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

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Title:
Evaluating the pathogenicity of Botryosphaeria ribis Gross. & Duggar on Melaleuca quinquenervia (Cav.) Blake in South Florida
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xvii, 152 leaves : ill. ; 29 cm.
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Rayachhetry, Min Bahadur, 1953-
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Melaleuca quinquenervia -- Diseases and pests -- Florida   ( lcsh )
Canker (Plant disease) -- Florida   ( lcsh )
Forest Resources and Conservation thesis, Ph. D
Dissertations, Academic -- Forest Resources and Conservation -- UF
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Thesis:
Thesis (Ph. D.)--University of Florida, 1995.
Bibliography:
Includes bibliographical references (leaves 143-151).
Statement of Responsibility:
by Min Bahadur Rayachhetry.
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Typescript.
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Vita.

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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.
















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









Results ........................................


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









Pathogenicity of Botryosphaeria ribis
vs Pestalotia. ............................ 60
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 ................................. 69
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 ................................. 88
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









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















LIST OF TABLES


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









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















LIST OF FIGURES


Figure Pae

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. ............................................. 37

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 .............................................. 37

2.8. Pycnidial stroma of B. ribis erupting from dead bark
of M. quinquenervia showing solitary and botryose
types of pycnidia. ..................................... 39

2.9. Section through a M .quinquenervia leaf showing a
solitary pycnidium of B. ribis erupting from epidermis.
Evidence of ostiole and pycnidial wall. ................ 39

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









2.12. Cross-section through a pycnidium of B. ribis showing
pycnidial wall composed of textura angularis, and
conidiophores. .......................................... 39

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
development. ........................................... 39

2.14. Macro- and microconidia from freshly squashed 7-day-
old pycnidium of B. ribis showing the microconidia and
macroconidia. ........................................... 39

2.15. Macroconidia of B. ribis from PDA germinated in water.
Evidence of mixture of aseptate and septate conidia
bearing germ tubes. .................................... 39

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. ...................... 80

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









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









4.8. Effects of defoliation on canker development on stems
of M. quinquenervia ramets inoculated with B. ribis. ... 111

4.9. Effects of defoliation on mortality of M. quinquenervia
ramets inoculated with B. ribis. ....................... 111

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. A M. 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









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















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.









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 60C for 3-day/wk was more effective than

6C for 6-day/wk and 0 (l)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.
















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,









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 Alternaria 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

(Alternaria 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











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 letters' 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 Saccardo's 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.











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.











Mycelial Growth and Morphology



Four artificial growth media, potato-dextrose-agar (PDA)

(Difco), cornmeal-agar (CMA) (Difco), 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 CanTM,

Greenwich, CT) after inoculation, randomized with respect to

location on the laboratory bench and maintained at 300 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(l)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 Am

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.











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

450C. 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.











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).

Growth rates (mm/day) in

Isolate no. 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.OA,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.OB,b 11.1A,a 5.7A,b
BR-8 12.7A,a 9.8A,b 14.3A,a 6.OA,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












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) pm in

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-1 BR-2 BR-3 BR-4 BR-5 BR-6 BR-7 BR-8

Mycelial color:
Potato dextrose agar B B B LB B B B B
Corn meal agar LYB LYB W LYB LYB W W W
Mycological agar LB LB LB LB LB T B B
Starch agar YG YG LYG YG YG YG YG LYG

Aerial hyphae:
Potato dextrose agar AM AM AM AS AM AL AM AM
Corn meal agar CS AS AS BS CS CM -- BS
Mycological agar AS AS AS AS AS AS AS AS
Starch agar CS CS CS DS BS DS DS

Pycnidial stroma:
Potato dextrose agar A D D A B N A A
Corn meal agar A D B A D N A B
Mycological agar D D B A C D B B
Starch agar B D C A 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=1-25; 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.












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 Am (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.












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 Zone of confrontation
pairs Pigment Mycelial color Sclerotia Micro- Macro- Teleomor- Groups
conidia conidia ph stage

BR-1xBR-2 none brown (2 f) (1) (1) none 1
BR-1xBR-3 none brown none none (1) none 1
BR-1xBR-4 none brown none (4) (1,4) none 1
BR-1xBR-5 none brown none none (1,5) none 1
BR-1xBR-6 brown dark brown (6 f) none (1) none 2
BR-1xBR-7 none dark brown none none (1,7) none 2
BR-1xBR-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,











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 300C 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 botryosee) 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) Am. 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.












Table 2.4. Range and average (parenthesis) diameter
pycnidial stroma on 2- to 4-week old culture of some
isolates.


(mm) of the
B. ribis


Average diameter (mm) in
Isolate
no. PDA CMA SA MA

BR-4 0.1-1.0(0.5) 0.1-0.5(0.2) 0.3-1.2(0.5) ----
BR-5 0.1-1.3(0.6) 0.1-1.0(0.4) ---- 0.1-1.0(0.4)
BR-7 0.1-1.2(0.5) 0.1-0.5(0.2) 0.1-0.5(0.2) ----
BR-8 0.1-3.8(2.3) ---- ---- ----


Table 2.5. Mean and range of dimensions (length and
the number of nuclei in macroconidia.


width) and


Dimensions (Am)

Isolate 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 18.0ab 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.,
from R. mangle) from 21-day-old
For each isolate, N=100. Means
each other at P=0.05, according


BR-1 through BR-6 from M. quinquenervia, and BR-7 and BR-8
pycnidial stroma from inoculated M. quinquenervia-leaves.
with same letters) are not significantly different from
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) Am and 1.3-2.5 (average 1.6) Am, 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 300C. None of the microconidia, either

in water under a coverslip or on WA, germinated at 16 h after

incubation at 300C.

In WA, germination of macroconidia began as early as 1 h

after incubation at 30-350C (Figs. 2.17, 2.18). Within 4 h at 20-

350C germination had concluded and was over and below 90% in BR-1

and BR-7, respectively. However, after 24 h at 20-400C, 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 (20-

300C) for germination of macroconidia opposed to a narrow range

(250C) for BR-7 (isolated from mangrove trees) (Table 2.6). In

both isolates, spore germination was insignificant at 5 and 450C.

At 450C, 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 0.0 3.7 91.2 94.8 98.3 90.3 46.5 0.0
1.7de 0.0e 11.0d 72.8ab 77.2ab 82.7a 72.0b 43.0c 0.Oe

BR-7 0.0 0.0 2.5 55.2 89.8 82.0 64.3 25.5 0.0
0.Of 0.Of 10.0e 48.Oc 71.5a 65.0b 53.4c 30.3d 0.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 -200C, whereas for

isolate BR-7 viability was lost after the same period of storage.









27

Initiation of the germination of macroconidia stored at -200C

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 5-450C was described by the following

equation derived through logistic regression:

G(Isolate BR-1) = -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 Am, respectively. The

measurements given for asci and ascospores of B. dothidea are 65-

140 x 16-21 and 13-35 x 6-14 Am, 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 5-400C

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

25-350C, 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 (450C), 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 -200C, 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 25-350C than outside

that temperature range. In both isolates, macroconidial

germination was at the least or absent at 50C, and viability was

lost at 450C.









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. 2.1-2.7



Figure 2.1.


Figure 2.2.


Figure 2.3.


Figure 2.4.




Figure 2.5.



Figure 2.6.



Figure 2.7.


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 Am.

Blended mycelial mat grown in PDB under
continuous shaking conditions (100 rpm). Note
hyphae containing chains of chlamydospore-like
cells (arrows). Bar=50 pm.

Cross-section through a multilocular pycnidial
stroma produced on PDA. Note numerous locules
(lo). Bar=500 Am.

Enlarged pycnidia from a portion of multilocular
stroma. Note macroconidia (arrow) and an obscure
ostiole (os). Bar=100 Am.

Pycnidial stroma on the leaf inoculated and
maintained in a sterile moist chamber. Note
solitary (arrows) and botryose (arrow heads)
types of pycnidia.

























&


-1 i. I













Figs. 2.8-2.16.



Figure 2.8.



Figure 2.9.




Figure 2.10.




Figure 2.11.


Figure 2.12.




Figure 2.13.





Figure 2.14.





Figure 2.15.




Figure 2.16.


Pycnidia and macroconidia of B. ribis associated
with artificial growth media and M. quinquenervia
leaves and stems.

Pycnidial stroma erupting from dead bark. Note
solitary (arrow) and botryose (arrow heads) types
of pycnidia.

Section through leaf showing a solitary pycnidium
erupting from epidermis. Note leaf epidermis
(arrow), ostiole (os), and pycnidial wall (arrow
head). Bar=50 pm.

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 Am.

Pycnidial stroma on leaf oozing macroconidial
cirrhi(arrows) on drying at room temperature.

A portion from the cross-section through a
pycnidium showing pycnidial wall (pw) composed of
textura angularis, and conidiophores (arrows).
Bar=25 Am.

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 Am.

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 Am.

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 im.

Germinating aseptate (arrow) and septate (arrow
heads) macroconidia showing one or more germ
tubes. Bar=50 Am.
























I .


ai .


\


r


E
-~---


4L

S*:Aa.


X4.













100-

90-

o 80-
- -
M 70-
--
60
G-

n 50-

S40-
0
o 30-
CD
20-
O-
10-
0-


1 0 5 2 '0 25

Incubation period (hours)


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 differences

in percentage of germination by 4 h after incubation at the

temperature ranges shown in this figure.


S 5C -- 0C --15C
-- 20C 25C --30C
--W- 35C y- 40C ---5C


0 5 10 15 20 25

Incubation period (hours)


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.


-- 5C 10C 15C
-y- 20C 25C --.. 30C
- 3- 35C ...- 40C 45C


100.

90.

80.

70.

60.

50.

40.

30.

20-

10
0















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









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 mammatum (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











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,











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.











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 Al of the 1% mycelial

suspension was placed on each wound. Following inoculation, the

petri dishes were sealed with Parafilm and incubated at 250C 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(l)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 11x104 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 250C 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 250C 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 50C 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 250C 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 4.0x104 spores/ml), Gr.3 (5.7xl04 spores/ml),

and Gr.4 (11.6x104 spores/ml). The ramets in a fifth treatment

group (Gr.5) were not injured but were inoculated with









57

macroconidial inocula at 11.6x104 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 270C 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 250C and ca 10 h fluorescent light cycle), and

evaluated for B. ribis colony development.











Statistical Analyses



Data from laboratory, greenhouse, and field experiments were

analyzed using similar statistical procedures. Total canker

length proximall + 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.













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
Isolate 4
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).











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 19.3a 21.3a 53.3a 27.8a 36.0a 35.7a
Hyphae 19.7a 23.3a 17.3a 43.0a 44.0a 43.0a 44.0a

QNte: 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 letters) 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











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.

Analysis of variance

Sources df 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 Mean canker length Clone Mean canker
number Greenhouse Field number length

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 66.33ab 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 letters) 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











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











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. Percentage 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 4.0x104 spores/ml), Gr.3 (injured, inoculated with 5.7x104
spores/ml), Gr.4 (injured, inoculated with 11.6x104 spores/ml, and Gr.5 (ramets not injured
but inoculated with 11.6xl04 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

Discussion



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 (Prunus 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. 3.1-3.4.





Figure 3.1.





Figure 3.2.




Figure 3.3.



Figure 3.4.


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.







1! 2







i ii










4 J.
I'

:..,










80






300







250







S200



a)



W 150

U

0



100
a)






50








1 2 3 4 5 6 7 8 9

Treatments


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.




Figure 3.6.



Figure 3.7.




Figure 3.8.



Figure 3.9.


Stems of M. quinquenervia ramets with wound alone
and wound inoculated with B. ribis, and
maintained under greenhouse conditions for 8
weeks.

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.














6






















'I 0








I.,
41
V\































BR-1 BR-2 BR-3 BR-4 BR-5 BR-6 BR-7 BR-8
Tree clones

Figure 3.10. Pathogenic interaction between B. ribis and M.
quinquenervia. Each bar represents the mean of canker length
of 21 stems.


MQ-1 MQ-2 MQ-3 MQ-4 MQ-5 MQ-6 MQ-7

Tree clones

Figure 3.11. Clonal susceptibility of M. quinquenervia towards
B. ribis. Each bar represents the mean of canker length of
24 stems.




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