of xylem water potential did not have a significant
effect on disease development (Figs. 4.1, 4.2). As the xylem
water potential reached -1.8 MPa before watering, the ramets in
the groups 3 and 4 began to drop older leaves at 4 to 5 weeks
after inoculation (Fig. 4.3) and 50 percent of the plants revealed
wilting symptoms. The wilted plants were girdled by the necrotic
cambium and phloem. A similar phenomenon of older leaf drop was
observed among European white birch seedling inoculated with B.
dothidea (Crist and Schoeneweiss 1975).
Susceptibility of poplars to C. chrysosperma (Pers.) Fr.
increased when callusing of wounds was inhibited by moisture
stresses (Puritch and Mullick 1975). In the present study, the

102
callus ridges were prominent among ramets of the groups 1 and 2,
and no mortality was observed in these groups. In groups 3 and 4,
callus ridges were initiated within a week but further growth was
arrested, and 40% of the ramets in both groups were girdled
resulting in dieback and eventual mortality of the whole plant.
Moisture stress appears to promote the fungal invasion,
colonization, and disease development in trees. Ramets of M.
quinquenervia, receiving daily watering treatment were able to
exert some degree of resistance to fungal growth through xylem and
phloem tissues. McPartland and Schoeneweiss (1984) studied the
hyphal morphology of B. dothidea in drought-stressed and non-
stressed symptomatic stems of B. alba. Their studies revealed
that hyphae in the xylem vessels of nonstressed plants were thin,
unbranched and had restricted growth (5 mm from the inoculated
wound at 7 weeks following inoculation), and the majority of which
had burst hyphal tips in contrast to thick, branched hyphae with
extensive growth in the vessels of drought-stressed plants.
In the present study, moisture-stress may have induced
changes in the level of certain chemicals among ramets in 7-day
(Gr.3) and 12-day (Gr.4) watering cycles and subsequently
predisposed tissues to support enhanced fungal growth. Stress
factors resulting in low carbohydrate level in the stem tissues
inhibit compartmentalization response and callus formation and
support active invasion of the stem tissues (Old et al. 1990) .
Puritch and Mullick (1975) found that nonsuberized impervious
tissue (NIT) formation is a nonspecific reaction to the wound
whose formation is delayed among drought-stressed plants. Lack of
NIT around the wound facilitates invasion of inner healthy tissues

103
by pathogenic organisms (Puritch and Mullick 1975) . A decrease in
starch and an increase in sugars have been reported in moisture-
stressed loblolly pines (Hodges and Lorio 1969). The increased
levels of sugars and sugar alcohols in the bark of peach trees
gave susceptible reactions to Cytospora canker (Rohrbach and
Luepschen 1968). Melaleuca lanceolata Otto seedlings water-
stressed under laboratory conditions produced an increased level
of L-proline and trans-4-hydroxy-N-methyl-L-proline (Naidu et al.
1987). Proline is widely regarded as one of the membrane
stabilizing agents (Ayres 1991) in a living cell. Water-stressed
plantlets of P. deltoides Bartr. ex Marsh, produced higher levels
of proline, alanine, and glutamine than the unstressed plantlets
(Belanger et al. 1990). Griffin et al. (1986) have proven that
these three amino acids stimulate the mycelial growth of H.
mamma turn ((Wahl.) Mill.
Low Temperature
Considerable literature exists on the effect of low
temperature on disease development among woody plants. However,
almost all of those studies are related to the effects of freezing
temperature on the creation of wounds, i.e., infection court for
the establishment of canker pathogens (Helton 1961 and 1962, Kable
1967, Schoeneweiss 1975, Schoeneweiss and Wene 1977) .
In the present study, continuous exposure of ramets for 16 h
caused stem freezing but did not result in cracks and ramet
mortality. Discoloration of sapwood was greater among ramets in
group 2 which received 3-day alternate exposure to 6°C in the cold

104
room and ca 3 0°C under the greenhouse conditions than the ramets
in other groups (Fig. 4.7). Therefore, it is assumed that the 3-
day alternate low and high temperatures favored the hyphal growth
through the xylem and affected the host resistance. On the other
hand, 6-day/week exposure of ramets (in group 3) to 6°C affected
the xylem-invasion potential of the fungus as well.
Stem exposure to 0( + l)°C for 16 h appeared to have some
effect in xylem discoloration due to the presence of B. ribis.
Schoeneweiss (1974) has shown an increased disease susceptibility
of tallhedge (R. frángula) to T. ulmea when the temperature was
reduced to -30°C. By differential exposure of European mountain
ash (Sorbus aucuparia L.) to low temperature treatments (up to
30°C), Wene and Schoeneweiss (1980) were able to predispose 2-
year-old stems to a canker causing isolate of B. dothidea.
Ramets maintained in a greenhouse 7 days per week at
3 0(±5) °C (Gr.l) produced callus and slowed the invasion-potential
of the fungus. Though callus formation was initiated among ramets
exposed to 6°C for 3-day/wk (Gr.2), 6°C for 6-day/wk (Gr.3), and
0(+l)°C for 16 h/wk (Gr.4), further development was arrested.
Visually detectable callusing was 100, 20, 0, and 20 percent among
ramets of M. quinquenervia in groups 1, 2, 3, and 4, respectively.
In general, the low temperature treatments appeared to inhibit
callus formation and thus reduce host resistance to B. ribis.
Wound-periderm formation in apple trees (Doster and Bostock 1988)
and wound response in the barks of three conifer species (Mullick
and Jenson 1976) have been reported to be much slower at lower
temperature in winter than in higher temperature in summer.

105
Defoliation
Defoliation has been documented to weaken and predispose
plants to invasion by various pathogenic organisms (Schoeneweiss
1967 and 1981, Wargo et al. 1973, old et al. 1990). Schoeneweiss
(1981) mentioned that defoliation at a critical period or repeated
defoliation over several years may result in predisposition of
plants to nonaggressive pathogens. In the present study,
defoliation for four cycles in an active growing period
(June/July) and subsequent wound inoculation with B. ribis
resulted in 80-100 percent mortality of the ramets. Luttrell
(1950) reported 80 percent mortality of 3-month-old American elm
seedlings within 6 months after inoculation with B. ribis
following complete defoliation. In the present experiments, the
necrotic tissues around the point of inoculations were similar to
those described for blackstem diseases caused by C. chrysosperma
on cotton wood (P. deltoides) (Schoeneweiss 1967).
Defoliation in scarlet red oaks caused by leaf rollers has
been reported to diminish the availability of the levels of
carbohydrates for growth (Staley 1965). Old et al. (1990) have
reported an extreme susceptibility of two Eucalyptus species to
invasion by B. ribis following defoliation. Reduced starch levels
in roots and stem tissues due to its conversion to glucose and
fructose has been reported for defoliated sugar maple trees (Wargo
1972, Wargo et al. 1973). Growth of some fungi is supported by
these reducing sugars (Wargo 1972). Additionally, these sugars
may have priority of being channelled in maintaining energy
balance for survival rather than being used for building physical

106
and chemical barriers around wounds. This phenomenon may explain
why completely defoliated ramets of M. quinquenervia in this study
were not able to resist quick girdling of the stems by B. ribis
when compared to those nondefoliated or partially defoliated
plants in which callus development was prevalent during spring and
summer inoculations.
Conclusions
The effects of low moisture, low temperature, and defoliation
stresses on disease progress among ramets of M. quinquenervia was
studied under greenhouse conditions. Low moisture contents in the
xylem rendered the ramets susceptible to increased levels of
canker formation and subsequent mortality. Alternate cycles of 3-
day exposure to (6°C) and 30 (±5)°C favors increased levels of
canker development compared to 6-day/wk at 6°C, 16 h/wk at 0
(±1)°C, and 30(±5)°C throughout. Defoliation levels of 0 and 50
percent did not affect canker development but complete defoliation
resulted in high mortality of ramets within 4 weeks after
inoculations with B. ribis isolates. All three stress factors
either reduced or prevented callusing of wounds among ramets of M.
quinquenervia inoculated with B. ribis.

107
—■—Group 1, —•—Group 2, —*—Group 3, —▼—Group 4
Observation numbers
Figure 4.1. Xylem water potential of differentially moisture-
stressed stems of M. quinquenervia ramets inoculated with B.
ribis. Upper and lower points for each group in each observation
represent xylem water potentials immediately before and 24 hr after
watering to field capacity. Groups 1, 2, 3, and 4 represent ramets
watered every 24, 72, 168, and 288 hrs, respectively.
Figure 4.2. Effects of low moisture on canker development in the
stems of M. quinquenervia ramets inoculated with B. ribis. Bars
with the same letter(s) are not significantly different at
P=0.05 (Scheffe's test). Treatment groups are same as in Figure
4.1.

Figs. 4.3-4
Figure 4.3.
Figure 4.4.
Figure 4.5.
.6. Effects of low moisture on canker disease in M.
quinquenervia caused by B. ribis.
Four groups (gr.) of ramets watered every 24
(gr.l), 72 (gr.2), 168 (gr.3), and 288 (gr.4)
hrs. Note progressive crown thinning (from gr.l
to gr.4) due to leaf-drop.
A stem showing a typical canker among ramets
wounded and inoculated with B. ribis and watered
every 24 hrs. Note callus ridge (arrow)
surrounding a canker.
A stem showing a typical canker among ramets
wounded and inoculated with B. ribis and watered
every 288 hrs. Note lack of callus and the
presence of blackened tissues (arrow).
Inner tissues in a typical stem canker among
ramets wounded and inoculated with B. ribis and
watered every 288 hrs. Note blackened bark
(arrow) and sapwood (arrow head).
Figure 4.6.

109

110
Figure 4.7. Effects of low temperature on stem canker development
on M. quinquenervia ramets inoculated with B. ribis. Treatment
groups 1, 2, 3, and 4 represent exposure to 30(±5) C for 7 days a
wk, 6 C for 3-day/wk, 6 C for 6-day/wk, and 0 (+1) C for 16-hr/wk,
respectively. Bars with the same letter(s) are not significantly
different at P=0.05 (Scheffe,s test).

Ill
a
Figure 4.8. Effects of defoliation on canker development on stems
of M. quinquenervia ramets inoculated with B. ribis. Groups 1
and 2 represent 0 and 50 percent defoliation. Bars with the same
letter(s) are not significantly different at P=0.05 (Scheffe's test).
Treatment groups
Figure 4.9. Effects of defoliation on mortality of M. quinquenervia
ramets inoculated with B. ribis. Treatment groups 1, 2, and 4
represent 0, 50, 100 percent defoliation plus B. ribis,
respectively; treatment group 3 represents 100 percent
defoliation plus wound alone.

CHAPTER V
HISTOPATHOLOGY OF Botryosphaeria ribis
IN Melaleuca quinquenervia STEMS.
Introduction
Reports on histopathological effects of B. ribis in the stem
tissues of other woody plant species of economic importance are
available in the literature (Biggs and Britton 1988, Brown and
Hendrix 1981, Milholland 1972). There are reports that B. ribis
is capable of colonizing both wounded and unwounded plants
(Luttrell 1950, Scrieber 1964, Brown and Hendrix 1981).
Milholland (1972) used five isolates of B. dothidea to inoculate
wounded and unwounded plants of different blueberry cultivars and
noted that the unwounded inoculations did not produce cankers,
whereas the wound inoculations resulted in cankers and stem
blight. However, Brown and Hendrix (1981) inoculated wounded as
well as unwounded apple trees with B. ribis and reported lesion
development in both cases. They observed that the fungal hyphae
advanced through disrupted cortical cells in wounded stems. In
the case of unwounded stems, infection took place through
lenticels, and the hyphae were restricted outside the callus
periderm formed by hyperplasia beneath the site of infection
(Brown and Hendrix 1981) .
112

113
Biggs and Britton (1988) inoculated peach trees with B.
dothidea and B. obtusa and reported intracellular hyphae of both
species in cortical parenchyma, callus parenchyma, xylem ray
parenchyma, vessels, and tracheids. However, the intercellular
hyphae were observed only in the phloem fibers, necrotic cortical
parenchyma outside the periderm, and in the ligno-suberized
parenchyma tissues on the callus surface.
In blueberry stems, tyloses and protrusions originating from
adjacent parenchyma cells occluded the xylem vessels, as the
fungus progressed down the stem intra- as well as inter-cellularly
through the different tissue types (Milholland (1972). Thus,
Milholland (1972) attributed blueberry blight to the occlusion and
impediment of the vascular lumen by the fungal hyphae. In apple
trees, Brown and Hendrix (1981) found that B. dothidea hyphae were
parallel with the vessels and tracheids in the wounded stemtissue
of the apple trees, but were infrequent in the xylem ray
parenchyma. They interpreted these attributes as regulating the
rapid proximal and distal colonization and the slower lateral
development of canker in apple stems. However, in peach trees,
the hyphal extensions of B. dothidea and B. obtusa were observed
in all tissue types at lateral and tangential margins of infection
(Biggs and Britton 1988). Also, Biggs and Britton (1988)
interestingly noted that, at the proximal and distal ends, the
fungal hyphae were found only in the lumen of vessels and
tracheids but were lacking in the parenchyma cells.
No information is available in the literature regarding the
histopathological relationship between M. quinquenervia and B.
ribis or the pathological anatomy of M. quinquenervia. The

114
histopathological information on B. ribis in stems of M.
quinquenervia will help in the understanding of the process of
pathogenesis involved at tissue and cellular levels. Therefore,
the specific objectives of these experiments were to assess 1) the
mode of the hyphal invasion and colonization in symptomatic
foliage and stems, and 2) the anatomical response of the host to
the infection by the fungus.
Materials and Methods
Preparation of Leaf Materials
Excised leaves (4th, 5th, and 6th, from the terminal buds of
the main stems) of M. quinquenervia ramets wound-inoculated with
1% (vol/wt) hyphal suspensions (see under "Inocula Preparation" in
Chapter III) of B. ribis isolates, and wounded but inoculated with
sterile distilled water alone (check) were incubated in a moist
chamber under sterile conditions for up to 21 days for use in
histopathological studies. Five necrotic leaves collected at 3,
7, and 21 days after inoculation, and five control leaves
collected 21 days after wounding and inoculation with sterile
distilled water were cut into 10 mm x 5 mm segments and fixed in
formalin-propionic acid (FPA).
Segments were dehydrated with tertiary butyl alcohol
followed by infiltration and embedding with paraffin (Paraplast X-
TRA, OXFORD, LABWARE, Division of Sherwood Medical, St. Louis, MO
83103, USA) (Jensen 1962). Leaf sections of 5- to -15 /¿m were

115
obtained using a rotary microtome and affixed onto glass slides
using Haupt's adhesive. Sections were dewaxed in three changes of
100% xylene then passed through a series of a mixture of xylene
and absolute ethyl alcohol (ETOH) (1:1), absolute ETOH, and 70%
ETOH. At this stage, the sections were stained with Pianeze's
IIIB (Vaughan 1914) and/or lactophenol-cotton blue. Ten sections
from each leaf segment were examined for fungal colonization of
leaf tissues and formation of pycnidial stroma. Photomicrograph
of representative sections were taken using light-microscopic
systems (Olympus/BMH or Nikon OPTIPHOT II).
Preparation of Stem Materials from Unstressed Plants
Stems of M. quinquenervia ramets (1.5 mm diameter and 2.0 mm
deep hole) were wound inoculated in September (fall) 1993 and
March (spring) 1994 with 1% hyphal suspensions (same as under
"Inocula Preparation" in Chapter III) of B. ribis isolates. In
the checks, similar wounds were made and were filled with sterile
deionized water. After 8 weeks, one stem segment (10 mm long, 5
mm wide, and 5 mm thick) was taken from the proximal and distal
margins of lesions on each of five check and B. ribis-inoculated
trees, with or without callus.
The stem segments were processed and stained using the
methods used for leaf tissues (see "Preparation of Leaf Materials"
in this Chapter). Three fresh stem segments from each of three
treatments, i.e., not wounded, wounded but not inoculated, and
wound inoculated with B. ribis, were tested for compounds such as
lignin, suberin, and tannin that may have been formed by the host

116
in response to wounding or fungal invasion of the tissues. Lignin
was detected using a combination of phloroglucinol (1% wt/vol in
70% ETOH) and 25% HC1 (Jensen 1962), and Sudan IV saturated in 95%
ethanol and glycerol 1:1 (v/v) was used to detect suberin (Jensen
1962, Daykin and Milholland 1990). Tannin and other phenolic
substances in the tissues were detected using ferric chloride and
ferric sulphate reactions (Johansen 1940). Ten sections per
segment were studied for host reaction or fungal invasion of
different tissue types. Photomicrographs of the representative
sections were taken using light-microscopic systems (Olympus/BMH
or Nikon OPTIPHOT II).
Preparation of Stem Materials from Stressed Plants
Three B. ribis inoculated stem segments of M quinquenervia
from each of low moisture (7-day watering cycle), low temperature
(exposure to 6°C for 3-day/wk), and complete defoliation (see
Chapter IV) treatments were processed and evaluated using the same
methods and techniques as described for "Preparation of Stem
Materials from Unstressed Plants." The sections were observed
under a light microscope to reveal differences in
histopathological conditions of stressed and unstressed stems of
M. quinquenervia that were inoculated with B. ribis using the same
methods and techniques.

Results
Host Response
Leaves
Every B. ribis inoculated leaf became necrotic within 7 days
and turned dark brown, but leaves wounded and inoculated with
sterile distilled water (checks) remained green as long as 3
weeks. No leakage of substances from the leaves was observed
during that period. A cross section of a leaf that was wounded
but not inoculated with B. ribis is shown in Figure 5.1.
Excised leaves, wound-inoculated with isolates of B. ribis,
developed rapid leaf discoloration of tissues expanding
centrifugally from the point of inoculation. As the leaf
discoloration progressed, brown-colored substances leaked from
leaves onto the moist filter paper underneath the leaves. The
leakage of substances from the leaf onto the filter paper may be
an indication of the penetration of oil chambers by fungal hyphae.
Some of these oil chambers are located in chlorenchymatous tissues
beneath the epidermis.
Stems
Melaleuca quinquenervia stems with wound alone (Fig. 5.2),
and wound plus B. ribis produced callus-ridges around wounds (Fig.
5.3). The physical and chemical responses of the host to wounding

118
alone and to wounding plus B. ribis inoculations are described in
the following paragraphs.
Callused. In the checks of both spring and fall
inoculations, the wounds were completely filled with callus
tissues that extended into the xylem (Figs. 5.2, 5.4). As a
hyperplastic response to the wounding outside the xylem, the
cambium produced several layers of relatively small but thick-
walled parenchymatous cells which comprised the callus tissues
(Fig. 5.4). After producing several layers of parenchymatous
cells, a cambial layer redifferentiated in the callus tissue and
produced xylem and phloem inward and outward, respectively (Fig.
5.4). Thus, a patch of hyperplastic parenchymatous cells remained
surrounded by xylem tissues towards the outer and inner sides.
Cells in the outer layer of callus at the interface of the
sapwood were filled with substances which reacted positively to
ferric chloride and ferric sulphate, and indicated the nature of
the cellular contents to be tanninoid. A substance reacting
positive to Sudan IV, presumably suberin or other fatty
substances, was detected in the cell walls of a few 1- to 3-celled
layers at the outer phelloderm of unwounded (Fig. 5.5) or wounded
stems (Fig. 5.6). It appeared that the Sudan-VI-positive
substances in these layers are constitutively produced among
healthy stems but are also inducible around the wound periderm.
In wounded stems, the layers of suberized cells were continuous
with the periderm of callus to the interface between callus and
discolored sapwood (Fig. 5.6). Also, some isolated groups of
parenchyma cells in the callus gave a positive reaction to the
suberin tests. Walls of all tracheids and vessels reacted

119
positively to phloroglucin-HCl, but those adjacent to the wound
and in the callus tissues reacted intensely (Fig. 5.6), suggesting
these cells to be heavily lignified. Additionally, groups of
parenchymatous cells and bast fibers in the callus also intensely
reacted to phloroglucinol (Fig. 5.6). Among healthy stems, these
cells stained light-pink, suggesting the presence of normal
amounts of lignin as expected.
Callusing as well as production of suberin, lignin, and
tannin by stems wound-inoculated with B. ribis were similar to
those observed among stems that were wounded but not inoculated.
Position of the suberized and ligno-suberized layers among
inoculated stems were also similar to those observed in
uninoculated wounds. Beneath points of inoculations on stems,
vessels and tracheids in several tangential compartments of xylem,
delimited by radial ray parenchyma cells, were heavily lignified
(Figs. 5.7, 5.8) as shown by the intense coloration following
phloroglucinol-HC1 treatment. Similarly, ray and pith parenchyma
cells adjacent to necrotic xylem stained bluish-green when treated
with ferric chloride or ferric sulphate. Such a positive staining
of xylem-ray and pith parenchyma with ferric salts indicated the
presence of tannin or other ergastic phenolic compounds in these
cells.
Despite callus formation, sapwood discoloration among B.
ribis inoculated stems extended to several millimeter beyond the
callus ridges. A progressive disruption of cambium and inner
phloem was observed at the canker margins behind the discolored
tissues (Fig 5.7). The periderm at the inner margin of newly

120
formed callus adjacent to the surface of sapwood became ligno-
suberized and enclosed tanninoid cells (Fig. 5.7).
After formation of several layers of hyperplastic parenchyma
cells, vascular cambium redifferentiated, and produced phloem and
xylem tissues (Fig. 5.7) similar to those produced among
uninoculated stems. Cells in these hyperplastic parenchyma and
xylem tissues were invaded by fungal hyphae and resulted in
necrosis and created an opening due to collapse of invaded tissues
(Fig. 5.8). In turn, the necrophylactic periderm was ligno-
suberized and may function to limit fungal growth to the inner
side of the callus opening (Fig. 5.8). Hyphal invasion of callus
tissues progressively caused necrosis and the collapsed tissues
created an opening in the center of the callus wedges (Fig. 5.8).
Also, a ligno-suberized periderm developed within callus wedge on
both side of the opening (Fig. 5.8). Tyloses were abundantly
present in the vessels of B. ribis inoculated stems (Fig. 5.9).
Noncallused. When the callus was absent around the wound,
sapwood became discolored relatively quickly and phloem and
cambium collapsed along vertical and tangential direction from the
point of inoculation and created a depression on the stem surface.
Phloem, cambium, and xylem tissues were blackened at the advanced
stages of necrosis and the canker margins were progressively
discolored, imparting brownish coloration. Several 1- to 3-celled
layers of suberized cells were observed in the phelloderm region
of the stems similar to those detected in callused stems. Fresh
pith in the stems inoculated with fungus at the cambial region
turned brown compared with a pale-green color in wounded but
noninoculated stems.

121
Stressed. The groups of M. quinquenervia ramets exposed to
low moisture and low temperature produced no visible or slight
callus around the wounds that were inoculated with B. ribis. In
general, all the stems that produced none or minimal amount of
callus showed the mode of fungal invasion of different tissues and
host reactions similar to those observed for noncallused wounds.
Fungal Invasion and Colonization
Leaf tissues
Primarily, the hyphae entered the mesophyll tissue of the
leaf through stomata (Fig. 5.10). Although not observed in the
cross-sections of the inoculated leaves, the hyphae may have
penetrated leaf directly through the epidermis. This hypothesis
is supported by the leaching of the leaf content onto the filter
paper as soon as visually detectable leaf necrosis occurred.
Following entry into the leaf, hyphae advanced through inter- as
well as intracellular spaces of mesophyll and vascular cells of
the midrib, and colonized the oil chambers (Fig. 5.10). Intra¬
leaf pycnidial stroma were initiated in those oil chambers. Those
stroma grew in size, became uni- or multilocular, resulted in
rupture of epidermis, and delivered macroconidia on to the leaf
surfaces (Fig. 5.11).

122
Stem tissues
Callused. Fungal hyphae were observed in the inter- and
intracellular spaces of hyperplastic tissues in callus (Fig.
5.12). At the advanced stages of hyphal invasion, parenchymatous
cells at the callus margin became necrotic and eventually
collapsed as the fungus continued to grow into the inner part
(Fig. 5.13). Hyphal invasion of xylem elements occurred beyond
the callus ridges.
Fungal hyphae were observed in the inter- and intracellular
spaces of the discolored cells of phloem tissues and pith
parenchyma. However, the detection of younger (unmelanized)
hyphae in these cells was difficult since these hyphae and the
cellular contents of discolored cells stained similarly with
Pianeze1s IIIB stain. Radial and tangential growth of hyphae in
sapwood occurred through inter- and intracellular spaces of the
xylem rays, tracheids, and vessels (Figs. 5.14, 5.15, 5.16).
Hyphae were observed in the lumens of parenchymatous cells (5.13),
xylem rays (Fig. 5.14), vessels and tracheids (Figs. 5.15, 5.16),
and cortical and pith parenchymatous cells. Hyphal invasion of
adjacent tracheal cells or vessels occurred through pits as
indicated by the constrictions at the point of emergence in the
lumen (Fig. 5.15). In the tracheal lumens, hyphae grew freely or
appressed to the tracheal wall (Fig. 5.15). Hyphal swellings
(Fig. 5.15) and branching (Fig. 5.16) often clogged the lumen of
the tracheal cells. In addition to the hyphae, vessels in the
infected xylem contained granular substances which stained red
with Pianeze,s IIIB stain.

123
Noncallused. Among noncallused stems, the fungal hyphae
were concentrated in the cambial region. Hyphae were also present
in the inter- and intracellular spaces of the cortex but were
difficult to detect since the melanized B. ribis hyphae were often
masked by the cellular remains of collapsed cells in these areas.
As in the case of callused stem, fungal growth in vascular tissues
occurred through inter- and intracellular spaces of the cells in
the xylem (Fig. 5.17) and pith. Partially submerged pycnidial
stroma emersed from the collapsed tissues of the stems (Fig.
5.17). Preliminary observations revealed that fungal colonization
of stem tissues exposed to low moisture, temperature, and
defoliation was similar to that described for noncallused wounds
of the stems.
Discussion
Histopathological relationship of B. ribis has been
established with some other woody plants. This is the first
histopathological description of the host-pathogen relationship
between M. quinquenervia and B. ribis both obtained from South
Florida and maintained under greenhouse conditions in the campus
of the University of Florida in North Central Florida.
Leaf Invasion and Colonization
Botryosphaeria ribis-inoculated leaves of M. quinquenervia,
maintained in moist chamber revealed a progressively expanding

124
tissue discoloration from the point of inoculation. The tissue
discoloration followed extrusion of brown substances from leaves.
This phenomenon is suggestive of B. ribis-initiated changes in
wall and membrane integrity of the cells in chlorenchyma and
mesophyll of the leaves. The hyphal entry into these leaves
occurred through wounds and stomata. Michailides (1991) found
similar stomatal penetration of the leaves and fruits of pistachio
(Pistacia vera L.) by germ tubes of the macroconidia of B.
dothidea. Present experiments (see Chapter II, "Simulated Storm
Damage and Inoculation with Macroconidia") revealed the inability
of B. ribis to cause lesions on intact leaves of M. quinquenervia
plants in the absence of free water on the plant surface.
Therefore, under field conditions, unlike on pistachio leaves
(Michailides 1991), B. ribis does not appear to have the ability
to cause leaf necrosis and to contribute to epidemic disease
development on M. quinquenervia trees.
Stem Invasion and Colonization
Callus formation around the wounds on trees is considered a
nonspecific resistance mechanism in the host for walling off the
pathogen (Biggs et al. 1983, Griffin et al. 1984). In the present
study, callus formation among uninoculated and inoculated wounds
on stems of M. quinquenervia varied from completely closed wounds
(no canker among uninoculated stems) to pronounced callus ridges
(around cankers of most spring inoculations) to slight callus
ridges (around cankers of most fall inoculations) to no visible
callus (around cankers of some spring and fall inoculations).

125
Stems without callus were quickly girdled by the fungus. These
observations are in agreement with Griffin et al. (1984) who found
similar variation in canker formation in clones of Populus
tremuloides Michx. inoculated with isolates of Hypoxylon mammatum
(Wahl.) Miller. They concluded that the variation in callus
formation is under strong genetic control for specific clone-
isolate interactions. Evidence (see Chapters III and IV)
indicates that, in M. quinquenervia-B. ribis combinations, the
variation in callus formation is not only under genetic control,
but, to a certain extent, is also responsive to environmental
conditions and host phenology. In this study, wounds were
completely closed (no canker) in the absence of the fungus while
cankers of various dimensions were formed in the presence of B.
ribis. These observations reveal the role of B. ribis in creating
cankers on the inoculated stems.
The formation of 1) ligno-suberized periderms surrounding
callus ridges, 2) patches of tanninoid parenchymatous cells in
callus, and 3) intense lignification of the walls of tracheids and
vessels beneath the wound, in response to both wounding as well as
wounding plus B. ribis, provide nonspecific physical as well as
chemical barricades in the host that deter fungal growth into the
existing and newly formed tissues. Vance et al. (1980) reported
lignin and suberin provide chemical barriers as a general
mechanism of resistance to the infection of plants by pathogenic
organisms.
Despite being ligno-suberized, the wound periderm at the
ventral side of the callus ridges (facing the sapwood) seems to be
an area from which fungal growth can continue in to the inner

126
portion of the callus. Biggs et al. (1983) reported similar
findings for Populus hybrid (NE-388) infected by C. chrysosperma.
At the advanced stages of hyphal invasion, parenchymatous cells at
the callus margin of M. quinquenervia stems became necrotic and
eventually collapsed as the fungus continued to grow into the
newly formed cells of the callus. Similar findings have been
reported for newly formed cells in the callus of B. dothidea-
invaded peach trees (Biggs and Britton 1988).
As a morphological response to colonization of tissues by B.
ribis, vessels in and adjacent to the infected region of the xylem
frequently responded with formation of tyloses and accumulation of
granular substances. These structures were rare in uninfected
stem tissues. These observations are in agreement with Milholland
(1972) who found that formation of tyloses and protrusions in the
vessels in blueberry stems was initiated by B. dothidea-invasion
of the tissue. Also, the formation of tyloses and gums in vessels
in response to B. dothidea and B. ribis infection has been
reported for almond (English et al. 1975) and mango (Ramos et al.
(1991) trees, respectively.
Fungal hyphae were observed in the inter- and intracellular
spaces of the discolored cells of phloem tissues and pith
parenchyma. However, the detection of younger (unmelanized)
hyphae in these cells was difficult since these hyphae and the
cellular contents of discolored cells stained similarly with
Pianeze's IIIB stain. English et al. (1975) have reported similar
difficulty in detecting mycelia in the phellogen or phellem using
Pianeze,s IIIB stain.

127
Radial, tangential, and vertical growth of hyphae in sapwood
occurred through inter- and intracellular spaces of the xylem
rays, tracheids, and vessels. These observations are not in
agreement with Brown and Hendrix (1981) who reported that hyphae
in xylem rays were less frequent than in vessels of B. dothidea
infected xylem in apple stems. However, these observations do
agree with English et al. (1975) and Biggs and Britton (1988) who
found that hyphal invasion by B. dothidea occurred in all cell
types and in all directions (tangential, radial, and vertical)
from the point of infection. In apple stems, rapid downward and
slow lateral development of cankers has been attributed to a
higher frequency of hyphae in vessels than in xylem rays (Brown
and Hendrix 1981). According to Milholland (1970), the hyphal
frequency in the xylem rays was crucial to the development of
cankers in blueberry stems.
Invasion of adjacent tracheal cells or vessels of M.
quinquenervia by hyphae of B. ribis occurred through pits and
intertracheal spaces as described by English et al. (1975) in
xylem tissues of almond trees infected by B. dothidea. The lumens
of tracheids and vessels contained one or more hyphae, with
localized swellings often occluding the cellular spaces (Fig.
5.15). Similar phenomenon has been reported for almond (English
et al. 1975) and apple (Brown and Hendrix 1981) stems infected by
B. dothidea. Such occlusions resulting from fungal hyphae,
tyloses, and other protrusions impede or restrict the flow of
water through the vessels and tracheids (Milholland 1972) and
possibly contribute to decline and dieback of stems above the
canker.

128
In noncallused stems from spring and fall inoculations of M.
quinquenervia with B. ribis, fungal hyphae densely colonized the
cambial region. Hyphae were also present in the inter- and
intracellular spaces of the cortex but were difficult to detect
since they were often masked by the cellular remains of the
collapsed cells in these areas. Fungal growth in vascular tissues
of stems with noncallused wounds was comparable to that in the
stems with callused wounds. However, the overall hyphal
concentration in the infected tissues of the stems with
noncallused wound was more than that observed in the stems with
callused wound. As a result the noncallused stems were quickly
girdled by the fungus.
Adverse environmental conditions appeared to inhibit callus
formation and enhance the conditions needed for growth and
development of B. ribis hyphae in the stem tissues of M.
quinquenervia (see Chapter IV). These observations are in
agreement with Doster and Bostoc (1988), who established that low
temperature treatments in almond trees inhibited lignin and
suberin production in bark wounds.
Partially submerged pycnidial stroma developed on the bark
of declining M. quinquenervia stems. Similar production of
pycnidial stroma has been reported for B. dothidea on almond trees
(English et al. 1975) and B. ribis on walnut trees (Rumbos 1987) .
Pycnidia produced on the bark were important sources of inocula
for disease development among apple trees (Drake 1971) and
pistachio (Michailides 1991) vines.
The overall histopathological evidence from this study
revealed that after successful infection through wounds, isolates

129
of B. ribis are capable of perpetuating in the stem tissues of M.
quinquenervia. However, the fungal growth through callus tissues
and the wood formed prior to infection appeared to be slower in
the stems with callused wounds than in the stems with noncallused
wounds. Similar phenomenon has been reported for pathological
relationships between Innonotus obliquus (Fr.) Pilat and paper
birch (Betula papyrifera Marsh) (Blanchette 1982), and
Phytophthora cinnamomi Rands and red oak (Quercus rubra L.) (Robin
et al 1992).
Conclusions
Histopathology of inoculated foliage and cankers on M.
quinquenervia stems revealed B. ribis to be pathogenic. Leaf
infection occurred through stomata and required free water on the
leaf surface to cause leaf necrosis.
Callusing was more pronounced around stem wounds made in
spring than in fall. Callus ridges of B. ribis inoculated wounds
on stems were surrounded by 2-3 celled suberized layers. These
layers were observed surrounding callus ridges of uninoculated
wounds and healthy barks on the stems. Cells in the callus ridges
were rich in tanninoid substances and lignin. The tissues in the
callus ridges were invaded by B. ribis hyphae. Hyphal entry into
the callus ridges appeared to occur through the ventral surface,
i.e., from the interface between callus ridges and sapwood.
Tissue discoloration and hyphal invasion in lateral and vertical

130
directions of phloem, cambium, and xylem occurred beyond the
margins of callus ridges.
Hyphal concentration was intense in the cambium and phloem
regions in noncallused wounds and the fungus was able to girdle
stems relatively quickly. Both in callused and noncallused stems,
hyphae invaded cortex, cambium, xylem, and pith. Hyphal growth in
these tissues occurred through inter- and intracellular spaces.
Callus did not limit fungal colonization of cells in newly
formed tissues or the tissues formed prior to wound inoculation.
The process of stem girdling by fungal hyphae was prolonged by
callus formation.

Figs. 5.1-5,
.7. Morphology and histology of M. quinquenervia
leaves and stems.
Figure 5.1.
A cross-sectional view of a healthy leaf stained
with Pianeze's IIIB. Note stoma (arrow),
epidermis (ed), chlorenchyma cells (cm),
mesophyll cells (mp), and oil chamber (oc).
Bar=100 /¿m.
Figure 5.2.
A wounded but noninoculated stem showing callus
(arrow) closing wound.
Figure 5.3.
A stem wound-inoculated with B. ribis. Note that
canker surrounded by callus ridges (arrow).
Figure 5.4.
A Cross-sectional view of a wounded stem stained
with Pianeze's IIIB. Note callus (cal) filling
the wound, vascular cambium (arrow head) and
xylem (xy) at the time of wounding, and patches
of ligno-suberized cells (arrows) in callus.
Also, note redifferentiated vascular cambium
(double arrows) and newly formed xylem (xy) in
callus. Bar=500 jixm.
Figure 5.5.
A cross-section through unwounded stem stained
with phloroglucinol-HCl and Sudan IV. Note
phelloderm (pd) and layers of suberized cells
(arrow head), cortex (cor), phloem (ph), and
xylem (xy) . Bar=50 0 /xm.
Figure 5.6.
A Cross-sectional view through a portion of 4-wk-
old wound on stem stained with phloroglucinol-HCl
and Sudan IV. Note suberized cell layers (arrow
head) surrounding callus ridges (cal), extending
to the ligno-suberized periderm (arrow) enclosing
tanninoid cells (ta) and ligno-suberized
parenchyma cells (lp). Also, note intensely
lignified xylem (lx) beneath the wound. Bar=500
/¿m.
Figure 5.7.
A longitudinal section (stained with Pianeze's
IIIB) through the margin of callus (cal) on a B.
ribis-inoculated stem. Note a gap (g) created at
the interface of xylem (xy) and callus due to
progressive necrosis of cambium (arrow) and
xylem, groups of ligno-suberized cells (arrow
heads) enclosing tanninoid parenchymatous cells.
Also, note re-differentiated cambium (double
arrows), and xylem (xy) in the callus. Bar=500
/x m.

132

Figs. 5.8-5,
.13. Morphology and histology of M. quinquenervia
leaves and stems inoculated with B. ribis.
Figure 5.8.
A cross-section through a 8-wk-old, B. ribis
inoculated wound of a fresh stem stained with
phloroglucinol-HCl revealing xylem (xy), and
callus ridge (cal). Note suberized (arrow) and
ligno-suberized parenchyma cells (arrow heads) in
and around the callus ridge. Also, note gaps (g)
in callus due to cell disruption caused by fungal
invasion from the ventral surface (interface
between xylem and callus ridge) of callus ridge.
Bar=500 /xm.
Figure 5.9.
A longitudinal section through the canker margin
of a B. ribis-inoculated stem. Note tyloses
(arrow head) plugging a vessel in the xylem (xy)
near disrupted vascular cambium (arrow). Bar=250
/xm.
Figure 5.10.
A cross-section through a leaf (stained with
Pianeze's IIIB) inoculated with B. ribis. Note
fungal hyphae (arrow heads) penetrating epidermis
(ed) through stomata. Also, note hyphae in
chlorenchyma (cm), mesophyll (mp), and oil
chamber (oc) . Bar=100 /xm.
Figure 5.11.
A cross-section through a leaf (stained with
Pianeze's IIIB) inoculated with B. ribis
revealing a fungal hypha penetrating through
stomata (arrow head), and a pycnidium (pyc)
emerging from the epidermis (arrow) . Bar=100 /xm.
Figure 5.12.
A cross-section through a callus ridge of a stem
canker caused by B. ribis. Note cells surrounded
by hypha (arrows) resulting in deterioration of
cellular integrity. Bar=100 /xm.
Figure 5.13.
A cross-section through a callus ridge of a stem
canker caused by B. ribis revealing intra- and
intercellular hyphae (arrows) in deteriorating
areas as shown in Figure 5.12. Bar=50 /xm.

134

Figs. 5.14-5.17.
Morphology and histology of M. quinquenervia
leaves and stems inoculated with B. ribis.
Figure 5.14.
Figure 5.15.
Figure 5.16.
Figure 5.17.
A longitudinal section through xylem from the
canker margin of a B. ribis-inoculated stem.
Note hyphal growth (arrows) along ray parenchyma
(rp) and tracheidal (tr) cells. Bar=50 /xm.
A longitudinal section through xylem from the
canker margin of a B. ribis-inoculated stem.
Note hyphae (arrows) growing vertically along
inner wall and apparently clogging tracheidal
cells. Also, note hyphae passing radially
(marked by constrictions) (arrow heads) from one
cell to another and localized hyphal swellings
beneath the arrow on the upper right-hand corner
of the figure. Bar=20 /xm.
A longitudinal section through xylem from the
canker margin of a B. ribis-inoculated stem.
Note a hyphae branching (arrow) in a tracheidal
lumen. Bar=20 ¿xm.
A cross-section through noncallused stem canker
caused by B. ribis revealing a pycnidium (pyc) in
phelloderm, hyphal concentration in cambium and
phloem (arrows), and hyphae (arrow heads) in the
lumen and intercellular spaces of vessels and
tracheids. Bar=200 /xm.

136

CHAPTER VI
SUMMARY AND CONCLUSIONS
Melaleuca quinquenervia (Cav.) Blake, a tree species from
Australia belonging to the Myrtaceae, has become a noxious weed in
natural ecosystems of South Florida. Mechanical and chemical
control of this weed are expensive and inefficient. Chemical
control methods also raise environmental concerns in the wetland
ecosystem of South Florida. The option of biological control
using insects and fungal pathogens has been considered as a long¬
term solution. Surveys for and evaluation of M. quinquenervia-
specific insects are in progress under the responsibility of USDA
ARS, Fort Lauderdale, FL. The School of Forest Resources and
Conservation, University of Florida, is currently evaluating the
biocontrol potential of an endemic fungus.
In 1989-1990, a fungus was consistently isolated from the
margins of cankers on declining M. quinquenervia trees from the
Loxahatchee National Wildlife Refuge in South Florida. Six
isolates of this fungus derived from canker margins of these trees
(BR-1 through BR-6) and two isolates obtained from red mangrove
galls (BR-7 and BR-8) were identified as the Fusicoccum stage of
Botryosphaeria ribis Grossenb. & Dug., and were studied for their
biological characteristics and virulence toward M. quinquenervia
in greenhouse conditions. Stress factors (low moisture, low
137

138
temperature, and defoliation) were evaluated for their effects on
canker development. Histopathological investigations were
performed to understand the host-pathogen relationship at the
tissue and cellular levels.
Mycelial morphology and growth rate of isolates in the
artificial media (cornmeal agar [CMA], mycological agar [MA],
potato-dextrose agar [PDA], and starch agar [SA]) and sporulation
intensity and dimension of conidiomata varied by isolates and
growth media. Except BR-6, the majority of isolates sporulated
best on PDA, SA, and excised leaves incubated in a moist chamber.
Conidiophores lining stromatal locules had bulbous bases and were
holoblastic among younger conidiomata.
Fresh macroconidia from younger conidiomata were fusiform,
truncate at the base, hyaline, and aseptate. Some of the
macroconidia developed 1-3 septa and pale coloration when retained
in dry conidiomata or germinated in water under coverslips.
Regardless of the number of septa, 1-5 germ tubes were produced
from polar as well as lateral sides of conidia. Microconidia were
produced by most isolates but they did not germinate at any time
and conditions. Macroconidial germination within 4-8 h was
greater at 25-35°C than outside that range. Macroconidial
germination of tested isolates was least or none at 5°C and their
viability was completely lost at 45°C. The teleomorphic stage was
not observed in any growth media or paired combinations of
isolates.
Based on the variation in mycelial, conidiomatal, and
conidial characteristics of the isolate(s) in different growth
media and microenvironment as well as the information in

139
literature, the so-called teleomorph(s) (B. ribis and B.
dothidea), and their anamorph(s) (Fusicoccum and Dothiorella), are
highly variable. The variation in their morphological
characteristics may have root in their adaptive ability in a given
host or microenvironment. Hence, their taxonomic relationships
need to be clarified further. I suggest that these two telomorphs
are synonymous and so are their two anamorphs.
Eleven monoconidial cultures within each of six isolates
(originally obtained from canker margin of M. quinquenervia during
1989-1990) were evaluated for differences in pathogenicity using a
leaf bioassay. Differences in virulence among monoconidial
cultures were insignificant. Melaleuca quinquenervia clones
varied in susceptibility towards isolates of B. ribis.
One monoconidial culture selected from each of six isolates
(derived during 1989-1990) and two monoconidial cultures of B.
ribis obtained from canker galls of mangrove trees from South
Florida were evaluated for virulence on M. quinquenervia in
greenhouse and field conditions. Correlation between the position
of the point of inoculation on the stem of M. quinquenervia ramets
and the canker length was significant, i.e., the middle segments
of trees were more susceptible to canker development than the
root-collar regions and top segments. However, the correlation
between the diameter of the stem at the point of inoculation and
canker length was weak.
The disease-inciting ability of hyphal and macroconidial
inocula was not significantly different. Cankers of similar
nature on M. quinquenervia, from which B. ribis isolates were
obtained from Loxahatchee National Wildlife Refuge, were also

140
reproduced on ramets in greenhouse conditions. All of the eight
original isolates evaluated were able to incite cankers on M.
quinquenervia stems of all clones, but BR-2 and BR-4 were
relatively more virulent isolates. Some of M. quinquenervia
clones were more susceptible to B. ribis isolates than others.
Canker development on the mid-section of the main stem was more
rapid than the root-collar. Fungal growth on the stem from the
point of inoculation progressed more rapidly in tissues distal to
the point of inoculation than in those proximal to the point of
inoculation. Callusing of wounds, across clones of M.
quinquenervia ramets, inoculated in spring, was pronounced when
compared to fall inoculations. Establishment of B. ribis on M.
quinquenervia stems required wounding to the depth of the sapwood
or some kind of injury causing stress to the tree.
Low xylem water potential predisposed ramets to increased
level of canker formation and subsequent mortality. Alternate
cycles of 3-day exposure to 6°C favored increased levels of stem
canker development than the exposures to 6-day/wk to 6°C or 16-
h/wk to 0 (+ 1) °C or 30(+5)°C. Defoliation levels of 0 or 50
percent did not have an effect on canker expansion but 100 percent
defoliation resulted in mortality of ramets within 4 weeks
following inoculations with B. ribis isolates. All the stress
factors evaluated either reduced or prevented callusing of stem
wounds of M. quinquenervia inoculated with B. ribis.
Histopathology of wound-inoculated foliage and stem canker
tissues on M. quinquenervia revealed B. ribis to be capable of
invading and perpetuating in the inner tissues. Leaf infection
through stomata required free water on the leaf surface and

141
further expansion of leaf lesion stopped (at < 100% relative
humidity and ca 25°C) under greenhouse conditions.
The callusing ability of M. quinquenervia stems varied from
season to season. Stems wounded in spring callused more
extensively than did those wounded in the fall. Callus ridges of
B. ribis inoculated stems were surrounded by suberized layers as
in the bark of healthy stems. Tissues in the callus ridges were
rich in lignin and tanninoid substances. These tissues in the
callus ridges were invaded by the hyphae of B. ribis. Fungal
invasion of callus tissues appeared to occur from the ventral
surface of the callus ridge, i.e., from the interface between
callus ridges and sapwood. Tissue discoloration and hyphal
invasion in lateral and vertical direction of phloem, cambium, and
xylem occurred beyond the margins of callus ridges.
In noncallused wounds, hyphal wedges were intense in cambium
and phloem regions, and the fungus was able to girdle the stem
relatively quickly. Hyphae invaded tracheids, vessels, and xylem
rays of both callused and noncallused stems. Hyphal growth in
these tissues occurred through inter- and intra-cellular spaces.
Callusing of wounds appeared to facilitate relatively prolonged
survival of M. quinquenervia trees but did not have an effect on
the ability of fungus to invade callus and sapwood of the infected
stems.
Thus, the pathogenicity of B. ribis on M. quinquenervia has
been established, as well as the wound-dependent nature of this
pathogen. Following establishment in the host, rapid disease
development was favored by abiotic (low moisture and low
temperature) and biotic (defoliation) stresses. Therefore, it

142
appears that B. ribis is a potential candidate for biological
control of M. quinquenervia; however, its successful application
may require a combination with other tree-stressing agents such as
insects (defoliators or stem borers), or environmentally friendly
chemicals that can defoliate trees.
Further investigations of the tree-killing efficacy under
field conditions, host range, and environmental safety of B. ribis
should be conducted before making decision to use this fungus as a
mycoherbicide.

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BIOGRAPHICAL SKETCH
Min B. Rayachhetry was born on January 1, 1953, in Marek
Katahare Goan, Dhankuta, Nepal. He graduated from Barpather High
School (Assam, India) in June 1968. He earned his Intermediate
Science (1970) and Bachelor of Science degrees (1973) from
Dibrugarh University, Assam, India. During 1973-1978, he worked
in the Department of Plant and Forestry Research, His Majesty's
Government of Nepal, in the capacity of an assistant botanist and
acting assistant scientific officer.
In 1978, Mr. Rayachhetry was accepted for master's degree in
botany in the Department of Botany, at Tribhuvan University, where
he earned an MS in botany in 1981. In 1981, he was promoted to
assistant scientific officer and was deputed to the Institute of
Forestry, Nepal, to teach botany, tree physiology, and forest
ecology. In 1984, he was admitted to the School of Forest
Resources and Conservation, at the University of Florida where he
earned his MS in forest resources and conservation with an
specialization in forest pathology, in 1987.
In February 1987, Mr. Rayachhetry resumed his teaching
responsibility in the Institute of Forestry where he taught forest
protection, natural resources management practice, and guided
undergraduate research projects. In June 1990, he returned to the
School of Forest Resources and Conservation for the PhD program in
forest pathology. After concluding PhD program, he hopes to
further his career as a forest pathologist.
He married Geeta Basnet in 1983 and has two sons. During
the course of his PhD program he was accompanied by his wife
Geeta, and sons Amit and Ajamber.
152

I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
George M. Blakeslee, Chair
Associate Professor of Forest
Resources and Conservation
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Roger ¡y J Webb
Associate Professor of Forest
Resources and Conservation
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
]-/â– /?) L l J
Lai
/) '
V7 I j jj t A' limes W. Kimbrough /
Professor of Plant Pathology
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Rhaghavan Charudattan
Professor of Plant Pathology

I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosop'
xofessor of Microbiology and
Cell Science
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Project Leader of the Aquatic
Plant Management Laboratory
USDA-ARS, Fort Lauderdale
This dissertation was submitted to the Graduate Faculty of
the School of Forest Resources and Conservation in the College of
Agriculture and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of
Philosophy.
May 1995
and Conservation
Dean, Graduate School

LD
1780
199^
UNIVERSITY OF FLORIDA
3 1262 08554 5464




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