The distribution, etiology, and importance of red mangrove diseases in Florida

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Title:
The distribution, etiology, and importance of red mangrove diseases in Florida
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Red mangrove diseases in Florida
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x, 81 leaves : ill. ; 28 cm.
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English
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Olexa, Michael T ( Michael Theodore ), 1947-
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Subjects / Keywords:
Mangrove plants -- Diseases and pests -- Florida   ( lcsh )
Mangrove swamps -- Diseases and pests -- Florida   ( lcsh )
Mangrove forests -- Diseases and pests -- Florida   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 76-80).
Statement of Responsibility:
by Michael T. Olexa.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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aleph - 000355710
notis - ABZ3963
oclc - 02526759
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Full Text








THE DISTRIBUTION, ETIOLOGY, AND IMPORTANCE
OF RED MANGROVE DISEASES IN FLORIDA



By

MICHAEL T. OLEXA


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



UNIVERSITY OF FLORIDA


1976





























To Mrs. Scott Carter and Vincent J. Traynelis












ACKNOWLEDGMENTS


I wish to express my appreciation and thanks to Dr. T. E. Freeman,

Supervisory Committee Chairman, for his guidance, understanding, and

critical assistance throughout this study.

I also wish to acknowledge the assistance of the other members

of my committee, Drs. J. W. Kimbrough, H. H. Luke, D. J. Mitchell,

D. R. Minnick and D. A. Roberts. Special thanks are due L. H. Purdy

and J. F. Gerber for their assistance in this project.

Appreciation is extended to B. Yokel for making available the

facilities of the Rockery Bay Marine Laboratory. I also wish to

thank Dr. Samuel Snedaker for the use of his field and laboratory

facilities.

Special thanks are extended to Drs. S. A. Alferi, J. Benny,

and J. Bezerra. Additional appreciation is also extended to Mr.

Robin Lewis and the Audubon Society for use of their facilities.

Appreciation is also given to June Hill, Raymond Martyn,

Sheryl Morey, Donald Samuelson, and Kathy Roberts for their

cooperative work in parts of this dissertation.











TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ..................................................... iii

LIST OF TABLES ................................................... ... vi

LIST OF FIGURES .... ................................. ....... ......... vii

ABSTRACT........................................................ ix

INTRODUCTION............................... ...................................................... 1

LITERATURE REVIEW................................................... 2

PHASE I: FOLIAR DISEASE INVESTIGATION............................ 14
Materials and Methods ......................................... 14
Collection........ .......... .. ... ..... .... .. ... .. 14
Isolation .................... ......... .................. 14
Identification................ .......................... 15
Pathogenicity Tests........................................... 15
Cultural Studies........................................... 17

Results......................................................... 18

Discussion.................................................. 23

.PHASE II: GALL DISEASE INVESTIGATION............................... 30
Materials and Methods......................................... 30
Distribution........... ......... ..... .... ...... ....... 30
Collection.......................... ...... ........ 30
Isolation............................................. 30
Pathogenicity Study............... ...... ............. 31
Histological Study............ ... ........ ....... 33
Cultural Study.................................. 33
Auxin Study................. .......................... 35
Soil Isolation............................. .... ...... 36
Entomological Study............................. 36

Results.......................***...* .......................... 37
Isolation.............. ............ ....... ............. 39
Pathogenicity Study........................ ....,..... 39
Histological Study........................................ 43
Cultural Studies......................... ............ ..... 43
Auxin Study................* ..... ...... ......... ..... ... 45
Soil Isolation.......... ............................ ... 45
Entomological Study.............. .................... 50







Page
Discussion................... ..... ..................... ..... 50

PHASE III: MANGROVE DISEASE ASSESSMENT......................... 56
Materials and Methods...................................... 56
Survey Areas........................................... 56
Selection of Specific Survey Locations.................. 56
Point Method............................................ 60
Foliar Disease Survey.................................. 60
Fallen Leaf Study...................................... 61
Gall Disease Survey.................................. 61

Results...................................................... 62
Foliar Disease Severity Assessment ..................... 62
Foliar Disease Incidence Assessment........................ 67
Gall Disease Severity Assessment....................... 67
Gall Disease Incidence Assessment...................... 67
Fallen Leaf Study.................. .................. 68

Discussion ............. .............................. 68

LITERATURE CITED..................................................... 76

BIOGRAPHICAL SKETCH............................... ........... 81











LIST OF TABLES


Page
1. Laboratory results of foliar pathogenicity tests conducted
on wounded and nonwounded red mangrove foliage.....................25

2. Results of field inoculations of wounded and nonwounded
stems, prop roots, and leaf scar areas............................41

3. Results of auxin and culture filtrate applications made
in a mangrove riverine and overwash forest........................46

4. Mangrove disease incidence assessment and severity results
expressed as percentages of units and tissue areas infected........64

5. Percentage of fallen leaves found infected with Anthostomella,
Cercospora and Pestalotia at each survey location .................. 69











LIST OF FIGURES
Page
1. World distribution of mangroves................................... 5

2. World distribution of Rhizophora man le............................ 7

3. Rhizophora mangle: General morphology............................. 9

4. Mangrove riverine forest...........................................12

5. Mangrove overwash forest..................................... ..12

6. Anthostomella rhizomorphae, leaf symptoms caused by and
morphological characteristics of the fungus........................19

7. Cercospora rhizophorae, leaf symptoms caused by and
morphological characteristics of the fungus........................21

8. Leaf spots associated with Pestalotia disseminata, and the
spores of three fungi frequently isolated from these spots ..........24

9. Gall distribution in comparison to minimum temperature
patterns: 1937-67................................................. 38

10. Symptoms caused by Cylindrocarpon didymum, and morphological
characteristics of the funous.....................................40

11. Inoculations of red mangrove with Cylindrocarpon didymum............42

12. Cross section of a red mangrove gall infected with
Cylindrocarpon didymum............................................44

13. Results of auxin applications to wounded stems.....................48

14. Synthesis of IAA by cultures of Cylindrocarpon didymum incubated
in Czapek's and modified Czapek's media at 25C ....................49

15. Results of a mite dissemination study..............................51

16. Map of Henderson Creek riverine and overwash forest sample sites....57

17. Map of Faka Union riverine and overwash forests and Fakahatchee
and East River controls.................................. ....58

18. Map of Barron River riverine and overwash forests..................59






Page
19. Surface area formulae and diagram depicting gall disease severity
assessment technique............................................. 63

20. Graphs of foliage disease severities and incidences among sites
surveyed ................... ..... .... ..... ..... .................. 65

21. Graphs of gall disease severities and incidences among sites
surveyed.................................. ............ ... ........... 66


viii






Abstract of Dissertation Presented to the Graduate
Council of the University of Florida in Partial
Fulfillment of the Requirements for the
Degree of Doctor of Philosophy


THE DISTRIBUTION, ETIOLOGY, AND IMPORTANCE
OF RED MANGROVE DISEASES IN FLORIDA

By

Michael T. Olexa

August 1976

Chairman: Thomas Edward Freeman
Major Department: Plant Pathology

A plant pathological-environmental study was made along Florida's

coastal and inshore marine areas. The objectives of the research

were to classify the major mangrove diseases in Florida, assess their

importance, and relate their incidence and severity to man-induced

factors stressing mangrove populations. The study consisted of three

phases:

I. Survey and pathogenicity studies of fungi isolated from

red mangrove leaves.

II. An etiological study of a gall disease of red mangrove.

III. Disease incidence and severity surveys of mangrove areas in

the Florida Everglades subjected to channelization, urbaniza-

tion, and heavy metal pollution.

Two pathogenic fungi, Cercospora rhizoohorae Creager and Anthostomella

rhizomorphae (Kunze) Berl. & Vogl., and several saproohytic fungi were

consistently isolated from mangrove leaves. Evidence indicates that

they may be important factors in leaf fall.

Cylindrocarpon didymum (Hartig) Wollenw. was consistently associated

with and isolated from gall tissue in red mangrove. Pathogenicity was







determined on the basis of field inoculations. Initial symptoms appeared

as roughened, callused tissue six months after inoculation. Cylindrocarpon

didymum could be reisolated readily from callused tissue. A perfect stage

of the fungus was not observed on living or non-living host tissue, and

all attempts to induce one in culture failed. Climatic data coupled

with field observations provided evidence that temperature was the limiting

factor in the northward distribution of the gall disease.

Significant differences in disease incidences and severities

existed in several areas subjected to varied man-induced factors

stressing mangrove populations.












INTRODUCTION


Mangroves are one of Florida's major natural resources. Unfortunate-

ly, mangrove swamps have in the past been consigned to the category of

wastelands (44). However, numerous values and benefits are obtained from

mangrove swamps. By acting as "buffer" zones, mangrove swamps greatly

reduce coastal hurricane damage (21, 44). Some researchers have suggested

that they are also active "land builders" (20). Mangroves are also ex-

cellent breeding, nesting, and overwintering grounds for numerous species

of birds. In Everglades National Park, there are approximately 99 species

of aquatic and wading birds that inhabit the mangrove swamps (44).

The economic importance of red mangrove in South Florida results

from its production of large quantities of detrital material upon

which is based an estuarine food chain. This fact has direct sport

and commercial fishing value (28, 44). Red mangrove-dominated

estuaries serve as nursery grounds for many marine species of economic

importance (44). Pink shrimp, black mullet, gray snapper, red drum,

pompano and blue crab utilize these areas as nursery and feeding

grounds.

In view of the widespread distribution and importance of mangroves

throughout the world and more specifically Florida, they have been

subjected to very little phytopathological study. The loss of mangroves

through plant disease could have a devastating effect on numerous

economically important marine organisms progressively up the food chain.

This alone would justify intensified plant pathological research in







these valuable nursing areas. Such research would also result in important

contributions in pest management. Pathological knowledge obtained about

this natural ecosystem could aid as a guide to disease control in managed

forests. In an effort to establish a foundational basis for future re-

search in the mangrove monoculture, a phytopathological study consisting

of three phases was initiated. Phase one involved a survey and patho-

genicity study of fungi isolated from red mangrove foliage. The second

phase entailed a study of a gall disease of red mangrove. The final phase

of research was conducted in an effort to determine if plant disease

could be utilized as an indicator of environmental stress. In this

final phase, disease surveys were made in mangrove forests of south

Florida subjected to varied parameters of man-induced environmental stress.


LITERATURE REVIEW

Mangroves

Historically, evidence of man's interest in mangroves spans many

centuries. The earliest reference to these plants is contained in the

chronicle of Nearchus in 325 B.C. Nearchus, commander of the fleet of

Alexander the Great, described the habitat of mangroves while'on a

voyage from the delta of the Indus River to Susa, Persia (12). Theophrastus

(305 B.C.), Plutarch (A.D. 70), Pliny (A.D. 77) and Arrian (A.D. 136), also

described these unique trees that had the ability to grow in salt water (12).

The term "mangrove" (West Indian derivation) is based on two different

concepts (21, 23, 70). First it refers to an ecological group of halo-

phytic evergreen species which belong to several unrelated families,

but possess similarities in their physiological characteristics and

structural adaptations to similar habitat preferences (21, 23, 54).

Secondly, it implies a complex of plant communities fringing along the







sheltered tropical shores. Schimper (58) defined "mangrove" to include

the formation below the high tide mark. Consequently, he and many others

have used the term "tidal forest" as a synonym of the mangrove vegetation.

True mangroves, however, may form only a part of the whole tidal zone;

they may occur from below the level of the highest tides, or on coasts

where there are no tides at all (21, 23, 54, 61). Other mangrove synonyms

include: mangrove swamp, tidal forest, and tropical mangrove woodland (69).

Mangroves belong to approximately twelve genera in eight different families.

The species in these families include:

Avicenniaceae Myrsinaceae

Avicennia sp. Aegiceras sp.

Chenopodiaceae Plumbaginaceae

Suaeda monoica Forsk. Aegialitis sp.

Combretaceae Rhizophoraceae

Laguncularia sp. Rhizophora sp.

Meliaceae Bruguiera sp.

Conocarpus sp. Ceriops sp.

Xylocarpus sp. Sonneratiaceae

Rubiaceae Sonneratia sp.

Scyphiphora sp. Acanthaceae

Acanthus sp.
In all, some 55 species have been reported (69).

Geographically, mangroves are one of the most widespread of all

coastal plant species, occupying 75% of the world's coastal areas

between latitudes 250N and 25S (23). World-wide distribution falls

into two zones: the first being the Indo Pacific zone which covers the

coasts of East Africa, the Red Sea, India, Southeast Asia, southern

Japan, the Philippines, Australia, New Zealand and the southeastern







Pacific archipelago as far east as Sanma and the second being the West

Africa-Americas zone including the Atlantic coasts of Africa and the

Americas, the Pacific coast of tropical America and the Galapagos Islands.

Distribution is generally restricted to the tropics, but mangroves have

been reported outside of tropical areas (23, 69). In the northern hemi-

sphere, mangroves are found on all coasts north to latitudes varying

between 240 and 310N. Spread has been observed as far as 3120'N in

southern Japan, to 300N in Florida and Bermuda, and to 24038'N in

California (23). In the southern hemisphere, their distribution on

the east and west coasts differs somewhat. They extend to the tropic

of Capricorn on the east coast of South America, to 300S on the east

coast of Africa, and to 290S on the coast of New South Wales. On South

America's west coast they can be found as far as 40S, and extend to 90S

on Africa's west coast. Temperature is apparently the principal

environmental factor that limits the geographical distribution of man-

grove species. They are considered tropical and subtropical trees.

The minimum air temperature at which they will survive is approximately

-4C (21, 44). Figure 1 depicts the general geographical distribution

of mangroves.

In relation to human value, mangrove is one of the most important

sources of timber, fuel posts, poles, railroad ties, and tannin in the

tropics. Mangroves also contain resins used as plywood adhesives; and

the bark, leaf shoots, and roots contain utilizable dyes. Some cultures

use mangrove hypocotyls as a food source (31). Due to their many and

varied uses, silviculture of mangroves has been practiced for decades

in southeastern Asia (23, 69).

Florida's coastline is predominantly composed of mangroves. Those
















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mangroves growing along Florida's coastline include red mangrove

(Rhizophora mangle L.), white mangrove (Laguncularia racemosa L.), and

black mangrove (Avicennia germinans L.) (21, 57). Of the three, black

mangrove is the most cold-resistant and survives throughout the Gulf

area of Florida from Pensacola (300N) to the Everglades and Keys (250N)

(57). It is found on the Atlantic coast as far north as St. Augustine

(50, 57). Less cold hardy, the red mangrove extends on the Gulf mainland

to Levy County and on the Atlantic to Volusia County (30, 57). Least

hardy of all, the white mangrove is sparsely distributed northward to

Brevard County, on the Atlantic and Hernando County on the Gulf.

Exceptions to these distributions are of the off-shore islands of Levy

County, the Cedar Keys. All three species are found on these keys

with Avicennia being the dominant species (57). South Florida's Ten

Thousand Islands region contains the most extensive mangrove forests

of the western hemisphere.

Red Mangrove

Red mangrove is the dominant species throughout south Florida (61).

The genus Rhizophora was first adequately described by Theophrastus in

305 B.C. (12). Linnaeus, in one of his earlier writings (Systema Nat.

1736), presented a vague conception of the limits of the genus Rhizophora.

He later recognized seven species of Rhizophora, which he iVcluded in a

separate genus. The seven species were R. conjugata, R. aymnorhiza, R.

candel, R. mangle, R. cylindra, R. corniculata and R. caseolares. All

species except R. mangle are native of the orient. Rhizophora mangle is

found in the West Africa-Americas zone (69). World distribution of R.

mangle is shown in Figure 2.

The seedling of red mangrove is morphologically distinctive and

























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easily recognized. The detached seedling, which serves as the propagule

for dispersal consists of the pencil-like hypocotyl, usually up to 20 cm

long, and a short plumule about 0.5 cm long (Fig. 3A). The plumule's

visible portion consists of a pair of cotyledonary stipules which enclose

the first pair of leaves (27). After establishment, the first years of

growth involve additions to the foliage without any major change in habit

or increase in height (57). Prop roots appear on the red mangrove by the

third or fourth year of growth (Fig. 3B) (57). The leaf is stiff, rather

leathery, with a shiny bright green adaxial surface and a light yellow

abaxial surface. It contains an acutely pointed tip, giving the broad

based leaf the appearance of an arrowhead (Fig. 3C) (57). The paired

flowers of the red mangrove are usually overlooked because they face

inward the plant. Flowering occurs in the spring, summer and fall.

Occasional flowers and partially developed seedlings are present during

winter and early spring (14, 57). Each flower has four sepals, four petals,

eight stamens and an inferior ovary with four pendulous, apically inserted

ovules (Fig. 3D). Little is known about their pollination biology (27).

Following root development, shoot development begins. Roots always

develop at the distal end of the hypocotyl. Primary roots are narrow

(1-3 mm diameter), taper little and branch soon after emergence. Sub-

terranean roots contrast in appearance and texture with aerial roots.

Aerial roots arise from the trunk and main branches of trees more than

3 years old, and serve as anchoring and presumably aerating organs (27).

They are of no significance in regeneration. The most characteristic

feature of R. mangle is the familiar prop roots so noticeable along

shorelines.
Mangrove Forest Types

In south Florida, mangroves can be found in one of five mangrove




























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forest types which are distinguished by the periodicity of inundation by

terrestrial runoff and tides (46, 61). These forest types are the basin,

dwarf, fringe, overwash and riverine forests (62). Two of the five forest

types were utilized throughout this study. These two mangrove types as

described by Snedaker and Pool (61) are:

Riverine forest: "The riverine type designates the tall floodplain

forests occurring along river and creek drainages. Although they are

usually separated from the drainage way by a shallow berm, they are

flushed by the daily tides. This forest type is often fronted by a

fringe forest occupying the slope of the drainage way. During the

summer wet season, water levels rise and salinity drops due to upland

terrestrial runoff. The riverine type consists of relatively straight-

trunked trees numerically dominated by R. mangle (with noticeably few,

short prop roots) and varying mixtures of A. germinans and L. racemosa.

Low surface water flow velocities preclude scouring and redistribution

of ground litter" (61). Figure 4 depicts a riverine forest.

Overwash forest: "The smaller low islands and finger-like projections

of larger land masses in shallow bays and estuaries are characterized by

the R. mangle dominated "overwash forest" types. Their positions and

alignments obstruct tidal flow and as a result this forest is overwashed

at high tide. The incoming tidal velocities are high enough to carry

with them any loose organic debris; the debris is dropped in the inner

bays as the velocity decreases and is not returned to the overwash

forest. The dense prop root system appears to be limited to the space

defined by the ground surface and wet season (April and September)

mean high water. The spatially limited prop root system plus the absence

of foliage below the canopy level give this forest type an appearance

of a symmetrical and regular architecture when viewed from within" (61).







Figure 5 depicts an overwash forest.

Red Mangrove Diseases

Relatively few diseases have been reported on R. mangle. The Index

of Plant Diseases (38) lists Anthostomella rhizomorphae (Kunze) Berl &

Vogl. as a pathogen of red mangrove. Vizioli (68) described another

member of the genus Anthostomella rhizophorae, as a parasitic fungus

collected in Puerto Rico on R. mangle. Thompson (66) reported a

Fusarium sp. on red mangrove in 1927. A primary foliage pathogen of red

mangrove in Florida, Cercospora rhizophorae Creager, was first collected

by plant inspectors, R. W. Swanson and R. T. McMillan, Jr. (47) and

later fully described by Creager (17). A Mycosphaerella sp. has also

been reported on red mangrove foliage (39). Olexa and Freeman (53)

identified Cylindrocarpon didymum (Hartig) Wollenw. as the apparent

causal agent of a gall disease of red mangrove in Florida. Batista

and others (8) collected and characterized two previously unreported

fungal pathogens of R. mangle in Brazil. The two fungi were Phomopsis

rhizophorae Batista et Maia, and Physalospora rhizophorae Batista et

Maia. Kohlmeyer (43) compiled a list of 17 species of marine fungi and

19 species of terrestrial fungi associated with Rhizophora hosts.

The following phytopathological study was designed to determine the

distribution, etiology, and importance of red mangrove diseases in Florida.

The study consisted of three phases. The first phase involved a survey and

pathogenicity study of fungi isolated from red mangrove foliage. The

second phase entailed a study of a gall disease of red mangrove. The

primary objective of this phase was to determine through inoculation studies

the causal agent of the disease. Mangrove diseases identified in the initial

phases of the study were assessed for incidence and severity in the third













Figure 4. Mangrove riverine forest.


Figure 5. Mangrove overwash forest.


Redrawn from Snedaker and Pool, 1973.
Bureau of Sport Fisheries and Wildlife Report.
Contract No. 14-16-008-605:C1-C13.





13


phase. The disease assessments were made in virgin and man-disrupted

mangrove forests in an effort to determine if significant disease

differences existed between these areas.












PHASE I: FOLIAR DISEASE INVESTIGATION


Materials and Methods

Collection

Disease surveys were conducted from June 1974 through June 1975 to

isolate and identify the primary foliar pathogens of red mangrove along

Florida's Atlantic and Gulf coasts. Surveys were conducted from Cedar

Key to Naples on the Gulf coast, from Daytona to Ft. Lauderdale on the

Atlantic coast, and in several locations in the Everglades and the Ten

Thousand Islands. Diseased leaves were photographed, placed in plastic

bags and transported to the laboratory for pathogen isolation, identi-

fication, and subsequent pathogenicity studies.

Isolation

Following microscopic examination, segments of 1 to 2 mm2 were

*removed from chlorotic margins of all leaf lesions. Segments were

then surface sterilized from 1 to 3 minutes in 0.5 to 1.0% sodium

hypochlorite (10-20% Clorox) and placed in petri plates containing

potato dextrose agar (PDA) or water agar. Pure cultures were then

obtained by transferring hyphal tips and spores to PDA. Fungal cultures

were maintained on PDA at room temperature in cotton plugged test tubes

and transferred at one month intervals. Isolation of several foliar

fungi was accomplished through the utilization of the Alferi technique

(2). This consisted of surface sterilizing the lesion area with 90%

ethanol and drying the area with a flammed scapel held in close proximity

to the lesion surface. Fungal mycelia and fruiting structures were then







individually removed with a moistened needle, and transferred to petri

plates containing PDA or water agar. Attempts were made to induce

sporulation in several cultures by transferring hyphal tips from the

advancing edges of fungal cultures to a host tissue medium and sub-

jecting the fungi to near ultraviolet (uv) radiation (310-400 mu). The

host medium was prepared by first triturating 70 g of red mangrove leaves

petioless removed) in 500 ml of distilled water in a Waring blender,

filtering the pulpy mass through cheesecloth and then through a Buchner

funnel. The liquid filtrate was retained and added to one liter of

distilled water which contained 20 g of agar. Hyphal tips were also

transferred to water agar and V8 juice agar and placed under uv light.

Identification

Foliar lesions were examined macro- and microscopically for the

presence of pathogenic organisms. Lesions were examined with a stereo-

scopic microscope and then stained with lacto-phenol cotton blue and

examined with a light microscope. When no pathogenic organisms were

observed, leaves were placed in a moist chamber at room temperature and

examined periodically for several days. Various phases of the fungal

isolates were photographed.

Pathogenicity Tests

Red mangrove seedlings for pathogenicity studies were maintained in

the greenhouse in polyethylene-lined 20 cm clay pots, filled to within

6 cm of the lip with an autoclaved mixture consisting of 50% sand and 50%

peat. Seedlings were watered weekly with tap water and maintained in

shaded areas of the greenhouse. Plants chosen for inoculation were

approximately 30 cm in height and had a minimum of six healthy non-

chlorotic leaves. Several inoculation techniques were employed in an





16

effort to fulfill the rules of pathogenicity. These inoculation techniques

were:

(1) Two wounded and nonwounded leaves of each plant were inoculated

with a mycelial and spore suspension. Mycelium and spores were scrapped

from the surface of petri plate cultures flooded with sterile distilled

water and applied with a brush to the proximal half of the leaf's abaxial

or adaxial surface. Distilled water was applied to two control plants.

Leaves were wounded by scratching the surface with a straight pin. Inocu-

lated and control leaves were on the same seedling and each was individually

labeled.

(2) Two wounded and nonwounded leaves of each plant were inoculated

with a 5 mm2 mycelial-impreganted block of agar cut from the advancing

edge of cultures maintained on PDA. Agar blocks were placed on control

leaves. Wounding was accomplished by attaching agar blocks to the leaf's

surface with a straight pin. Both leaf surfaces were inoculated.

(3) The final inoculation technique consisted of fastening diseased

plant parts from field samples onto the leaf surfaces of two wounded and

nonwounded leaves. Diseased plant parts were attached with masking tape.

Inoculum was attached to the leaves of two controls. As with the second

technique, wounding consisted of attaching inoculum to the leaves with

a straight pin.

Following inoculation, all plants were covered with plastic bags for

three days. All inoculations were repeated twice. In an effort to verify

inoculum viability, inoculum for each technique and isolate was placed on

concave slides and observed hourly for spore germination and mycelial

growth. In all pathogenicity tests, disease progression was observed

twice a week.







Cultural Studies

Studies were conducted to determine optimum growth temperatures of

foliar isolates. Fungal disks of one cm in diameter were cut from the

advancing mycelium of fresh cultures of each of the fungal isolates, and

placed in the center of petri plates containing 20 ml of PDA. Four plates

of each isolate were then incubated at temperatures of 10, 15, 20, 25, 30,

35C and colony diameter was measured every 48 hours. Several foliar

isolates were also evaluated for growth at various temperatures on PD-yeast

broth. The liquid medium consisted of 5.0 g/l of Difco yeast extract and

24 g/l of Difco PD broth. Following preparation, 30 ml of the medium were

placed in a number of 250 ml Erlenmeyer flasks. All flasks were divided

into lots of 21, and each group inoculated with fungal disks (one cm in

diameter) of a specific foliar isolate. Each group was then incubated

at temperatures chosen from the previous plate study. Within each group,

three flasks were removed every three days for a 21 day period. On

removal, each fungal mat was washed with distilled water in a Buchner

funnel on preweighed filter paper and dried in an oven for 68 hours at

750C. The preweighed paper weight was then subtracted from the combina-

tion weight to determine the weight of the fungal mat.

Additional experiments were conducted to determine if the fungal

isolates produced ethylene which could be a factor in leaf drop. Five

PDA cultures of each foliar isolate were placed under a bell jar con-

taining a four week old tomato seedling. Two other bell jars, each

containing individual tomato plants served as controls. Oranges infected

with Penicillium digitatum Link. were placed under one control, while

nothing was placed under the third. A 50 ml beaker of a saturated

solution of potassium hydroxide was placed under each of the bell jars.







Jars were sealed at their bases and plants observed for signs of epinasty

and leaf fall.

Results

The fungi most consistently isolated from foliar lesions included

Anthostomella rhizomorphae (Kunze) Berl. & Vogl. and Cercospora rhizophorae

Creager. A fungus tentatively identified as Pestalotia disseminata Theum.

was consistently associated with characteristic leafspots. An Alternaria

sp. and a Cladosporium sp. were also frequently isolated from necrotic

areas associated with A. rhizomorphae, C. rhizophorae, and P. disseminate.

Anthostomelia rhizomorphae was observed and isolated from leaf

specimens collected along both coasts. Its northern-most distribution

was found at Tampa Bay on the west coast, and Mosquito Bay on the east

coast. The fungus seemed to appear in greater abundance in south Florida,

and was predominantly found during the winter and early spring. In the

field, it appeared identical to A. rhizomorphae as described by Stevens

(64). Its cultural characteristics were similar to those described by

Martin (48).

Leafspots caused by this fungus were pale to yellowish and greatly

swollen under field conditions. They presented a general appearance of

insect galls (Figure 6A & B). Spots were 0.5 to 1 cm in diameter, or

when marginal were more or less extended.

Microscopic observation of diseased areas in leaves revealed thin

walled perithecia approximately 700 u in diameter and colorless. As the

perithecia matured, a clypeus formed which occupied the space between the

perithecia and the lower epidermis (Fig. 6C). Paraphyses were numerous,

threadlike, and hyaline (Fig. 6D & E). Asci were oblong, thin walled, and

8 spored (Fig. 60 & E). Spores were oblong 24-40 X 14-17 u, one celled

and brown at maturity (Fig. 6D & E).





































Figure 6. Leaf symptoms caused by Anthostomella rhizomorDhae, and
morphological characteristics of the fungus. (A) Leafspots on
adaxial surface of leaf; (B) Leafspots on abaxial surface of leaf;
(C) Perithecium with clypeus (CL): (D-E) Paraphyses (PA), Ascus (AS),
Ascospores (SP) (Figures 6 C & E from Vizioli, Some Pyrenomycetes of
Bermuda. Mycologia 15:107-119).


P







Perithecia of A. rhizomorphae placed on PDA produced colonies 25 mm

in diameter after seven days growth. Colonies were white and felt-like

with compact hyphae. Growth was partly zonated with one or two zones

developing in each colony. Spore formation was not observed and all

attempts to induce sporulation in culture was unsuccessful. Optimum

growth in liquid media occurred at 250C. The fungus was not found to

produce ethylene.

Cercospora rhizophorae was isolated from mangrove leaves in all

coastal areas surveyed. The fungus appeared in greater abundance in

the early spring and summer.

The fungus was observed fruiting on the adaxial leaf surface on

relatively large leaf spots (Fig. 7A & B). Fruiting structures occurred

in mass, discernable as ashen to dark olivaceous brown mats (Fig. 7B).

Symptoms appeared as chlorotic spots or large lesions reaching a diameter

of 5 to 25 mm. Lesions were usually bordered with reddish brown or in

some cases yellowish halos.

When viewed microscopically, conidiophores were noted to arise from

erumpent stromata in dense compact fascisles on the adaxial leaf surface

(Fig. 7C). The number of conidiophores in each fascicle was variable,

but 50 or more were not uncommon. Individual conidiophores were pale to

olivaceous at the proximal end and hyaline at the tips. Conidiophores

contained one to several septa, branched occasionally and possessed

rounded or irregular tips (Fig. 7D). They measured 30-50 X 3-5 u.

Conidia were pale to olivaceous in coloration and contained from 1 to 11

septa (Fig. 7E & F). Conidial size varied from 25-120 X 3-5.6 u.

Tufts of spores transferred to PDA produced ashened colored colonies

10 mm in diameter after seven days growth, and optimum growth in liquid














































Figure 7. Leaf symptoms caused by Cercospora rhizomorphae, and morpho-
logical characteristics of the fungus. (A) Leafspot on adaxial surface
of leaf; (B) Leafspot on abaxial surface of leaf, with fruiting
structures (FS); (C) C. rhizophorae. Stroma and Conidiophores;

(D) Conidiophores; (E-F) Conidia.







media was observed at 30C. Sporulation was not observed in culture, but

occurred when leaves were inoculated with fungal mycelium. Ethylene was

produced by the fungus.

A Pestalotia sp. tentatively identified as P. disseminata Thuem. was

isolated throughout the study from mangrove leaves in all survey areas.

Symptoms were expressed as leafspots containing dark centers surrounded

by chlorotic margins (Fig. 8A & B). The fungus was often found fruiting

on the abaxial leaf surface.

Microscopic observations revealed fruiting structures which ruptured

the epidermis or displaced it completely exposing a black conidial mass,

75-150 u, and sometimes up to 250 u in diameter. Conidia were 5-celled,

elliptic and tapering at the base. Conidia measured 19-25 X 6-8 u and

contained 2 to 4 hyaline apical appendages (Fig. 8C).

Spores transferred to PDA produced white, felt-like colonies

interspersed with numerous dark masses of spores. Colonies measured

80 mm after seven days of growth. Optimum growth occurred at 250C.

Ethylene was not produced by this fungus.

An Alternaria sp. was frequently isolated from the necrotic

areas associated with the previously described fungi.

Microscopic examination of pure cultures revealed short conidiophores

bearing branched chains of conidia. Conidia containing both cross and

longitudinal septa were clavate and measured 25.5 46.5 X 7.5 13.5 u

(Fig. 8D).

Spore formation was observed in culture. Spores placed on PDA

produced light gray colonies 71 mm in diameter after seven days growth.

The optimum growth temperature was found to be 250C. Ethylene was not

produced by the fungus.







Cladosporium sp. was also frequently isolated from necrotic areas

associated with species Anthostomella, Cercospora and Pestalotia.

Microscopic observations revealed tall dark conidiophores clustered

with ovoid conidia. Conidia measured 7.5 13.5 X 4.5 6.0 u (Fig. 8E).

Spore formation was observed in culture. Spores placed on PDA

produced dark gray colonies 49 mm in diameter after seven days growth.

The optimum growth temperature was found to be 20C. This fungus

produced ethylene.

Pathogenicity Tests

Cercospora rhizophorae and A. rhizomorphae were pathogenic on

mangrove leaves (Table 1).

Four weeks after inoculation with a mycelial-spore suspension of C.

rhizophorae, chlorotic spots were observed. After 48 days, the spots

were approximately 10 mm in diameter and resembled symptoms on leaves in

the field. Symptoms were reproduced and the fungus recovered with each

inoculation. Wounded mangrove leaves were not affected by the fungus.

Controls remained disease free.

Six days after inoculation with A. rhizomorphae, irregular lesions

developed on wounded tissues which extended approximately 5 mm beyond

the margins of the agar blocks. Lesions were dark brown in coloration

and contained chlorotic borders. A fungus with cultural characteristics

resembling A. rhizomorphae was recovered from the margins of the lesions.

Controls remained disease free. Symptoms were similar to those found

in the field, except that perithecial formation did not occur.
Discussion

With the exception of short morphological descriptions and incidental

notes regarding foliar pathogens, there have not been any extensive studies










ix.i


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... I .. ". i,, -


I BE
3BE


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Figure 8. Leafspots associated with Pestalotia disseminata, and the
spores of three fungi frequently isolated from these spots. (A-B)
Leaf symptoms associated with Pestalotia disseminata; (C) Spores of
Pestalotia disseminate; (0) Spores of an Alternaria sp.; (E) Spores
of a Cladosporium sp.












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26

on the foliar diseases of red mangrove in Florida. Therefore, it was the

primary purpose of this research to locate, characterize, and assess the

importance of the major foliar pathogens of red mangrove in Florida.

Information gathered during the study was further utilized in the final

phase of the research project.

Three species of fungi were consistently isolated from mangrove foliage

during the course of this study. These included: Anthostomella rhizomorphae,

Cercospora rhizophorae, and Pestalotia disseminata.

Identification of A. rhizomorphae was based on the works of Martin

(48), Stevens (64), and Vizioli (68). Although the three descriptions

of A. rhizomorphae were somewhat diversified, Stevens described the fungus

consistently isolated in the field during these studies. The identity of

A. rhizomorphae will no doubt change with future publications because the

genus Anthostomella is presently undergoing revisions (10). The fungus is

listed as parasitic on red mangrove (38) and has been reported in Florida

(39), Bermuda (68) and Puerto Rico (64).

Greenhouse studies contrasted markedly with field observations and

showed this fungus to be a weak pathogen. These inoculation results can

best be explained by the adverse climatic conditions experienced in the

greenhouse. During the inoculation trials, daily greenhouse temperatures

often fluctuated between 3 and 470C. Inoculation were unsuccessful in

all but the agar block technique. Positive results were noted only

when agar blocks were applied to wounded tissues. These results were

viewed with discretion, for an accumulation of metabolites of either

a pathogenic or nonpathogenic fungus could well have produced similar

necrotic effects. The fact that field symptoms were not reproduced

casts some doubt as to the validity of these inoculation trials. However,







field observations and the publications of several investigators (48, 64,

68) leave little doubt as to the pathogenicity of A. rhizomorphae on mangrove.

Despite the widespread distribution of A. rhizomorphae throughout the

state and its abundance in the southern portions of Florida, it does not

appear to present a serious threat to the state's red mangrove population.

Cercospora rhizophorae was first described by Creager (17) and has

been reported as the only species of Cercospora known to occur on the

genus Rhizophora. The fungus is listed as a pathogen of red mangrove (39)

and has not been reported in areas other than Florida.

In 1964, McMillan (47) noted the high degree of pathogenicity of

C. rhizophorae toward R. mangle. Inoculation trials in the field confirmed

these pathogenicity studies. Positive results were obtained only when

mycelial and spore inoculum was applied to nonwounded tissue. Disease

was not noted on injured leaf tissue. Interpretation of these results

are at best speculative, but in response to injury the host may have

produced biochemical inhibitors which checked the advance of the fungus.

Adverse laboratory conditions experienced during the inoculation trials

are thought responsible for the inconsistent results of the pathogenicity

studies.

Inoculated leaves fell prematurely. This observation coupled with

the ability of C. rhizophorae to produce ethylene suggests that the fungus

may well be an important factor in leaf fall. Although the fungus was

shown to be a severe pathogen of red mangrove, its distribution and

abundance throughout the state suggests that it is presently of little

threat to Florida's mangrove forests.

Pestalotia disseminata was identified with the use of Guba's (33) key.

The fungus was first reported on leaves of red mangrove at Little River,







Florida in 1918 (33). A fungus identical to P. disseminata was found on a

Eucalptus sp. in Coembra, Portugal, but was not reported as being patho-

genic. Guba (33) considers these two fungi as synonyms. During the

study, P. disseminata was consistently associated with and isolated from

characteristic leafspots. Although inoculation tests failed to prove

pathogenicity, there is considerable debate in the literature in regard

to the pathogenicity of the genus Pestalotia. Some have found the genus

to be saprophytic, while others studying its pathogenicity on the same

and closely related hosts have found it to be a parasite of some economic

importance (15, 16, 36, 71). Guba (33) states that very little, if any,

importance can be attached to published reports crediting species of

Pestalotia as being plant parasites. There are, however, a number of

reports confirming the ability of Pestalotia spp. to at least act as

secondary pathogens. Cortez (16) found that P. palmarum Cooke was

distinctly a wound parasite capable of causing the gray blight of

coconut in the Philippines. White (71) determined that P. palmarum

wasn't able to infect healthy tissues, but could follow other pathogens

or gain entrance through injured areas. Pestalotia disseminata isolated

from mangrove leaves was more often than not isolated from healthy non-

wounded leaves. Howarth and Chippendale (36) found that Pestalotia

macrotricha Kleb. grew in wounded tissue but that it was unable to

extend such infection into healthy tissue. Again, extensive field

observations of P. disseminata dispute these observations. It can thus

be seen that there are numerous divergent opinions regarding the

pathological relationship of this genus to its host plants.

In view of its prevalent association with characteristic leafspots,

P. disseminate was noted as one of the possible pathogens of R. mangle








in this study. It was found to be the most widespread of the three fungi.

Despite its statewide distribution and abundance, it was not considered

to pose a serious disease threat to R. mangle.

In summation, three fungi were consistently isolated from mangrove

foliage. It appeared that P. disseminata was found throughout the year

with A. rhizomorphae occurring most frequently during winter and spring,

followed by an abundance of C. rhizophorae in the summer and fall. These

fungi appear to have a definite yearly succession pattern, and although

pathogenic, may well serve a beneficial role in Florida's mangrove

ecology.












PHASE II: GALL DISEASE INVESTIGATION


Materials and Methods

Cylindrocarpon didymum (Hartig) Wollenw. was previously reported as

the apparent causal agent of a gall disease of red mangrove (52, 53).

PART 1

Distribution

Surveys were made along the Gulf and Atlantic coasts from June through

August of 1974 to determine the northern-most distribution of the gall

disease. Climatic data (13, 41, 49) coupled with field observations were

used to determine the northern-most distribution of the gall disease.

During the survey, gall samples were distributed with various state and

local agencies who were requested to report occurrences of the disease.

Collection

Galls were collected along both coasts during the survey. Galls

were placed in plastic bags, marked as to location and transported to

the lab.

Isolation

Three isolation techniques were utilized:

(1) Galls were divided into two groups. One group was surface steri-

lized for two minutes in 0.5 to 1.0% sodium hypochlorite (10-20% Clorox)

and thoroughly rinsed with sterile distilled water. The second group was

not surface sterilized. Both groups as well as noninfected stems were

placed in a mist chamber and observed twice each week for the presence of

fungi. Fungi observed on the galls were transferred to either PDA or Difco







corn meal agar (CMA).

(2) Gall fragments were placed in moist chambers and observed for

several days (67). Fungi present on the galls were transferred to either

PDA or CMA media. Stems without symptoms were also placed in the mist

chamber.

(3) Gall sections (2 mm2) were sterilized by immersion in 0.5 to 1.0%

sodium hypochlorite for two minutes and placed in petri dishes containing

either PDA or CMA. Four sections were placed in each plate.

Pathogenicity Study

Several inoculation techniques were utilized in attempts to demonstrate

pathogenicity. Inoculations were made on mangroves in Wood River and Faka

Union forests. Inoculation techniques were as follows:

(1) Mycelial and spore suspension. Inoculum was prepared by flooding
petri plates containing eight-day-old cultures of C. didymum with sterile

distilled water and scrapping the cultures with a spatula. Inoculum was

then placed in 500 ml Erlenmeyer flasks, chilled on ice and transported

to the field. Wounded and nonwounded stems, leaf scar areas and develop-

ing prop roots were inoculated. Wounding consisted of scratching the outer

bark with a scalpel. Inoculum was applied with a brush, and distilled water

was applied to the controls. Inoculated and noninoculated areas were then

wrapped with moistened cheesecloth and masking tape. Ten trees were

inoculated in each forest type. Wounded and nonwounded stem and prop root

controls were on the same tree.

(2) Disk inoculation. Inoculations were made by removing bark cores

with a number 12 cork borer, inserting a disk impregnated with mycelia and

spores and reinserting the core. Controls were bored, cores removed and

then replaced. Inoculations and controls were wrapped with masking tape

and labeled. Each forest type contained five inoculated trees and five






controls. Both stems and prop roots were inoculated.

(3) Gall tissue inoculation. Galls were collected and divided in

the field. Gall segments were inserted into wounds and also placed

against the outerbark of nonwounded stems and prop roots in each of five

trees. Symptomless stems were placed into wounds and against the outer-

bark of five controls. Inoculations were duplicated at each of the

sites. Inoculations and controls were on the same tree.

Greenhouse inoculations. Mangrove seedlings were collected in

the field, soaked in tapwater for 14 days and potted. Two weeks later

44 plants, all approximately 30 cm in height and possessing at least four

firm nonchlorotic leaves, were selected for the experiment. Seedlings

were divided into wounded and nonwounded groups and subjected to two

inoculation techniques:

In the first inoculation technique, a mycelial and spore suspension

was applied to wounded and nonwounded hypocotyls. Hypocotyls were wounded

with a scalpel. Eight wounded and nonwounded seedlings were inoculated,

and distilled water was applied to three wounded and nonwounded controls.

Inoculated and noninoculated areas were wrapped with masking tape and

labeled.

In the second technique, wounded and nonwounded hypocotyls of 16

seedlings were inoculated with triturated gall tissue. Triturated

symptomless stem tissue was applied to three wounded and three nonwounded

controls. Inoculated and noninoculated areas were then wrapped with

masking tape and labeled.

In another inoculation experiment, eight petri plates containing

seven day old cultures of C. didymum were flooded with sterile distilled

water and scrapped with a spatula. The inoculum was poured into a 500

ml beaker. Seven seedlings, which had been maintained for six months

under greenhouse conditions, were carefully uprooted and their root







systems washed. Root systems of four seedlings were dipped in the inoculum

and three controls were dipped in sterile distilled water. All seedlings

were replanted and foliage was examined weekly. Six months after inocula-

tion, seedlings were uprooted and examined.

PART 2

Histological Study

Galls were first studied under the dissecting microscope and

observations recorded. Additional observations were made under light

and electron microscopes. Prior to histological study, galls were

placed in moist chambers and the emerging fungus positively identified

as C. didymum.

Galls were sectioned and fragments were prepared for examination.

Fragments were placed in distilled water for several hours, transferred

to 1.0% sodium hypochlorite (10-20% Clorox) for 15 minutes, agitated,

and rinsed with distilled water. They were then stained for ten minutes

in lacto-phenol cotton blue, rinsed in distilled water, and mounted in

a 20% gum arabic water solution. Sections were then cut to a thickness

of 16 microns with a cryostat, examined, and photographed.

Gall tissues were prepared for electron microscopic examination by

a technique modified by Samuelson (56).

Cultural Studies

The optimum growth study was carried out on a liquid medium as

previously described.

Perfect Stage Study

Attempts were made to locate and induce a Nectria stage of C. didymum

for further pathogenicity studies. Numerous decaying galls were collected

seasonally from dead and fallen trees in the Everglades and Ten Thousand







Islands and examined for specimens of Nectria.

A fungal isolate identified only as "Verticilliurm4-Acremonium" was

obtained from the University of Miami's Institute of Marine and

Atmospheric Sciences. The fungus was reported to have been isolated as

a Nectria from the hypocotyl of a red mangrove seedling. Attempts were

made to induce a Nectria stage through inoculations, and determine

through anastomosis if C. didymum and the isolate were both imperfect

stages of the same Nectria.

Eight mangrove seedlings were selected for inoculation. Four

hypocotyls were painted with a mycelial and spore suspension of C. didymum,

and four with suspensions of the Miami isolate. Each group contained two

seedlings wounded with sandpaper. Inoculated areas were approximately

6 mm2. Distilled water was applied to wounded and nonw ounded controls.

Inoculated seedlings and controls were covered with plastic bags for

three days following inoculation.

Blocks of PDA and CMA were cut from petri plates For the anastomosis

experiment. Blocks of PDA and CMA were then individually placed on steri-

lized glass slides. Bits of mycelium of Cylindrocarpon and the Miami

isolate were removed from the advancing mycelial edges of pure cultures and

placed in close proximity of the agar. Cylindrocarpon fragments placed in

close proximity of each other, and fragments of the Miam.i isolate oriented

in the same manner, were utilized as controls. Coverslips were then

placed on the agar blocks, and slides were placed in a moist chamber.

Microscopic observations for anastomosis were made every six hours.

In another experiment designed to induce a Nectria stage, 1-angrov?

wood chips were placed in culture tubes containing either PDA or -i-rgrov'

media which was prepared as previously described. Two tubes of mangrove





35

media and four of PDA were then inoculated with C. didymum. Tubes were

observed weekly for the presence of a Nectria stage.

Auxin Study

Field applications. Indole-3-acetic acid (IAA), giberillic acid

(GA3) and C. didymum culture filtrates were applied to stems and develop-

ing prop roots under field conditions. Indole-3-acetic acid solutions

were prepared in concentrations of 100, 500, 1000, 2000, and 4000 parts

per million (ppm). Gibberillic acid was prepared at the concentration

of 300 ppm. The culture filtrate was obtained by growing C. didymum in

PD-yeast broth media for seven days. Mats were removed, crushed with a

mortar and pestel, and fungal pulp and media were filtered through a

Buchner funnel. Filtrate and solutions were placed under refrigeration

and transported to the field. Cotton pads saturated with each of the

above solutions were applied to wounded and nonwounded stems and prop

roots. Distilled water was applied to wounded and nonwounded controls.

Duplicate applications were made at the Wood River riverine and Faka Union

overwash forests.

Greenhouse applications. The same concentrations of indole-3-acetic

acid and GA3, as well as the C. didymum filtrate were applied to a five

year old mangrove tree. Each application was made once on wounded and

nonwounded stems. Distilled water was applied to the controls. Controls

and inoculations were wrapped with masking tape and labeled.

Colorimetric estimation of IAA. Additional experiments were con-

ducted to determine if C. didymum was capable of synthesizing IAA. Agar

plugs cut with a #6 cork borer and impregnated with C. didymum were individually

placed in several 500-ml Erlenmeyer flasks. Each flask contained 30 ml of a

modified Czapek's solution (3.0 g NaNO3, 1.0 g KH2PO4, 0.5 g MgS04'7H20, 0.5 g






KCL, 5 mg FeC13 and sucrose, 30.0 g per liter) or a tryptophan medium

prepared by omission of sodium nitrate from Czapek's solution and

substitution of 1.0 g L-tryptophan per liter as the sole source of

nitrogen. Additional flasks of non-inoculated Czapek's solution and

tryptophan medium were used as controls. At 12-hour intervals, 1 ml

of filtrate from each inoculated flask and control was removed,

added to 2 ml of FeCl3-HC104 reagent, and assayed for IAA colorimetrically

(29).

Soil Isolation

A study was made to determine if C. didymum was present in mangrove

soils. Henderson Creek riverine and overwash forests were sampled at

locations of previously constructed gridlines. Twelve samples were

collected at each site with a number ten cork borer. Core samples were

taken to a depth of 70 mm. Core samples were subdivided into three depth

regions; 0-20 mm (A), 20-40 mm (B), and 40-70 mm (C). Samples were assayed

for C. didymum by sprinkling four allotments of soil from each region onto

a PDA-Tergitol Streptomycin (TS) medium (63). Controls consisted of pure

cultures of C. didymum mixed with mangrove soils and sprinkled onto the

PDA-TS medium. Plates were placed under continuous fluorescent lighting

and examined daily. Colony number and presence of C. didymum were recorded.

PART 3

Entomological Study

In the initial stages of the gall disease investigation, an entomologist

was taken to several gall infested mangrove forests. Diseased trees were

examined and galls were collected for further entomological examination.

Throughout the study galls were collected seasonally from diseased trees,

dissected in the field and lab and examined for insect instars. Galls






were also seasonally collected from debris and living hosts in the

Faka Union overwash forest and the Wood River and Faka Union riverine

forests. These were placed in Burlese funnels for insect and mite

extraction.

In another aspect of the entomological study, several hours were

spent during the winter, spring, summer and fall observing insect

activity in the Faka Union overwash forest. Observations were also

made in the Wood River riverine forest during the winter and early spring.

Dissemination study. Experiments were made to determine if fungal

feeling mites could serve as agents of dissemination. Mites extracted

with Burlese funnels were rinsed several times in distilled water and

placed (with a camel's hair brush) into petri plates containing pure

cultures of C. didymum. Four days later the petri plates were placed

on a slide warmer and mites driven out of the cultures. The mites were

collected and transferred to petri plates containing acidified PDA.

Several mites from each sample were surface sterilized with 80% ethanol

and externally and internally examined. They were then crushed, added

to sterile distilled water and streaked onto acidified PDA. Plates were

examined twice each week for the presence of C. didymum.

Results

Mosquito Lagoon and Tampa Bay were found to be the northern most

areas of gall occurrence on the east and west coasts, respectively

(Fig. 9-1). Both localities were in low elevations (9-1). Distributional

limits of the disease coincided with winter minimum temperature patterns of

0, -20 and -40C (Fig. 9-2, 3 & 4). Tree heights and trunk diameters in

these localities were noticeably smaller than those mangroves observed

in areas south of the geographical limits.








Cold
Locations


So Locations Locations
















o 0 M


Figure 9. Gall distribution in comparison to minimum temperature

patterns: 1937-67. 9(1) Northern distributional limits of a gall

disease of Rhizophora mangle; 9(2) Total number of hours 00C and

lower; 9(3) Total number of hours -20C and lower; 9(4) Total

number of hours -4C and lower.







Isolation

Cylindrocarpon didymum was consistently isolated from galls collected

along the states inshore marine and coastal areas. The fungus was observed

emerging from both surface and nonsurface sterilized galls (Fig. 10). In

the third isolation technique, a Pestalotia sp. was occasionally noted

among the Cylindrocarpon colonies. The fungus was not observed emerging

from nongalled stems and prop roots.

Pathogenicity Study

The results of field inoculations are summarized in Table 2. Gall

development was observed only on nonwounded stems inoculated with the

mycelial and spore suspension. Seven percent of the inoculations were

positive. Positive results were observed six months after inoculation

as roughened callused areas (Fig. 11A). In addition, the periderm of

several nonwounded inoculated areas of stems and prop roots was cracked

and lenticels were conspicuously prominent both in color and size (Fig.

11B & C). Cylindrocarpon didymum was reisolated from the lenticels

and callused areas. Galls did not develop on wounded stems, prop roots,

and leaf scar areas in the overwash and riverine forests. Controls

remained disease free. Disk inoculations were found contaminated with

numerous Hymenopteran and Isopteran insects. Accurate readings could

not be taken and the experiment was abandoned. Segmented gall inocu-

lations were unsuccessful.

No infections resulted from any of the greenhouse inoculations.

Examination of all inoculation sites revealed no evidence of excessive

callus tissue formation. Mangrove root systems dipped in inoculum

appeared normal when compared to the controls (Fig. 11D). The fungus

was not reisolated from any of the inoculation sites. Unlike Hart's




40
















A B






















from gall tissue maintained in a mist chamber for 12 days; (C-E)
Mycelia, conidia, and conidiophores of Cylindrocarpon didymum.
Figure D bottom from Barnett & Hunter, Fungi Imperfecti, Burgess
Pub. Co. 241 p.























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Figure 11. Inoculations of red mangrove with Cylindrocarpon didymum.

(A) Positive result manifested as roughened callused area; (B-C)

Enlarged lenticels and cracked bark frequently observed on nonwounded

inoculated stems; (D) Negative results of mangrove root dip

inoculation.







(34) experiment with Cylindrocarpon radicicola Wr., callus tissue forma-

tion was not observed on mangrove roots.

PART 2

Histological Study

Observations made with the stereoscopic microscope revealed numerous

cankered areas (Fig. 12B). Mean length of nondiseased lenticels was found

to be 1 mm while those within the cankered areas measured from 3 to 8 mm.

The pathogen appeared to affect the continuous phellogen layer of the

periderm, and the vascular cambium. In some instances the bark was ob-

served to separate from the xylem (Fig. 12A). Disruption of the

secondary phloem was also noted. Observations of infected lenticels

with the compound microscope disclosed fungal mycelium in close proximity

of the phellogen layer (Fig. 12E). This was not observed in noninfected

lenticels (Fig. 12D & F). Attempts to observe fungal hyphae in the

bark and vascular cambium with a light microscope were unsuccessful.

Fungal hyphae were, however, observed (with the electron microscope) in

the phloem in close proximity of the vascular cambium (Fig. 12G & H).

Cultural Studies

Optimum growth of C. didymum occurred at 250C. All attempts to

locate or initiate a Nectria stage proved unsuccessful. Nectria was

not observed to develop on hypocotyls inoculated with either C. didymum

or the Miami isolate. When viewed microscopically in culture, distinct

lines of demarcation were observed between the Miami isolate and

Cylindrocarpon colonies. Nectria development did not occur in culture

tubes containing both isolates and mangrove woodchips immersed in either

PDA or mangrove media. Although Nectria was not seen on the numerous

galls examined in the field, a fungus identified as C. didymum was















































Figure 12. Cross section of a red mangrove gall infected with Cylindrocarpon
didymum. (A) Bark separation from the xylem; (B) Enlarged lenticel; (C)
General anatomy of a lenticel; (D and F) Noninfected lenticel; (E) Lenticel
infected with Cylindrocarpon didymum; (G and H) Electron micrograph of
fungal tissues in close proximity of the vascular cambium. Fungal tissues
are marked by arrows.






periodically isolated from decaying galls.

Auxin Study

The results of field auxin applications are summarized in Table 3.

In the wounded stems which received IAA, GA3 and culture filtrate appli-

cations, callus formation and enlargement were observed six months after

initiation of the experiment (Fig. 13A & B). In addition, lenticels of

several wounded and nonwounded stems and prop roots became conspicuously

prominent both in color and size. Several of the application sites

somewhat resembled positive field inoculations. Such results were

noted in both the overwash and riverine forests. In the overwash forest

11% of the stems were significantly affected by the applications as

compared to 6% in the riverine forest. Culture filtrate applications

produced significant callus formations in 6% of the overwash and 25%

of the riverine treatments. Contrary to the findings of Ghouse and Yunus

(26) on Melia azedarch L. little size contrast was noted among the various

applications on mangrove (Fig. 13A & B).

Contrast in size, callus formation, and lenticel activity was not

noted among the greenhouse applications and controls. The fungus

synthesized IAA in vitro (Fig. 14).

Soil Isolation

Cylindrocarpon didymum was sporatically isolated from soils collected

in the Henderson Creek riverine forest. The fungus was isolated from

samples collected 3, 6, and 18 meters in the forest interior, and from all

three designated soil layers. Cylindrocarpon didymum was not isolated

from samples collected in the Henderson Creek overwash forest. Of the

1492 colonies examined on the TS medium, four were identified as C. didymum.

Cylindrocarpon grew profusely in the control plates.


















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Figure 13. Results of auxin applications to wounded stems. A (1-4)
IAA concentrations of 4000 ppm, 500 ppm, culture filtrate and control.
Arrows mark enlarged lenticels. B (1-6) IAA concentrations of 4000
ppm, 2000 ppm, 1000 ppm, 500 ppm, culture filtrate and control.





49



AUXIN(IAA) SYNTHESIS
BY
CYLINDROCARPON DIDYMUM
IN VITRO


12 24 36 48 60 72 84 96 108 120 132
INCUBATION TIME (HOURS)
-.--- IAA SYNTHESIS W/TRYPTOPHAN
I ** *e IAA SYNTHESIS WO/TRYPTOPHAN










Figure 14. Synthesis of IAA by cultures of Cylindrocarpon didymum

incubated in Czapek's and modified Czapek's media at 250C.








PART 3

Entomological Study

With the exception of several Collembola species, mites representative

of several families were consistently isolated from the galls. Of primary

concern were mites of the mycophagous family Tarsonemidae. Under con-

trolled laboratory conditions members of this family were found capable

of disseminating the fungus. Cylindrocarpon didymum spores ingested

by mites and later removed from their digestive tracts were found viable

when placed on acidified PDA (Fig. 15). Attempts to isolate the fungus

from mites collected in the field were unsuccessful. During seasonal

field observations, pronounced insect feeding activity and oviposition

were not observed on mangrove stems and prop roots.

Discussion

In 1973, Olexa and Freeman (53) described a gall disease of red

mangrove (Rhizophora mangle L.). The causal agent was not demonstrated

conclusively, but Cylindrocarpon didymum had been consistently isolated

from diseased tissue. It was, therefore, the primary objective of this

study to demonstrate Koch's postulates with this fungus and to assimilate

information on the etiology of the disease.

Several investigators (4, 9, 11, 37, 45, 59, 73, 75) have postulated

that the interactions between host, climatic and site factors may exert a

strong influence on intensity of canker diseases caused by facultative

parasites. This was shown for this disease by the results of field

inoculation trials. Field inoculations closely resembled symptoms

observed under natural conditions. Pathogenicity was not demonstrated

in the laboratory.

Field inoculations were carried out on wounded and nonwounded tissue.












































Figure 15. Results of a mite dissemination study. (A) Petri plate of

PDA containing mites transferred from pure cultures of Cylindrocarpon
didymum; (B) Microscopic examination of a mite removed from the plate

in Fig. A; (C) Internal examination; (D) C. didymum growth on acidi-

fied PDA following removal of the fungus from the mites digestive tract;

(E) Area C enlarged, arrows designate a spore and a chlamydospore.







Infection was obtained on the nonwounded but not wounded tissue. Failure

to achieve positive results with field wound inoculations may have been

due to the presence of chemical inhibitors produced by the host in

response to wounding. Hubbes (37) reported the presence of two such

substances in wounded aspen bark that inhibited the growth of Hypoxylon

pruinatum (Klot.) Cke. One was identified as pyrocatechol, a phenolic

compound also reported in the bark of other tree species. Occurrence of

inhibitory chemicals in mangrove bark is presently unknown. However,

mangrove bark has for decades been utilized as a commercial source of

polyphenol tannins. These compounds may have inhibited the growth of

C. didymum in wounded tissues. Hubbes (37) also reported that the

concentration of these inhibitors was higher during the fall and winter

months than during the spring and summer growing season. In the present

study, mangroves were inoculated during the winter season during the

period of decreased physiological activity (74). Thus, seasonal fluctu-

ations in the concentrations of these inhibitor chemicals could explain

the unsuccessful results encountered with wound inoculations.

Failure to achieve infection of mangrove seedlings with C. didymum

under greenhouse conditions could also be related to the presence of

inhibitory compounds. French and Oshima (25) observed that green bark

tissue markedly inhibited the germination of Hypoxylon ascosoores and

suggested the presence of inhibitory substances. This could also account

for the absence of galls on mangrove seedlings and leaf scar areas

during field inoculation trials.

Since growth hormones such as IAA and GA3 have been implicated in

numerous gall diseases (32, 65), an attempt was made to stimulate galls

using different concentrations of IAA and GA3 on both wounded and







nonwounded tissues. In addition, since C. didymum is capable of

synthesizing IAA in culture (Fig. 14), a culture filtrate was also

used. Excessive callus formation occurred under field conditions,

but not in greenhouse trials. Formations developed on wounded but not

nonwounded field applications. These findings contrasted with those

observed during the pathogenicity studies. Since lenticels of non-

wounded sites were the only areas affected by the auxin applications,

it appears that the rhytidome prevented contact of the solutions with

the cork and vascular cambiums. This hypothesis was further sub-

stantiated by the results observed from wound applications. Here,

when the cambial layers were exposed to the direct contact of the

growth regulators and culture filtrate, excessive callus formation

occurred. All wound application sites were of uniform size, and there

was no difference in the magnitude of callus formation. It is,

therefore, suggested that the unusually high concentrations of IAA

had an inhibitory effect on the stimulation of cambial activity.

The number of callus formations in the riverine forests as compared

to overwash forests varied little.

Callus formation did not occur with greenhouse applications.

Application trials in the greenhouse were effected by temperatures

which often went above 28C. Arzee and others (5) noted that phellogen

activity was decreased when growth regulators were applied to Robinia

pseudacacia L. maintained under temperatures which exceeded 280C.

The ability of C. didymum to synthesize IAA in culture (Fig. 14)

was of significance for it added additional evidence in support of

this fungus as the causal agent of the gall disease. In his list of

canker and gall inducing organisms capable of synthesizing IAA, Gruen








(33) listed several Nectria spp. This is of significance since some

Cylindrocarpon spp. have been shown to have Nectria perfect stages.

A perfect stage for Cylindrocarpon didymum has not been reported.

The complete etiology of the gall disease is still speculative.

From inoculation experiments and auxin applications it was determined

that the galls developed slowly and at least six months were required

for the appearance of symptoms. The optimum climatic conditions for

infection to occur were not determined. The greatest susceptibility

of the red mangrove to C. didymum may occur in the fall and winter and

could be related to the low turgor pressure normally occurring at this

time. Bier (11) found bark turgor level was directly correlated with

host resistance to canker pathogens and that high turgidities indicated

favorable resistance to diseases. He also noted that infection with

facultative parasites occurred only when relative bark turgidities

were below 80%. Through microscopic observations, it was apparent

that infection generally occurred through the lenticels. Isolation

of C. didymum from enlarged lenticels in the field and during inocula-

tion trials further suggested these sites as areas of pathogen entry.

French and Oshima (25) established that phellem stimulated the

germination of ascospores of HyVoxylon. This could account for the

frequent isolation of C. didymum near the phellem and phellogen areas

of the lenticels. Microscopic observation of the fungus in enlarged

lenticels and near the vascular cambium of the gall tissues added

additional evidence establishing C. didymum as the causal agent of

the disease.

Results of laboratory studies provided evidence implicating mites

as possible agents of dessimination of the disease. Although mites







collected in the field were not observed to contain C. didymum, they

were shown capable of dissemination of C. didymum in laboratory

experiments. Cylindrocarpon didymum and mycophagus mites were also

isolated from similar habitats and a majority of the galls observed

in the field were located in areas favorable for mite habitation (18).

Temperature was undoubtedly one of the most important factors

influencing the occurrence and severity of the disease. The northern

most distribution of the gall disease occurred in areas subjected to

low winter minimum temperature patterns.

In severe cases, the disease appeared to structurally weaken the

host and predispose it to wind damage. This situation, however, was not

commonly observed in the field. In view of the widespread distribution

of the disease throughout the state, and observations of minimal host

mortality in areas with severely diseased trees, C. didymum appears as

an unlikely threat to Florida's red mangroves. It is concluded, however,

that C. didymum is the causal agent of the gall disease of red mangrove

in Florida.












PHASE III: MANGROVE DISEASE ASSESSMENT


Materials and Methods

Disease incidence and severity surveys were made in several areas

of the Everglades and Ten Thousand Islands. Areas surveyed were sub-

jected to varied parameters of man-induced environmental stress. The

primary objective of the study was to determine if significant differences

in disease incidence and severity existed between surveyed areas. Within

each area, two sites, a riverine and overwash forest were sampled. Prior

to the initiation of field work, statisticians of the Institute of Food

and Agricultural Sciences were consulted as to the validity of survey

techniques. A preliminary survey was also made, and statisticians

were again consulted in order to determine valid sample sizes.

Survey Areas

Two riverine forests, the Fakahatchee and East River, and an

overwash forest common to both were selected as controls. Experi-

mental areas of study and parameters of stress included:

(1) Henderson Creek riverine and overwash forests-------urbanized.

(2) Faka Union riverine and overwash forests---------channelized.

(3) Barron River riverine and overwash forests--heavy metal pollution.

In all, five areas and nine sites within the areas were chosen for

disease assessment (Figs. 16, 17 & 18).

Selection of Specific Survey Locations

Selection of survey locations at various sites was made in the

following manner:


















































4-



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Figure 18.


Barron River riverine and overwash forests.
sites are marked by arrows.


Miami


Survey


1;4 c N-


os ee Wal~
,voro







(1) As each site was approached, a guide was requested to land the
boat at his discretion.

(2) The landing site was immediately marked with a wooden stake, and

three of ten slips of paper were randomly chosen from a container. Each

slip of paper represented intervals of three meters from the landing site.

(3) Intervals were then walked off from the boat's starboard side, and

marked with brightly colored tape.

Trees were chosen for disease assessment using a modified quarter

method referred to as the point method (60).

Point Method

Grid lines were erected at interval markers. A maximum of 36 meters

of line was utilized per each site. Line sizes were of 12, 18 and 36

meters and utilized according to terrain availability. Three meter

intervals were marked along each grid line. Specific points along the

line were then chosen by the roll of four dice (each dot representing 1.5

meters) and marked with wooden stakes. The tree or trunk area (as with

some overwash forests) nearest the wooden stake was then selected for

disease incidence and severity assessment.

Foliar Disease Survey

Seven trees were selected at each site. Four "runs" of branches

were samples from each of the selected trees. A "run" of branches

consisted of a main branch and all the laterals and sub-laterals on it.

Run selection was made as follows:

(1) Trees were approached from the seaward side.

(2) The tree's canopy as viewed from above had been previously divided

into eleven segments (runs) on a circular plastic coated paper.

(3) Two dice were then tossed and four runs corresponding to the

first four numbers rolled on the dice were selected for sampling.








(4) Ten leaves were then randomly selected from the marginal area of

the run and disease incidence and severity assessed.

Disease severity assessment. Disease severity was assessed as the

area of plant tissue affected by disease expressed as a percentage of the

total leaf area (40). Leaves of each run were first placed in labeled

plastic bags and transported to the lab. Diseased areas were then out-

lined and coded with a felt tipped pen, and xeroxed. Xeroxed leaves

were cut out, weighed, diseased areas removed and leaves reweighed.

Severity of sites within areas was then determined. Severity of

specific foliar pathogens was determined in the same manner.

Disease incidence assessment. Disease incidence was assessed

according to the number of plant units (leaves) infected, expressed as

a percentage of the total number of units assessed (40). Disease

incidence of sites within areas was then assessed.

Fallen Leaf Study

A study was made to determine if a relationship existed between

foliage disease and premature leaf fall. One hundred fallen leaves

were randomly collected from each of the nine survey sites. Fifty

leaves were then randomly selected from each lot. Leaf-disease

incidence at each survey site was then recorded.

Gall Disease Survey

Fourteen trees were chosen at each site for gall disease severity

and incidence assessment.

Gall disease severity assessment. Circumferances were recorded at

and 2.4 meters above the "spread point" of each tree. "Spread point"

refers to the area of major prop root emergence. Circular and linear

galls within this area were counted and dimensions recorded. Tree







segments were converted to trapezoids. Surface areas of all galls and

trapezoids were then tabulated. Disease severity was assessed as the

surface area of plant tissue affected by disease expressed as a per-

centage of the total surface area. Surface area formulae and diagrams

of the procedure are depicted in Figure 19.

Disease incidence assessment. The number of trees containing galls

at each site were recorded. Disease incidence was assessed according to

the number of trees infected, expressed as a percentage of the total

number of trees assessed.

Final results of all assessments were then programmed to determine

if a statistically significant difference (5% level of significance) in

disease severity and incidence existed between sites among the areas

surveyed.

Results

The results of the disease incidence and severity surveys are

numerically summarized in Table 4. Comparison of disease severity

and incidence among sites within areas are depicted in Figures 20

and 21.

Foliar Disease Severity Assessment

Site 1 (Fig. 20A): Disease severity differed significantly

between control area 5 and the other areas surveyed. In comparison,

a significant difference in disease severity was not found to exist

among areas 1, 2, 3, and 4.

Site 2 (Fig. 20B): Disease severity of area 4 differed signifi-

cantly in comparison to the other survey areas. This was the only

statistically significant difference.

Of the total disease severity recorded, 38% of the foliage was





63




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CINC. dSEV.

60.3 1.52


65. 1.72


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FOLIAGE

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Table 4. Mangrove disease incidence assessment and severity results
expressed as percentages of units and tissue areas infected.


aRiverine forest.

boverwash forest.


cIncidence.
dSeverity.
dSevety.


SITE 1:


SITE 1:




SITE 2:


55.7 1.58


54.3 1.04







SEVERITY COMPARISONS


10.0 -
9.0 -
8.0-
7.0 -
6.0-
5.0-
4.0
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2.0
1.0-


INCIDENCE COMPARISONS


100.
90.
80.
70.
60.
50.
40.
30.
20.
10.


3> >
- 3J 3::k 3=- 3b
% w\ ( -Pb U1


Figure 20.


SITE 2
D


30 0 xM :9:
m W m m
S3 3 3
-J I%3 co -


Foliage disease severities and incidences among sites
surveyed.


aRiverine forest

b0verwash forests


SITE 1
A


10.0
9.0
8.0
7.0.
6.0 .
5.0 -
4.0
3.0-
2.0.
1.0-


SITE 2
B


100.
90.
80.
70.
60.
50.
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30.
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SEVERITY COMPARISONS


bSITE 2

B


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INCIDENCE COMPARISONS


3> 3a >
m m rm
- N3 ( L
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Figure 21.


Gall disease
surveyed.


severities and incidences among sites


aRiverine forests.

bOverwash forests.


10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0


10.0
9.0
8.0
7.0
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100.
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SITE 2
D


100. -
90. .
80. -
70. -
60. -
50. -
40. -
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~







covered by A. rhizomorphae, 18% with C. rhizophorae, and 44% with

P. disseminate.

Foliar Disease Incidence Assessment

Site 1 (Fig. 20C): The only significant difference in disease

incidence occurred between control area 5 and the other survey areas.

The greatest and least amount of recorded incidence was noted among the

control areas.

Site 2 (Fig. 20D): The greatest amount of disease was recorded in

control area 4 and differed significantly from area 3. Disease inci-

dence in areas 1 and 2 also differed significantly from area 3, but not

with the control. No difference was noted among areas 1 and 2.

Gall Di sease Severity Assessment

Site 1 (Fig. 21A): A significant difference in disease severity

was not detected between areas 3 and 4, and between areas 1, 2, and 5.

Disease severity was significant between area 3 in comparison to areas

1, 2 and 5.

Site 2 (Fig. 21B): There was a significant difference in disease

severity between area 1 and the other surveyed areas. Disease severity

differences were not significant among areas 2, 3 and 4.

Gall Disease Incidence Assessment

Site 1 (Fig. 21C): No significant difference in disease incidence

was detected among the various areas surveyed. In general, disease

incidence in the various areas was extremely high and was often recorded

at 100%.

Site 2 (Fig. 210): Here, too, a significant difference in disease in-

cidence was not detected among the various areas surveyed. Again, disease

incidence in many areas was extremely high and was often recorded at 100%.








Fallen Leaf Study

Results of the fallen leaf study are shown in Table 5, As noted,

the least amount of disease incidence was recorded in the Henderson

Creek overwash forest. In general, disease incidence was extremely

high and often approached 100%.

Discussion

The purpose of this phase of study was to determine if mangrove

disease incidences and severities differed significantly between virgin

and man-disrupted mangrove forests. Areas were chosen on the basis of

field observations, publications (1, 6, 7, 19, 35, 51, 55, 72) and per-

sonal communications, and designated according to obvious disruptive

factors present. These factors included heavy metal pollution (35),

channelization and urbanization. No doubt, disease incidence and

severity in these areas are also influenced by numerous other factors

other than the ones mentioned. Nevertheless, the criteria chosen for

this study were the most apparent influences present in these areas.

It is the objective of this discussion to briefly describe the areas,

and interpret the disease trends observed in reference to available

knowledge about mangrove susceptibility in response to these man-induced

environmental stresses.

Barron River riverine and overwash forests. Upper limits of the

Barron River are modified into a canal system connected to intensified

agricultural areas. The canals modify surface drainage by providing

direct and rapid discharge of water from these cultivated areas into

coastal waters (35). The riverine forest sampled was located approxi-

mately two kilometers north from the mouth of the river, and the overwash

forest was located in Chokoloskee Bay (Fig. 18). In 1971, Horvath and






69






















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others (35) sampled this area for heavy metals in an effort to make a

quantitative appraisal of the impact of Big Cypress land development on

the distribution and abundance of heavy metals in the Everglades

estuaries. Unusually high concentrations of certain metallic compounds

were found in the Barron River and Chokoloskee Bay. Lead concentrations

were found to be the highest reported at that time for coastal waters in

the United States (Barron River--12.0 ug/l, Chokoloskee Bay--41.8 ug/1),

Copper concentrations were also noted to be excessively high (6.7 ug/l).

These copper concentrations approached values which were shown in laboratory

experiments to reduce the survival of newly hatched amphipods and inhibit

photosynthesis in phytoplankton (35). Disease incidence assessments in

this area were generally less in comparison to control sites. This was

most apparent among the results of the gall disease incidence assessments

in the riverine and overwash forests (Fig. 21C & D), and gall severity

assessments in the riverine forest (Fig. 21A). Similar results were

recorded with foliage incidences and severities assessed in both

riverine and overwash forests (Figs. 20A-D). The exact effects of these

heavy metal concentrations on red mangrove physiology and a mangrove/

pathogen relationship are presently unknown. The significance of

lower levels of disease observed in this area can at best only be

speculated. If disease does in fact play a role in nutrient cycling

and the system must consistently maintain a threshold level of leaf

fall for food chain stabilization, then less disease could have serious

ecological and economic implications. Serious implications would result

from a reduction of marine life of ecological and commercial importance.

Faka Union riverine and overwash forests. The Faka Union River was

at one time channelled for the purpose of home site construction. The







river was widened by this construction which influenced the macro- and

micro-climates of the area. Here too, disease incidences and severities

in the sites surveyed were generally lower when compared to the control

sites. In all instances (with the exception of the East River control

site) foliage incidences and severities in the riverine and overwash

forests were less in comparison to the Fakahatchee controls (Fig. 20A

& D). With the gall disease assessments, the most pronounced differences

in results were the severity assessments in the riverine and overwash

forests (Fig. 21A & B). Gall disease severity in the riverine site

was approximately equal to that of the East River control and far less

than the Fakahatchee control (Fig. 21A). The greatest disease severity

recorded in an overwash forest was that of the Faka Union site (Fig, 21B).

The high severity of disease recorded here is interpreted as a predisposing

effect in the host in response to changes in patterns of sedimentation

caused by channelization. Prop roots are the mangroves most vulnerable

component. Odum and Johannes state (51) that T. 0. Kolehmainen observed

dead mangroves adjacent to recently constructed dikes where prop roots

had been covered with fresh sand. In the present study, it was observed

that prop roots in the Faka Union overwash forest were often coated with

a fine layer of silt. Whether this is of significance in the predispo-

sitioning of these trees to the gall disease remains to be demonstrated,

In general, less disease was found in areas where heavy metal

concentrations were high and those which had been channelized. This by

no means signifies that a reduction in plant disease is beneficial to

the system. Although less disease may be of benefit to an individual

host it may well serve as a detriment to the entire system by depleting

the system of a constant detrital nutrient source.








Henderson Creek riverine and overwash forests. Henderson Creek is

located near a large mobile home complex. The creek is utilized as a

source for the disposal of treated and untreated sewage. The disease

assessments were made during the dry winter season. During this season,

the population of the mobile home complex increases sharply. Subsequently,

the sewage effluent deposited in the river increases. The riverine forest

sampling site chosen for this area was located approximately one kilometer

from the source of discharge. The overwash forest, was an estimated two

kilometers further south (Fig. 16). Some of the highest disease incidences

and severities were recorded in this area (Fig. 20A & C: Fig. 21A, C & D).

It was also noted that the foliage and gall disease severities in the

riverine forests surpassed the values recorded in the control forests

(Figs. 20A & 21A). Joyner (42) has stated that the greatest danger of

sewage runoff to plant life is that of increased salinities which reach

toxic levels. Since red mangrove have been reported to tolerate salini-

ties up to 25 ppt, this fact would have little bearing on the mangrove

populations. It can be speculated, however, that nutrient input imposed

by the sewage effluent predisposes the mangroves to disease. Published

information concerning the reaction of mangroves to increased levels of

nutrient input is scarce. The fact that such high levels of disease

severity were noted in this area justifies further research into its

possible effects. Researchers (22) are presently investigating the

possibilities of utilizing cypress domes in tertiary sewage treatment

programs. Consideration should be given to the possible effects of

these increased nutrient inputs on the development of disease and

epidemics. It is strongly advised that further pathological studies

be conducted before this practice is fully implemented.







Fakahatchee and East River riverine and overwash forest. The

control areas like the other areas surveyed were south of US 41 (Fig. 17).

The highway's influence on these untouched natural areas is not known

and thought to be minimal by some investigators (74). Other than the

aforementioned unknown or minimal influence of the highway the controls

were apparently free from any man-induced environmental stress. Foliage

disease incidence and severity was greatest at the overwash site (Fig. 20

B & D), followed by the Fakahatchee riverine and least in the East River

riverine forest (Fig. 20A & C). Generally, the greatest amount of disease

severity and incidence (with the exception of the Henderson Creek area)

occurred in the control areas. These findings were somewhat unexpected.

An additional note was the difference observed among the riverine sites.

In both of these sites, disease incidences and severities contrasted

markedly. This trend was observed both in the gall and foliage survey (Fig.

20A & C: Fig. 21A & C). It is not known to what degree, if any, incidences

and severities fluctuate during the seasons or over a period of several

years in these control areas. In the riverine forests, time of sampling

may have been a major factor in the observed results. In order to

sufficiently interpret these findings a study would have to be conducted

over a several year period to determine if disease is indeed higher in

these undisturbed regions.

A large number of leaves collected at each of the nine survey sites

were found to be diseased. This finding and the fact that under laboratory

conditions inoculated leaves fell prematurely poses an interesting question.

To what effect does the pathogen influence leaf fall? That is to say,

does disease incidence and severity cause an increase in either the rate

or amount of leaf fall? Although field and laboratory observations provide








only circumstantial evidence, the possibility exists that pathogens

influence leaf fall. It would also be of interest to determine if

various nutritional elements, K, P, and N, are localized near infection

sites. If such is the situation, are these elements redistributed in

the leaf, or acquired by the host in response to infection? Available

literature concerning fungal organisms in the role of nutrient cycling

pertains only to the decomposition of mangrove detrital material, and

not the role of plant pathogens (3, 24). This should be broadened to

cover pathological effects.

This phase of study has shown that significant differences in disease

did exist among the areas surveyed. The next logical step, as previously

mentioned should be one of long term sampling of these areas to determine

if these trends remain constant or fluctuate. Of equal importance would

be an investigation to determine to what extent disease influences leaf

fall. If through such studies a constant leaf fall is noted only in the

control areas, then these ecosystems could be utilized as standards of

comparison for monitoring environmental stress along Florida's coastal

areas.

Conclusion

Florida's mangrove forests can be regarded as natural units or

ecosystems in which the mangrove populations coexist and interact with

other biotic communities and edaphic environments. If we could imagine

the mangrove system as a large box into which the pathogens have been

placed, the following questions of those pathogens might be asked:

(1) Why are they there?

(2) Could the system get along without them?







(3) What is the functional role of these pathogens in the total systems

approach to ecological and plant pathological studies?

These questions are presently unanswered. Plant pathologists and

ecologists, however, could benefit from the answers and knowledge

obtained from further pathological study in the mangrove forests of

south Florida.

Knowledge gained in this study has provided foundational information

for future phytopathological research in mangrove ecosystems. Information

gathered from such future work would contribute to improved pest management

programs in agro-ecosystems, and also provide approaches to disease control

in managed forests.

The finding that disease incidence and severity differed significantly

between virgin and man-disrupted mangrove forests enhances the feasibility

of utilizing plant disease in monitoring environmental quality within

natural ecosystems.












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80


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BIOGRAPHICAL SKETCH


Michael Theodore Olexa was born 12 March 1947, in Morgantown,

West Virginia. He attended grade school and high school in the town

of his birth. He graduated from Morgantown High in May of 1965, and

then entered West Virginia University. He received the degree of

Bachelor of Arts with honors in general biology in August of 1969.

Shortly after graduation he entered military service. After serving

two years of active duty, he entered graduate school at the University

of Florida in January of 1972.

He is a member of the American Phytopathological Society,

Mycological Society of America, and Gamma Sigma Delta Agricultural

Honorary.






I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.



T. Edward Freeman,
Chairman
Professor of Plant Pathology



I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.



J mes WI Kimbrough
rofes or of Botany



I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.



Herbert H. Luke --
Professor of Plant Pathology



I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.



David J. Miothell
Associate Professor of Plant Pathology







I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.


/


p-


4)anit1 R Minnick
Assist t Professor
/4


of Entomology


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.


Daniel A. Roberts
Professor of Plant Pathology



This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate Council, and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.


August, 1976



Dean, College
U


of Agridy1ture


Dean, Graduate School


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UNIVERSITY OF FLORIDA

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