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    Front Cover
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    Front Matter
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    Preface
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    Table of Contents
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Full Text
'.i


PROCEEDINGS
THIRD MEETING
of the
INTERNATIONAL CO tN
LETHAL YELLOVII


PUBL. FL-78-2
AGRICULTURAL RESEARCH CENTER
INSTITUTE OF FOOD AND AGRICULTURAL SCIENCES
UNIVERSITY OF FLORIDA
FORT LAUDERDALE, FLORIDA
1978









THIRD MEETING

OF THE

INTERNATIONAL COUNCIL

ON


LETHAL YELLOWING


SPONSORS


Palm Beach County Board of Commissioners
Palm Beach County Cooperative Extension Service
Palm Beach Garden Club
Palm Beach Civic Association
Palm Beach Kiwanis Club
Florida Nurserymen and Growers' Association


OFFICERS OF I. C. L. Y.


H. Romney, Chairman
A'Brook, Vice-Chairman
A. Addison, Vice-Chairman
B. Ennis, Jr., Vice-Chairman
Chiarappa, Secretary


COORDINATOR

Mrs. Marie Rey


October 30 November 3, 1977

Jupiter Hilton Hotel
Palm Beach County, Florida


I j













PREFACE


The third meeting of the International Council on Lethal Yellowing (ICLY)
was held in Jupiter, Palm Beach County, Florida, October 30-November 3,
1977. It was organized by the Officers of ICLY and the scientists who are
working on the lethal yellowing (LY) disease of palms at the University of
Florida's Agricultural Research Center in Ft. Lauderdale. The meeting was
sponsored by the Palm Beach County Board of Commissioners, Palm Beach County
Cooperative Extension Service, Palm Beach Garden Club, Palm Beach Civic
Association, Palm Beach Kiwanis Club and Florida Nurserymen and Growers'
Association. It was coordinated by Mrs. Marie Rey, a charter member of the
Save the Palms Committee.

The purpose of the workshop-type meetings was to (1) exchange technical in-
formation on recent progress in research on LY; (2) describe current emphases
of various researchers; (3) examine the soundness of research approaches and
techniques; (4) identify promising new lines of research for future emphasis;
and (5) explore opportunities for increased collaborative work between indi-
vidual scientists and laboratories. The meeting brought together most of the
principal scientists of the world who are conducting research on LY. In ad-
dition, scientists who work in problem areas related to some aspect of the LY
research programs also participated. Many scientific fields of expertise were
represented among the participants including agronomy, biochemistry, botany,
entomology, forestry, genetics, horticulture, microbiology, plant pathology,
plant physiology and weed science.

This report includes a generalized summary of the meeting, abstracts of papers
discussed at sessions on the basic biology of mycoplasma diseases, disease
diagnosis, mycoplasma isolation and culture, vectors, disease control and at
a special session on emerging disease problems in palm culture. Rapporteur's
reports follow each group of abstracts dealing with one of the above subject
matter areas.

Each member of the lethal yellowing research team at ARC-Ft. Lauderdale con-
tributed substantially to the editing and organizing of this report. The
cost of publication of the report was covered by contributions of the Palm
Beach County Board of Commissioners and other sponsors.

Special thanks are due the sponsor and the governmental organizations who
financed the travel costs of participating scientists. This fine financial
support, the assistance given by many interested citizens of Palm Beach County,
and the excellent technical contributions by the participating scientists made
the meeting highly productive and worthwhile to all who attended.

W. B. Ennis, Jr.











TABLE OF CONTENTS



Preface W. B. Ennis, Jr. . . . . . .ii

Progress in Lethal Yellowing Research:
The Third International Conference H. M. Donselman ...... 1

INTRODUCTORY SESSION

Distribution and Impact of Lethal Yellowing
in Florida G. W. Gwin . . . . ... .. 5
Distribution and Impact of Lethal Yellowing
in the Caribbean D. H. Romney and H. C. Harries . . 6
The Cape St. Paul Wilt Disease in Ghana:
The Present Position, E. A. Addison . . . 7

SESSION I BASIC BIOLOGY OF YELLOWS DISEASES

Introduction . . . . . . 8
Comments on the Host Family of Lethal Yellowing and Suggestions
for Replacing Susceptible Species H. E. Moore, Jr .. 8
The Biology and Current Classification of Mycoplasmas J. G. Tully 8
Morphological Aspects of Mycoplasmalike Organisms in
Plants R. E. McCoy . . . . . 9
Mechanisms of Pathogenicity of Plant Mycoplasmas M. J. Daniels 9
New Findings in the Biology of Plant Yellows Disease K. Maramorosch 9
Summary T. A. Chen . . . . . . 10

SESSION II DISEASE DIAGNOSIS


Introduction . . . . . .
Kaincope Disease and Kribi Disease, Two West African
Coconut Yellows Associated with the Presence of
Mycoplamalike Organisms M. Dollet . . .
Further Studies on Root Symptoms of Lethal
Yellowing S. J. Eden-Green . . . .
Is Coconut the Primary Host of Lethal Yellowing
Disease? H. C. Harries . . . .
Possible Interaction Between Lethal Yellowing and
Bud Rot. H. C. Harries . . . .
Natural Symptom Remission and Total Recovery After
Lethal Infection H. C. Harries . . .
An Investigation of Xylem Pressure in Coconut Palms
with Reference to Lethal Yellowing J. McDonough and


. . 12


. . 12

. . 12

. . 13

. . 13

. . 13


M. H. Zimmermann . . . . . .
Electron Microscopy of Palm Species with Suspected
Lethal Yellowing D. L. Thomas . . . . .
Preliminary Studies upon Lethal Yellowing and the Distribution
of MLO in Coconut Palms H. Waters and I. Osborne . .
In Vivo MLO Morphology by Serial Sectioning of Lethal
Yellowing Infected Coconut H. Waters and P. Hunt . .
Profiles of MLO Based upon an Understanding of Known
Morphologies H. Waters and P. Hunt . . . .
A Model of MLO Distribution within a Sieve Element P. Hunt and
H. Waters . . . . . . .








Table of Contents continued


Implications of Elucidating MLO Morphology in Sieve
Tubes P. Hunt and H. Waters . . . .... 15
Serology: Preparation of Antisera from Infected Host
Antigen R. F. Whitcomb . . . .... .16
Summary H. Waters . . . . ... ..... .16
Summary R. E. Davis . . . . ... ..... .17

SESSION III MYCOPLASMA ISOLATION & CULTURE

Introduction . . . . ... . . 18
Development of Biochemical Methods for Detection
of Mycoplasmas H. G. Basham . . . .... 18
Identification of Mycoplasmas Cultured from
Plants M. J. Daniels . . . ..... .... 19
Cell Wall-Free Prokaryotes Harbored in Flowers R. E. Davis . 19
Attempts to Extract and Culture Mycoplasmas from
Coconut Palms S. J. Eden-Green . . . .... 20
Attempts to Culture Mycoplasmalike Agents at
Fort Lauderdale R. E. McCoy . . . .... 20
Summary J. Tully . . . . ... ..... 20
Summary I. Osborne . . . . ... ..... .21

SESSION IV VECTORS

Introduction . . . . ... . . 22
Current Attempts to Transmit Lethal Yellowing Disease
in Jamaica A. J. Dabek . . . .... .22
Lethal Yellowing Transmission Tests in Jamaica,
1975-1977 S. J. Eden-Green . . . ... 22
Feeding Sites of Leafhoppers and Planthoppers on
Plant Tissues J. B. Fisher . . . .... 23
Survey of Homoptera Associated with Palms in
Florida F. W. Howard ......... ......... .......... 24
Vectors Studies in Florida J. H. Tsai . . . ... 24
Strategies of Disease Vectors and Their
Hosts R. F. Whitcomb . . . . . 25
Summary G. H. Kaloostian . . . . ... .25
Summary F. M. Eskafi . . . . ... ..... 26

SESSION V DISEASE CONTROL

Introduction . . . . ......... .27
New Palms for South Florida H. M. Donselman . . ... .27
Present Effort in Preserving South Florida's
Palms H. M. Donselman . . . .... .28
Progress in the Development of Control of Lethal Yellowing:
Foliar Insecticide Applications F. W. Howard . ... 28
Recent Advances in Antibiotic Treatment of Lethal
Yellowing R. E. McCoy . . . .... .29
Susceptibility to Lethal Yellowing Disease in
Global Perspective H. C. Harries . . .... 29
Plant Health & Quarantine Problems Arising in the
International Transfer of Coconut Genetic
Resources H. C. Harries . . . .... 30









Table of Contents continued


Can Tall Coconut Varieties be Selected with Lethal
Yellowing Resistance Equal to that of the
Malayan Dwarf? H. C. Harries . . . . 31
Guidelines to Nursery Practice for Malayan Dwarf and
Maypan Coconut Seed D. H. Romney . . .... 31
Summary M. Dollet, F. W. Howard and S. J. Eden-Green ...... 31

SESSION VI EMERGING PROBLEMS IN PALM CULTURE

Lethal Disorders of Unknown Cause in the Date
Palm J. B. Carpenter . . . . ... ... 33
Flagellated Protozoa Associated with Marchitez Sorpressiva
of Oil Palm in South America M. Dollet . . ... .34
Transmission of Sudden Wilt Disease of Oil
Palm G. Martinez-Lopez . . . .... .34
Observations of the Phloem Inhabiting Flagellate
Phytomonas in Palms M. V. Parthasarathy . . 35
Symptomology of Hartrot Disease in Coconut and
Oil Palm W. G. van Slobbe . . . ... 35
Phytomonas Flagellates in Coconut (Hartrot Disease,
Marchitez Sorpressiva) W. G. van Slobbe . . ... .36
Phloem Inhabiting Phytomonas Protozoan from Diseased
African Oil Palms D. L. Thomas . . . ... 37
Wilt Disease of Coconuts in Trinidad H. Waters ......... 37
New Diseases of Coconut Palms in Tanzania K. G. Steiner . .. 37
Should the ICLY Concern Itself with other
Pathogens? H. C. Harries . . . . ... 38
Summary L. Chiarappa . . . . . .. 38

BUSINESS MEETING . . . ... . ... .40

REGISTRANTS . . . . ... . . . 42















PROGRESS IN LETHAL YELLOWING RESEARCH:


THE THIRD INTERNATIONAL CONFERENCE


Returning visitors to South Florida notice a difference in the landscape but are not
usually aware of the cause. Residents have heard that a disease is destroying coco-
nut palms but few understand the magnitude of the problem. Researchers probe for a
breakthrough in a problem that has baffled scientists for two decades yet the
mysterious decline of our palm trees continues, threatening not only South Florida's
landscape, but palms throughout the tropical and subtropical areas of the world.

Lethal yellowing is a disease that destroys palm trees. Once the disease has pro-
gressed to a point where the symptoms are visible, the tree can rarely be saved.
Recorded history indicates that lethal yellowing may have been in the Caribbean area
since the 1800's. It has only been in the past 20 years that lethal yellowing has
reached epidemic proportions. Various proposals have been made on why lethal yel-
lowing has remained a relatively inactive disease for so many years and only recently
became a serious threat to our palm trees. All of the 24 palms on the susceptible
list are exotics, not native to Florida or the West Indies. All of the Florida
native species of palms appear to be highly resistant to lethal yellowing. Increased
movement of plants and people from country to country through better transportation
may have moved the insect thought to spread the disease into new areas. Weather pat-
terns or hurricanes could have picked up the vector and transported it great distances.
Regardless of the reason, lethal yellowing is here and is causing millions of dollars
worth of damage each year. Concern about a solution for the LY problem brought to-
gether over 60 scientists from throughout the world at a meeting held in Palm Beach
County, Florida.

The International Council on Lethal Yellowing (ICLY) was formed soon after the disease
became a serious problem in Florida. In 1973 a group of scientists met at Fairchild
Tropical Gardens in Miami, Florida and decided that a cooperative effort between
scientists and countries would result in an earlier solution to lethal yellowing. Two
years later, in 1975, ICLY met in Jamaica at a similar conference. To discuss the most
recent research findings, to generate ideas for improving lethal yellowing research,
and to establish priorities for future research the Third ICLY Meeting was held Oct. 31,
through Nov. 3, 1977 at the Jupiter Hilton Hotel in Palm Beach County.

Leading off the program was a presentation by Dr. Luigi Chiarappa, Senior Plant Patholo-
gist, Plant Protection Service, Food and Agriculture Organization of the United Nations,
about the world-wide implications of lethal yellowing. In his travels around the world,
Dr. Chiarappa stated that the coconut palm deserves the common name "tree of life", a
name which people of many different cultures have assigned to this important plant.
Ecologically the coconut palm thrives in areas unsuitable for other crops. Salt spray,
sandy soil, moderate rainfall, and low fertility are tolerated by mature coconut palms
which, in turn, provide the inhabitants of tropical countries with almost every need.
Human life would be intolerable, and often impossible, without it. It provides food,
drink, oil, medicine, fiber, timber,thatch, mats, fuel and domestic utensils. The oil
is used for cooking, illumination, lubrication and making soap. It plays a prominent
role in the customs and folklore of the people. Each and every part is useful to man
in one way or another. It is not surprising that J. W. Purseglove, Tropical Crops
Specialist for the overseas development administration of Great Britain, describes the
coconut as 'mankind's greatest provider in the tropics'.














"The implications of lethal yellowing on a world-wide basis are staggering".
Dr. Chiarappa made the point that "Already in Africa, villages are sacrificing
animals to appease the Gods destroying their trees of life. India, although still
free from lethal yellowing, has a similar variety of coconut palm and is one of the
largest coconut producers in the world". Dr. Chiarappa's assessment of the world-wide
implications almost make the destruction of Florida's ornamental palms appear insignifi-
cant, but it does reaffirm the importance of the research conducted by the University of
Florida at the Fort Lauderdale Agricultural Research Center and by the Coconut Industry
Board in Kingston, Jamaica. Not only will the residents and tourists of Florida benefit
from such a program but also millions of people throughout the tropics.

The first technical discussion of the ICLY Meeting dealt with the basic biology of
yellows-type diseases. Much was learned from the presentations of pioneers in the study
of mycoplasma-caused diseases in humans from people such as Dr. Joseph Tully from the
National Institutes of Health and from studies of insect vector transmitted plant disease
by Dr. George Kaloostian at the United States Department of Agriculture in Riverside,
California. Other scientists who spoke in this session brought the participants up-to-
date on knowledge concerning these related diseases. The discussion provided insight
into how solutions were found to other mycoplasma diseases, such as corn stunt and
citrus stubborn diseases, and provided ideas applicable to lethal yellowing research.

In the same session Dr. Harold Moore from Cornell University spoke of the great diver-
sity in the palm family and the fact that lethal yellowing affects at least 7 of the
15 major groups of palms. His expertise in plant taxonomy suggested new test plants
for scientists to work with and added information necessary to the selection of new
resistant palms for South Florida.

In a special session on disease diagnosis Dr. Michel Dollet from France described two
diseases of coconut palms in West Africa which appear to be the same as lethal yellowing.
Further studies of palms found to be susceptible to lethal yellowing were presented by
Dr. Darryl Thomas from the Ft. Lauderdale Lethal Yellowing Team. In this past year
Dr. Thomas has verified the existence of mycoplasma-like bodies (MLO's) in 4 new palms.
The most important of these is the Chinese Fan Palm (Livistona chinensis). Dr. H. Waters
and Dr. Peter Hunt, of Jamaica, presented evidence that electron micrographs of lethal
yellowing MLOs can be misleading. Rather than the spherical shape usually ascribed to
MLOs they are actually varied in shape with offshoots creating a complicated anastomsing
network of filaments.

One of the most challenging aspects of lethal yellowing research has been in trying to
culture the disease-causing agent in the laboratory. Success in doing this would have
far-reaching effects, for it would allow rapid testing of antibiotics for controlling
the disease and provide material to induce artificially the disease in palms being
tested for resistance. Latest attempts by members of both the Jamaican and Ft. Lauder-
dale LY team have incorporated coconut palm sap into the growing media used in their
experiments. Mycoplasmas similar to the one thought to be causing lethal yellowing
have been grown in this growth media. New attempts are being made to refine this
growth media so it will support colonies of the mycoplasma associated with lethal
yellowing.














According to Dr. J. Tsai, Entomologist at the Fort Lauderdale Agricultural Research
Center, "Discoveryof the vector of lethal yellowing would lead to new methods of
control of the disease and provide techniques of artificially inducing the disease
in resistant palm breeding programs". It was agreed that the vector is probably an
insect that feeds on the phloem sap of the tree since this is where the MLOs are
found and also, that the insect can move great distances. Outbreaks of lethal
yellowing 100 miles from other known diseased trees have been seen in Florida.
Possibly air currents move the vector long distances.

Oxytetracycline injections can protect a palm from lethal yellowing as long as the
injections are continued every 3-4 months for the life of the tree. New techniques
for injections have been developed, including injection into the tree at the base of
the leaf, thus avoiding wounding of the tree trunk. Well supervised injection pro-
grams in Florida have been able to save up to 90% of the palms injected and allowed
time to establish replantings with resistant palms.

New resistant coconut varieties are being tested for their adaptability to South
Florida through cooperative research between the Jamaican plant breeder, Mr. Hugh
Harries, and Dr. Henry Donselman of the Ft. Lauderdale Research Team. Dr. Donselman
is developing a trial garden in Ft. Lauderdale to determine lethal yellowing resistance
in over 100 species of palms new to South Florida. "One of the best solutions to lethal
yellowing is to plant resistant varieties of palms, and we can do that right now. We
know that many palms, such as the 'Malayan Dwarf' coconut, queen palm, solitaire palm,
pigmy date palm, royal palm, and our native palms are highly resistant to the disease.
Thus the future of Florida's tropical landscape can be assured if we begin replanting
with these palms now".

Other disease control methods discussed were insecticide applications to suppress
vector populations. Such studies are currently being conducted by Dr. Bill Howard
in South Florida. To date insufficient evidence has been collected to warrant wide-
spread application of insecticides to palm trees.

The importance of palms in maintaining Florida's tropical landscape was brought to the
attention of the visiting scientists during a field trip to Palm Beach. Here they not
only saw the beautiful city and county plantings but also the extension collection of
rare palms maintained by Mr. Bill Mowry, President of the Palm Beach Palm Society and
one of the oldest collections of rare palms in Palm Beach at the estate of Mr. Michael
Burrows. It was pointed out on the trip that many of the palms dying from lethal yel-
lowing such as the date palms, windmill palms, and Chinese fan palms grow as far north
as Tallahassee and across the Gulf States westward to California. Thus, lethal yel-
lowing is of concern outside South Florida as well.

The last session of the ICLY Meeting was devoted to emerging disease problems in palm
culture. Other palms, such as the oil palm, which produces 18 times more vegetable
oil/acre/year than soybeans, are affected by diseases which threaten plantations
throughout the tropics. Research support in these areas is necessary if we are to
develop these important crops to their economic potential.














Members of ICLY devoted an evening of discussion to future emphasis of lethal yellowing
research. The following items have been designated as the most important areas for
concentrated effort in the next 2 years:

A. Culturing the mycoplasma from host palms and insects.
B. Determine means of transmission of lethal yellowing.
C. Explore possible alternate host plants other than palms that
may carry the lethal yellowing disease agent.
D. Determine how lethal yellowing kills palms by looking at the
physiology of palms.
E. Develop a rapid disease diagnosis method.
F. Intensify breeding and screening for disease resistance in
palms (including species other than coconut).
G. Re-test seed transmission experiments to verify that lethal
yellowing cannot be spread by seed and find methods of
assuring that healthy palms do not harbor the disease causing
agent.

The goals set by members of ICLY will be challenging but are necessary if we are to
totally understand this disease and develop effective programs to combat it. The en-
thusiasm and optimism shown by the scientists at the Third ICLY Meeting demonstrated
their willingness to help combat a most serious disease not only in Florida but
world-wide. Progress depends heavily on continued research funding, and support
from concerned citizens. Mrs. Marie Rey, Coordinator for this Meeting, deserves a
great deal of credit in coordinating arrangements for a most successful meeting.
Much of the travel and room expenses were paid for by the Institutes of the individual
scientists. Funding for the meeting was also provided by generous donations from the
Palm Beach County Board of Commissioners, Palm Beach Garden Club, Palm Beach Civic
Association, Palm Beach Kiwanis Club, Chase Federal Bank of Miami; Mr. R. Rich, and
the Florida Nurserymen and Growers Association. Through such support scientists will
be able to insure the future of Florida's palms and ultimately find a solution to
lethal yellowing-a worldwide crisis.


H. M. Donselman










INTRODUCTORY SESSION


DISTRIBUTION AND IMPACT OF LETHAL YELLOWING IN FLORIDA. George H. Gwin, FDACS,
Division of Plant Industry, PEC, 1360 Old Cutler Rd., Miami, Florida 33158. During
the past 6 years, LY has attacked the Florida mainland, playing havoc with not only
'Jamaican Tall' coconut palms, but 21 other species of palms as well (1). The first
U. S. epidemic of this disease occurred in Key West and the lower Florida Keys during
1955-69. After apparently running its course, a certain complacency was felt, but
when it struck Coral Gables on the mainland late in 1971, the reaction by the public
and the news media reminded one of 'Pearl Harbor'. Within one year the death toll of
the coconut palms reached 1,200; in 2 years it surged upwards to 20,000, and the third
year to over 100,000. Now we have estimated that 375,000 of the coconut palms have
succumbed, and thousands of the other species, as well. Several of these other species,
such as Veitchia merrillii, and the Phoenix spp., are almost as commonly planted as the
coconut palms.

As a follow up of aerial and ground surveys conducted by the Division of Plant Industry
(DPI) personnel, the State contracted for the removal of over 12,000 diseased palms be-
fore turning this phase of the program over to the municipal agencies affected, who in
turn removed thousands. The remainder were removed by the property owners themselves.

In June 1974, the U. S. Environmental Protection Agency approved a label for oxytetra-
cycline as a formula to be used in combating this palm pestilence. By late fall it
was on the retail market, sold at garden centers and plant nurseries. The DPI let
contracts for the purchase of this antibiotic in large wholesale quantities for resale
to other governmental agencies throughout the 7 counties under quarantine. This service
continues, and is currently being administered from the DPI office in Fort Lauderdale.
Based on the amount of this chemical bought by the State, we estimate that 1 million
injections have been applied. This was accomplished through the cooperation of over
60 governmental agencies. Due to a complexity of factors, too numerous to recite at
this time, many of these programs were doomed to failure from the start. However,
many other programs were successful and are worthy of special mention, for example:

Key Biscayne Chamber of Commerce
City of Bal Harbor
City of Miami Beach
City of Palm Beach
Collier County
Monroe County

The majority of these agencies have saved over 75% of their total palms, and others
have instigated effective programs in spite of the disease having been rampant in
their respective areas before the antibiotic and injection equipment were available
to the public. Monroe County, headed by its dedicated Extension Agent, is a good
example of the latter (2).

In the early stages of this current epidemic, thanks to work done by the Coconut
Industry Board of Jamaica, the LY-resistant Malayan palm variety was discovered and
made available to Florida for under-planting on a large scale. They report having
shipped over 750,000 of these seed coconuts to Florida, of which we estimate 300,000
have survived the rigors of our growing conditions. (Personal communication with
Mr. Astor Goodison, Chairman, Coconut Industry Board)









To complicate matters, during January 1977 our Florida palms were hit with record
breaking, freezing temperatures which did extensive damage to them as well as to
all our tropical plants and to the multi-million dollar vegetable crop. This set-
back served only to increase the general economic slump which the whole United
States has been experiencing for the past several years. This is suggested as a
possible limiting factor which adversely affected many governmental budgets; specifi-
cally, the LY injection and replanting programs fall into this category.

Of the tropical palms most affected by the cold weather, the Malayan Dwarfs top the
list. Many were killed, and others severely damaged. The severe discoloration of
the fronds has discouraged future plantings with this variety. This same damage has
also made it more difficult to recognize the LY symptoms.

Whereas no new countries have been reported as infested, as recently as September 15,
1976, 3 new cases of LY were confirmed in Stuart (Martin County), making it the
northernmost find of LY in the State. As has been customary, our DPI personnel, in
cooperation with the local County Extension Agent arranged for the removal of these
diseased palms and subsequent injections. No new cases have been reported from this
area. Since the original LY cases were found in Collier County October 9, 1974, on
the west coast of Florida, only 1 additional case an untreated palm was found
September 5, 1975. This latter cause brought the Collier County palm loss to a total
of 8, which is an outstanding example of what can be accomplished by early detection,
and by having the chemical, tools, technical know-how, and EPA approval in hand before
the pest makes its appearance.

References

1. Division of Plant Industry, Revised Lethal Yellowing Quarantine List of
susceptible palms, June 1977, J. K. Condo, Chief, Bureau of Plant Inspection.
2. Monroe County Extension Service, Raymond H. Zerba, Jr., Extension Horticulturist,
Monthly, Lethal Yellowing Newsletters, 1977.

DISTRIBUTION AND IMPACT OF LETHAL YELLOWING IN THE CARIBBEAN. D. H. Romney & H. C. IHarri<
Coconut Industry Board, P. 0. Box 209, Kingston 10, Jamaica. Any review of lethal
yellowing disease of coconuts in the Caribbean must begin by listing the Bahamas, the
Cayman Islands, Cuba, Haiti, Dominican Republic and Jamaica as the only territories
known to be affected. A report of possible lethal yellowing from Trinidad in 1975 can
now be discounted (Waters, 1977). Palms on the Mexican island of Cozumel (after Florida,
the closest part of the continental coast to the disease in Cuba) were found in 1977 to
have symptoms partially resembling lethal yellowing: one dying palm between Limon (on
the coast) and Turrialba in Costa Rica did not have typical symptoms; both these cases
are under investigation.

Reports from Haiti in 1946, 1960 and 1970 describe spread of the disease from the north
to the west coast but no details are known; some Malayan Dwarf seed has been imported
from Jamaica. Disease appears not to have reached the main coconut growing areas of
the Dominican Republic; a recent visit by Mazzani, as an FAO coconut consultant, might
provide up to date information: replanting schemes under consideration there include
dwarf and hybrid material. No new information is available concerning the extent of
the disease in Cuba but replanting with Malayan Dwarf has begun: it is hoped that the
presence of other resistant varieties and the possibility of hybrid production is to
be investigated soon. On the Cayman Islands, the disease is not known to be very









active: it is not widely realized that there has been a source of resistant tails
(reputedly from Ecuador) on Grand Cayman for many years. The Bahamas have imported
dwarf seed but we have no new information on the extent of the disease. It might be
best to consider Bahamas with Florida since it is a decorative and not an agricultural
plant in both places.

In Jamaica the only commercial area that can be considered disease-free is in the
isolated valleys inland from Montego Bay: even in areas where disease has not been
active for many years occasional cases occur. Copra production has continued to be
low although factors other than the disease contribute to this. Needless to say the
'Malayan Dwarf' planted since 1966 under the LY Rehabilitation Scheme prove very
popular as a source of "jellies" rather than copra (an increasing market) and are
prone to loss by theft. The present rate of replanting stands at 1,059,729 plants
during 1975-76, of which 49,480 were 'Maypan'. More than 60% of all plants were
distributed by the Coconut Industry Board under the LY Rehabilitation Scheme. Chemo-
therapy to protect susceptible tails has not been adopted for both economic and en-
vironmental reasons. One increase in lethal yellowing activity that can be reported
with satisfaction is that the research staff involved in full time investigation in
Jamaica now stands at five.

Reference

Waters, H. (1977) Phloem-inhabiting protozoan of coconut in Trinidad. 3rd ICLY
Meeting, Florida.

THE CAPE ST. PAUL WILT DISEASE IN GHANA: THE PRESENT POSITION. E. A. Addison, Crops
Research Institute, P. 0. Box 3785, Kumsai, Ghana. Since it was observed in 1932,
the Cape St. Paul Wilt disease has remained the most important disease of coconut
palms in Ghana. In 1968 the disease appeared on the west coast at Cape Three Points,
a distance of about 450 km. from the nearest point of the disease occurrence in the
east. It has now jumped to Princes Town which is about 10 km. northwest of Cape
Three Points.

The etiology of Cape St. Paul Wilt has defied discovery but recent investigations in
electron microscopy have revealed the presence of MLO in diseased material.

Results of variety resistance trials in the endemic area at Dzelukope in the west in-
dicate that the Malayan Dwarf and the red Cameroon Dwarf coconuts are all as susceptible
to the disease as the local tall coconut palm.

The variety trials have been extended to the Cape Three Points area where environmental
conditions are more suitable for coconut cultivation the the Dzelukope area to further
test the apparent lack of resistance of the Malayan Dwarf coconuts under Ghanaian
conditions.









SESSION I BASIC BIOLOGY OF YELLOWS DISEASES

Chairman: W. B. Ennis, Jr. Rapporteur: T. A. Chen

INTRODUCTION. Lethal yellowing is a member of the yellows group of plant diseases.
Knowledge of the basic concepts and mechanisms of action of this large and diverse
disease group is a prerequisite to an understanding of the LY disease. Factors
involved in production of disease include the susceptible host plant, the pathogen
which incites the disease and the vector which carries the pathogen from plant to
plant. This section discusses these factors in the general sense.

COMMENTS ON THE HOST FAMILY OF LETHAL YELLOWING AND SUGGESTIONS FOR REPLACING
SUSCEPTIBLE SPECIES. H. E. Moore, Jr., L. H. Bailey Hortorium, Cornell University,
Ithaca, New York 14853. The palms or Arecaceae are most often associated with the
Cyclanthaceae and Pandanaceae, each in its own order, and sometimes with the order
Arales in contemporary phylogenetic schemes, and evidence has been brought forward
to suggest a relatively primitive position among the monocotyledons. The family
includes many diverse types and levels of evolution. Lethal yellowing attacks
without evident pattern -- species of genera in seven of 15 major groups have been
affected. Replacements for affected species should be sought among self-cleaning
palms of rapid growth adapted to seasonal climates and low elevations, and in Florida,
to limestone-derived soils and some cold. Possible candidates for introduction and
use are suggested.

THE BIOLOGY AND CURRENT CLASSIFICATION OF MYCOPLASMAS. J. G. Tully, National Institute
of Allergy & Infectious Diseases, Bldg. 7, Rm. 200, National Institutes of Health,
Bethesda, Maryland 20014. The information explosion on mycopiasmas which has occurred
in the past 10-15 years is undoubtedly due to the realization of the role these organisms
play in human and animal diseases, to their potential value as models to study a number
of biologic phenomena, and to recent and rapidly-expanding information on their etiologic
role in various plant diseases. This presentation will attempt to show how these wall-
free prokaryotes differ from other prokaryotic organisms and the basis and rationale for
their current classification. International agreement successfully developed minimum
standards for the description of new species within this taxonomic scheme (class Mollicute
and these standards have not only received unusual acceptance but have also greatly im-
proved the quality of taxonomic studies on new agents. Some attention is given here to
pertinent areas of current mycoplasma research, as they relate to the central theme of
this conference. These include some consideration of the morphology of selected myco-
plasmas, particularly variations in shape expressed under different growth conditions
and after certain preparative procedures. General comments on mycoplasma nutrition will
emphasize that while there has been much success in cultivating new spiroplasmas we still
are far from having a reasonable understanding of the nutritional needs of mycoplasmas.
Recent observations on tetracycline resistance in some mycoplasmas also suggests that
we must exercise some precaution in trying to interpret the possible nature of some
plant disease agents on the basis of antibiotic sensitivity tests. These precautions
might also extend to more practical efforts now underway on field experiments in the
chemotherapy of plant diseases namely, what are the possibilities of inducing anti-
biotic resistance or of selecting out antibiotic resistant populations during these
trials? Finally, some comments on mycoplasma-host relationships, as they are currently
viewed with acholeplasmas and spiroplasmas, will be presented. Discussion will include
some observations on pathogenic spiroplasmas and demonstrate some of the relationships
between virulence and prolonged artificial cultivation of new agents.









MORPHOLOGICAL ASPECTS OF MYCOPLASMALIKE ORGANISMS IN PLANTS. R. E. McCoy, Univ.
of Florida, 3205 S.W. 70 Ave., Ft. Lauderdale, FL 33314. The proliferation of
literature on plant infecting mycoplasmalike organisms (MLO) in the past decade
has provided an extensive, but sometimes confusing, picture of the relationship
of these organisms to their host plants. Specific points of confusion have con-
cerned the tissues in which these organisms reside, and in distinguishing the
cellular types of the phloem. The presence of MLO's in sieve elements has been
well documented, however reports of their presence in phloem parenchyma or companion
cells are disputed. Most reports of MLO in parenchyma either do not state the criteria
used to distinguish these cells from sieve elements, or the bodies reported as MLO are
indistinguishable from vesicles in endoplasmic reticulum cisternae, perinuclear space,
or invaginations of the plasmalemma observed in healthy and virus infected plants.
For MLO to occur in parenchyma they would have to survive in a cell environment vastly
different from that in the sieve element, and they would have to pass through plasmodes-
mata, a highly unlikely phenomenon.

Confusion also exists in regard to the morphology of MLO observed in situ in sieve
elements. Without special preparative methods, histological preparations of phloem
tissues yield a highly disturbed view of sieve element structure. This has resulted
in many misinterpretations of MLO morphology in plants, particularly the passage of
MLO through sieve pores. Pressure disruption of sieve elements results in plugging
of sieve pores with callose, p-protein, cellular debris, and MLO's if they are present.
Micrographs of MLO squeezing through callose occluded sieve pores are simple artifacts
of fixation. Most MLO should be able to pass through "functioning" sieve plate pores
without restriction, moving in the sieve tube flow.

MLO in plants may be spiral, filamentous, beaded, or roundish polymorphic bodies, 0.3
to 1.2 um in diameter. Deviating morphotypes including "elementary bodies" are evi-
dence for older infections and a degenerating cultural environment.

MECHANISMS OF PATHOGENICITY OF PLANT MYCOPLASMAS. M. J. Daniels, John Innes Institute,
Colney Lane, Norwich NR4 70H, England. To investigate the mode of action of a
pathogen on a plant it is essential to be able to study the properties of the parasite
and the host in isolation and the interaction between them. In the case of the yellows
group of diseases caused by mycoplasma-like organisms choice is limited to a handful of
diseases caused by spiroplasmas, since only these pathogens have so far been cultured
in vitro. Most work has been carried out with Spiroplasma citri, the causal agent of
citrus stubborn disease, which in many herbaceous plants causes a sudden wilt and death.
Evidence will be presented showing that S. citri produces phytotoxins which may account
for some of the observed symptoms of infected plants. Methods for detection and assay
of the toxins will be described. The corn stunt spiroplasma does not cause wilting of
infected plants, and preliminary results indicate that it does not produce a similar
toxin. Other possible mechanisms of action were briefly mentioned.

NEW FINDINGS IN THE BIOLOGY OF PLANT YELLOWS DISEASES. Karl Maramorosch, Waksman
Institute of Microbiology, Rutgers The State University, P. O. Box 759, Piscataway,
N. J. 08854. The recognition in 1967 that plant yellows diseases are associated with
wall-less prokaryotes has resulted in (1) extensive studies of the morphology of myco-
plasma-like organisms (MLO) in plant and vector tissues in situ; (2) in chemotherapy
of yellows-type diseases, and (3) in the cultivation of several isolates belonging to
the new family of Mollicutes, Spiroplasmataceae. Apart from the wall-less MLO, two
types of walled microorganisms, sometimes called rickettsia-like (RLO) have been de-
tected either in the phloem, or in the xylem of diseased plants.


1









During the past two years considerable progress has been made in several laboratories
where spiroplasmas:have been studied. It was found that spiroplasmas can cause diseases
of plants, invertebrates, and vertebrates. The cultured microorganisms have been com-
pared by serological and chemical means and their antibiotic spectrum determined. Re-
cently spiroplasmas have been isolated from numerous species of naturally infected
plants in New Jersey, the Washington, D. C. area, and California. In 1975 we have
isolated a spiroplasma from plants naturally infected with aster yellows in New Jersey.
The culture has been deposited in the American Type Culture Collection and made avail-
able to colleagues in Denmark and England. The aster yellows spiroplasma resembles
serologically the spiroplasma isolated from the cactus Opuntia tuna monstrosa ( S.
kunkelii ATCC 2954) leading to the speculation that the ornamental cactus might have
been infected with a strain of aster yellows. Both isolates appear constantly asso-
ciated with a rod-shaped virus. No spiral forms have been detected by darkfield
microscopy in crude plant sap and electron microscopy of thin sections only rarely
gave an indication of spirals in diseased tissues. We now postulate the working
hypothesis that other MLO diseases, including lethal yellowing of palms, might be
caused by spiroplasmas.

SUMMARY T. A. Chen, Rapporteur. During the last ten years the study of plant yellows
disease has taken a giant step forward. Plant pathologists now recognize that, beside
fungi, bacteria viruses, and nematodes, mycoplasmas and rickettsialike prokaryotes
are also involved in plant diseases. There are over 100 different plant diseases
suspected to be caused by such agents. As Dr. Moore also mentioned this morning, of
the 15 major groups of palms at least seven are known to be susceptible to lethal
yellowing. Except for the citrus stubborn disease and the corn stunt disease, evidence
for mycoplasma etiology of plant yellows disease is provided only by the resemblance of
the associated agents to mycoplasmas through electron microscopy and by their disap-
pearance upon tetracycline treatments.

The successful isolation and cultivation of spiroplasmas from stubborn infected citrus
trees and stunt infected corn plants, and subsequent demonstration of their pathogeni-
city, have not only established that the spiroplasma is a new plant pathogen, but the
spiroplasma is also recognized as a newly discovered microorganism. As a result, a
new family, Spiroplasmataceae, and a new genus, Spiroplasma, were created in the class
Mollicutes.

Just like many other scientific achievements, the discovery of plant MLO was made acci-
dentally. If it were not for a veterinarian pointing out the presence of MLO in the
dwarf mulberry plant tissues, we plant pathologists might still be looking for viruses
in the yellows diseases today. After the discovery, attempts have been made in many
laboratories around the world to isolate and cultivate these agents. Some claims prove
to be mistaken and otherscannot be repeated. Up to date spiroplasmas are the only forms
to be isolated and culturedin vitro. And Spiroplasma citri and corn stunt spiroplasma
are the only isolates proved to be plant pathogens. Although Koch's postulates have
not been fulfilled for all other yellows diseases, it is evident that the diversity of
prokaryotes associated with plant yellows diseases is indeed very wide.

Unlike animal and human mycoplasmas, the spiroplasmas and most other MLO's associated
with the yellows diseases of plants are intracellular pathogens. They are transmitted
from plant host to host in nature by insects and multiply both in the plant and insect
cells and tissues. In order to produce a disease syndrome, a three-way interaction is
necessary in which host plants, specific insect vectors and the mycoplasma must all
play an important role. Disease of a plant may be observed, MLO may be detected in









in such plants and, in some cases, spiroplasma may even be isolated and cultured.
Yet, if a vector cannot be found, the study of such a disease would be incomplete.
This missing link would not only make it difficult to study the etiology of the
disease, but also render it almost impossible to prevent the spread of the disease.

Mycoplasma are known to exert a myriad of effects on animals cells. Just to give
you a few examples: they can deplete essential arginine from animal cell cultures;
they are capable of stimulating nucleic acid synthesis; certain mycoplasmas can alter
the morphology of host cells and cause chromosome aberrations; some of them can pro-
duce peroxides which lead to the hemolysis of erythroceytes; some species can enhance
or inhibit the replication of animal viruses; and it has also been demonstrated that
mycoplasmas can induce the production of interferon in animal cells. The yellows
diseases are characterized by the general chlorosis of the plant, dwarfing or stunting,
the growth of adventitious shoots, stimulated growth of dormant buds, virescence of
flowers, sterility of reproductive organs and the abnormal witches-broom growth habit.
As far as we know, plant spiroplasmas and MLO's are the inhabitants of phloem tissues
and generally they are restricted in their plant habitat, to exert such profound effects
to the entire plant. It is especially interesting to note that in the case of lethal
yellowing, very small numbers of MLO can be found in the phloem. If they are indeed
the causal agent how can they produce such profound effects in such a large plant? As
Dr. Daniels mentioned today, toxins of low molecular weight produced by MLO may be one
of the answers. Plant growth regulators are also suggested as the possible pathogenic
substances produced by the yellows agents. However, all of these hypotheses need
further work.

In the recent development of MLO research new spiroplasmas have been isolated and cul-
tured from plants such as cactus, bermudagrass, and, as Dr. Maramorosch just mentioned,
lettuce. In addition, it was also found that spiroplasmas can cause diseases of honey-
bees and mammals such as mice and rabbits. These reports have placed spiroplasma in a
new prospective. However, it is very dangerous to generalize today that all yellows
diseases are caused by spiroplasmas. We must treat these unsolved yellows problems with
an open mind and not be discouraged by occasional failures. As Dr. Tully mentioned,
Mycoplasma pneumoniae can be isolated and cultured much more efficiently in SP-4 medium
than the conventional regular medium which has been used for culture of this mycoplasma
for many, many years. That means that our knowledge on the nutrition of mycoplasma as
a whole is still very limited. By improving our technique and by gaining more knowledge
about the nutritional requirements of mycoplasmas, I am sure that some day, and I hope it
is in the near future, those other MLO's can be isolated and cultured. I therefore,
share with all the other speakers today in applauding ICLY, through which we have the
opportunity to get together to exchange our knowledge, and we hope that all the contri-
butions made here will have very fruitful results in the near future.









SESSION II DISEASE DIAGNOSIS

Chairman: D. L. Thomas Rapporteurs: R. E. Davis & H. Waters

INTRODUCTION. Research on LY has been severely hampered by the inability to culture the
pathogen or transmit the disease. However, there is an even more basic deficiency in
our knowledge of LY the inability to make a rapid, simple and positive diagnosis of
the disease. For LY determination in coconut, we rely on symptomatology and the
laborious process of electron microscopy. Even with this, there is no certainty that
the LY-like diseases of coconut in Africa are indeed LY, nor is their even complete
assurance that LY in Jamaica is the same as that in Florida. Palm species other than
coconut have been reported as suspected hosts of LY but final proof is still lacking.
There is much interest in searching for herbaceous alternate hosts of LY, but it is
speculative as to how LY infection would be determined in these plants. Faster and
more precise methods of LY identification, be they physiological, serological or
microscopic, would be of immense value in accelerating LY research both in the field
and in the laboratory.

KAINCOPE DISEASE AND KRIBI DISEASE, TWO WEST AFRICAN COCONUT YELLOWS ASSOCIATED WITH
THE PRESENCE OF MYCOPLASMA-LIKE ORGANISMS. M. Dollet, Virology Department, I.R.H.O.
Compared Pathology Research Station, 30380 ST. CHRISTOL-LES-ALES, France. In West
Africa there are two lethal yellowing diseases of coconut, each found within well-
defined geographical limits: Kaincope disease in Togo and Ghana, and Kribi disease
in the South of the Cameroons. These two disease zones are about 1500 km apart, and
differ quite considerably in their climatic regime and ecological conditions. In
1975 MLO were discovered by electron microscope in coconuts infected with Kaincope
disease. Along with this study, work was started on Kribi disease, whose development
and epidemology closely approach those of Kaincope. A study of the symptoms showed
that the external ones were the same in both cases: premature nut fall, yellowing
and drying of the leaves, slowing up of growth and rapid death of the tree. In both
cases, as soon as the first outward signs of the disease appear, serious pathological
anomalies are noted in the young inflorescences still enclosed in the spathes: yel-
lowing then browning of the male flowers followed by necrosis of the inflorescence.
Sometimes, light blackening of the young ovules in these inflorescences can be seen,
and the endocarp of the little nuts also takes on a grey colour.

The study by electron microscope of samples of inflorescences from the Kribi region
revealed MLO, elements surrounded by a trilamellate unitary membrane, the cytoplasmic
content with ribosomic granules and nuclear fibrilla of DNA. They are of variable form
and certain figures can be interpreted as phases of multiplication by budding or of
division. They have been found in diseased TypicalTall and Cameroon Red Dwarf coconuts.
In the present state of our observations, it seems that the MLO are more numerous in
coconuts infected with Kribi disease than in those with Kaincope where, on the contrary,
we find many more plastids containing particles of phytoferritin The long leaps made
by the disease, leaving islets of coconuts untouched, remain an epidemological mystery,
like the resistance of the Benin coconut groves to Kaincope disease.

FURTHER STUDIES ON ROOT SYMPTOMS OF LETHAL YELLOWING. S. J. Eden-Green, Coconut Industry
Board, Box 204, Kingston 10, Jamaica. Surface roots were induced by banking moist coir
dust around the boles of over 100 apparently healthy coconut palms growing in a diseased
area in St. Thomas, Jamaica. Aerial symptoms in palms that developed lethal yellowing
were recorded weekly and roots were examined by removing samples at one or two week in-
tervals. A preliminary analysis of data from 24 palms which developed the disease showed
a close correlation between the onset of progressive frond yellowing, which characterizes
the rapid decline of diseased palms, and the development of symptoms in the root system
studied.








IS COCONUT THE PRIMARY HOST OF LETHAL YELLOWING DISEASE? H. C. Harries, Coconut
Industry Board, P. 0. Box 204, Kingston 10, Jamaica. In addition to Cocos nucifera,
twenty-one palm species are listed as susceptible to LY. Are they all as susceptible
as the 'Jamaica Tall'? This variety of coconut represents the original type that
probably evolved on isolated islands, possibly in the southwest Pacific or Indian
Oceans. Are the other palm species from similar origins also highly susceptible?
Can any of the species listed be considered resistant in the same way that the
'Malayan Dwarf' coconut is resistant because very few die? Malayan and other con-
tinental southeast Asian coconuts represent types that were selected under culti-
vation. Disease resistance appears to have been a criterion for selection. Do palm
species from continental Asia show less susceptibility than those from the islands?

If an MLO disease did occur in Asia perhaps it still does. It would be a minor disease
of limited extent because of resistance and perhaps because of natural control of the
vector. Symptoms could be easily overlooked. In the Caribbean and West Africa lethal
yellowing is obvious because the original source of susceptible germplasm was very re-
stricted yet the area planted was very extensive. An ideal situation for an epidemic
as cereal breeders well know. Coconuts were introduced into the Western Hemisphere
nearly 500 years ago and they established widely and quickly. This would not have been
so if a lethal disease were present. If the first report of the disease, by the Marquis
of Sligo in Grand Cayman in 1834, is correct this is ten years before coconuts became
commercially important in the Far East and seventy years before commercial interest
followed in the Caribbean. Disease would therefore have been less likely to arrive
on coconut planting material than on some other palm or, more probably, on another crop
and particularly one that is not seed propagated.

POSSIBLE INTERACTION BETWEEN LETHAL YELLOWING AND BUD ROT? H. C. Harries, Coconut
Industry Board, P. O. Box 204, Kingston 10, Jamaica. Bud rot occurs sporadically and
is more common in some locations than in others. It is of minor importance, in com-
parison with LY and although control with fungicide is possible, it may not be economic.
The 'Malayan Dwarf' appears to be more susceptible to bud rot and, as more are planted,
the disease might become more troublesome.

Both diseases occur in a hybrid seed garden, with the uncommon arrangement of 357
yellow 'Malayan Dwarf' interplanted with 39 tall x tall F1 hybrids, surrounded on all
sides by a further 362 such hybrids. By 1975 losses to LYoccurred almost entirely
amongst the tall hybrids, both those interplanted and at all points on the perimeter;
22% in comparison with 1% in the dwarf plot. The situation is reversed for bud rot;
21% in the dwarfs compared to 3% in the hybrids.

The symptoms of bud rot and LY in bearing palms are quite distinct. However, when
attacks are co-incidental, a possible interaction between the varietal susceptibility
to the two diseases might occur since both diseases ultimately kill the palm by des-
troying the growing point. In areas of active LY, all palms are exposed to the vector.
Under high infection pressure the dwarf palms and possibly many of the hybrids have a
degree of resistance that normally ensures survival. If, however, the palm is simul-
taneously attacked by the bud rot organism, it may be less able to survive the joint
attack. The development of symptoms will reflect the resistance or susceptibility to
each disease; amongst LY susceptible palms, LY symptoms will predominate but LY re-
sistant palms will show bud rot symptoms.

NATURAL SYMPTOM REMISSION AND TOTAL RECOVERY AFTER LETHAL YELLOWING INFECTION. H. C.
Harries, Coconut Industry Board, P. O. Box 204, Kingston 10, Jamaica. In 1974 natural
symptom remission was identified on 34 palms that had survived up to five years after LY
was first seen. MLOs were found (in one sampled) but the final cause of death could not









be simply attributed to any single factor. It is now believed that a case of
complete recovery has been recorded. During a routine inspection in May 1976,
a 'Jamaica Tall x Rennell Tall' F1 hybrid, planted in January 1966, was found to
have LY symptoms. No symptom had been observed at earlier inspections. Closer
examination in November showed no leaf yellowing, no spear leaf necrosis, a recently-
opened, normal inflorescence, developing fruit on bunches ? and 3 and maturing fruit
on four of the older bunches. But bunches 4, 7, 9 and 10 had no set nuts; only the
blackening and drooping branchlets that distinguish lethal yellowing. The palm was
inspected by members of the Board's Research Department, the ODM Lethal Yellowing
Team and the U.W.I. Botany Department. The appearance was considered consistent
with unilateral symptoms of LY. Young, unopened inflorescences that were removed
appeared normal, and electron microscopy did not reveal MLO in them. The JT x RT
hybrid has lost between 57 and 92% of the palm here and other sites. If this repre-
sents infections and recovery such events must be rare. The low frequency of such
an event is masked by the difficulty of finding any conclusive symptoms because the
palms appear healthy. The converse, a dying 'Malayan Dwarf' stands out like a sore
thumb. Moreover, symptoms might not recur. If the chances of resistance being over-
come once are small then twice might be negligible. This palm may not only therefore
be expected to survive, it might also be considered to have resistance approaching
that of the dwarf, and will be watched carefully. If MLO diseases occur in countries
where varieties are predominantly not highly susceptible this situation may be fre-
quent but unnoticed.

AN INVESTIGATION OF XYLEM PRESSURE IN COCONUT PALMS WITH REFERENCE TO LETHAL YELLOWING.
J. McDonough and M. H. Zimmermann, Harvard University Forest, Petersham, Massachusetts
01366. The Scholander pressure chamber was used to measure diurnal cycles of xylem
pressures in coconut palms during Florida's dry season (Oct. to March). Healthy trees
showed lowest pressures shortly before noon. In LY diseased trees diurnal pressure
fluctuations were much less pronounced and midday minima were higher. This pattern
indicates decreased transpiration (probably closed stomata). It was observed at least
two weeks prior to development of symptoms currently used to diagnose LY. Xylem-pressure
measurements may thus be useful as an early disease detection device.

In contrast, measurements on a coconut of the 95% resistant Dwarf Malay variety with
symptoms of wilt showed lower than normal xylem pressures throughout the day. This
indicated that the disorder was not due to LY but resulted from excessive water loss,
a symptom associated with wilt disease.

ELECTRON MICROSCOPY OF PALM SPECIES WITH SUSPECTED LETHAL YELLOWING. D. L. Thomas,
Agricultural Research Center, University of Florida, 3205 S.W. 70 Ave., Fort Lauderdale,
Florida 33314. Members of several palm species within heavily infected LY areas of
south Florida have died with a decline similar to LY. Tissue samples from young, un-
emerged frond bases of declining palms were surveyed electron microscopically for the
presence of MLO because LY infection was suspected. MLO were observed in the mature
sieve elements of declining members of Allogoptera arenaria, Arenga engleri, Arikuryroba
schizophylla, Borassus flabellifer, Caryota mitis, Chrysalidocarpus cabadae, Corypha
elata, Dictyosperma album, Gaussia attenuata, Hyophorbe verschaffeltii, Latania sp.,
Livistona chinensis, Nannorrhops ritchieana, Phoenix canariensis, P. dactylifera, P.
reclinata, P. sylvestris, Pritchardia affinis and Trachycarpus fortune. MLO were not
found in 15 healthy control palms. The MLO were unevenly distributed in the majority
of the palms. Over 50 percent of the sieve elements in some species contained MLO while
other species had MLO in less than 1% of their sieve elements. In some sieve elements
the MLO were confined to areas near the cell wall but more frequently they completely









filled the cell lumen. Their shape varied from almost spherical to filamentous.
When MLO were in high concentrations they often were embedded in an electron-dense
matrix of unknown composition. The constant association of MLO with declining members
of the above species and the absence of MLO in healthy controls suggest that these
various palm declines are identical to LY. Final proof must still await transmission
studies.

PRELIMINARY STUDIES UPON LETHAL YELLOWING AND THE DISTRIBUTION OF MLO IN COCONUT PALMS.
H. Waters and I. Osborne, ODM Lethal Yellowing Research Team, P. 0. Box 204, Kingston
10, Jamaica. Coconut Industry Board, P. O. Box 204, Kingston 10, Jamaica. In LY-infected
coconut palms, MLO are found in spear leaves, roots and inflorescences but only when the
distal parts of these organs are necrosed. MLO's have also been detected in flag leaves
but appear to be restricted to the yellow regions. Quantitative estimates of MLO in-
cidence in these sites are presented. These preliminary findings are discussed in re-
lation to the accessibility of MLO to insect vectors and the implications for research
workers attempting culture of the MLO.

IN VIVO MLO MORPHOLOGY BY SERIAL SECTIONING OF LETHAL YELLOWING-INFECTED COCONUT.
H. Waters and P. Hunt, ODM Lethal Yellowing Research Team, P. 0. Box 204, Kingston 10,
Jamacia, and Botany Department, University of the West Indies, Kingston 7, Jamaica.
An analysis of serial sections of sieve elements from roots of LY-affected palms re-
vealed that MLO could be grouped on the basis of their three dimensional structure
into five classes. Among filamentous types, we recognized filiform, cylindrical and
catenulate forms; among irregularly lobed types, saccate and erythrocyte-like forms
were recognized. Branching may occur in all but the saccate forms. It is unlikely
that different morphologies are the result of fixation or other artefacts since all
forms may be found in a single sieve element.

PROFILES OF MLO BASED UPON AN UNDERSTANDING OF KNOWN MORPHOLOGIES. H. Waters and
P. Hunt. ODM Lethal Yellowing Research Team, P. O. Box 204, Kingston 10, Jamaica,
and Botany Department, University of the West Indies, Kingston, 7, Jamaica. In
certain instances even single MLB profiles can be interpreted as diagnostic of
morphology. A section through the branch point of a filiform organism presents a
unique profile and this is also true of a section across the isthmus of a strongly
catenulate organism. The dumbbell-shaped profile is characteristic of erythrocytic
forms and also the branching of cylindrical organisms. Only rarely do circular pro-
files reflect a spherical morphology. An understanding of morphology allows a more
critical assessment of previously published interpretations of MLO profiles. This is
particularly important where profiles have been interpreted as indicative of repro-
duction.

A MODEL OF MLO DISTRIBUTION WITHIN A SIEVE ELEMENT. P. Hunt and H. Waters. Botany
Department, University of the West Indies, Kingston 7, Jamaica and ODM Lethal Yellowing
Research Team, P. O. Box 204, Kingston 10, Jamaica. A three dimensional reconstruction
of all the MLO present in ten serial transverse sections of a sieve element revealed
patterns of distribution related to known morphologies.

IMPLICATIONS OF ELUCIDATING MLO MORPHOLOGY IN SIEVE TUBES. P. Hunt and H. Waters.
Botany Department, University of the West Indies, Kingston 7, Jamaica and ODM Lethal
Yellowing Research Team, P. O. Box 204, Kingston 10, Jamaica. The implications of
understanding the morphologies of whole MLO in diseased coconut roots are discussed
in relation to the following points:









(i) Are the observed profiles of MLO in other parts of diseased palms and in
other diseases consistent with the morphologies demonstrated in roots?

(ii) How do these morphologies relate to those described for culturable
Mycoplasmas?

(iii) Do the different morphologies represent different phases in the growth
of MLO and their colonisation of a sieve tube? If so, what is the sequence and which
forms (if any of those that we have described) are best suited for migration within
the palm or for acquisition by insects?

(iv) What are the size ranges of the MLO's? What are the smallest and mean
volumes? How many organisms are present in 1 cm3 of infected tissue and how many
might we expect to extract or obtain in a toddy?

SEROLOGY: PREPARATION OF ANTISERA FROM INFECTED HOST ANTIGEN. R. F. Whitcomb,
USDA, ARS, Beltsville, MD, 20705, USA. Antisera against wall-free prokaryotes
(mycoplasmas) are best prepared against cultivated microorganisms. Many diseases,
however, are incited by mycoplasmas which have resisted cultivation attempts.
Antisera against such agents, if they can be prepared at all, must be directed
against organisms isolated from diseased plant or insect material. Antisera pre-
viously prepared against corn stunt organisms from diseased corn can now be shown
to have high activity in growth inhibition, deformation, and ring precipitin tests
against cultivated microorganisms. This retrospective demonstration suggests that
preparation of antisera against "noncultivable" organisms is feasible, and offers
the hope of disease diagnosis, even if the biological agent cannot be cultivated in
cell-free media.

SUMMARY H. Waters, Rapporteur. These comments are to be considered in conjunction
with the abstracts provided by the various authors to summarize what we felt were the
main points of the reports we had today. M. Dollet reported that Kribi and Kaincope
were symptomologically related and the symptoms are similar to lethal yellowing. My-
coplasma organisms are associated with Kribi and Kaincope.

Dr. Eden-Green demonstrated that there was a correlation between the symptoms of
leaves and the onset and progression of root death. We suggest that the roots pro-
vide a good site for the nondestructive sampling of infected tissue with time.

Mr. Harries asked the question, "Is Coconut the Primary Host of Lethal Yellowing
Diseases?'. The conclusion arrived at was that coconut is not the primary host, but
rather that lethal yellowing has followed the introduction of the coconut to different
parts of the world. The conclusion to his second paper was that it is possible that
infections by the bud rot pathogen may predispose a palm to lethal yellowing if the
pathogen is available in the area and the converse may also be true. In answer to
his third question it appears that occasional examples of natural remission from LY
can occur and perhaps the "escapes" that one sees, especially in Jamaica, represent
naturally remitted palms.

Ms. McDonough discussed xylem pressure in relation to LY and suggested that these could
be used to identify LY palms two or hopefully more weeks prior to symptom expression.
The lethal yellow affected palms have higher pressures, about five bars compared with
15 bars in healthy palms. We thoughtthat perhaps a comparison between stomatal behavior
in relation to these pressure differences may provide a useful technique for the pre-
symptom diagnosis of LY.









Darryl Thomas indicated the wide palm host range for MLO and highlighted the obser-
vation of electron dense matrices within which MLO appeared to be embedded. The
nature of the matrices varied both within and between species. Clearly if his cor-
relation between MLO's and the dense matrix could be confirmed and correlated with
the appearance of MLO then perhaps we have a method whereby we can screen some palms
for MLO's by light microscopy without going through the complexities of EM work.

SUMMARY R. E. Davis, Rapporteur. The reports by Drs. Waters and Hunt focused on the
morphology of mycoplasmalike organisms (MLO) associated with lethal yellowing disease.
These very extensive studies have some important implications for interpretations of
bodies often previously thought to represent dividing forms of MLO. However, there
may be a need for rigorous examination of effects of various physical factors on the
morphology of MLO in vivo. Such factors might involve osmolarity of fixatives, for
instance. In any case, the work of Drs. Waters and Hunt leaves no doubt about the
highly pleomorphic nature of the plant inhabiting MLO.

The question of mode of passage of MLO through sieve pores is an important one, but
perhaps not an exceedingly difficult one. The plant-inhabiting MLO may fragment
or in fact may elongate and actually grow through sieve pores. Moreover, the complex
forms described by Drs. Waters and Hunt might represent advance stages of cell infec-
tion. A further point raised in the discussion suggested the possibility of two
different morphological populations in LY-infected plants.

We think it has been appropriate to begin the program by a session on diagnosis of
disease. Part of the discussion questioned the importance of diagnosis of lethal
yellowing disease. However, I think we agree that correct diagnosis will be a neces-
sary prelude to successful control of lethal yellowing. Perhaps to the trained eye,
unequivocal diagnosis of lethal yellowing is already a practical reality. But as a
novice to palm pathology, I have the impression that correct diagnosis, and particu-
larly early diagnosis of this disease, seems not to be an easy task. We recognize
that vastly different causal agents can induce similar symptoms in a given plant.
For example, we now know that the complex long diagnosed as corn stunt based on
symptomotology is really a complex of three diseases caused fundamentally by different
agents. These include a spiroplasma, an apparently nonhelical mycoplasmalike organism,
and a virus. The geographical ranges of these agents overlap and two or three different
agents could presumably be found in a single naturally infected plant. Could similar
circumstances complicate resolution of the lethal yellowing problem?

Areas for increased research effort should include cultivation in vitro. Even though
the concentration of mycoplasmalike organisms might be somewhat lower in roots than
other parts of the LY-infected plant, roots may well provide a useful source of MLO
for culture. In some plants there may be important differences in metabolic products
accumulated in leaves and roots. Could such products influence the success of estab-
lishing in vitro cultures? Finally, might xylem sap be used in development of an in
vitro assay for LY based on stomatal closure?









SESSION III MYCOPLASMA ISOLATION & CULTURE

Chairman: R. E. McCoy Rapporteurs: J. G. Tully & I. Osborne

INTRODUCTION. The lack of identification of the causal agent of LY underscores our
basic need to isolate or cultivate this organism. An understanding of the taxonomic
status of the LY pathogen and its relationship to other similar organisms is one aspect
by which we would gain if it were cultured. More importantly, cultivation of this
organism would provide us a tool for the indepth study of the biology of this enigmatic
disease for the evaluation of control agents, and, in addition, would greatly assist
the search for a vector. This problem is not limited to LY, of course, but is common
to the majority of MLO associated plant diseases. The isolation and culture of these
agents is a basic need in the field of plant pathology that is only beginning to be
met since the culture of the spiroplasmas causing corn stunt and citrus stubborn disease:
The report on the aster yellows agent is of particular significance here also. Progress
in the culture of plant MLO's is painfully slow and difficult. However, as seen in
the following abstracts, the challenge is being met.

DEVELOPMENT OF BIOCHEMICAL METHODS FOR DETECTION OF MYCOPLASMAS. H. G. Basham,
Agricultural Research Center, University of Florida, 3205 S. W. 70 Ave., Ft. Lauderdale,
Florida 33314. One of the major impediments to progress in research of the lethal yel-
lowing disease of palms is the lack of a relatively simple and rapid assay for the
presence of mycoplasma-like-organisms (MLO) in culture and in diseased plant and insect
tissue. Although there are a number of techniques available, most fail to meet the
above criteria because (1) they are too slow, (2) they depend upon detection of a pro-
duct not uniquely associated with MLO, or (3) they are not sensitive enough to handle
the small concentrations of MLO in diseased palms. Initial efforts to develop a suitable
assay for MLO in this laboratory have included attempts to detect enzymes uniquely as-
sociated with tissues containing MLO, serology, and detection of unique species of
ribonucleic acid (RNA) in diseased palms. In all cases, no product or activity which
could be directly associated with the presence of MLO was found. This may have been
due to low titers of MLO in diseased tissue and/or oxidative reactions initiated when
tissue extracts are made from palms. Work in the RNA system is continuing.

The procedure which shows the most promise to date is based on an assay for production
of H202 by microorganisms (Cohen & Somerson; J. Bacteriol.: 98, 543; 1969). Hydrogen
peroxide production by flavoprotein oxidases at the end of the electron transport chain
is a common process in many MLO. Such H202 production is not knownto occur in plant or
animal tissues. The assay is extremely sensitive and could be made specific for MLO-
generated H202 if appropriate inhibitors can be found. In the assay, MLO preparations
are incubated with catalase (an enzyme which catalyzes the reaction: 2 H202 02 + 2H20)
preparations are incubated with catalase and glucose. The MLO generate H202 as one
of their terminal metabolic products in this system. In the presence of the catalase
inhibitor 3-amino-l,2,4-triazole (AT), a molecule of catalase is irreversibly inhibited
when it complexes with a molecule of H202. In this system then, respiratory activity
of MLO (i.e. generation of H202 is measured as an increasing inhibition with time of
the catalase included in this reaction mixture (RM).

A typical RM contains 1.2 x 103 units catalase, 100 umoles AT, 100 umoles glucose,
ca. 107 organisms which have been washed in phosphate buffered saline (PBS = 0.1 M
phosphate at pH 7.4), all in 2 ml PBS. The RMs are incubated at 30 C. At time zero
and at various time intervals up to 60 min, 0.1 ml aliquots are withdrawn, incubated
for 10 min. in 0.45 M ethanol, then diluted to 10 ml with PBS. Activity of catalase
in the diluted samples is determined with an oxygen electrode by measuring the initial
rate of 02 evolution in 1.2 ml of the diluted sample in the presence of 1.5 umoles









H202 as substrate. Percent inactivation of catalase is determined by comparison
of each rate with that in a matched sample containing heat inactiviated organisms.

Experiments to date, utilizing Acholeplasma laidawii in a mid-log growth phase
(24 hr. 30 C, Razin's medium), show that 5 x 10b organisms/ml RM give 50-70% inhi-
bition in 60 min. Useful range of the assay depends on the amount of catalase in
the RM, but calculations suggest that the assay could detect as few as 10 organisms/ml.
Inhibition is roughly proportional to numbers of organisms, but values are too variable
to serve as an accurate indicator of numbers of organisms.

Preliminary experiments with crude plant extracts were unsuccessful due to catalase
inhibition in controls. Rotenone, which should inhibit H202 production in MLO, is
not utilizable in this system because of its low solubility in the RM. Work is con-
tinuing to overcome these difficulties.

IDENTIFICATION OF MYCOPLASMAS CULTURED FROM PLANTS. M. J. Daniels, John Innes Institute,
Colney Lane NR4 7UH, Norwich, England. On many occasions mycoplasmas have been cultured
from yellows-diseased plants. Two questions have immediately confronted the experi-
menters. 1. Is the cultured organism the aetiological agent of the disease? 2. What
is the relationship of the new isolate to other known mycoplasmas? The first question
is answered by insect/plant inoculations. This paper is concerned with the second pro-
blem. Easily tested properties such as sterol dependence and fermentation reactions
serve to place mycoplasmas in large groups, but further identification demands access
to costly antisera to a large number of reference strains. An alternative approach is
suggested by the observation that gel-electrophoretic patterns of mycoplasma cell pro-
teins in sodium dodecyl sulphate are characteristic of the species and highly repro-
ducible. Experiments will be described aimed at developing a computer-based comparison
of an unknown pattern with previously encountered reference patterns, with a view to
the recognition of a small number of knowimycoplasma strains with patterns similar to
the unknown. The "short list" can then be tested by conventional methods.

CELL WALL-FREE PROKARYOTES HARBORED IN FLOWERS. R. E. Davis, Plant Virology Laboratory,
Plant Protection Institute BARC-West, ARS, U.S.D.A., Beltsville, MD 20705. Studies
on the ecology of cell wall-free prokaryotes revealed their occurrence in flowers of
spring-blooming plants. MLO and spiroplasmas were isolated and cultivated in vitro
from non-surface sterilized floral parts of several species. Tulip tree (Liriodendron
tulipifera L.) flowers were found frequently to harbor MLO or spiroplasmas; however,
neither MLO nor spiroplasmas were detected within tulip tree tissues by ultrathin
section electron microscopy or by attempts to cultivate organisms from surface steri-
lized plant parts. The organisms obtained in culture were apparently harbored on sur-
faces of floral parts. The findings indicate a newly revealed ecological niche for
cell wall-free prokaryotes. The role that nectar may play in the survival and dissemin-
ation of these agents could have important implications for possible modes of trans-
mission of pathogenic MLO and spiroplasmas. In vitro, the spiroplasma isolates from
flowers, as well as several known plant and insect pathogenic spiroplasma strains, ex-
hibited a striking translational locomotion when in contact with a solid surface or
when in media of elevated viscosity (0.8% methyl cellulose, 1500 centipoise). It
would be of interest to explore whether such motility enhances spread of pathogenic
spiroplasmas within susceptible host tissues. Since conditions on some plant surfaces
may favor translational locomotion, one might also inquire whether this motility renders
certain spiroplasmas capable of entering plants in the absence of penetration of host
tissues by insect vectors. The occurrence of cell wall-free prokaryotes in flowers
suggests the possibility of previously unsuspected mechanisms for their transmission.









ATTEMPTS TO EXTRACT AND CULTURE MYCOPLASMAS FROM COCONUT PALMS. S. J. Eden-Green,
Coconut Industry Board, P. O. Box 204, Kingston 10, Jamaica. Following early failure
to isolate mycoplasmas associated with lethal yellowing on conventional media, ex-
traction of MLO was attempted from whole coconut tissues using a freeze-thaw technique.
Electron Microscope (EM) examination of centrifuge pellets from both diseased and
healthy extracts showed large concentrations of vesicular bodies, 0.1-0.8 um dia.,
often enclosing varying amounts of ribosome-like and filamentous material. These
structures were presumably of host origin, and precluded positive identification of
MLO in extracts. EM examination of phloem sap (toddy) from detached crowns of diseased
and healthy "Jamaica Tall" palms showed no consistent differences indicating the pre-
sence of MLB in the former, save for a small sample obtained from one palm in advanced
stages of disease.

Phloem sap collected over a 4-day period supported growth of Acholeplasma laidlawii,
Spiroplasma citri and Mycoplasma fermentans when supplemented with combinations of
serum lipoprotein (serum fraction) and yeast extract. Filter sterilized samples from
both diseased and healthy palms often showed an acid pH drift, associated with
anerobiosis, during incubation, but light microscope observations were not interpreted
as evidence for the growth of MLO. In four notable exceptions, Acholeplasmas, tenta-
tively identified as A. axanthum, were recovered from phloem sap collected simultaneously
from two healthy and two diseased palm hearts. Further attempts to isolate MLO by
inoculating phloem sap-based media with diseased palm tissues gave one further isolate
resembling A. ananthum, but no other MLO. Preliminary studies indicated that these
isolates were not acquired by leafhoppers following membrane feeding, but survived
when injected into insects.

The field collection and comparative yields of phloem sap from inflorescences of
several coconut palm varieties was described.

ATTEMPTS TO CULTURE MYCOPLASMALIKE AGENTS AT FORT LAUDERDALE. R. E. McCoy, Univ. of
Florida, 3205 S.W. 70 Ave., Ft. Lauderdale, FL 33314, USA. Attempts to cultivate
possible mycoplasmal pathogens from plants at Fort Lauderdale have centered around a
coconut phloem sap medium (McCoy, 1976. Proc. Soc. Gen Microbiol 3:155). Although the
corn stunt and citrus stubborn spiroplasmas have been demonstrated to grow well in this
medium, no non-helical mycoplasma has been cultured from plant or vector tissues. Cul-
tures are monitored for growth by light microscopy. If membranous bodies or ghosts are
observed, passage of the culture is attempted. In addition, a small aliquot of broth
may be passed through a cellulose acetate filter of a 0.2 um pore size. The filter is
then embedded and thin-sectioned for electron microscopy. Mycoplasmas may be detected
in this manner and morphologically verified.

The mycoplasmalike organism associated with lethal yellowing has been maintained in
diseased coconut inflorescence pieces grown in a tissue culture medium (Schwabbe et al.
Wye College, University of London) for periods as long as 6 weeks. Presence of MLO's
was verified by electron microscopy of thin sections of inflorescence pieces sampled
from the shaken tissue culture broth. No differences in growth rate have been detected
between healthy and diseased inflorescence pieces grown in this medium, even though
MLO's were detected in sieve-tube elements of diseased tissue pieces.

SUMMARY J. Tully, Rapporteur. In summary of what we have heard this morning, I think
we began by hearing some important new observations on the isolations of mycoplasmas
and spiroplasmas from the tulip tree and I think this has very important implications
for accurate information on the epidemiology of these organisms and their role in plant
diseases. It appears that some of these organisms require cholesterol. Others appear
to be more typically acholeplasmas. There are certainly spiroplasmas on the tulip tree









flower that are apparently related to the honey bee spiroplasma. Some of the
organisms also can grow at 370 C and their rapid growth in culture is also quite
significant. Of course it is very important in these studies to establish the
actual taxonomic species and to be able to get some indication of the other host
distributions. We also heard some of the problems in biochemical methods for the
detection of mycoplasmas in infected plant tissues and these certainly are fought
with difficulties, particularly if you might have bacterial action and mycoplasmas
in the same preparations. The efforts reported with the catalase-hydrogen peroxide
system with purified mycoplasmas I think is a step forward. We also heard work on
the identification of mycoplasmas by polyacrylamide gel systems using a computer-
assisted system although, of course, the ultimate identification of mycoplasmas must
be established through serological means. However, the information presented here
would also suggest the utility of this method as a preliminary step in identification.
We also heard this morning on attempts to extract phloem sap and to use this material
to prepare culture medium for testing infected and healthy palms. Electron microscopy
of the phloem showed mycoplasmalike bodies in both healthy palm tissues, albiet not
in large numbers, and in diseased material. There was important information on the
problems in collection of phloem sap and stated difficulties in the yield of sap from
specific palm varieties and in highly infected trees. The isolates of Acholeplasma
axanthum that were recovered here are interesting and certainly have implications to
interpretation down the road a piece. This early work was from the group in Jamaica.
We have also heard from attempts to culture mycoplasma from the Ft. Lauderdale group
and there are a lot of problems here with low yields of MLO in some of the phloem
tissues cultured. There are advances being reported in tissue culture procedures
to supplement cultural attempts and this looks very hopeful, particularly for as-
saying the antibiotic sensitivity of the infectious agent, temperature requirements
and so forth. And finally we heard also of preliminary studies on the in vitro
stability of the aster yellows agent and the value of an assay technique in exploring
various effects of pH, osmolarity, and the addition of oxidation reduction regulators.
This appears to be a useful means to define the stability of the organism from the
infected insect tissue.

SUMMARY I. Osborne, Rapporteur. In my attempt to follow Dr. Tully's expert summary
of the papers presented on mycoplasma isolation and culture, I would like to make some
comments on my understanding of what has been said this morning. On the question of
MLO detection in palms, we have been presented with a number of potentially very sensi-
tive, rapid methods of detecting the presence of MLO's in suspected palms, palms sus-
pected of being infected with MLO's, by biochemical comparison of these palm tissues
taken from known healthy and diseased palms. It would seem that these techniques
would be of immeasurable value if we are to make meaningful estimations or evaluations
as to whether or not any particular healthy looking palm is indeed healthy, considering
what is believed to be the long incubation period of the lethal yellowing disease. From
what we have seen so far the attempts to isolate the causative agent of the lethal
yellowing disease, they have been largely based on the assumption that the organism
would be readily expelled from the phloem tissues of palms in the phloem exudant. How-
ever, in the light of information presented by Drs. Waters and Hunt on MLO's size, shape,
and distribution, it would seem that these techniques need to be reassessed if we are
to assume that forms described so aptly by Drs. Waters and Hunt are not degenerate forms
or that the virulent forms of the organisms are not vastly different in morphology.
Going back to the attempts at the culturing of unknown LY MLO's, it has been demonstrated
that most known mycoplasmas will grow on palm toddy or supplemented toddy, although
Dr. Tully has pointed out that these mediums may not contain the necessary complement
of steriods required for growing some MLO's. However, from papers presented, it seems
that attempts to culture the unknown palm MLO's have been based on their recovery from
the phloem exudant which again brings to mind the papers of Drs. Waters and Hunt. Per-
haps the successful maintenance of MLO's in infected palm tissue as outlined by Dr. McCoy
lends hope to the possibility that MLO inoculations for culture media can be obtained
through the use of tissue slices.









SESSION IV VECTORS

Chairman: J. H. Tsai Rapporteurs: G. H. Kaloostian & F. M. Eskafi

INTRODUCTION. Since 1955, a number of researchers have been engaged in the search
for a LY vector. Soil and airborne vectors were investigated by earlier workers.
The discovery of MLO in palm tissues affected by LY has simplified the search for
the vector. The current vector research emphasis in both Jamaica and Florida is
placed on investigating and testing the phloem feeders of palms. Various approaches
to testing the suspected vectors are presented in this section.

CURRENT ATTEMPTS TO TRANSMIT LETHAL YELLOWING DISEASE IN JAMAICA. A. J. Dabek, Coconut
Industry Board, P. 0. Box 204, Kingston 10, Jamaica. Despite the trials and tribulation!
of the past, current attempts to transmit the mycoplasmic coconut LY disease in Jamaica
centres on insects, more specifically the Homoptera. In contrast to the emphasis on
members of the palm-feeding Fulgoroidea given between 1972-1976, however, my own search
has been centred on the Cidadelloidea. There are at least 3 reasons for this: (1) the
failure to transmit the disease with Fulgorids (although some palm feeding representa-
tives of this group still remain to be tested); (2) other plant diseases suspected to
be caused by mycoplasmas are usually vectored by the Cicadelloidea; (3) the field rate
of spread of lethal yellowing in Jamaica, at the height of a natural field infestation
(c. 4%/month) could agree with the apparently rare occurrence of the Cicadelloidea on
palms.

On present information, it is impossible to say whether the natural vector is a common
or uncommon species, and possibly more than one species may be involved. Accordingly,
in my preliminary vector experiments, I have selected ubiquitous and abundant species
(Hortensia similis (Walker), Tylozygus fasciatus (Walker)), common but more sporadically
distributed species (Carneocephala sagittifera (Uhler), Chlorotettix sp.) and rare
species (Acinopterus sp (?), Xerophloea sp.?). All are potential vectors since all
occur in diseased areas.

All of the species were given acquisition feeds of 1-7 days on attached and detached
necrotic spear leaves and/or detached rachillae from diseased palms. They were then
transferred to various grasses or parafilm M-covered 5% sucrose solution for periods
which varied from 5-38 days to give any acquired mycoplasma a chance to incubate in
the insect tissues. Finally, the surviving insects were caged on the spear leaves of
young, non-bearing Jamaica Tall test palms and their survival time noted. It is as
yet too early to expect any symptom development. Tests with these and other members
of the Cicadelloidea are continuing.

(? = identification yet to be confirmed by an expert).

LETHAL YELLOWING TRANSMISSION TESTS IN JAMAICA, 1975-1977. S. J. Eden-Green, Coconut
Industry Board, Box 204, Kingston 10, Jamaica. Root acquisition feeding transmission
tests with Haplaxius sp. (Cixiidae) and direct introductions of Proarna hilaris
(Cicadidae) were concluded with the transfer of almost 900 adult Haplaxius to 7 test
palms and 2460 adult Proarna to some 38 test palms. No transmissions were noted and
can now be expected.

In subsequent work, mainly with Cicadelloidea, two series of field acquisition tests
were carried out by enclosing insects on either green (series I) or yellow (series II)
attached fronds of diseased palms for 3-4 days and incubating for 10-14 days on grasses
before transfer to test palms. Species tested and transferred, each to 5 test palms,
were: Chlorotettix spp. (a mixture of C. minimus + C. viridius; 274 in ser. I, 169 in
ser. II); Graminella spp. (20 in ser. II); Hortensia similis (30 in ser. I) and









Metascarta histrio (39 in ser. I). Tests with C. nigromaculatus, Protalebrella
braziliensis and Sanctanus fasciatus were attempted but the insects failed to
survive to transfer. In addition, Haplaxius adults were given field acquisition
feeds in series II and 485 were transferred to 5 test palms where many survived
for over two weeks in cages allowing access to pulvinus tissues.

Leafhoppers were also injected with palm extracts though likely to contain LY MLO
and allowed similar incubation and transmission feeds. "Inoculum" consisted of
fresh droplets of sap squeezed from the freshly cut surface of diseased coconut in-
florescence spike, below necrosed regions (series III), or phloem sap collected from
palms in advanced stages of disease and concentrated by centrifugation (series IV).
Species tested and transferred, each to 5 test palms were: Chlorotettix spp. (120
in ser. III; 31 in ser. IV); Haplaxius sp. (12 adults in ser. IV, injected as nymphs)
and Spangbergiella vulnerata (99 in ser. III).

None of the test palms in series I-IV has shown symptoms of lethal yellowing, only
those in series II and IV are still within the suspected incubation period.

FEEDING SITES OF LEAFHOPPERS AND PLANTHOPPERS ON PLANT TISSUES. J. B. Fisher, Fairchild
Tropical Garden, Miami, Florida 33156 and J. H. Tsai, Agric. Research Center, Univ. of
Fla., Fort Lauderdale, Florida 33314. The mouth parts of the leafhoppers and plant-
hoppers are highly modified for piercing plant tissues and extracting sap for food. At
the same time, they are involved in acquisition and inoculation of plant pathogens.
Haplaxius crudus (Van Duzee), Graminella nigrifrons (Forbes), Macrosteles fascifrons
(Stal), Oncometopia nigricans (Walker), Peregrinus maidis (Ashmead), and Spangbergiella
sp. are considered to be possible vectors of lethal yellowing of coconut palms. These
insects are currently used in various transmission trials. Their feeding sites are of
particular significance to vector studies because the lethal yellowing causal agent is a
phloem-restricted prokaryote. Test insects were caged in a small area of coconut leaves
and roots. Plant tissues were fixed and serially sectioned. The salivary sheaths left
behind by the insects were deeply stained with safranin (counter stained with fast green).
Test probings and stylet penetrations were examined under a light microscope. Approxi-
mately 5% of over two thousand serial sections were found to contain various lengths of
salivary sheaths.

The stylet penetrations were intercellular and intracellular. The formations of salivary
flange were often noted in short probes. Occasionally, phloem feeders fed on xylem.
Xylem feeders also probed on phloem. Many branched salivary sheaths were observed in
the sections. Stylet penetrations appeared to be at random as evidenced by salivary
sheaths directing opposite to vascular bundles.

Insect Plant/Organ Tissue feed upon

Haplaxius crudus (nymphs) Coconut/root phloem ( xylem)
Haplaxius crudus Coconut/leaf phloem
Graminella nigrifrons Coconut/leaf phloem
Macrosteles fascifrons Coconut/leaf phloem
Oncometopia nigricans Coconut/leaf xylem (+ phloem)
Peregrinus maidis Coconut/leaf phloem
Spangbergiella sp. Coconut/leaf phloem ( xylem)
Spangbergiella sp. St. Augustine grass/leaf phloem ( xylem)









SURVEY OF HOMOPTERA ASSOCIATED WITH PALMS IN FLORIDA. F. W. Howard, Agricultural
Research Center, University of Florida, 3205 S.W. 70 Ave., Ft. Lauderdale, Florida
33314. Auchenorrhyncha leafhopperss sensu lato) were collected from palms in Florida
with the objective of identifying a possible vector of LY, which attacks coconut palms,
Cocos nucifera L., and apparently 23 other species of palms. Collections were made from
350 palms of 40 different species distributed in numerous localities, mostly in the LY-
infected area of southern Florida. Data comparing the relative abundance of species of
Auchenorrhyncha were obtained by sampling from 60 Manila palms, Veitchia merrillii
(Beccari) H.E. Moore, in an area where LY infection progressed throughout a 10-month
study period.

Haplaxius crudus (Van Duzee) (Cixiidae) appears most likely to be the vector of the LY
pathogen, based on its distribution, frequency in collections, apparent host range,
feeding behavior, and phyletic relationships with vectors of similar disease organisms.
Cedusa sp. (Derbidae) and Idioderma virescens Van Duzee, (Membracidae), were collected
less frequently on palms. Species rarely collected on palms were Omolicna sp. (Derbidae)
Bothriocera sp., Nymphocixia vanduzeei Muir (Cixiidae), Sogatella kolophon meridiana
(Beamer), Chloriona slossoni (Ball) (Delphacidae), Clastoptera undulata Uhler (Cercopidae
Protalebrella braziliensis (Baker), Stragenia robusta (Uhler), Empoasca sp., Rabela
tabebuiae (Dozier), Chlorotettix minimum Baker, an unidentified species of Typhlocybinae
and an unidentified species of Deltocephalinae (Cicadellidae), and Ormenaria rufifascia
(Walker) (Flatidae).

VECTOR STUDIES IN FLORIDA. J. H. Tsai, Agric. Res. Center, University of Florida, 3205
SW 70 Ave., Fort Lauderdale, Fla., 33314. Attempts to transmit LY were based on the
premise that the pathogen is an MLO. The emphasis in the vector studies was placed on
Haplaxius crudus (Van Duzee) (Homoptera: Auchenorrhyncha). Besides direct collecting
and feeding of H. crudus from diseased palms to healthy palms, partially purified and
crude phloem sap from diseased palms was artificially fed and also injected by needle
into planthoppers. The binomics of this insect has been studied both in field and labor-
atory conditions. The longevity study of H. crudus was extended to several palm species
(Arenga engleri, Arikuryroba schyzophylla, Caryota mitis, Chrysalidocarpus cabadae,
Livistona chinensis, Phoenix dactylifera, Phoenix reclinata, Pritchardia affinis, and
Pritchardia remota which appear to be susceptible to lethal yellowing.

An intermediate insect was employed in hopes of building the titre of LY causal agents.
The partially purified sap and extract of ground meristematic tissues of diseased palms
was injected into Macrosteles fascifrons (Stal) and Graminella spp. Later the inocula
from the ground insects were injected into H. crudus.

Attempts were also made to transmit LY from diseased to healthy palms by either feeding
or injecting M. fascifrons and Graminella spp. Other plants such as aster, periwinkle,
celery and corn were tested as possible alternate hosts for an LY causal agent. The
longevity of Agallia albidula (Uhler), Draeculacephala sp., Spangbergiella sp., Prosapia
bicincta (Say) (Cercopidae), and Cedusa sp. (Derbidae) was also studied on Cocos nucifera
and Veitchia merrillii.

A preliminary result obtained from one experiment showed that the removal of the unopened
inflorescences from Veitchia palms has reduced the % of trees succumbing to LY over a
14-month period. It appears that the inflorescence visiting insects could be involved
in transmission of LY agents.









STRATEGIES OF DISEASE VECTORS AND THEIR HOSTS. R. F. Whitcomb, USDA, ARS, Beltsville,
MD. 20705. Animal populations are generally limited by a number of independent con-
straints. The susceptibility of populations to these constraints is governed by the
life history strategy of each species, which evolved in response to competing selective
pressures. Most crop pests are r-selected strategists: i.e., species selected for
maximization of reproductive effort and dispersal. Many may be fugitive species,
specifically adapted for utilization of temporally unstable resources. In contrast,
most of the insect species that account for total diversity in field and forest eco-
systems are more specialized, with lower reproductive rates and less emphasis on dis-
persal. Viewed in this light, the mechanisms by which dispersing r-selected crop pests
select host plants for feeding and oviposition are of special interest. Management
techniques for temporal and physical isolation of annual crop plants from reservoirs
of pest insects might be developed from such insights. However, protection of valuable
long lived plants will require different techniques, because the degree of isolation
needed to completely protect such plants may be unattainable in field situations. Re-
cent work has shown that climax plant species usually maximize defense mechanisms
against insects and disease organisms. These observations point to increased expendi-
tures of effort in searches for resistance, tolerance or immunity to insects and disease.
In some cases, limitation of potential pest species in nature may be conferred by spatial
separation of individual plants; in such cases resistance to virus or mycoplasma disease
may not be an important part of the species gene pool. In any case, a major thrust of
research on ornamental plants should be towards attainment of a diversity of species
and ecotypes, and avoidance of monocultures.

SUMMARY G. H. Kaloostian, Rapporteur. I would like to comment on the presentations
by Drs. Eden-Green, Dabek, and Fisher. Standard procedures are being followed in the
manipulation of vector species in the Homopteran group. If we agree that the lethal
yellowing agent is a mycoplasma, sharp-shooter leafhoppers should be given less emphasis
or even ignored. I would not use them myself.

Two days of acquisition feeding period on diseased palms should be sufficient for those
species that cannot feed on palms without high mortality. These species should be trans-
ferred to their preferred hosts for the incubation feeding period. The insects then
should be on the preferred hosts for the incubation feeding period for 20 to 25 days.
After the incubation feeding period, they can be caged on the test plants and held on
these plants for the transmission feeding period of ten or more days or until the insects
are dead. Better results can be expected if 100 or more insects are used per test. Tests
should be run in a series of five or ten plants per series with appropriate checks.

In the general discussion it was pointed out that attempts should be made to culture MLO's
from suspect insect vectors as has been done from palms. This is important even if the
vector is as yet unidentified. It is possible that the agent of lethal yellowing may be
cultured from the insects inspite of the unsuccessful attempts with diseased palms.

Attempts should also be made to mass rear suspect species in the greenhouse for use in
vector tests. There has been no mention of the use of eriophyid mites as suspect species.
Eriophyid mites may be present in large numbers on palms and the undercover on other
plants. I would suspect that in this area and Jamaica, and all through the lethal yellowing
areas there may be ten or 20 new species present. In the Philippines we found about 60
new species and nine new genera on the palm and weeds under the palms. Eriophyids are
good suspects, and sometimes they do feed in the phloem. They are relatively easy to
collect and manipulate in tests.









Personnel and Equipment: Research scientists should have good technical assistants
for the routine details involved in vector work. Equipment and facilities should be
provided for insect rearing space separate from test plant holding areas. Continuity
of effort is very important. Monetary support should be given until the search is
complete. Withdrawal of support or change in personnel can often disrupt an outstanding
research effort.

SUMMARY F. M. Eskafi, Rapporteur. To answer a point that Dr. Kaloostian brought up,
I have been testing one species of eriophyid mite in Jamaica. I have taken the mites
that are under the calyx of 1 to 2 inch coconuts where they are most abundant. I have
done three different kinds of transfer of these mites: (1) with little cages on leaf-
lets; (2) in between the spear leaves; and (3) by taping separated calyxes on the fruit
of the same size on coconuts in a disease-free area. It is difficult to find these
mites and the nuts on the diseased trees at the same time. As soon as the tree comes
down with the disease most of the small nuts fall down. However, I have been able to
find a few diseased trees having damaged fruits. It will take another four or five
months before the results show anything.

Dr. Howard conducted a survey of Homoptera associated with species of palms. The data
indicated that the likely vector would be Haplaxius, based on its distribution, fre-
quency in collections, apparent host range, feeding behavior, and phyletic relation-
ships with vectors of similar disease organisms.

Dr. Waters stated that the distribution of MLO in phloem of palms showed that the pin-
nate axis of the spear leaf had MLO present in 50% of the samples with different amounts
of necrosis. He also stated that yellow flag-leaves contained 23 to 35% MLO and this wa
the first report of such a case. Roots had about one-fourth MLO as that of the spear
leaves, and there was a direct relation between the percent sieve element infected and
the number of MLO in it. The highest number of MLO was about 80 days after the onset
of the disease.

Dr. Tsai discussed insects that he had tested by membrane feeding, injection and direct
acquisition. In other tests field collected insects from diseased areas were tested.
Results are pending. The numbers of Haplaxius caught by rotary nets showed that year-
round populations were present with peaks in late summer and early spring. Fifty per
cent of the population lived 8-12 days on test palms, with great variations for different
palms. Removal of flowers resulted in a decrease in LY incidence. A list of insects
visiting flowers was presented which included one homopteran, Idioderma.

I want to mention three points that make me suspicious that the vector may be a species
of eriophyid mite. First, this species has never been tested, although four species
were tested by Drs. Heinz and Schuilling. Secondly, the spread of the disease indi-
cates that it is caused by an inactive arthropod because it starts with one jump from
one end of the island to the other, which is undoubtedly done by man's help or trans-
portation, and then it spreads very slowly around. This indicates the spread is by
an arthropod which is very slow-moving. However, I suspect that a microscopic mite
would desiccate in a long migration by wind. Therefore, it would have to be a slow
transmission from tree to tree or a short distance movement by the wind. Thirdly, as
long as the trees are under screen or if they are under the canopy of large trees
they do not get infected. When the top is off the cage or when the bottom or the sides
of the cage is raised, it does not get infected. This suggests that wind disseminates
the vector. According to the farmers, the incidence of lethal yellowing disease in
Jamaica started about the same time that the amount of damage by the eriophyid mites
on the fruit started showing economic significance. The removal of the floral parts
by Dr. Tsai reduced the number of cases of lethal yellowing. This indicates the pos-
sibility that eriophyid mites are involved. As the fruit becomes larger the number
of mites increases. It reduces again when the fruit becomes mature or badly damaged.









SESSION V DISEASE CONTROL

Chairman: F. W. Howard Rapporteurs: M. Dollet & I. Osborne

INTRODUCTION. There is no permanent cure knowifor LY. Antibiotics bring about a
remission of the disease, but must be administered periodically to be effective.
Removal of diseased trees has not effectively stopped the spread of LY. Insecticide
control of the vectors would probably not be practical on a large scale because of
undesirable environmental consequences that could result, and the cost of insecticidal
treatments repeated indefinitely could be prohibitive.

The planting of a diversity of palm varieties or species that have natural resistance
to the disease is the best means of combating LY. The 'Malayan Dwarf' and 'Mapan'
coconut varieties have shown a high degree of resistance and have been extensively
planted in Jamaica. Many species of ornamental palms are resistant, and can be planted
in Florida.

Although host plant resistance is the best means presently available for dealing with
LY, researchers are continuing to investigate new antibiotics, insecticides, and other
chemicals and methods of application. Replacement palms are being evaluated and horti-
cultural and plant protection practices developed. Quarantine practices are an additional
important aspect of LY control.

NEW PALMS FOR SOUTH FLORIDA. H. M. Donselman, Agricultural Research Center, University
of Florida, 3205 SW 70 Ave., Ft. Lauderdale, Florida 33314. The sub-tropical environment
of South Florida allows the cultivation of over 500 species of palms. Residential and
commercial plantings can utilize only the 10-15 species commonly available from the
nursery industry. Now that LY is threatening many of the more common palms in culti-
vation, the tropical atmosphere, so effectively created by palms, is in jeopardy. To
insure the future of Florida's palms and maintain the unique setting necessary for the
tourist trade and residents, new palms resistant to lethal yellowing must be developed.

The coconut palm is generally recognized as the symbol of the tropics. Over 50% of the
susceptible 'Jamaican Tall' coconut palms have died on the east coast of Florida from
lethal yellowing in the past five years. During that same period of time an estimated
750,000 seed nuts of the resistant 'Malayan Dwarf' coconut have been imported from
Jamaica. Accounting for loss due to lack of seed viability, cultural problems, and an
unusually cold winter last year-. about 250,000 'Malayan Dwarfs' are now thought to be
established in landscapes or available for planting in South Florida.

Because the cultural requirements of 'Malayan Dwarf' coconut palms are greater than
'Jamaican Tall' varieties, education of the public as to the care of this coconut is
necessary. 'Malayan Dwarfs' appear to be less cold tolerant and require more fertili-
zation and water, at least for the first 2-3 years. For this reason there has been
great interest in the new hybrid 'Maypan' coconut. The hybrid vigor of this palm
may make it a more suitable replacement for our dying 'Jamaican Tall' coconuts.
Availability of the 'Maypan' will depend upon importation of hybrid seed nuts from
Jamaica after the demand of their coconut industry has been met. .The Division of
Forestry in Florida is also planting a hybrid seed orchard in Miami which could begin
producing hybrid coconuts in 2-3 years for distribution for public plantings. Other
coconut palms showing some resistance to lethal yellowing such as the 'Panama Tall'
and Fiji Dwarf', should be tested in trial plantings throughout South Florida.









The Christmas Palm, Veitchia merrillii was at one time the most common homeowner
palm in South Florida. Already over 35% of these palms have died along the southeast
coast. A resistant replacement palm of similar stature and ease of culture must be
found to replace this palm. Additional palms are needed for several of the other 23
dying palm species. Experiments involving over 120 species of palms new to Florida
are now underway at the Agricultural Research Center in Fort Lauderdale. Research is
necessary to determine germination methods, cultural requirements, and rate of growth
of these palms. An area approximately one hectare in size is currently being developed
to maintain an outdoor palm nursery and trial garden. Until a method of artificially
transmitting lethal yellowing is developed, resistance will be determined through
natural selection. Highly susceptible palms will be interplanted with the test plants
to increase the incidence of lethal yellowing in the trial.

PRESENT EFFORT IN PRESERVING SOUTH FLORIDA'S PALMS. H. Donselman, University of
Florida, ARC, 3205 S.W. 70th Ave., Ft. Lauderdale, Florida 33314. One of the first
injection replanting programs to come into existence was in Miami Beach, Florida.
The success of this program is shown by the presence of thousands of healthy coconut
palms and young, resistant 'Malayan Dwarf' coconuts growing alongside. Ninety percent
of the coconut palms in Dade County that were not injected have died.

The most successful injection program has been in Collier County on the west coast of
South Florida. Ly was reported here in 1974. A removal program was initiated for
diseased trees and all healthy susceptible palms in the immediate area were injected
with antibiotic. There has been no new incidence of lethal yellowing in the county.

Perhaps the largest injection and replanting program is in Palm Beach, Florida. Their
program includes the injection of 40,000 coconut palms. This past year only 150-200
trees in the program died. Lethal yellowing occurs to the north of Palm Beach County
in Martin County and is presently very active immediately to the south, in Boca Raton,
Florida.

A different type of injection and replanting program has been developed in Monroe County.
The Extension Service of Monroe County provides a monthly update of lethal yellowing
developments to interested citizens and also supplies information and antibiotics to
the local clubs. The clubs, in turn, sell the antibiotic at cost to the citizens who
can also obtain, by leaving a deposit, injection equipment. The replacement with re-
sistant 'Malayan Dwarf' coconuts is also emphasized and young plants may be obtained
for a nominal cost from the clubs. This type of program is especially well suited
for smaller municipalities or unincorporated cities.

Successful injection programs depend on public support and the support of city and
county officials. Unsuccessful programs are usually the result of negligence on the
part of the people doing the injecting.

PROGRESS IN THE DEVELOPMENT OF CONTROL OF LETHAL YELLOWING: FOLIAR INSECTICIDE
APPLICATIONS. F. W. Howard, Agricultural Research Center, University of Florida,
3205 S.W. 70 Ave., Ft. Lauderdale, Florida 33314. Foliage of Manila palms, Veitchia
merrilli, were sprayed with insecticides biweekly in a LY infected residential section
of Hollywood, Florida, for a period of 10 months. The treatments were (1) Diazinon
AG500 at 13 ml./10 1., (2) Cygon 400 at 26.4 ml./10 1., both insecticides sprayed to
runoff, and (3) untreated check. About 500 palms were in each treatment in October,
1976, at which time spraying commenced. Throughout the study period, insects were
collected from Tanglefoot spread on 5 pinnae of 60 palms per treatment. The experiment
is still in progress, and statistical analysis of data has not been completed, but upon









inspection of raw data it appears that Diazinon, but not Cygon, reduced the numbers
of Haplaxius crudus (Van Duzee). There was a total of 80 H. crudus adults in the
samples from Diazinon-treated areas compared to 125 from the untreated check in the
spring samples. In the summer samples, there was a total of 132 H. crudus in samples
from Diazinon-treated areas, compared to 245 from the untreated check. However, at
the end of 10 months, no differences were detected between treatments and controls
in LY incidence or the number of trees surviving.

RECENT ADVANCES IN ANTIBIOTIC TREATMENT OF LETHAL YELLOWING. R. E. McCoy, Univ. of
Florida, 3205 S.W. 70 Ave., Ft. Lauderdale, FL. 33314, USA. Injection of oxytetra-
cycline solutions into palm petioles is a promising technique that eliminate the
permanent injury produced in the palm trunk by currently used injection methods.
The petiole base affords a large enough volume of tissue to drill a three inch deep
hole for making an injection by either the Mauget injector or by gravity flow. High
pressure injection techniques do not work since the tissue splits easily, allowing
the solution to leak out. Although oxytetracycline residues are greatest in the in-
jected fronds, the technique was therapeutically effective against lethal yellowing.
Six of 12 treated palms stopped all sysmptom development for at least 4 months, and
only 2 of 12 palms showed no effect of the treatment.

In November 1976, a recount was made of the number of coconut palms remaining in the
Country Club Prado of Coral Gables. This area had been under continuous treatment
since October 1973, and the first 16 months results were published (McCoy, et al.
1976 Phytopathology 66:1148-1150). At the beginning of the test 103 palms were
present in the treated area of which 17% were affected by LY. An adjacent control
area had 65 palms with 28% displaying active LY symptoms. After 16 months, 97% of
the palms in the control area were diseased and 40% of the treated palms had lethal
yellowing. After an additional 20 months, not one new case of lethal yellowing had
appeared in the treated group of palms, while 100% of the control group and other
surrounding palms were lost.

An additional prophylactic test was conducted in Plantation, Florida in which 100
symptom-free coconut palms were selected in two disease foci. In each group of 50
trees, 25 were treated prophylactically for a period of 13 months and 25 served as
controls. At the end of the test, 70% of the control palms had developed lethal
yellowing while only 6% of the treated palms had done so.

This evidence, plus the fact that no new lethal yellowing cases had developed in the
northern and western counties that had recorded outbreaks in the past 3 years and
where preventive treatment programs were strictlyfollowed, demonstrates the effective-
ness of oxytetracycline as a preventive treatment for lethal yellowing.

SUSCEPTIBILITY TO LETHAL YELLOWING DISEASE IN GLOBAL PERSPECTIVE. H. C. Harries,
Coconut Industry Board, P. O. Box 204, Kingston, 10, Jamaica. In Jamaica, coconut
varieties from many parts of the world are exposed to LY. Over periods of seven to
eleven years control plots of the 'Jamaica Tall' variety within each experimental
field, and entire commercial plantations in close proximity, were eradicated by the
disease. Replanted 'Jamaica Tall' seedlings in experimental plots also died. Re-
planting commercial fields with the resistant 'Malayan Dwarf' variety is completely
satisfactory in terms of disease resistance. Many foreign varieties show as high a
rate of susceptibility as the 'Jamaica Tall'. Some varieties show an intermediate
level of resistance; few show the high degree of resistance of the 'Malayan Dwarf'.
If the estimated areas planted to coconuts across the world are tabulated with the
potential lethal yellowing losses of the varieties predominating in those areas, the
global threat of this disease can be assessed (Table 1).


I









Region Estimated Area Ly Loss Factor Estimate Losses


Southeast Asia 58.5 50 26
South Asia 24.6 90 22
Pacific Islands 7.8 75 6
Africa 4.1 90 4
America 5.0 80 4

Total 100.0 62


An estimated loss that approaches two thirds of the world's coconuts is serious.
Those who are complacent that the disease will never reach them should remember
that this was thought by the farmers in eastern Jamaica before 1961 and by certain
countries in West Africa before 1975. Certainly, for the major coconut growing
countries it is not an immediate threat. However, it does raise the question of
the advisability of introducing susceptible varieties into areas where they do not
now occur and the suitability of certain hybrid combinations. Resistance to LY
also has a bearing on the identification and distribution of coconut varieties.

PLANT HEALTH & QUARANTINE PROBLEMS ARISING IN THE INTERNATIONAL TRANSFER OF COCONUT
GENETIC RESOURCES, H. C. Harries, Coconut Industry Board, P. O. Box 204, Kingston,
10, Jamaica. Plant quarantine regulations in almost all countries where coconuts
grow prohibit the introduction of coconut planting material from Jamaica and from
other countries where LY occurs. These regulations have been relaxed within those
countries which already have the disease to allow seed of the 'Malayan Dwarf' and
other resistant varieties to be introduced from Jamaica. It seems improbable that
anyone would ask for susceptible varieties, such as the 'Jamaica Tall' to be made
available. Even though there is no proof that the disease is seed-transmitted, who
would accept the risk? Moreover, the planting of disease-susceptible varieties will
increase the losses when an outbreak does occur. Should this concern those countries
in the Far East which are introducing seed and pollen of susceptible varieties and
seed of hybrids with one susceptible parent? The Coconut Industry Board has been
asked to send dwarf and hybrid seed to countries where there is no lethal yellowing.
So far the commercial pressures to do so have been resisted.

Yet the most important planting material is not available outside Jamaica. The
'Malayan Dwarf' is not the most important because it already occurs in most coconut
growing countries. It is simple for any concerned organization to identify and mul-
tiply 'Malayan Dwarf' in advance of any disease outbreak. What cannot be multiplied
are the resistant individual palms that make up only a small or medium part of the
tall coconut populations of Asia and Oceania because these palms cannot be identified
without exposure to the disease. After exposure has killed half or more who would
risk taking planting material from the remainder? Yet, if these are truly resistant,
they could become first class sources of breeding material, and conceivably, commercial
planting material.

To overcome this problem a form of post-entry quarantine can be devised for MLO, viroid
and other disease of coconut. Made possible by embryo culture, it now remains for the
pathologists to devise satisfactory screening methods. For example, embryos collected
in large numbers in a diseased area (and possibly even from individual diseased palms)"
can be maintained in laboratory culture for 16 weeks and grown on in screened glass-
houses for some months before they need to be planted. If in this time it can be es-
tablished that no MLO, viroid, virus or other pathogen, can be found when adequate
samples are tested (using electron microscope examination, serology, nucleic acid
extraction and analysis, and transmission (vector or otherwise) to suitable test plants)
it could be considered safe to plant the embryo-cultured seedlings.









CAN TALL COCONUT VARIETIES BE SELECTED WITH LETHAL YELLOWING RESISTANCE EQUAL TO
THAT OF THE MALAYAN DWARF? H. C. Harries, Coconut Industry Board, P. 0. Box 204,
Kingston 10, Jamaica. Exposure to LY infection in the field and death or survival
over an indefinite number of years is the only way that susceptibility or resistance
can be recognized. Groups of highly resistant, less resistant and susceptible
varieties have been categorized this way. Under conditions where LY disease is very
active, there are presumably many factors and all palms, tall or dwarf, susceptible
or resistant, are exposed to repeated attack. If, for instance, palms carry factors
for vector resistance, they are protected. However, if this resistance is overcome
palms that also carry further factors, such as resistance to MLO multiplication, will
survive. If there are a number of independent factors, then the 'Malayan Dwarf' may
be presumed to carry a full complement. Resistance is high in each palm and remains
high at each generation because of the variety's natural self-pollinating habit. The
'Jamaica Tall' would carry new factors, if any, and is highly susceptible. The inter-
mediate and variable levels of resistance shown by other tall varieties is thus at-
tributed to the possession of some factors for resistance by particular individual
palms. In F1 hybrids factors in each parent may combine.

Some tall palms that carry the pathogen resistance factors but not the vector resis-
tance factors, may survive after showing signs of infection. However, if the pathogen
resistance factors also break down then these palms would die. Since a few typical
'Malayan Dwarf' do die, breakdown of resistance factors must be a possibility. Since
so few die, even over a period of many years, it is encouraging to think that the
resistance has occurred butthe cross-pollinating nature of tall varieties ensures that
these characters remain assorted between individuals of one generation and the next.
Theoretically, a few tall palms could have the same complement of resistance factors,
and therefore the same degree of resistance, as the 'Malayan Dwarf'.

GUIDELINES TO NURSERY PRACTICE FOR MALAYAN DWARF AND MAYPAN COCONUT SEED. D. H.
Romney, Coconut Industry Board, P. 0. Box 204, Kingston 10, Jamaica. In Jamaica,
over half a million 'Malayan Dwarf' and 'Maypan' ( Malayan Dwarf x Panama Tall' F1
hybrid) coconut seedlings are planted annually. These cultivars are superseding
the traditional 'Jamaica Tall' due to their high resistance to lethal yellowing
disease. The guidelines to nursery practice given will produce seedlings, in quantity,
of good genetic and horticultural quality and without undue expense. The major cri-
teria described were:

Marking of pure 'Malayan Dwarf' mother palms in extensive stands;
regular supervised reaping; rejection of immature, over-mature,
puny or damaged seeds; siting of nurseries near the area to be
planted, in full sunlight and fenced against animals; prompt
setting of seeds after reaping, horizontally orientated and with
the less mature being set separately from the more mature; use of
mulch, lifting, by cutting the roots with a spade, when the seedlings
are 45-90 cm. high; recognition and rejection of off-types at lifting;
prompt planting.

Superficial damage to the seednuts due to gall-mite (Aceria guerreronis) was shown to
be of no detriment to germination. Pest, disease, and weed control in the nursery was
outlined.

SUMMARY Drs. Dollet, Howard and Eden-Green, Rapporteurs. Dr. Donselman reported
that some programs are being carried out by concerned municipalities in Florida.
The recommended program consists of injecting LY susceptible palms with antibiotics,
interplanting with resistant species and varieties, and removing palms killed by the









disease, especially the "telephone pole"-like trunks of dead coconuts. Dr.
Donselman emphasized that (1) injection programs have worked in communities where
they have been well supervised; (2) injections of antibiotics do not offer a per-
manent solution. It is a method that buys time, keeping diseased palms alive while
disease-resistant palms can be planted and grown; (3) a diversity of palms should be
planted, rather than a single variety, such as Malayan Dwarf. Although the Malayan
Dwarf is a good palm, it is more desirable to develop a diversity of palms than to
convert our landscape to a single kind of palm.

Hugh Harries indicated that there are possibilities of developing tall varieties
with the appearance of the 'Jamaica Tall' variety coconut so common in Florida by
plant breeding. However, this would be a very great and lengthy task.

Dr. Howard described an experiment in which attempts are being made to control the
the spread of LY by applying insecticides to palm foliage. Results are not yet
conclusive.

Dr. McCoy described experiments with oxytetracycline injections of coconut and
other palms. His studies show that these injections are very effective when done
properly by well-trained, conscientious personnel. The failure of the injections
in some areas may be due to improper techniques, especially failure to follow an
injection schedule.

He has been studying the effectiveness of injecting palm petioles, rather than trunks,
so as to cause less injury to the tree. Results are encouraging.

Dr. Romney described nursery practices that should be followed in growing Malayan
Dwarf and Maypan coconut palms. An important element that should never be neglected
is good supervision of nursery employees to insure that good nursery practices are
being followed.

Bill Theobold described the Florida Division of Forestry's role in combating the LY
problem. They are growing coconut palm varieties that will be new to Florida. Mr.
Theobold recently made studies of coconut varieties in Jamaica and was impressed
with the aesthetic qualities of such varieties as the Fiji dwarf coconut, the Red
Spicata dwarf coconut, and the Panama Tall coconut. The Division's role in intro-
ducing new varieties of coconut will be confined to public lands.









SESSION VI EMERGING PROBLEMS IN PALM CULTURE

Chairman: M. V. Parthasarathy Rapporteur: L. Chiarappa

LETHAL DISORDERS OF UNKNOWN CAUSE IN THE DATE PALM. J. B. Carpenter, Agricultural
Research Service, U. S. Department of Agriculture, Indio, California 92201. In 1975,
a warning on the potential danger of introducing LY into date palm growing areas was
given at the Third FAO Conference on the Improvement of Date Production, Processing
and Marketing. The danger for those areas lies not in the introduction of diseased
date palms, but in the introduction of diseased, vector-laden ornamental palms which
might serve as foci of infection for spread of LY to date palms.

Three minor, but lethal, disorders of unknown cause have been reported on date palms.
Each has symptoms that bear some similarity to those reported for LY or other diseases
associated with mycoplasmas.

Rapid Decline or Rhizosis. Rapid decline was considered responsible for the death of
several hundred date palms in California during the period 1933-1950. Since 1950,
only a few trees affected with rapid decline have been seen.

Symptoms of rapid decline in fruiting palms usually appear in the summer months.
In female palms, the fruit on 2 or 3 bunches shatters or shrivels, the fruit strands
discolor and wilt, and later other fruit bunches are affected. Leaf symptoms develop
in two main patterns and may occur simultaneously with the onset of fruit damage or
may be delayed. Usually, at the time fruit drop occurs, the tightly folded central
or terminal leaves are dead, white and stiff. Otherwise, the pinnae of the lower
leaves turn chocolate, or reddish brown in color. The lowest leaves die and death of
foliage progresses upward to the terminal leaves. Similar leaf symptoms occur on male
palms. After symptoms appear, progress of the disease is rapid. Palms may die within
a few weeks or months. The offshoots, or basal sprouts on the palms, do not always
die when the parent plant perishes. Rapid decline has been found in palms of several
varieties. Control measures, other than destruction of affected trees, are unknown.

Faroun is a minor, fatal date palm decline of unknown cause reported from Mauritania.
Female and male palms are affected. The first symptom is a failure of apparently
normal palms to flower for one or two seasons before foliage symptoms appear. Af-
fected trees become conspicuous when they assume a parasol form as the old and middle
rank leaves become positioned somewhat horizontally. Subsequent new leaves and their
pinnae are reduced and distorted, until the central portion of the crown becomes a
grotesquely stunted rosette. These symptoms are accompanied by abortion of the
axillary buds, which accounts for failure of the palms to flower. Offshoots are af-
fected on some declining palms. Internally, palms in advanced stages of decline have
numerous brown gum pockets and long, dark-colored cracks in the crown tissues. Similar
cracks occur in leaf bases near the point of attachment to the trunk. No fungal or
viral pathogens have been associated with faroun. Under the cultural practices em-
ployed, nutritional disorders were not suspected. Faroun is generally fatal and af-
fected palms die within 2 to 4 years after the first appearance of symptoms.

Al-wijam is a minor date palm disorder of unknown cause in Saudi Arabia where it occurs
in otherwise good palm plantings. The symptoms of the disorder are stunted growth,
loss of vigor, failure to bear fruit, and gradual drying of the foliage which pro-
gresses from the old to the young leaves. Nixon reported a symptom that suggested a
virus disease: "on the midribs of the younger leaves, as seen from the outer side,
there is usually a faint, narrow, yellow, longitudinal line". Al-wijam occurs on
palms of several varieties. Growers are cautioned to avoid using offshoots from af-
fected trees or from other trees in the same planting.









FLAGELLATED PROTOZOA ASSOCIATED WITH MARCHITEZ SORPRESSIVA OF OIL PALM IN SOUTH
AMERICA. M. Dollet, Virology Department, I.R.H.O. Compared Pathology Research
Station, 30380 St. Christol-Les-Ales, France. Marchitez sorpressiva is a sudden
wilting of oil palms more than 2 years old which has been rife in Colombia since
1963. The disease later broke out in Peru (1970) and thereafter in Ecuador (1973),
causing numerous losses. The first visible symptom is bro'-ning of the tips of the
leaflets of the lower leaves. This browning spreads, then the youngest leaves be-
come pale green and tighten round the spear. At this stage, the root system is
found to be in an advanced state of decay. In the last stage of the disease all
the leaves on the palm are completely dried up and take on a characteristic silver-
grey hue.

The electron microscope study of samples of inflorescences from palms with early
symptoms of the disease, taken in Peru and Colombia, reveals the presence of a
large number of flagellated protozoa identifiable as Trypanosomatidae. These
organisms are localized solely at phloem level and move from one sieve tube to
another via the pores, undergoing considerable elongation. They are also visible
by optical microscope, particularly in the roots where they often fill the entire
cell space. We can distinguish slender forms with a flagellum, more compact forms
also with a flagellum, and round organisms with no apparent flagella which may cor-
respond to a Leishmania form. From these different forms it can be speculated that
the organism goes through a complete cycle in the palm.

The protozoa can be kept alive in coconut water, either pure or with different media
added.

The presence of these flagellated organisms in great numbers and the exclusion of all
other germs in palms with early symptoms, and their specific association, whatever
the geographical background with marchitez symptoms, makes us think that they are at
the origin of the disease. It would be very interesting to compare this disease with
the only other two intraphloem flagellated protozoa diseases known: coconut hartrot
and "phloem rot" of the coffee bush, which are also rife in South America.

TRANSMISSION OF SUDDEN WILT DISEASE OF OIL PALM. Gerado Martinez-Lopez, Virus
Laboratory, Instituto Colombiano Agropecuario, Apartado Aereo 151123, Bogota,
Colombia, S. A. Sudden wilt disease, marchitez sorpresiva, is a serious disease
of oil palm in Colombia and is of unknown etiology. It is characterized by the
quick death of the diseased plants. The first symptoms are the brown appearance of
the lowermost fronds, an abnormal number of unexpanded spear leaves and loss of the
glossy appearance characteristic of healthy fruits. Very rapidly the leaves are in-
volved and in less than three months the whole palm is dead. The disease has been
more severe in areas where Panicum maximum Jacq. is the main weed and where a poor
cover of Pueraria phaseoloides (Roxb.) Benth is maintained. Associated with P.
maximum the development of the nymphs of Haplaxius pallidus Caldwell (Homoptera:
Cixiidae) is observed. This insect lives as adult on oil palm. In studies oriented
to determine the role of this insect in the spread of marchitez, 1395 young adults
of H. pallidus were exposed to diseased oil palms for a feeding acquisition period
of 42 hr. After this, the 1059 surviving insects were used, in groups of 6 to 19 per
plant, to inoculate 100 palms, each five years old, remaining on them for an average
of 13.1 days. A group of 100 palms, side by side with the inoculated ones, was used
as a healthy control. Observations during 12 months on these groups of palms indi-
cated that 13 of those inoculated developed the symptoms of the disease between 98
and 260 days after the beginning of the inoculation period with an average incubation
period of 132.4 days. Only two of the control plants became infected. The incubated
period in the oil palm was similar when healthy plants were injected in the trunk with









undiluted extracts from the inner soft mass of young leaves and leaf bases of
diseased plants. By this procedure it was possible to reproduce the symptoms in
8 out of 20 plants (40%) in a preliminary experiment. In a second experiment 17
out of 50 plants (34%) developed the symptoms in less than 150 days and 28 (56%)
in less than 174 days. None of the control plants became infected. These results
indicate that H. pallidus is the vector of an unknown pathogen that can be trans-
mitted by injection with the sap from infected plants.

OBSERVATIONS ON THE PHLOEM INHABITING FLAGELLATE PHYTOMONAS IN PALMS. M. V.
Parthasarathy, Botany, Genetics and Development, Cornell University, Ithaca,
New York 14853. Trypanosomatid flagellates of the genus Phytomonas were examined
from coconut, oil and Maripa palms affected by hartrot in Surinam. The flagellates
were 12-22,/ in length not including the flagellae. There was no significant dif-
ference in the average length of the organism in different species of palms. Many
of the flagellates appeared twisted. Almost all flagellates observed were promasti-
gotes. Several binary and multiple fissions were also observed. The uniformity in
average length and the similarity of ultrastructure of the flagellates infecting the
three species of palms suggest that the organisms belong to the same species of
Phytomonas. The flagellates remained alive for up to 60 minutes in sap squeezed out
of phloem. Although the organisms exhibited sinuous movements they did not display
any obvious locomotion. However, when fresh longitudinal sections of sieve tubes
heavily infected with the flagellates were examined with a phase contrast microscope,
some of the organisms showed apparent locomotion presumably due to the crowded con-
dition in the sieve tube combined with the sinuous movements of flagellates. 3%
concentrations of antibiotics and drugs such as penicillin, streptomycin, oxytetra-
cycline, tryparsamide and Berenil had no apparent effect on the flagellates. Sieve
elements that contained the flagellates did not show any drastic changes in their
structure although heavily infected ones often had an electron-dense material between
the plasmalemma and cell wall. Plastids with starch granules also appeared to be rare
in sieve elements infected with the flagellates. Preliminary observations suggest
that the flagellates induce premature collapse of protophloem in immature inflores-
cences of oil palms affected by hartrot.

SYMPTOMOLOGY OF HARTROT DISEASE IN COCONUT AND OIL PALM. W. G. van Slobbe, Agr.
Expt. Sta., P. 0. Box 160, Paromaribo, Surinam. The external symptoms of hartrot
in both coconut and oil palms develop rapidly. It takes from 2 to 4 weeks from
earliest symptoms until complete browning of the canopy. Recovery of diseased palms
has not been observed. This wilting disease can be described as a degeneration of
all projections of the palm, moving in the direction of the trunk: browning of the
tips of the pinnae progressing from top to base of each individual leaf extending up-
wards in the canopy; dropping of the nuts and desiccating of the bunches and male
flowers in oil palm; decaying of the young spear leaves, root tips and secondary
roots.

Between both palm species the following differences exist: the brown color of the
leaves in oil palm is more pronounced than in coconut; sometimes the roots of oil
palm possess a characteristic smell while occasionally cavities are found in the
trunk of diseased oil palms only; the oldest unopened inflorescences in oil palm
putrefy completely while in coconut they dry up starting with brown necrotic patches
on the spikes. Before the canopy of coconut turns completely brown, the spear rot
has already affected the growing point, while in oil palm after browning of the canopy
the apex still seems to be healthy.

The caterpillar Sagalassa valida Walker affects the roots of oil palm in Surinam but
no affected coconut roots have been found.









PHYTOMONAS FLAGELLATES IN COCONUT (HARTROT DISEASE, CEDROS WILT) AND OIL PALM
(HARTROT DISEASE, MARCHITEZ SORPRESIVA). W. G. van Slobbe, Agr. Expt. Station,
P. 0. Box 160, Paromariba, Surinam. Phytomonas flagellates occur in the latex-
bearing cells of some plants. Recently only one disease was known that was
probably caused by these organisms, phloem necrosis in coffee, detected in 1929
in Surinam by Stahel (5). Symptoms appeared five months after grafting diseased
roots on the roots of healthy coffee trees. The disease occurred also in Guiana,
Brazil, Colombia and probably in El Salvador.

Coconut: Hartrot is the Dutch name for heart rot. Though this name is misleading
because the disease is a wilt, it has been used for 70 years in Surinam. Sometimes
this disease has been called Bronze Leaf Wilt, Unknown Disease, Lethal Yellowing
and Coronie Wilt. Between 1918 and 1933 about 25,000 of 65,000 palms were affected
by the disease in Coronie, the main coconut district. At other locations most of
the palms planted on clay soils succumbed to hartrot disease soon after they came
into bearing. Until now coconuts are growing in Coronie and some affected trees
can be seen. Since September 1975 we have known that flagellates occur in the phloem
of coconut palms affected by this disease (3). Subsequently they were found in coco-
nuts affected by Cedros Wilt in Trinidad (6). By squeezing a few drops of liquid out
of the tissue by means of an ordinary pair of tongs, one can observe the flagellates
alive by a magnification of 400 x. Dwarf coconuts, showing the first symptoms of the
disease have been carefully investigated. Flagellates were found in every inflore-
scence (number -7 up to +13), in the inner and outer spathes, and in the roots.
Flagellates also occur in the soft tissue at the base of the young leaves.

Oil Palm: African Oil Palm, Elaeis guineensis was introduced into Surinam at a later
date (4). Some years after planting many palms died, due to a wilting disease of un-
known etiology. Since the second half of 1976 we have known that flagellates occur
in the phloem of diseased palms. Subsequently flagellates were detected in oil palms
affected by marchitez sorpresiva in Peru (1), Contrary to our experience with the
coconut palm it is difficult to detect flagellates in the oil palm by means of light
microscopy, because only a few occur in the young unopened inflorescences by squeezing
liquid out of the stele of roots which are not yet degenerated one can find many
flagellates. Also many occur in the stalk of the nearly ripe bunches.

Epidemiology: Epidemiologic analysis of the disease indicates that the apparent in-
fection rate seems to be independent of the number of diseased palms. As we do not
even know the vector or the host plant, one may not say that a diseased palm cannot
be a source of infection.

Vector and hostplant: Sometimes we find flagellates in the midgut and the salivary
glands of bugs (Hemiptera: Pentatomidae) caught near the trunk of diseasedand healthy
coconut palms. It is known that flagellates occur in other insects. The vectors of
the African sleeping disease Trypanosoma tsetse flies, Glossina spp. (Diptera) and
Leishmaniasis is transmitted by sandflies, Phlebotomus spp. (Diptera).

Flagellates occur in the latex-bearing cells of some species of Euphorbiaceae,
Asclepiadaceae, Apocynaceae and Urticaceae and in the phloem of Coffea sp. (Rubiaceae)
and palms including Maximiliana maripa. Any plant species can possibly be a host.

Control: Application of Endrin near the trunk prevents the oil palm from becoming
affected by m.architez sorpresiva in South America (2). There are indications that
Endrin will work in coconut too, but this is not yet known because the treatments
were initiated in May 1977. Since January 1977 we have applied Sevin in the leaf
axils of dwarf coconuts every two weeks. This study is being continued.









References:

(1) Dollet, M., Giannotti, J. & M. Ollagnier, 1977. C. R. Acad. Sc. Paris,
t. 284 Serie D: 643-645
(2) Lopez, G., Genty, P. & M. Ollagnier, 1975. Oleagineux 30 (6):243-250
(3) Parthasarathy, M. V., 1976. Principes 20:59
(4) Schut, B., 1976. Surin. Landb. 24 (1): 42-48
(5) Stahel, G., 1931. Phytopathol. Z. 4:65-82; Ibid. 1932. 5:539-544;
Ibid. 6:335-357
(6) Waters, H., 1977. Yearly Progress Report on Coconut Breeding 1976 FAO:
27-28

PHLOEM INHABITING PHYTOMONAS PROTOZOAN FROM DISEASED AFRICAN OIL PALMS. D. L.
Thomas, Agricultural Research Center, University of Florida, 3205 S.W. 70 Ave.,
Ft. Lauderdale, Florida 33314. Tissues from two African oil palms with marchitez
sorpresiva symptoms and one palm without the disease were collected from areas
near Santo Domingo de los Colorados, Ecuador. Samples were excised from unopened
inflorescences, unemerged frond bases, and from areas of the trunk near the apical
meristem. Mature sieve tubes from the diseased trees contained uni-flagellated
protozoa which were classified on the basis of their ultrastructure as members of
the genus Phytomonas belonging to the family Trypanosomatidae. The distribution
of the flagellates was uneven throughout the tissues surveyed. Only 17 percent
of 139 vascular bundles contained the organisms. Two forms of the protozoa were
observed within the same cells throughout the examined material. The first form
was a normal promastigote with electron-dense cytoplasm. The second form was ir-
regularly globose and had a more electron-transparent cytoplasm with fewer cellu-
lar components. Intermediate stages between these two forms, as well as dividing
protozoa, were occasionally observed. Infrequently protozoa were seen which ap-
peared to be passing through sieve pores. No phloem necrosis was noted in any
specimen examined. The etiologic role of the protozoa was not determined but
this study supports similar reports from Peru, Colombia and Surinam which indicate
that protozoa might be involved in the disease syndrome.

WILT DISEASE OF COCONUTS IN TRINIDAD. Henry Waters, ODM Lethal Yellowing Research
Team, P. O. Box 204, Kingston 10, Jamaica. Protozoan flagellates of the genus
Phytomonas were found in inflorescence, stem, leaf, spear and cabbage samples
from coconut palms exhibiting symptoms of a wilt disease in Trinidad. No such
organisms appeared to be associated with apparently healthy palms. Affected
palms were identified from all the major coconut growing areas in Trinidad but
none could be found in western Tobago. Losses to wilt are heavy (Ca. 15,000 palms
in the last two years) in the Cedros area but are apparently lighter in the Toco
and Manzanilla areas. The symptomology and disease progression are illustrated
and a description of the presumed pathogen is given. Similar organisms were also
found in the roots of an adjacent diseased coffee plant.

NEW DISEASES OF COCONUT PALMS IN TANZANIA. Kurt G. Steiner, Biologische
Bundesanstalt, Messeweg 11/12, 33 Braunschweig. The coconut industry is
threatened in many African countries by pests and diseases. In West Africa,
particularly Togo and Ghana, diseases corresponding to LY have destroyed thousands
of hectares of coconut palms in the last twenty years. Very little is knowiof the
situation on the east coast of Africa. In 1972 a lethal bole rot, caused by the
fungus Marasmiellus cocophilus Pegler was reported to have destroyed many hectares
of coconut palms in some parts of Kenya and Tanzania. A survey on pests and dis-
eases of coconut palms was carried out in September 1976 by the German Agency for
Technical Cooperation. Three major diseases were.identified: lethal bole
rot, LY, and an unknown disease. Lethal bole rot could be found only in Kenya









north of Mombasa, where it has destroyed many hectares of palms and is still
spreading.

In the Tanga region of Tanzania an "unknown disease" was found that is associated
with Rickettsialike organisms. This disease spreads in foci. The largest and
oldest foci are near the border of Kenya, but smaller ones can be found over nearly
all the region. This disease starts with the abortion of nuts, like lethal yel-
lowing. Then the outer fronds of the palms become necrotic and die. The fronds
are, however, never discolored or reduced in size. The palms die approximately
two years after the appearance of the first symptoms. When the first symptoms
appear on the aerial parts, most of the smaller roots have already decayed and
only the main ones remain. In the stem below the growing point a discoloration
of parenchyma and vascular bundles was observed. In younger palms the symptoms
can also appear in the stem directly above the bole. No pathogenic fungi, bac-
teria or nematodes could be detected in diseased roots or stem tissue other than
the Rickettsialike organisms.

In southern Tanzania from ca. 50 kms north of Daressalaam south to Pande, palms
exhibiting symptoms of LY were found. The disease has been most destructive south
of Daressalaam in the regions of Kibiti and Kilwa. Ground surveys could be carried
out only north of Daressalaam. Here the disease probably started 3-5 years ago.
Several trees were cut down and symptoms of LY could be clearly identified. MLO
have yet been detected in phloem tissues. Therefore further investigations will
be necessary to prove if the disease is really identical to LY.

SHOULD THE ICLY CONCERN ITSELF WITH OTHER PATHOGENS? H. C. Harries, Coconut Indus-
try Board, P. O. Box 204, Jamaica. LY was treated as a regional disease until ICLY
was formed. Even now the group tends to be Caribbean oriented but this will change
as African concern increases. Other coconut diseases, cadang-cadang, root wilt, and
leaf scorch appear to be considered as national problems, as does red ring, despite
its regional extent. Now we have new problems with Aceria & Phytomonas. All coco-
nut pests and diseases pose serious global threats.

If pests and diseases continue to spread, we shall one day see LY in Asia and Oceania
and cadang-cadang in America. Before this happens, a truly international and multi-
disciplinary approach is required for coconut research. Can ICLY outlast the commit-
ment shown by the urban coconut growers of the USA and act as a nucleus for co-ordin-
ated coconut pathology research and control?

SUMMARY L. Chiarappa, Rapporteur. We are one hour and ten minutes behind schedule
and I must accelerate the proceedings of this meeting. I have the unique opportunity
here of doing the job of two rapporteurs in half of the time! You have been here for
the past three days and you must be tired of discussions although it did not seem to
be so from the discussions we had here over the past three hours. This discussion
was indeed very lively so I will try to make my reporting very short and especially
sweet.

I do not think that the inclusion of Kaincope' disease in this session was intentional.
This is not one of the emerging diseases in palm culture. I believe that the work
underway in West Africa is extremely important to clarify simularities and dissimu-
larities with lethal yellowing in Florida and the Caribbean. There are at least two
new diseases of unknown etiology being reported from Tanzania: a lethal bud rot and
a still unnamed, "unknown disease". The latter appears to be of great interest be-
cause it seems to be associated with a rickettsialike organism. The presence of
what appears to be lethal yellowing in Tanzania is also of great importance because
it expands the geographical area of the disease and the geographical scope of ICLY.









Moving to the other problems that we have considered there are three diseases of
unknown etiology of the date palm: rapid decline of California, the faroun of
Mauretania and the al-wijam of Saudi Arabia. I am sure that Dr. Carpenter will
rush to California to start looking right away for mycoplasmas and flagellates in
date palms. Moving away from the coconut and date palms to the oil and other palms,
we have seen the serious crop losses caused in Latin America by marchitez sorpres-
siva in Colombia, Peru, Ecuador and by hartrot in Surinam. The etiology of these
diseases remains still insufficiently clear, and possibly under these names we are
dealing with two or more diseases. There are two important aspects that emerge from
recent work in Latin America and the Caribbean area. One is the association of fla-
gellate organisms to diseases of oil and coconut palms. Second is the possibility
that other crops such as coffee could also be involved. Very important is the fact
that an insect vector, Haplaxius palidus has been shown to be the vector of one of
these diseases. This should encourage the insect vector men in Florida and Jamaica
to intensify their research of lethal yellowing. The utilization of Phytomonas as
a guide in the culturing of MLO's has been suggested. I feel that there may be some
better, closer models already available. Altogether it seems from this session that
much is to be learned from studying of diseases of other palms and that this cross
fertilization will be a useful future undertaking for ICLY.









BUSINESS MEETING

INTERNATIONAL COUNCIL ON LETHAL YELLOWING

JUPITER, PALM BEACH COUNTY, FLORIDA

November 1, 1977

The meeting was called to order by Dr. D. H. Romney, Chairman of the Third Meeting
of the International Council on Lethal Yellowing (ICLY). He expressed appreciation
to the organizing committee at the University of Florida's Agricultural Research
Center in Ft. Lauderdale for developing meeting and program plans, to Mrs. E. M. Rey
for the excellent local arrangements and coordination of many activities and functions
that added to the success of the meeting. He also acknowledged the sponsors who pro-
vided financial support for the meeting and made possible the attendance of several
out-of-country scientists who contributed to the technical discussions, and to others
who contributed to the success of ICLY-3. The Chairman indicated that the agenda in-
cluded the ICLY newsletter, publication of meeting proceedings, place of next meeting,
inclusion of palm diseases other than lethal yellowing (LY) in ICLY activities and
programs, priorities for future research emphasis, and election of officers.

ICLY NEWSLETTER. There was discussion of the need to continue issuing an ICLY News-
letter periodically. It was agreed that this was a job for the Secretary. As Dr. L.
Chiarappa planned to be away from his office in Rome for several months it was agreed
that newsworthy items should be sent to Mr. H. C. Harries in Jamaica who would collate
the information and forward it to the Secretary. These are to be included in the
yearly progress report on Coconut Breeding issued by FAO.

PUBLICATION OF PROCEEDINGS. There was agreement that a report of the meeting should
be prepared as promptly as feasible to send to the sponsors of the ICLY-3 meeting.
This would include abstracts of the meeting.

Alternatives for publishing the abstracts of ICLY-3 meeting were considered. Three
alternatives were: (1) Oleagineaux, (2) Principes or (3) by the University of Florida.
It was agreed that the feasibility of publishing in these outlets would be checked
in an appropriate manner by Drs. Dollet, Parthasarthy and Purdy, respectively. After
this was done, the final decision on where to publish would be left to the discretion
of the Executive Committee. Individual authors should take initiative to publish any
full-length papers on research discussed at ICLY-3.

PLACE OF NEXT MEETING. Consideration was given to scheduling the next meeting in West
Africa, so that the disease situation in the coconut palm might be assessed first hand
by the scientists who would be attending an ICLY meeting. Funding for such a meeting
in West Africa was discussed and was deemed to pose a problem, especially the travel
costs. It was decided that the Fourth Meeting of ICLY should be held in Florida, pro-
bably in the Fall of 1979. It was suggested that the Executive Committee consider
arranging for an ICLY representative to attend the next meeting of the FAO Technical
Working Party on Coconut Production, Protection and Processing that is scheduled for
Africa. This representative should stay longer to assess the disease situation and
stimulate local interest in collaborative work between workers in West Africa,
Jamaica, USA and possibly other countries.









INCLUSION OF PALM DISEASES OTHER THAN LETHAL YELLOWING. There was considerable
discussion of whether ICLY should consider palm diseases other than LY. It was
decided to continue concentrating activities principally on LY, but to include
other threatening diseases of palms for consideration and possible discussion at
future meetings.

PRIORITY AREAS FOR FUTURE LY RESEARCH. The following areas were identified for
future research emphasis:

1. Culturing of mycoplasmalike organisms from host palm tissue
and suspected insect vectors.
2. Transmission of the disease (including survey of potential
vectors in West Africa and other areas where LY occurs).
3. Search for possible alternate hosts of LY.
4. Breeding and evaluating for disease resistance in coconut
and other palms.
5. Physiology of palms.
6. Rapid disease diagnosis methods for LY.
7. Evaluation of seed transmission of LY and methods to insure
disease-free seed.

ELECTION OF OFFICERS. The following were elected to serve as officers of ICLY until
the next Meeting of the Council:

Chairman W. B. Ennis, Jr.
Secretary L. Chiarappa
Executive Committee E. A. Addison
M. Dollet
G. Martinez-Lopez
D. H. Romney
H. Waters

GERMPLASM TRANSFER. There was discussion of the need for ICLY to assist in efforts
to discourage the movement of palm seed and plants collected in areas infested with
LY. ICLY went on record as commending the Palm Society and Fairchild Tropical Garden
for imposing a moratorium on exporting seed obtained from LY affected areas.

The meeting was adjourned by Chairman Romney after announcements on plans for the
field trip and program for November 2, and the wrap-up session on November 3, 1977.










REGISTRANTS FOR THIRD MEETING

OF INTERNATIONAL

COUNCIL OF LETHAL YELLOWING

1. INTERNATIONAL

Dr. E. A. Addison, Ghana
Dr. Luigi Chiarappa, Italy
Dr. Andrew J. Dabek, Jamaica
Dr. Michael J. Daniels, United Kingdom
Dr. Michel Dollet, France
Dr. S. J. Eden-Green, Jamaica
Dr. Fred Eskafi, Jamaica
Ing. Carlos Garzon F., Ecuador
Mr. Astor Goodison, Jamaica
Mr. Hugh C. Harries, Jamaica
Dr. Peter Hunt, Jamaica
Dr. R. H. Kenten, United Kingdom
Dr. Gerardo Martinez Lopez, Colombia
Mr. I. Osborne, Jamaica
Professor A. F. Posnette, United Kingdom
Mr. D. H. Romney, Jamaica
Mr. Mark Schuiling, Brazil
Dr. Kurt George Steiner, West Germany
Ir. W. G. Von Slobbe, Surinam
Dr. Henry Waters, Jamaica

2. UNITED STATES

Dr. John B. Carpenter, Indio, California
Dr. T. A. Chen, New Brunswick, New Jersey
Dr. R. E. Davis, Beltsville, Maryland
Dr. James L. Hilton, Beltsville, Maryland
Dr. George H. Kaloostian, Riverside, California
Dr. Karl Maramorosch, New Brunswick, New Jersey
Ms. Janet McDonough, Petersham, Massachusetts
Dr. Harold E. Moore, Ithaca, New York
Dr. Howard D. Ohr, Riverside, California
Dr. M. V. Parthasarathy, Ithaca, New York
Dr. William R. Phelps, Atlanta, Georgia
Dr. William H. Sites, Asheville, North Carolina
Dr. P. Barry Tomlinson, Petersham, Massachusetts
Dr. Joseph Tully, Bethesda, Maryland
Dr. Martin H. Zimmermann, Petersham, Massachusetts

3. FLORIDA

Dr. S. A. Alfiari, Jr., DPI, Gainesville
Dr. J. L. App, University of Florida, Gainesville
Ms. Sandra K. Austin, Pine Jog Envir. Sci. Ctr., W. Palm Beach
Dr. H. G. Basham, ARC, Ft. Lauderdale
Ms. Elisabeth Beeman, ARC, Ft. Lauderdale










FLORIDA continued

Mr. William P. Brook, Wildwood Tropical Nursery, Dania
Dr. Pauline Calloway, Extension Service, Gainesville
Mr. Joseph Caporale, Public Works, Lake Park
Dr. W. J. Carpenter, University of Florida, Gainesvillc
Ms. Pat Cortez, Extension Service, Palm Beach
Mr. L. J. Daigle, Extension Service, Miami
Mr. John F. Dance, Parks & Recreation, Lake Worth
Dr. H. M. Donselman, ARC, Ft. Lauderdale
Dr. W. B. Ennis, Jr., ARC, Ft. Lauderdale
Dr. Jack B. Fisher, Fairchild Tropical Garden, Miami
Mr. George H. Gwin, DPI, Miami
Mr. Brian Houha, ARC, Ft. Lauderdale
Dr. F. W. Howard, ARC, Ft. Lauderdale
Mr. C. E. Hutcheson, Extension Service, Palm Beach
Mr. Glen Hutchinson, ARC, Ft. Lauderdale
Dr. F. G. Maxwell, University of Florida, Gainesville
Dr. R. E. McCoy, ARC, Ft. Lauderdale
Mr. Michael Moore, DOF, Ft. Lauderdale
Ms. Ronelle Norris, ARC, Ft. Lauderdale
Ms. Betti Patterson, ARC, Ft. Lauderdale
Mr. Charles Poucher, DPI, Winter Haven
Dr. L. H. Purdy, University of Florida, Gainesville
Mr. Donald Richardson, Pine Jog Envir. Sci. Ctr., W. Palm Beach
Mr. Carter P. Seymour, DPI, Gainesville
Ms. Alexis Smith, University of Florida, Gainesville
Mr. John Tamsberg, DOF, Ft. Lauderdale
Mr. William Theobald, DOF, Ft. Lauderdale
Dr. D. L. Thomas, ARC, Ft. Lauderdale
Dr. J. H. Tsai, ARC, Ft. Lauderdale
Mr. Harlo E. Von Wald, DPI, Miami
Mr. T. Watrous, University of Florida, Gainesville
Mr. Lewis E. Watson, Extension Service, Ft. Lauderdale
Dr. S. H. West, University of Florida, Gainesville
Mr. C. Whittier, Palm Springs
Mr. Robert B. Whitty, Extension Service, Stuart
Ms. Donna Williams, ARC, Ft. Lauderdale
Mr. Charles T. Woods, University of Florida, Gainesville




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