Citation
Dasheen mosaic virus of cultivated aroids and its control by seed propagation and culture of shoot tips

Material Information

Title:
Dasheen mosaic virus of cultivated aroids and its control by seed propagation and culture of shoot tips
Creator:
Hartman, Robert Dale, 1948- ( Dissertant )
Zettler, F. William ( Thesis advisor )
Edwardson, J. R. ( Reviewer )
Hiebert, E. ( Reviewer )
Knauss, J. F. ( Reviewer )
Purcifull, D. E. ( Reviewer )
Sheehan, T. J. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1974
Language:
English
Physical Description:
xii, 80 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Araceae ( lcsh )
Dissertations, Academic -- Plant Pathology -- UF
Mosaic diseases ( lcsh )
Plant Pathology thesis Ph. D
Orange County ( local )
Symptomatology ( jstor )
Taro ( jstor )
Seedlings ( jstor )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Dasheen mosaic virus (DMV) was recovered from species representing 13 genera in the Araceae, but did not infect seedlings of « species in 1 7 other families. The following results of this investigation support earlier reports that DMV is a member of the "potato virus Y" group of plant viruses: 1) . mean particle length of 782 nm was recorded, 2) characteristic cylindrical inclusions were observed and 3) the virus proved serologically related to potato r and tobacco etch' viruses. Surveys indicate that DMV is prevalent in commercial plantings of aroids and induces serio, losses. Infected plants were encountered in all Caladiu - °rtUlanU ' m - (cal^->> Colocasia esculenta (taro) and Kanil- soma sagittifolnnm (cocoya*) fields surveyed in Florida and in all stock beds of Dieffenbachia. nicta (dieffenbachia) xn ornamental foliage plant nurseries visited. Based on these surveys, it appears that DMV has become uniformly established in certain cultivars of caladiu. and dieffenbachia. The deleterious effects of DMV on yields of caladium, dieffenbachia, Philodendron selloum and Zantedeschia elliottiana were assessed in controlled experiments. Weight losses of ZfO, 63, 30 and 59% and reductions in leaf area of 33, 66, 30 and 51% v/ere recorded, respectively. Virus-free plants of caladium, dief fenbachia, and Zantedeschia spp. were obtained through zee J. propagations, although caladium and dieffenbachia progeny differed from parental plants. Excised shoot-tips of caladium, cocoyam and taro v/ere successfully cultured aseptically and free of DMV on the revised medium of Murashige and Skoog. Callus of all three species proliferated into numerous plantlets on this medium and plantlets survived transplanting from culture to soil. Caladium plantlets assumed characteristic foliar variegation within three months after transplanting and attained maturity within 6-7 months.
Thesis:
Thesis (Ph. D.)--University of Florida, 1974.
Bibliography:
Includes bibliographical references (leaves 74-79).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Dale Hartman.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030179754 ( AlephBibNum )
ACJ0100 ( NOTIS )
77942037 ( OCLC )

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Full Text












DASHEEN MOSAIC VIRUS OF CULTIVATED AROIDS AND ITS
CONTROL BY SEED PROPAGATION AND CULTURE OF SHOOT TIPS







By

ROBERT DALE HARTMAN


A: [.i .J E ;:' ,' i I ;r 7- :E F .^I -^t i l E '.. !. -: ,;:. i, I_ ( ii ",, 'i'; _L ,_,i
"i : '- ,,i E; i i:IK:;' F -i.,.: T .L.:
ih F 1-1Tir ,L'LC r LF -'- r F- r: FiL-'. F-Hi


i4.4





























Veil your face, 0 foolish experimenter,
overconfident in your mischievous straw!
You thought that you had created a new
type of instrumentalist; and you have
obtained nothing at all. The Cricket
has thwarted your schemes: he is scraping
with his right fiddlestick and always will.
Your sorry science tried to make a left-
handed player of him. He laughs at your
devices and settles down to be right-handed
for the rest of his life.

-- J. Henri Fabre




























Figure 1. (Frontispiece) In vitro culture of cala-
dium, Caladium hortulanun 'Candidum',
(right) and fully developed plant (left)
transferred from culture to soil 6 months
previously.













ACKNOWLEDGEMENTS


The author wishes to extend sincerest appreciation to

Dr. F. W. Zettler for his support, guidance, encouragement

and time that he has so generously given throughout this

study. Special thanks are also extended to Drs. J. R. Ed-

wardson, E. Hiebert, J. F. Knauss, D. E. Purcifull and T.

J. Sheehan for their support in specific areas of this study

and for their general guidance given throughout this study.

Also, special thanks are due Mrs. Thelma C. Carlysle, USDA

Research Technician for her assistance in producing the

scanning electron micrographs of the caladium apical shoot-

tips.

Sincere appreciation is due the members of the Depart-

ment of Plant Pathology and especially all the personnel of

the Plant Virus Laboratory whose assistance helped make this

work possible.

Lastly, the author wishes to thank his wife for her con-

tinual support and love so generously given throughout this

study and for her typing expertise and critical comments

which facilitated the completion of this dissertation.












TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS . . . . . . . . . v

LIST OF TABLES. . . . . . . . . . viii

LIST OF FIGURES .................. ix

ABSTRACT .. ...... ....... ... .... xi

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

MATERIALS AND METHODS . . . . . . . . 5

Characterization of Dasheen Mosaic Virus . .. 5
Host-range Determinations. . . . . .. 5
Transmission Electron Microscopy . . . 6
Leaf extracts. . . . . . . . 6
Sectioned material ........... 6
Scanning Electron Microscopy ........ 8
Light Microscopy . . . . . . . 8
Purification of Virus and Virus-induced Cyto-
plasmic Inclusions. . . . . . . 8
Serology . . . . . . . . . 9
Distribution and Effects of Dasheen Mosaic Virus 11
Surveys. . . . . . . . . .. 11
Symptom Expression . . . . . ... 12
Quantitative Effects . . . . . . 13
Elimination of Dasheen Mosaic Virus . ... 13
Seed.Propagation . . . . . . . 13
Shoot-tip Culture. . . . . . . ... 14

RESULTS . . . . . . . . ... . . 17

Characterization of Dasheen Mosaic Virus . .. 17
Host-range Determinations. . . . . .. 17
Transmission Electron Microscopy . . .. 24
Leaf extracts. . . . . . . ... 24
Sectioned material . . . . . ... 24
Light Microscopy . . . . . . .. 27
Purification of Virus and Virus-induced Cy-
toplasmic Inclusions. . . . . . ... 27
Serology . . . . . . . . . 34
Distribution and Effects of Dasheen Mosaic Virus 37
Surveys. . . . . . . . . ... 37
Symptom Expression . . . . . . .. 37
Quantitative Effects . . . . . ... 45







Page

Elimination of Dasheen Mosaic Virus . . ... .49
Seed Propagation ............... 49
Shoot-tip Culture............... 55

DISCUSSION .. . .. . . .. . .. . .. 63

LITERATURE CITED. .................. 74

BIOGRAPHICAL SKETCH . . . . . . .... 80













LIST OF TABLES


ABLE Page

1 Araceous plants susceptible to dasheen
roaic. virz .., . ................ 18

2 Plants not infected with dasheen mosaic
virus following manual inoculations ... 21

3 Systemic symptoms expressed on cocoyam
leaves formed after inoculation of plants
with dasheen mosaic virus . . . .. 44

4 Effects of dasheen mosaic virus on fresh
weights and leaf sizes of caladium, dieffen-
bachia, Philodendron sellout and Zantedeschia
elliottiana plants. . . . . . ... 48












LIST OF FIGURES


FIGURE

1 In vitro culture of 'Candidum' caladium and
fully developed plant transferred from cul-
tale to soil . . . . . . . .

2 Thin sections of DMV-infected caladium leaf
tissue showing pinwheel, circular, bundle and
laminated aggregate inclusions . . . .

3 Stained epidermal leaf cells of caladium con-
taining cytoplasmic inclusions and negatively
stained particles of dasheen mosaic virus . .

4 Ultraviolet absorption spectrum of a partial-
ly purified preparation of dasheen mosaic
virus from dieffenbachia . . . . . .

5 Laminated aggregate and tubular inclusions
and particles of dasheen mosaic virus purified
from infected caladium and dieffenbachia leaves

6 Serological reactions of dasheen mosaic virus
to potato virus Y and tobacco etch antisera .

7 Dasheen mosaic virus symptoms in dieffenbachia
and Philodendron selloum . . . . . .

8 Dasheen mosaic virus symptoms in cocoyam and
'Candidum' caladium leaves . . . . .

9 Foliar symptoms of dasheen mosaic virus ex-
pressed by dieffenbachia plants during a 12
month study period under two temperature re-
gimes . . . . . . . . . . .

10 Dieffenbachia spadix during pollen shed and at
various stages after pollination . . . .

11 Caladium spadix prior to pollen shed, at pollen
shed, and at various stages after pollination

12 Dieffenbachia seedlings 12 months after germi-
nation. . . . . . . . . . .


"Frontispiece"


26


29



31


33

36


40


42



47

51








FIGURE Page

13 In vitro culture of shoot-tips used to elimi-
nEte dasheen mosaic virus and other phytopatho-
gens from infected aroids. . . . . ... 60






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


DASHEEN MOSAIC VIRUS OF CULTIVATED AROIDS AND ITS
CONTROL BY SEED PROPAGATION AND CULTURE OF SHOOT TIPS


By
Robert Dale Hartman

June, 1974

Chairman: Dr. F. W. Zettler
Major Department: Plant Pathology


Dasheen mosaic virus (DMV) was recovered from species
representing 13 genera in the Araceae, but did not infect

seedlings of 45 species in 17 other families. The follow-

ing results of this investigation support earlier reports

that DMV is a member of the "potato virus Y" group of plant

viruses: 1) a mean particle length of 782 nm was recorded,

2) characteristic cylindrical inclusions were observed and

3) the virus proved serologically related to potato Y and

tobacco etch viruses. Surveys indicate that DKV is preva-

lent in commercial plantings of aroids and induces serious

losses. Infected plants were encountered in all Caladium

hortulanum (caladium), Colocasia esculenta (taro) and Xan-

thosoma sagittifolium cocoyamm) fields surveyed in Florida

and in all stock beds of Dieffenbachia picta dieffenbachiaa)

in ornamental foliage plant nurseries visited. Ba;:.- :i-

these surveys, it appears that DMV has become unif'..or

established in certain cultivars of caladium and C: :'f5r.a:t.i.








The deleterious effects of DMV on yields of caladium, dieffen-

bachia, Philodendron sellout and Zantedeschia elliottiana were

assessed in controlled experiments. Weight losses of 40, 63,

30 and 59% and reductions in leaf area of 53, 66, 30 and 51%

were recorded, respectively. Virus-free plants of caladium,

dieffenbachia, and Zantedeschia spp. were obtained through

seed propagatic., although caladium and dieffenbachia pro-

geny differed from parental plants. Excised shoot-tips of

caladium,cocoyam and taro were successfully cultured aseptically

and free of DMV on the revised medium of Murashige and Skoog.

Callus of all three species proliferated into numerous plant-

lets on this medium and plantlets survived transplanting from

culture to soil. Caladium plantlets assumed characteristic

foliar variegation within three months after transplanting

and attained maturity within 6-7 months.











INTRODUCTION


The family Araceae includes about 105 genera and 1400-

1500 species of plants which are primarily of tropical or

subtropical distribution, although several species occur in

temperate climates. Species in eight genera are indigenous
to the United States. The typical aroid inflorescence is a

spadix subtended by a large, often showy spathe. These plants

are mostly monoecious and dichogamous.

Many aroids are economically important. In Florida,
species of Aglaonema, Dieffenbachia, Philodendron, Scindap-

sus and Syngonium account for over half of a $25,700,000/

year foliage industry (16). Approximately 96% of the world's

commercially produced caladiums are also grown in Florida

(39). Cryptocoryne spp. are of considerable significance
to the aquarium plant industry of Florida, and another aqua-

tic aroid, Pistia stratiotes, ranks as one of the world's

most noxious waterweeds.

On an international basis, perhaps the most significant
aroid genera are those containing edible species. The .:.:-

yams (Xanthosoma spp.) and taros (Colocasia spp.) are :'1rrj-

hydrate staples in many tropical and subtropical areas tr.r:,urh-

out the world. In Florida approximately 3000 acres of re

former are produced for the Cuban populace (49,54). 01' i.:r







importance as food crops are species of Alocasia, Amornho-

phallus, and Cvrtosnerma. These are grown principally in

Japan, parts of Southeast Asia, and in the South Pacific.

Most horticulturally important aroids are propagated

exclusively by vegetative means. Many aroid species are

sterile (51) and Hill described taro as having "been cul-

tivated f^r so lor. that it never flowers" (38). A few

aroids, however, are readily seed propagated including some

species of A:aloneaa, Anthurium and Philodendron. For many

aroids, special techniques must be employed to achieve

fertilization (46). Since, however, most ornamental aroids

are heterozygous cultivars, progeny do not necessarily re-

flect parental types (46,71).

Continued vegetative propagation has enabled certain

phytopathogens to become prevalent in stock plantings of

many aroids. Viruses can become especially serious under

these circumstances and indeed infections of tomato spotted

wilt virus in Zantedeschia spp. and cucumber mosaic virus in

Arum italicum are well documented (22,45,66,67). Virus-like

diseases have also been reported in species of Amornhoohallus

(10,12), Anthurium (34,35,70), Philodendron (65) and P. stra-

tiotes (53). In addition, a virus causing the "chirke disease"

of large cardamon was found to be sap-transmissible to the

aroid Acorus calamus (56).

In 1970, Zettler et al. (74,75) reported a filamentous

virus infecting Colocasia esculenta in Florida which proved

mechanically transmissible to seedlings of Philodendron







sellout and Zantedeschia elliottiana. Unlike the filamentous

anthurium virus described by Herold, it failed to infect to-

bacco. This virus was designated as dasheen mosaic virus

(DMV) and was characterized as belonging to the Potato Y virus

group of Brandes (7) and Brandes and Bercks (8) in: (i) being

aphid transmitted in a stylet-borne manner by aphids, (ii) having

a mea.. particle length of 750 nm, and (iii) inducing characteris-

tic cylindrical inclusions. An isolate of DMV was later charac-

terized by Alconero (1) as having a (i) dilution end point be-

tween 1/100 and 1/1000, (ii) thermal inactivation point in

10 minutes between 60 and 65 C and (iii) longivity in vitro of

75 hours at 26 C.

Since its discovery in 1970, DMV has been reported from

Zantedeschia spp. in the Netherlands (69) and from taro and/or

cocoyam in the British Solomon Islands (23,40,41,42), Trinidad

(42), Venezuela (17), Puerto Rico (2), Hawaii (2), the Fiji
Islands (32) and Japan (3). In addition, a mosaic disease of

Philodendron selloum in California (65) is assumed to be in-

duced by DMV (H. R. Hill, personal communication).

Two bacilliform viruses of taro have been described in
the British Solomon Islands (23,40,42). These viruses in

combination with DMV reportedly kill infected plants causing

a condition referred to locally as "alomae". Unlike DMV,

however, neither bacilliform virus has been detected outside

the island of Malaita. Alomae-infected plants were not

detected elsewhere in the Protectorate, nor were they ob-

served in material from Trinidad or Ghana.







The purpose of this study was to further characterize

DMV, assess its distribution and importance among aroids in

Florida and to investigate practical control measures for

this virus. Attempts were also made to identify other viruses

of aroids in Florida. Portions of this dissertation have been

published elsewhere (26,27,28,29,30,31).












MATERIALS AND METHODS


Characterization of Dasheen Mosaic Virus


Host-range Determinations

All host range determinations were based upon manual

transmission trials using inoculum expressed from symptoma-

tic leaves and diluted with deionized water. Six hundred

mesh carborundum was used as an abrasive. Unless otherwise

noted, all test plants were grown from seed. Two or more

leaves were inoculated per test plant. At least 4 P. sel-

loum seedlings were inoculated after all host range trials

to insure that the inoculum used was infectious, except in

one instance when two caladium seedlings were used subsequent

to inoculation of Rhoeo discolor. Test plants were maintained

for at least 14 days following inoculations. Regardless of

symptom expression, attempts were made to recover virus from

test plants. In recovery trials, at least 3 P. selloum seed-

lings were rubbed with extracts from inoculated leaves and

with extracts from leaves which developed subsequent to ino-

culation.

Whereas aroids were tested at various times throughout

the year, all non-aroids were inoculated August-October when

greenhouse temperatures were 20-30 C. At least 5 itrL. iui

of each plant species were inoculated except that cni -,







and 2 plants of Nicotiana sylvestrus, N. 7lutinosa and N.

clevelandii were used, respectively.


Transmission Electron Microscopy

All plant material was examined using a Philips Model

200 electron microscope operating with an accelerating vol-

tage of 60 KV. Size determinations were made by comparing

projected electron micrographs with projected micrographs of

a.54,864 line/inch diffraction grating.


Leaf extracts

All material used for examining virus particle morpho-

logy was obtained from negatively stained leaf extract pre-

parations. Mounts were prepared by dicing a small piece of

symptomatic tissue (ca. 3mm2) in several drops of 1-2% phos-

photungstic acid (PTA), pH 6.8, contained on a glass slide.

The diced tissue was mixed briefly with the PTA and a droplet

of the mixture was placed on a specimen grid coated with

Formvar and strengthened with arc-vaporized carbon. After

one minute, excess fluid was absorbed with filter paper.

Specimens were then examined with the electron microscope.

A similar procedure was used for preparing virus-induced

cytoplasmic inclusions for examination except that leaf ex-

tracts were stained in 1% ammonium molybdate, pH 6.5, rather

than PTA (55).

Sectioned material

For electron microscopic observations of virus-induced






cytoplasmic inclusions, 2mm2 sections of leaf tissue were cut

from Caladium hortulanum 'Candidum' and Dieffenbachia ricta

'Exotica' leaves with symptoms. The leaf sections were placed

in a 6% solution of glutaraldehyde in cacodylate buffer at

pH 7.2. The tissue was placed under vacuum for 5 minutes and
fixed for 1-3 hours at 4-5 C. The tissue was then washed

twice for 15 minutes each with cacodylate buffer and post

fixed for 1.5 hours at 23 C with 2% osmium tetroxide. The

tissue was washed twice for ten minutes each with buffer

and deionized water, respectively. The tissues were pro-

gressively dehydrated in a series of increasing concentra-

tions of ethanol to 100%. At 70% ethanol they were stained

for 5-10 hours in 2% uranyl acetate at 3-4 C. The tissue

was subsequently transferred through two changes of 100%

acetone for 15 and 30 minutes, respectively, at 23 C. The

embedding plastic was an Epon 812, Araldite "M" mixture as

described by Mollenhauer (47). The embedding procedure was

done in three steps of 30, 70 and 100% plastic contained in

acetone for 1 hour each after which it was placed in a vacuum

oven at 60 C to remove the acetone. The tissue was placed

in a drying oven overnight at 60 C and further polymerized

in an oven at 80 C for 1-2 days.

Tissues were sectioned with glass knives mounted on a

Sorvall Model MT-2 ultramicrotome. They were transferred

to acid-cleaned, uncoated specimen grids and stained for 15

minutes in 0.5% aqueous uranyl acetate, rinsed in water, a'r

subsequently stained for 5 minutes in lead citrate (- i. Tr.







sections were then rinsed in deionized water and allowed to

dry prior to examination with the electron microscope.


Scanning Electron MicroscoTy

Apical shoots of 'Candidum' caladium were prepared for

examination with a Cambridge Mark II stereoscanning electron

microscope operating with an accelerating voltage of 10-20 KV.

Shoot tissue comprising the apical dome and 1-2 leaf primor-

dia were dissected and mounted on a 12mm diameter aluminum

specimen stub covered with a thin layer of silver base paint.

The specimens were examined as fresh material rather than

being coated with metal.


Light Microscopy

Epidermal strips were removed from the abaxial surfaces

of DMV-infected and healthy leaves of caladium, dieffenbachia

and Colocasia esculenta (taro) and prepared for examination

with a light microscope according to the procedures described

by Christie (13,14) for staining virus-induced inclusions.

Epidermal strips were placed in 5% Triton X-l00 for 5 minutes

and subsequently stained for 10 minutes in calcomine orange

and "Luxol" brilliant green. The strips were rinsed briefly

in 95% ethanol and mounted in Euparal on a glass slide.


Purification of Virus and Virus-induced Cytomlasnic Inclusions

Attempts were made to purify virus particles and cyto-

plasmic inclusions from leaves of 'Candidum' caladium and

'Exotica' dieffenbachia with symptoms and from entire symp-






tomatic shoots of the latter. All DMV-infected plants were

obtained from commercial propagators in Orange County. All

purification procedures were conducted at 4 C and 500g of

tissue were used in each purification run.

The virus purification procedure was as described by

Hiebert and McDonald (36) except that the final step in-

volved three rather than one cycle of differential centri-

fugation.

The procedure used to purify the cytoplasmic inclusions

induced by DMV was as described by Hiebert et al. (37) and

modified by Hiebert and McDonald (36).

Serology

Attempts were made to obtain antiserum specific to DMV.

Virus purified from 'Exotica' dieffenbachia leaves was emul-

sified (1:1, v/v) with Freund's incomplete adjuvant and in-

jected intramuscularly into a rabbit. Only one injection

was made. The blood was collected 26 days after injection,

incubated at 37 C in a water bath, and allowed to clot. The

serum fraction was collected, placed in glass ampules, and

frozen. Antigens were prepared by triturating 2 g of symp-

tomatic or healthy 'Candidum' caladium leaf tissue with a

mortar and pestle in 2 ml of deionized water. The triturated

sap was then expressed through cheesecloth and centrifuged

at approximately 2600 g for 30 minutes. The supernatant

was then discarded and the pellet resuspended in 1 ml of

water. In this trial, the gels consisted of 0.8% Noble agar,








0.5% sodium dodecyl sulfate (SDS), and 1% sodium azide in

water (24).

The Ouchterlony agar double-diffusion technique was

used in all serological studies. The respective antisera

were placed in center wells and the antigens in peripheral

wells. Test plates were incubated 1-4 days at 25 C and ob-

served periodically. In all tests, normal serum and leaf

extracts from healthy plants were used as controls.

In other trials, attempts were made to determine the

serological relationships of DMV to other viruses of the

potato Y virus group. These tests were conducted with anti-

sera to pepper mottle virus, potato virus Y, tobacco etch

virus and turnip mosaic virus, obtained from D. 7. Purcifull

(Department of Plant Pathology, University of Florida, Gaines-

ville). Antigens of DMV were prepared by triturating 4 g

of symptomatic or healthy leaf tissue of P. selloum with a

mortar and pestle in 8 ml of water. The triturated sap was

then expressed through cheesecloth and centrifuged at approxi-

mately 2600 E for 30 minutes. The supernatant was discarded

and the pellet was resuspended in 8 ml of a 3% aqueous pyrroli-

dine solution. Antigens were either derived from P. selloum

seedlings which had been inoculated 8 or 56 days previously

with a DIN isolate originally from 'Candidum' caladiums or

from pooled leaf tissue from P. selloum seedlings which had

been inoculated 13 days previously with DMV isolates from

various naturally infected caladium varieties 'Itocapus',

'John Peed', 'Dr. Groover', 'Avalon Rose', 'W. B. Halderman'






and 'Crimson 'ave'. Antigens to the other PVY-type viruses

were obtained from D. Batchelor (Department of Plant Patho-

logy, University of Florida). These antigens were prepared

by triturating 2 g of leaf tissue in 4 ml of water. The

sap was then expressed through cheesecloth, pipetted into

vials and freeze dried. When virus samples were needed,

4 ml/vial of a 3% pyrrolidine solution was added. The so-

lution was stirred and allowed to stand for 30 minutes prior

to use. The gels used in these tests consisted of 0.6% Noble

agar, 0.05M Borate buffer at pH 8.2 and 0.02% sodium azide.


Distribution and Effects of Dasheen Mosaic Virus


Surveys

Surveys were conducted from 1969-1971 in an attempt to

assess the prevalence of DMV in Florida. These surveys in-

cluded foliage nurseries in Orange and Dade counties, cala-

dium fields in Highlands and Orange counties, Xanthosoma

sagittifolium cocoyamm) fields in Dade County, an aquarium

plant nursery in Palm Beach County and experimental and orna-

mental plantings of taro at the University of Florida, Gaines-

ville. In addition, aroid samples were solicited from areas

outside continental United States, and assessed for DMV in-

fection. All plants received were maintained in isolation

and destroyed upon completion of tests.

Infections of DMI were assessed on the basis of symptoms

expressed and recovery of virus, to at least 1 of 4 or more

manually inoculated seedlings of P. selloum. Certain samples








were further checked by electron microscopic examinations of

negatively stained leaf extracts.


Symptom ExTression

Throughout the course of this study, various aroids in-

fected with DMV were maintained in greenhouses and periodically

examined for symptoms expressed.

In one experiment, 70 rooted cane pieces of 'Exotica'

dieffenbachia were obtained from a foliage nursery in Orange

County, Florida. All were cuttings of the same developmental

stage when collected, and all except 4 exhibited foliar dis-

tortion and mosaic. Half of the plants were placed in a green-

house where the temperature varied from 20-40 C, whereas the

others were maintained in a different greenhouse at 24-30 C.

The light intensities of both greenhouses were equivalent and

plants in both greenhouses were routinely watered and ferti-

lized. Each plant was observed routinely from April 20, 1971

to March 1, 1972 and the symptoms expressed on each leaf re-

corded. On July 19, half the plants in each greenhouse were

transplanted from 4 to 6 inch pots and examined for any re-

sultant effects on symptom expression.

In a similar experiment, 6 cocoyam plants rendered free

of DKV through tissue culture were selected and manually ino-

culated with DM 1. Thereafter, these plants were maintained

for 5 months in a greenhouse at 20-40 C. Throughout this time,

these plants were checked periodically for foliar symptoms.








Quantitative Effects

The effects of DMV on growth of caladium, dieffenbachia,

P. sellout and Z. elliottiana were assessed after manual ino-

culation. All planting stock used was propagated from seed

and maintained in isolation until inoculated. Plants of P.

sellout and Z. elliottiana were inoculated as seedlings and

an equal number of non-inoculated seedlings served as con-

trols. Seedlings of caladium and dieffenbachia were main-

tained in isolation until they attained a size suitable for

vegetative propagation. They were then vegetatively propagated

and genotypically identical pairs were selected for compari-

sons between inoculated and non-inoculated plants.

Inoculated and non-inoculated plants were maintained in

pots on greenhouse benches for observation. All data except

for Z. elliottiana were recorded within a 12 month period.

Results for Z. elliottiana were recorded 2.5 years after

inoculation during which time the corms were harvested, stored,

and replanted twice. Fresh corm weights, leaf areas (maximum

length X maximum width) and petiole lengths were recorded for

caladium and Z. elliottiana. Fresh shoot weights, leaf areas,

and shoot heights were recorded for dieffenbachia and total

fresh plant weights and leaf areas were recorded for P.

sellout.


Elimination of Dasheen Mosaic Virus

Seed Propagation

Seed of caladium, dieffenbachia, and Zantedeschia spp.








were produced by intraspecific crosses. These crosses were

made between plants of 'Candidum' caladium, 'Exotica' dieffen-

bachia, Z. albo-maculata, Z. elliottiana, or Z. rehmannii.

All plants used in the crosses were shown to be infected with

DMV based on symptoms and transmission to seedlings of P.

selloum. Dieffenbachia plants were grown in a stock bed in

a fiberglass house at Apopka and pollinated May-June, 1971.

The caladiums were grown in Gainesville either in a green-

house or outdoors and pollinated May-June, 1972. The Zante-

desclia spp. were grown in a greenhouse at Gainesville main-

tained at 24-30 C and pollinated May-June, 1971. The polli-

nation procedures were similar to those described by McColley

and Miller (46) for philodendron. All crosses were made be-

tween 6-10 a.m. or 5-8 p.m. Pollen was collected daily with

a camel-hair brush and transferred within 48 hours after

shedding to neighboring receptive blooms of different plants.

Bloom receptivity was indicated by the increased stickiness

of the stigmatic surfaces of the spadix. Bloom receptivity

of caladium and dieffenbachia was further indicated when the

spathe began to unfurl revealing the distal portion of the

spadix. Prior to pollination, the spathe was cut away from

the spadix, discarded, and the pollen was gently applied to

the proximal ovulate portion of the spadix with a brush.


Shoot-tin Culture

Attempts were made to culture the following aroids in

vitro: 'Candidum' and 'Frieda Hemple' caladium, taro, cocoyam,

'Exotica' dieffenbachia, Aelaonema modestum (chinese evergreen),






P. sellout, Cryotocoryne cordata and G. nevillii.

In this study, the "revised" medium of Murashige and
Skoog (50) was used except that (i) meso-inositol (i-inositol

dihydrate) was substituted for myo-inositol, (ii) Edamin was

omitted and (iii) each liter of medium contained 8 rather

than 10 g of agar. The kinetin level employed was 1.0 mg/l

of medium and the indole-acetic acid level was 15.0 mg/l.

Shoot-tips from all plants were excised and treated se-
quentially as follows: shoots were (i) trimmed to 1.5 cm3,

(ii) rinsed in flowing tap water, (iii) submerged for 10

minutes in 0.52% sodium hypochlorite, (iv) trimmed further,

(v) submerged for 5 minutes in 0.26% sodium hypochlorite and

(vi) rinsed briefly in sterile distilled water. With the aid

of a dissecting microscope, each shoot was then trimmed until

only the apical dome and one or two leaf primordia remained

(Fig. 13A,B). Finally, each shoot was transferred, cut sur-

face down, to the surface of 15 ml of solidified medium con-

tained in a 30 ml screw-cap vial. All cultures were main-

tained at 21-25 C and provided 12 hours of daily light (ca.

750 ft-c) from incandescent and fluorescent bulbs.

Caladium, cocoyam and taro plantlets which had differen-
tiated in culture were separated from undifferentiated callus

and transplanted individually to sterilized sand/peat (1:1)

contained in 7.5 cm clay pots. All potted plantlets were

placed in insect-proof cages in a growth room and observed

for virus symptoms before being transplanted to larger pots

and transferred to greenhouses. Differentiated Croytocoryne




16



spp. plantlets were transferred to sterilized sand contained

in 3.8 1 wide-mouth glass jars.

Special precautions were taken to ascertain the presence

or absence of infected material. Plants were maintained for

at least 3 months in observation cages before being trans-

ferred to greenhouses. Samples were further checked for DMV

infection by electron microscopic examination of leaf dip

preparations and/or through manual transmission attempts to

seedlings of P. sellout.
















RESULTS


Characterization of Dasheen Mosaic Virus


Host-range Determinations

Dasheen mosaic virus proved to have a wide host range

within the Araceae and was recovered from members of 9 of

the 12 tribes and 13 of the 16 genera represented (Table 1).

The following aroids did not become infected when manually

inoculated as seedlings: Aglaonema commutatum, Anthurium

bakerii, A. crisoim"arinatum, Peltandra sp., P. stratiotes

and Snathiohvllum floribundum. None of the non-araceous

plant species including Tetraronia exnansa proved susceptible

to DMV when manually inoculated (Table 2).

Neither host range differences nor differences in symp-

tom expression were noted when single isolates from 'Candidum'

caladium, 'Exotica' dieffenbachia and taro were compared.

All three isolates infected manually inoculated seedlings of

Anthurium scandens, caladium, dieffenbachia and P. sellout,

and all three isolates induced identical symptoms in P. sel-

loum seedlings inoculated in recovery trials.













4U m U:
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HO P Hs u s+ OH
SE en E- ^ H 0-


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cno
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Table 2. Plants not infected with dasheen mosaic virus follow-
ing manual inoculationsa



Family Species Cultivar


Aizoaceae

Amaranthaceae



Amdryllidaceae

Bromeliaceae

Chenopodiaceae




Commeliniaceae

Compositae

Convolvulaceae

Cruciferae

Curcurbitaceae




Gramineae


Tetragonia exoansa

Amaranthus tricolor

Gomphrena globosa

Amaryllis sp.

Aechmea pubescens

Beta vulgaris

Chenopodium amaranticolor

Chenomodium quinoa

Rhoeo discolor

Zinnia eleEans

Ipomoea ourpurea Scarlet

Brassica nerviridis Tender green

Cucurbita cepo Small sugar

Curcurbita peno var.
melopoeo Early Prolific
Straightneck

Avena sativa Red Rustproof

Lolium sp. Ryegrass

Pennisetum typhoideum Pearl

Sorghum vulgare Georgia 615

Triticum aestivum Michigan Amber

Zea mays Golden Cross
Bantam










Table 2 continued


Family Species Cultivar


Iridaceae

Labiatae

Leguminosae


Liliaceae



Phytolaccaceae

Solanaceae


Gladiolus hortulanus

Ocimum basilicum

Arachis hypogaea



Cassia occidentalis

Cassia tora

Cyamonsis tetraqonoloba

Desmodium canum

Glycine max

Phaseolus vulparis


Pisum sativun


Vicia faba

Vina unguiculata


Allium cepa

Lilium lonFiflorum

Phytolacca americana

Cansicum frutescens

Datura stramonium

Lyconersicon esculentun

Nicotiana clevelandii

Nicotiana plutinosa


Florunner
Early Runner
Dixie Runner









Bragg

Red Kidney
Michelite 62

Alaska
Little Marvel

Longpod

Early Ramshorn
Blackeye

Yellow Bermuda





California Wonder



Manalucie







Table 2 continued


Family Species Cultivar


Nicotiana hybrid

Nicotiana sylvestris

Nicotiana tabacum Samsun SN

Petunia hybrid

Physalis floridana

a All plants tested were inoculated as seedlings. In every
instance attempts were made to recover DIV to P. selloum
2-6 weeks after inoculation.

b Nicotiana hybrid (N. clevelandii x N. glutinosa) developed
by S. R. Christie T15).








Transmission Electron Microscopy

Leaf extracts

Electron microscopic examinations of negatively stained

leaf dip preparations of various aroids exhibiting mosaic symp-

toms revealed the presence of filamentous rods (Fig. 3B) simi-

lar to those reported by Zettler et al. (74,75). Measurements

of virus particles from cocoyam plants infected with a DIV

isolate originally from 'Candidum' caladium were similar to

those previously reported for DMV in taro (74,75); i.e., 77%

of 95 particles measured were 726 to 815 nm long with a main

maximum at 801 nm and a mean length of 782 nm. Such particles

were not seen in extracts from healthy cocoyam plants.

Striated inclusions were seen in leaf extracts of 'Can-

didum' caladium and 'Exotica' dieffenbachia leaves stained with

ammonium molybdate. These inclusions resembled those reported

by others for viruses of the potato Y virus group (20). Tubes

and laminated aggregates were observed in leaf extracts from

both caladium and dieffenbachia. No apparent differences in

specific inclusion morphology were noted between these species.

Sectioned material

Ultrathin sections of 'Candidum' caladium infected with

DMV revealed the presence of pinwheel, circle, bundle and la-

minated aggregate inclusions (Fig. 2) similar to those described

by Zettler et al. (74,75) for DMV-infected taro plants. Of the

sections examined, the relative numbers of tubes seemed to be

much greater than the number of pinwheels or laminated aggre-

gates. Virus particles were commonly found in close association




























Figure 2. A,B) Thin sections of a caladium leaf infected
with dasheen mosaic virus showing pinwheel (PW),
circle (C), bundle (B) and laminated aggregate
(LA) inclusions. Scale line of A is 900 nm and
B is 450 nm. Note close association of virus
particles with inclusions.




























44







with these cytoplasmic inclusions (Fig. 2).

Light Microscopy

Amorphous cytoplasmic inclusions were seen in stained

epidermal strips taken from 'Candidum' caladium, 'Exotica'

dieffenbachia and taro when examined with a light microscope

(Fig. 3). Neither nuclear nor nucleolar inclusions were ob-

served in any of the epidermal strips. No amorphous cytoplas-

mic inclusions were observed in epidermal strips from healthy

plants of caladium, dieffenbachia and taro.

Purification of Virus and Virus-induced Cytoplasmic Inclusions

DMV particles were partially purified from dieffenbachia

shoot and leaf tissue (Fig. 5C). This material had a maximum

ultraviolet absorbtion at 260 nm in aqueous 0.02M Tris buffer,

pH 7.4, and 10 3 Cleland's reagent. The UV absorption spec-

trum from 230-360 nm was very similar to that reported for

other PVY type viruses (Fig. 4). The 260/280 ratio of ca.

1.507 (not corrected for light scattering), however, is a
value higher then that recorded for other flexous-rod shaped

viruses (personal communication, E. Hiebert).

Attempts to purify particles of DMV from caladium were

unsuccessful. A green mucilaginous pellet resulted from the

first high speed centrifugation. The purification procedures

were discontinued at this step since the pellet proved diffi-

cult to resuspend and contained a relatively low concentration

of virus particles based upon electron microscopic examination.

DMV-induced cytoplasmic inclusions were partially purified

















E>c
a >.
0 0C
S-l (3
) CS-P

a) 0( 0


mE

CH *O


0l





El E
Hoc





crdo






E S8
'CS







o5d
CQ z -4



oa CO


hoEm

0M 0 C0
0 CO H-
HC > U
0 H
aEr)=

H P -il
CC
TJ C' C




^r md (t
*^*H 4->
k > Q ^









rl










4t%~C


,~F OP































Figure 4. Ultraviolet absorption spectrum (not corrected
for light scattering) of dasheen mosaic virus,
purified from 'Exotica' dieffenbachia and con-
tained in 0.02 M Tris buffer and 10-2M Cleland's
reagent.



























































WAVELENGTH in nm












) 0 0=
4i- Ei C r-H

C3 -t c Ct *-H







dm0
E~o i- c






z O- 0 r-
H O- a) Or










S-i0 0
H0 ) 0a)O
bo (0 D-d







0 4U

-Ci r- ci E a









H 0 C
( H (n r-4-'
C d OH ai-i 4 2






















do
CO 30-
0 i'2 0 CD













a- C CL>-
t 42 to u H (











(C
40

-4
b43 3 '





:I








from symptomatic caladium and dieffenbachia leaves and dieffen-

bachia shoots. Striated plate-like inclusions as well as

striated tubular inclusions were observed from both dieffen-

bachia and caladium tissue. iThereas plates predominated in

dieffenbachia extracts (Fig. 5A), tubes predominated in ex-

tracts of caladium (Fig. 53).


Seroloay

No reactions were noted between center wells containing

serum prepared from DM% and peripheral wells with extracts

from healthy or DI,-infected caladium leaves.

Precipitin reactions formed between peripheral wells

with extracts of D=V-infected P. sellout leaves and center

wells containing antisera to TEV and PVY (Fig. 6). Never-

theless, the homologous reactions were much stronger and spurred

over the heterologous reactions. The heterologous reactions

were stronger when DNV antigens were prepared from P. sellout

plants inoculated 10-13 days prior to extraction than from

plants inoculated 56 days earlier.

No reactions were observed between antisera of pepper

mottle or turnip mosaic viruses and extracts from DM:-infected

P. sellout leaves. Nor were any reactions observed between

any of the antisera wells and peripheral wells containing ex-

tracts of healthy tobacco or P. sellout leaves.


























Figure 6. Serological relationship of dasheen mosaic virus
(DMV) to potato Y (PVY) and tobacco etch (TEV)
viruses. The center wells contain: (A) PVY an-
tiserum, (B) TEV antiserum, (C) Normal serum.
The peripheral wells in each pattern were filled
with pyrrolidine treated tissue extracts from:
(1) healthy tobacco, (2) PVY-infected tobacco,
(3) and (5) DMV-infected Philodendron selloum,
(4) TEV-infected tobacco, and (6) healthy P.
selloum.









b -L~ 2


Z1 I3




6 -1 2
8:


'4'



6`
i. C
5 11 ~ 3







Distribution and Effects of Dasheen Mosaic Virus


Surveys

Symptoms of DMV infection were observed in all commer-

cial, ornamental and experimental aroid plantings surveyed

in Florida. Areas surveyed included: 4 and 1 caladium fields

in Highlands and Orange counties, respectively, 4 cocoyam

fields in Dade County, 1 experimental and 2 ornamental taro

plantings in Alachua County, 1 aquarium plant .nursery in Palm

Beach County, and 3 and 6 foliage nurseries in Dade and Orange

counties, respectively. Dasheen mosaic virus was recovered

to seedlings of P. sellout, and filamentous particles were

observed in leaf dip preparations from the following aroids

obtained in these surveys: A. modestun, C. cordata, caladium

('Candidum', 'Caroline Whorton', 'Crimson Wave', 'Dr. Groover',

'Exposition, 'Frieda Hemple', 'Miss Chicago', 'White Christmas'),

taro, an ornamental Colocasia sp., Dieffenbachia amoena and

dieffenbachia ('Exotica', 'Perfection') and cocoyam.

Dasheen mosaic virus was also recovered to P. selloum and

virus particles observed in taro samples received from India,

Hawaii, and the Fiji Islands, and in cocoyam samples from Tri-

nidad. A sample of Xanthosoma brasilensis from Guadeloupe

likewise proved infected with DMV.

Synotom Exnression

Foliar mosaic symptoms were observed in all plants from

which DMV was recovered (Table 1). Dasheen mosaic virus, how-

ever, was rarely isolated from tissues without such symptoms.








The foliar mosaic pattern of taro and cocoyam was typically

associated with the major veins and resembled a "feathery"

appearance (Fig. 8B), a characteristic noted by others (2,17,

22,25). A "vein mosaic" pattern (6) was typically expressed

on the first leaves of P. sellout seedlings to show symptoms

after inoculation with DMV (Fig. 7C). On subsequently formed

leaves, however, this veinal pattern was not apparent (Fig.

7D).
Prominent foliar distortion symptoms were frequently

noted for certain aroids, including 'Exotica' and 'Perfection'

dieffenbachia (Fig. 7A), Philodendron pittieri, P. selloum,

Zantedeschia aethioica, Z. albo--aculata and Z. elliottiana.

Distortion symptoms induced by DKV in dieffenbachia included

epinasty and, in many instances, a marked disruption of the

vertical symmetry of the unfurled leaf (Fig. 7A). As such

leaves became fully expanded, torn regions resulted due to

uneven expansion rates of maturing lamellar tissues.

Whereas DMV symptoms of some aroids were not conspicuous

and did not markedly deter from their attractiveness, other

aroids were severely affected, particularly 'Exotica' and 'Per-

fection' dieffenbachia and Z. elliottiana. Among variegated

aroids such as caladium and dieffenbachia, DMV-induced mosaic

symptoms supplanted inherited patterns, which detracted from

their attractiveness (Fig. 7B, 8A).

Several authors have observed that leaves with symptoms

appear intermittently among infected plants (2,75). This phe-

nomenon was apparent for most aroids observed in this study,







c 0
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CN E E -P




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r4O *0






4P d Pi
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including caladium, cocoyam, dieffenbachia, P. sellout and taro,

Infected plants of certain species, however, such as P. nittieri

expressed symptoms continually throughout the observation

period.

In one experiment involving cocoyam plants inoculated with
DIV, successively produced leaves were examined for expressed

symptoms (Table 3). For all six plants, mosaic symptoms were

apparent on the first leaf to appear following inoculation.

Thereafter, leaves with and without symptoms were produced on

each of the plants, and, with one exception, leaves with symp-

toms appeared in succession following the emergence of symptom-

less leaves. The duration of this intermittent expression of

symptoms among aroids was not assessed in this study, although

it was noted that plants of P. sellout inoculated with DMV as

seedlings in October, 1969, still intermittently produced

leaves with and without mosaic symptoms 4.5 years later.

An unsuccessful attempt was made during this investiga-
tion to correlate certain environmental conditions with the

appearance of symptoms using DMV-infected 'Exotica' dieffen-

bachia plants. Both temperature and pot size, as it relates

to growth rate, were studied. The periodicity of symptom ex-

pression was identical at both temperature regimes tested,

although plants growing in the greenhouse where temperature

fluctuated between 20 and 40 C averaged 16.2 leaves per plant

compared to only 12.6 leaves per plant for the plants growing

where the temperature was controlled between 24-30 C (537 and

429 leaves per 33 and 34 plants, respectively). At both














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regimes, the lowest proportion of leaves with symptoms emerged

June-September whereas leaves with the highest proportions

emerged October-December and April-May (Fig. 9). This symp-

tom periodicity data generally correspond with observations
of 'Exotica' dieffenbachia plants grown commercially at

Apopka, Florida (Knauss, personal communication). Similarly,

no effect on symptom expression was noted in this experiment

as a result of transplanting plants from 4 to 6" pots, although

plants transferred to larger pots grew more vigorously than

their counterparts in smaller containers: at 20-40 C and 24-

30 C, an average of 16.8 and 13.2 leaves per plant were pro-
duced in 6" pots compared to only 15.6 and 11.9 leaves per

plant in 4" pots, respectively (303 and 238 leaves for 18

and 18 plants and 234 and 191 leaves for 15 and 16 plants,

respectively).

Quantitative Effects

Plants infected with D-V proved stunted in comparison
to their healthy counterparts (Table 4) regardless of whether

or not mosaic symptoms were evident on plants when recorded.

Fresh corm weights and leaf areas (maximum length x maximum

width) of 69 caladium plants were 40 and 53% less than heal-

thy plants, respectively. Corm weights and leaf areas of 8

Z. elliottiana plants were likewise reduced 59 and 51%, re-

spectively. Similarly, fresh shoot weights and leaf areas

of 27 infected dieffenbachia plants were 63 and 66% less than

controls; and total fresh plant weights and leaf areas of 36

P. selloun plants were 30 and 30% less, respectively (Table 4).





























Figure 9. Symptoms of dasheen mosaic virus expressed in
newly emerged leaves of 'Exotica' dieffenbachia
plants in relation to date of emergence and
temperature regime at which plants were main-
tained. Thirty-four and 33 plants were main-
tained at 24-30 and 20-'0 C, respectively.
Bars indicate the percentage of leaves with
symptoms according to the order of foliar
succession.

































































AUG C
MONTH








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All these results were highly significant at the 1% level.


Elimination of Dasheen Mosaic Virus

Seed Prownaation

Dieffenbachia fruits, although cream colored during most

of their development, became red when ripe approximately 10-

12 weeks after pollination. Dieffenbachia fruits did not de-

hisce but remained loosely attached to the spadix (Fig. 10).

A spadix bore 15-50 ovaries, each containing a single round

seed 5-6 mm in diameter which was green when mature. Seeds

germinated within 20 days after they were removed from the

fruit and planted in moist peat.

Unlike dieffenbachia, caladium fruits ripened 5-6 weeks

after pollination and abscissed from the spadix (Fig. 11).

The exposed fruit surfaces remained green throughout their

development without apparent color change at the time of ab-

scission. The exposed surfaces of the ovary walls were cream-

colored at maturity. Ovulate portions of the spadix contained

approximately 200 seed-bearing ovaries, each containing 1-14

oval seeds which were 1-1.5 mm in length and light tan in

color. As many as 1500 seeds were obtained from a single

spadix. Seeds were removed from the fruit and planted in

moist peat. Seeds germinated readily 8-10 days after planting.

Seed germination rate decreased markedly, however, when seeds

were dried and stored 2 weeks at 23 C and 53% relative humidity.

Fruits of Zantedeschia spp. also remained green throughout












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53








their development, but did not abscise from the spadix at

maturity (4-6 weeks after pollination). Each fruit contained

one seed.

Seedlings were maintained in a greenhouse at about 60%

shade in isolation front commercial aroid stock. Plants were

transferred from peat to a steam-sterilized soil/perlite mix

contained in clay pots and treated routinely with a liquid

formulation of 20-20-20 fertilizer.

Although parental 'Exotica' dieffenbachia, 'Candidum'

caladium, and Zantedeschia spp., plants used in this study

were virus-infected as evidenced by symptoms observed at the

time of pollination, none of the more than 500 resulting dief-

fenbachia, 1000 caladium and 100 Zantedeschia progeny displayed

virus symptoms, indicating that the seedlings were virus-free.

A small percentage of the dieffenbachia seedlings exhibited

albinistic tendencies and died shortly after germination. The

juvenile foliage of the surviving dieffenbachia progeny was

non-variegated and variegated leaves did not develop until 4-

8 months after germination. Similarly, juvenile leaves of

caladium were homogeneously green and variegated leaves did

not develop until about 3 months after germination.

A year after germination a marked variation was noted

among the 207 randomly selected dieffenbachia seedlings which

had grown to 30-34 cm in height. Fifty-three of the progeny

exhibited none of the white variegation typical of the 'Exotica'

parents. The foliar patterns of the remaining progeny varied

from those having relatively localized areas of white to those







having white patterns exceeding in area that of the original

parents (Fig. 12A). A few plants exhibited a degree of foliar

coloration unlike the 'Exotica' parents. Marked differences

in leaf shape and apical dominance were also noted among the

progeny. Whereas some of the leaves had dimensions similar

to those of the parents, others were somewhat more lanceolate.

Axillary buds of some progeny grew only after the apical meri-

stem was removed whereas the axillary buds of other progeny

proliferated regardless of the presence or absence of the

apical shoot (Fig. 12B).

Little variation was noted among the 100 randomly selected

caladium seedlings 8 months after germination. Fifty-one per-

cent of the progeny had predominately white leaves with 22 and

27% having predominately green or intermediately white leaves,

respectively. Forty-one percent had striped petioles with 55

and 4% having predominately green or purple petioles, respec-

tively. The parent 'Candidum' plants by comparison have pre-

dominately white leaves with striped petioles. Minor differen-

ces were noted in leaf shape or apical dominance between the

'Candidum' parents and the progeny.

All seedlings of Zantedeschia spp. resembled the parents

from which they were derived. These data correspond closely to

those obtained by Ryohitsu (60) for the self-pollinated progeny

of yellow, white and pink Zantedeschia spp.

Shoot-tip Culture

Attempts to culture pathogen-free plants of caladium,
taro and cocoyam were apparently successful. Forty of 50,






























Figure 12. Dieffenbachia seedlings 12 months after ger-
mination. Note differences in foliar varie-
gation patterns (A) and in degree of shoot
proliferation (B).








I
I, ,


1l


I~f

Cr


6 A,








3 of 6, 4 of 6 and 6 of 6 shoots of 'Candidum' and 'Frieda

Hemple' caladium, taro and cocoyam, respectively, developed

in vitro without apparent microbial contamination. Shoot

tips, originally cream colored, became green within 2 weeks

after excision. Within 8 weeks, callus masses developed and

differentiated numerous shoot buds. 7Within 18 weeks, many

plantlets with roots and shoots developed from these buds

(Fig. 13C). Plantlets of all three species survived trans-

fer to soil and developed rapidly into vigorously growing

plants (Fig. 133).

Every 3 months each caladium culture generated 10-20

plantlets suitable for transplanting and enough callus for

an additional 10-20 cultures. 'Candidum' caladium callus

has been maintained in culture for at least 1.5 years without

apparent adverse affects expressed by plantlets after trans-

fer to soil.

All attempts to culture dieffenbachia, chinese evergreen

and philodendron were unsuccessful either because microbe-free

shoot tips could not be obtained or because they failed to grow

in vitro. Six of 60, 2 of 2 and 2 of 3 shoot-tips of dieffen-

bachia, chinese evergreen and philodendron, respectively, were

without apparent microbial contamination. However, none of

these developed callus or differentiated into plantlets. Shoot-

tips, originally cream colored, became green 1-2 weeks after

excision, but eventually became necrotic and died. Similar

attempts with C. cordata and C. nevillii were successful; how-

ever, both of these species failed to proliferate in culture.




























Figure 13. (A,B) Scanning electron micrograph of cala-
dium apical shoot showing first (1) and se-
cond (2) leaf primordia and apical dome (a).
Scale line is 0.166 nim. C) Six-week-old cul-
ture of 'Frieda Hemple' caladiums on Mura-
shige-Skoog medium. D) Cocoyam plant 6 weeks
after transfer to soil from culture. Scale
line is 5.4 cm. E) Mature pathogen-free
'Candidum' caladiums grown under greenhouse
conditions.





















-1"~



r ,
h'r: .~ rL I


v?






Three of 10 and 2 of 5 shoot tips of C. cordata and C. nevillii,

respectively, were without microbial contamination and became

green 1-2 weeks after excision but differentiated only one

plantlet per shoot tip.

Attempts to culture dieffenbachia on other media were

likewise unsuccessful. Three of 20 and 2 of 20 apical shoots

proved microbe free when placed on Vacin-Went (68) and Knudson

C (43) medium, respectively. However, as with the Murashige-
Skoog medium, none of the 5 shoots survived in culture.

Plantlets of 'Candidum' caladium and taro obtained from

some shoot tips in culture proved to be infected with DMV.

Virus-infected plantlets were readily detected in vitro and

as plants growing in soil. These observations were confirmed

through inoculations of P. selloum seedlings and electron mi-

croscopy. Plants of both caladium cultivars, taro and cocoyam

derived from other shoot-tips, however, proved free of virus

and were used in continued propagations.

Virus-free plants of 'Candidum' and 'Frieda Hemple' cala-

diums developed typically variegated leaves 10-12 weeks after

transplanting into soil. Some of the 'Candidum' caladiums

flowered 6-7 months after transplanting and produced corms 4-

5 cm in diameter with leaves 20-27 cm wide and 25-32 cm long

(measured from lamella tip to point of petiole attachment)

(Fig. 13E). After 1 year, these plants had corms ca. 9.5 cm

in diameter with a fresh harvest weight of ca. 200 g. Mature

plants were identical to their commercial counterparts, except

that they appeared to be more vigorous. In one experiment,







14 DMV-free and 14 DMV-infected plantlets were transplanted

from culture to soil and maintained under identical growing

conditions. After 6 months, corm weights of healthy plants

were significantly greater (t value = 2.6; P = <5%) than those

from diseased plants (56.9 and 40.2 g/corm, respectively).

Approximately 350 virus-free 'Candidum' caladium plant-

lets have been growv to maturity since this study was initiated.

These plants were propagated in a screened greenhouse on raised

asbestos-cement benches previously sterilized with a 5% commer-

cial formulation of sodium hypochlorite. The plants were rou-

tinely treated with either nicotine sulfate or malathion for

insect control and twice with Truban (5-ethoxy-3-trichloromethyl-

1,2,4-thiadiazole) for the control of pythiaceous fungi. During

their development, none of the plants ever had symptoms of D.V.

At harvest (ca. 6 months after planting), none of the plants

had apparent discolored or necrotic roots or corms.












DISCUSSION


This study provides additional evidence that DMV is a

member of the potato Y group of viruses as originally described

by Brandes (7), supplemented by Edwardson (18), and recognized

by' the International Committee on Nomenclature of Viruses (25,

72). The mean length of 782 nm for particles in leaf extracts
from DKV-infected cocoyam plants corresponds closely to the

values of 759 nm for particles in cocoyam by Debrot and Ordos-

goitti (17) and 750 and 751 nm for particles in taro by Zettler

et al. (74,75) and Debrot and Ordosgoitti (17), respectively.

Similar flexous rod particles were found in leaf extracts of

DMV-infected plants representing 12 other aroid genera inves-

tigated in this study. Similarly, cylindrical inclusions as

described by Edwardson et al. (20) were observed in extracts

and/or thin sections of leaves from DMV-infected plants of

caladium, dieffenbachia and Z. elliottiana. These inclusions

closely resembled those described by Zettler et al. (74,75)

for DMV-infected taro except that, in dieffenbachia, laminated

aggregate inclusions were more prevalent than tubular inclu-

sions.

The relatedness of DMV to potato Y and tobacco etch viruses

as indicated by serological data further supports the conten-

tion that DMV is a member of this virus group. These serological








results were confirmed by other workers who found that DHV and

7 other viruses considered to be members of this group reacted

positively to antiserum of tobacco etch D-protein (62). At-

tempts to obtain an antiserum specific to D;V were not success-

ful in this study. This is presumably due to relatively low

virus titers in the leaf tissues used for purification and to

a relatively low purity of the final injected viral prepara-

tion, as indicated by the relatively high 260/280 value of

1.59 recorded.

Herold (34,35) reported a mean particle length of 754 nm

for a virus of Anthurium andraeanum which was reported to be

transmitted by white flies and manually transmissible from

tobacco to other solanaceous plant species. Although DMV

was recovered from and inoculated to Anthurium spp. in this

study, transmission to tobacco was never accomplished. In

addition, transmissibility by white flies is not considered

to be a property typical of other members of the potato Y

virus group. Hence, it is concluded to be unlikely that

Herold's virus and DMV are synonymous.

The relation of DIN to other aroid viruses is difficult

to judge considering the general paucity of information pro-

vided by these previous studies. Raychaudhuri and Ganguly

(56) for example reported that the aphid-transmissible
"Chirke" disease of cardaton was manually transmissible to

the aroid Acorus calamus. However, further information that

would enable a correlation to be made between this virus and

DMV was not provided. Also, in this study, neither cardamon







nor Acorus sp. were available for inoculation with DMV.

Dasheen mosaic virus was shown to have a wide host range

within the Araceae. Although DMV infected species in 13 of

the 16 aroid genera tested none of the inoculated 45 species

of 17 other plant families became infected. These results,
however, do not necessarily indicate that DMV is not synony-

mous with any other previously described members of the po-

tato Y virus group. The plant species included in the host

range were not all-inclusive with respect to all 103 members

of the group listed by Edwardson (19).

James et al. (40) reported that DMV "occasionally" in-

fected plants of T. exuansa, although they apparently did not

effect recovery of DMV back to any seedling aroid. Repeated

efforts to infect T. exnansa with DMV in this study were un-

successful despite precautions taken to insure inoculun vi-

ability. If susceptible, T. exoansa must be considered a

marginal host, and certainly less satisfactory than seedlings

of P. selloum as indicators for DMV.

No evidence for virus inactivators or inhibitors among

aroids was noted in this study, as DMV was readily recovered

to P. selloum seedlings. In addition to DMV, an isolate of

cucumber mosaic virus from Connelina sp. manually inoculated

to caladium seedlings was readily recovered to tobacco in-

dicator plants (unpublished data of the author). The appa-

rent lack of inhibitory substances is not surprising consider-

ing the study of Simons et al. (63) who showed that extracts

of neither Dieffenbachia secuine nor Monstera deliciosa







contained substances inhibitory to the transmission of tobacco

mosaic virus.

Since its recognition in 1969, DKV has been recovered from

various locations including the United States (75), Europe (69,

75), the South Pacific (23,32,40,42), the Caribbean (2,42),
South America (17) and Japan (3). In this study, specimens

from India, Hawaii, the Fiji Islands, Trinidad and Guadeloupe

were received, in addition to samples from Florida, and de-

termined to be infected with DMV, thus confirming the inter-

national distribution of this virus. This widespread occurrence

of DMV presumably reflects: 1) the antiquity of aroids as cul-

tivated plants, 2) their obligatory means of vegetative pro-

pagation, 3) their extensive dissemination as popular culti-

vated plants and 4) the facility of DKV to be transmitted by

aphids. The widespread occurrence of DMV in commercial cala-

diums is further abetted by certain circumstances peculiar to

this industry. According to grower estimates, 96% of the world's

caladiums are from Florida, 76% of which are produced in High-

lands County on less than 600 acres (39). This crop is re-

strictively cultivated on acidic muck soil and with a minimum

of crop rotation or isolation. Considering that the variety

'Candidum' comprises over 30% of the total production and that

all these plants are derivatives of a single plant developed

before the turn of the century (33), the ubiquity of DMV in-

fections noted in this study for caladiums was seemingly in-

evitable.

This research shows DMV to be a serious pathogen of aroids.








Foliar symptoms are marked in some aroids, particularly

'Exotica' and 'Perfection' dieffenbachia. These symptoms in

dieffenbachia merit grower concern, and, in some instances,

entire beds have been abandoned due to foliar disfigurement.

Symptoms in most aroids, however, are relatively inconspicuous

-or absent altogether- and incited a minimum of concern,

likely accounting for the relatively little attention devoted

to aroid viruses. Of particular interest in this study was

the, intermittency of expressed symptoms for most aroids, but

absence of apparent symptoms does not indicate recovery from

virus. This fact was indicated in prolonged and controlled

observations of several aroids, in particular cocoyam,

'Exotica' dieffenbachia, and P. sellout. In every instance,

leaves with pronounced mosaic symptoms were expressed follow-

ing periods when only symptomless leaves had developed. For

this reason, it is concluded that most aroids naturally in-

fected with DMV are unreliable indicators of infection.

Several authors (10,12,23,40,41,42) have used vegetatively

propagated aroids in virus studies, and concluded that symp-

toms expressed on inoculated plants indicated successful trans-

mission. Such results should be viewed with caution unless

workers can assure that plants were not already infected when

inoculated. This investigation did not divulge reasons for

the intermittency of symptom expression, although it was con-

cluded that temperature was not likely to be a major contributing

factor since leaves of 'Exotica' dieffenbachia expressed symp-

toms synchronously at two different temperature regimes. A






more likely factor in effecting symptom expression might be

photoperiodic response. The author is not aware of published

reports citing such a factor being responsible for intermit-

tent symptom expression, although Reinert and Kasperbauer (57)

have shown that the phytochrome system affects the multipli-

cation of tobacco ringspot virus; and as reviewed by Butler

and Downs (9), the phytochrone system has been implicated as

having a major role in the development of photoperiodically

sensitive plants. Accordingly, studies should be undertaken

to ascertain the role of light in masking symptoms; the possi-

bility exists that for certain foliage plants, symptoms can

be masked by artificially manipulating day length. Similar-

ly, such manipulation could also be employed on a practical

basis to subdue virus multiplication prior to dissection of

shoot tips for tissue culture, or, conversely, to augment

virus multiplication to increase the reliability of DMI

assays.

Quantitative losses due to DMV are more substantial than

qualitative losses although they are probably far less appa-

rent, when plants are ubiquitously infected. In controlled

experiments with plants of caladium, dieffenbachia, P. selloum

and Z. elliottiana, fresh plant weights were reduced as much

as 63% and no less than 30%. Similarly, leaf areas were re-

duced 30 to 66% even when expressed foliar symptoms were not

apparent.

These studies show conclusively that practical control of

DMV is contigent upon the elimination of virus from propagating






stock. Two approaches for obtaining virus-free plants were

given serious consideration in this study: seed propagation

and shoot-tip culture. Alconero (1) attempted heat elimi-

nation of DMV from cocoyam but failed to inactivate the virus

at temperatures the plant would withstand. Success with mo-

dification of this technique may be successful with other

aroids, although relatively few viruses of the potato Y

group have been controlled in this manner (52). Moreover,

heqt therapy is unlikely to completely eliminate other patho-

gens, including species of Pythium, Sclerotinia, Fusarium and

Rhizoctonia known to be pathogenic to certain aroids.

Seed propagation is an effective means of eliminating

most phytopathogens (4). Seed of many aroids were collected

during this study (Table 1) and in no instance was there any

evidence for seed transmission, even when seed was collected

from plants known to be infected with DNV. Certain aroids,

particularly P. sellout, are normally seed propagated, and

resultant progeny closely resemble parental plants. As this

study shows, however, progeny of some aroids, such as cala-

dium and dieffenbachia, differ markedly from parental plants.

For such aroids, therefore, this technique cannot be used on

a practical scale to eliminate DMV from commercially desirable

cultivars such as 'Candidum' caladium or 'Exotica' dieffen-

bachia. Nevertheless, results with caladium and dieffenbachia

suggests a rich -but ignored- genetic potential for deriving

improved horticultural varieties. For example, a need exists

for "color" among foliage plants, such as that provided by the









golden green variegated pothos (Scindaosus aureus). A concerted

breeding program with Dieffenbachia spp., could enrich the in-

dustry considerably by infusing it with color, as this study

suggests. Similarly, horticulturalists have expressed in-

terest in the possibility of developing less poisonous variants

of dieffenbachia than are currently available (5).

The genetic potential of caladiums has previously been

demonstrated as evidenced by the 2000 or more named varieties

developed by such workers as Nehrling, Mead and Leitze (33).

Whereas these evaluations were based principally upon foliage

characteristics, incorporation of other factors such as cold-

hardiness and disease resistance could be considered under the

auspices of a future concerted breeding program.

Theoretically, shoot-tip culture affords the most ideal

means of eliminating DMV and other phytopathogens while re-

taining the genetic integrity of the parental stock. In cer-

tain instances, this technique has the considerable added ad-

vantage as a means to mass propagate plants under aseptic

conditions (48). This research resulted in successfully cul-

turing in vitro caladium, cocoyam, C. cordata, C. nevillii and

taro aseptically. This appears to be the first report of the

in vitro culture of aroids. Interestingly, with the exception

of the Cryntocoryne spp., the success reported herein involved

plants of the tribe Colocasieae whereas failures involved other

tribes in the Araceae. Although shoot-tips of both Cryto-

corvne spp. differentiated into single plants which survived

transfer to sand, neither species formed callus or proliferated







in culture indicating that the tissue culture techniques used

in this study, while ideally suited for the Colocasieae, will

have to be modified for members of other tribes. Attempts to

culture dieffenbachia, A. nodestum and P. selloum were not

successful either because of contamination, or because ex-

plants failed to survive in culture. It is possible that pro-

liferation for these plants could be effected through the use

of growth regulators such as 2,4-D, as shown by Carter et al.

(l1) to be essential for the proliferation of oat callus;

Pathogen-free plants derived from tissue culture may

effectively be used in pathogenicity trials with specific

disease agents. Plants of 'Candidum' caladiums were used,

for example, in determining that Pythium myriotylum was a

significant root pathogen whereas three other species of

Pythiun were not (59).

Tissue culture may also be used as a valuable precaution-

ary tool for those endeavoring to introduce exotic species or

cultivars of caladium, cocoyam or taro into new areas. Leon

(44) emphasized the dangers of inadvertently importing patho-

gens in vegetative plant parts and announced the need for de-

fining areas where pathogen-free material could be obtained.

Although some pathogens, such as DMV, may already be distri-

buted world-wide, others may have a restricted range. Thus,

culturing new accessions of these aroids in vitro, and screening

plantlets before their release, would greatly minimize importing

dangerous new pathogens accidentally.

The rapid proliferation of caladium, cocoyam and taro in


I -t







culture indicates that these plants can be propagated at an

unprecedented rate. A newly developed caladium cultivar, for

example, cannot be made available commercially until suffi-

cient saleable stock can be produced which, according to

grower estimates requires ca. 6-9 years during which time

enough material has been accumulated to plant 0.5 acres with

ca. 20,000 seed pieces. This study shows that at least 10-

20 plantlets and 10-20 new cultures can be generated in vitro

every three months. Realizing that each plantlet can attain

maturity 6 months later, it would be theoretically possible

to produce sufficient pathogen-free stock within three years

to supplant the entire caladium industry of Florida consisting

of an annual production of 40-50 million corms. A similar

approach was considered for narcissus by Stone (64) who di-

vulged the feasibility of replacing the entire narcissus stock

of the Isles of Scilly with virus-free material.

Regardless of technique used to eliminate DIV and other

pathogens from aroid planting stock, procedures should be

adapted to prevent the re-establishment of pathogens. Pre-

sumably certification programs similar to those established

for numerous other crops, such as potatoes and chrysanthemums,

could be developed and standards appropriate for specific

aroids established. Regretably, no such programs for aroids

have been developed in Florida or elsewhere despite theireconomic

importance. Ironically, DMV is firmly established in the co-

coyam and taro accessions of the Federal Experiment Station at

Mayaguez, Puerto Rico (2), and at the Saman Kocho Experiment






Station in Venezuela (17). Presumably higher standards of

disease freedom should be initiated at research centers.

Once released, it is logical to assume that pathogen-

free plants will become re-infected. This factor does not

negate the wisdom of certification programs, however, and

many, such as the bean seed program of New York, have with-

stood the test of time. Indeed, although DWM is aphid-trans-

mitted, rates appear to be low and approximate the findings

of Saladini and Zettler (61) for their observations of un-

expectedly low incidences of sugarcane mosaic virus infec-

tions in St. Augustinegrass plantings in Florida. Zettler

(personal communication) noted that only 2 of 112 and 34 of

157 Philodendron sellout seedlings became infected with DMV
during test feedings (involving five aphids per test plant)

by individuals of Aphis craccivora and Myzus oersicae allowed

single acquisition probes, respectively. These results are

in marked contrast to the results with other potato Y type

viruses of such plants as cowpea (73) and pepper (76), which

have relatively high rates of aphid transmission.













LITERATURE CITED


1. Alconero, R. 1972. Hot water treatments of corms of
Xanthosoma spp. infected with dasheen mosaic virus.
Plant Dis. Rep. 56:320-321.

2. Alconero, R., and F. W. Zettler. 1971. Virus infec-
tions of Colocasia and Xanthosoma in Puerto Rico.
Plant Dis. Rep. 55:506-508.

3. Arai, K., Y. Doi, and K. Yora. 1970. Notes on the
filamentous virus found from mosaic of taro. Ann.
Phytopathol. Soc. Japan 36:373 (Abstr.).

4. Baker, K. F. 1957. The U. C. system for producing
healthy container grown plants. Calif. Agr. Exp.
Sta. Ext. Service Manual 23. 332 p.

5. Barnes, B. A., and Lauretta E. Fox. 1955. Poisoning
with Dieffenbachia. J. Hist. Med. Allied Sci. 10:
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BIOGRAPHICAL SKETCH


Robert Dale Hartman was born in Miami, Florida on March 31,

1948. In June, 1966 he was graduated from Hialeah High School

located in Hialeah, Florida. In September, 1966 he entered

Miami Dade Junior College where he was awarded an A.A. degree

in June, 1968. He subsequently entered the University of Florida

where he received his B.S. degree in agriculture with majors in

plant pathology and entomology and graduated with High Honors

in June, 1970. He was supported in his upper division work by

an Academic Scholarship and a National Defense Loan. He enrolled

in the Graduate School of the University of Florida in the De-

partment of Plant Pathology in January, 1971 and began work

toward his Ph.D. degree in plant pathology with a minor in Or-

namental Horticulture. His Ph.D. work was supported by a NDEA

Title IV Fellowship and a departmental assistantship.

He is a member of Phi Kappa Phi, Gamma Sigma Delta, The

American Phytopathological Society and The Florida State Horti-

cultural Society.

He was married to the former Linda Diane Rockwell on October

25, 1969 and has a three year old daughter, Sandee Lynn.

He is currently in the Army Reserve serving as a pay special-

ist with the rank of Sp. 5. His term of enlistment will terminate

April, 1976.







I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scqs and quality,
as a dissertation for the degree of Doc f Philosophy.





F. William Zettler, Chairman
Associate Professor of Plant
Pathology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.





R. Edwardson
Professor of Agronomy


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.





E. Hiebert
Assistant Professor of Plant
Pathology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.





J. F. Knauss
Assistant Professor of Plant
Pathology






I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.





D. E. Purcifull
Associate Professor of Plant
Pathology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.





T. J-. Sheehan
Professor of Ornamental
Horticulture





This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate Council, and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

June, 1974


Dean, College of Agriculture





Dean, Graduate School




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PAGE 1

DASHEEN MOSAIC VIRUS OF CULTIVATED AROIDS AND ITS CONTROL BY SEED PROPAGATION AND CULTURE OF SHOOT TIPS By ROBERT DALE HARTMAN A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 197^

PAGE 2

Veil your face, foolish experimenter, overconfident in your mischievous straw! You thought that you had created a new type of instrumentalist; and you have obtained nothing at all. The Cricket has thwarted your schemes: he is scraping with his right fiddlestick and always will. Your sorry science tried to make a lefthanded player of him. He laughs at your devices and settles down to be right-handed for the rest of his life. — J. Henri Fabre

PAGE 3

Figure 1. (Frontispiece) In vitro culture of caladiura, Caladiun hortular.un •Candidurn', (right) and fully developed plant (left) transferred from culture to soil 6 months previously.

PAGE 5

ACKNOWLEDGEMENTS The author wishes to extend sincerest appreciation to Dr. F. W. Zettler for his support, guidance, encouragement and time that he has so generously given throughout this study. Special thanks are also extended to Drs. J. R. Edwardson, E. Hiebert, J. F. Knauss, D. E. Purcifull and T. J. Sheehan for their support in specific areas of this study and for their general guidance given throughout this study. Also, special thanks are due Mrs. Thelma C. Carlysle, USDA Research Technician for her assistance in producing the scanning electron micrographs of the caladium apical shoottips. Sincere appreciation is due the members of the Department of Plant Pathology and especially all the personnel of the Plant Virus Laboratory whose assistance helped make this work possible. Lastly, the author wishes to thank his wife for her continual support and love so generously given throughout this study and for her typing expertise and critical comments which facilitated the completion of this dissertation.

PAGE 6

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS v LIST OF TABLES viii LIST OF FICUKZ3 . , ix ABSTRACT x i INTRODUCTION 1 MATERIALS AND METHODS 5 Characterization of Dasheen Mosaic Virus .... 5 Host-range Determinations 5 Transmission Electron Microscopy 6 Leaf extracts 6 Sectioned material 6 Scanning Electron Microscopy 8 Light Microscopy 8 Purification of Virus and Virus-induced Cytoplasmic Inclusions 8 Serology 9 Distribution and Effects of Dasheen Mosaic Virus 11 Surveys 11 Symptom Expression 12 Quantitative Effects 15 Elimination of Dasheen Mosaic Virus 13 Seed. Propagation 13 Shoot-tip Culture lb RESULTS 17 Characterization of Dasheen Mosaic Virus .... 17 Host-range Determinations 17 Transmission Electron Microscopy 2.1+ Leaf extracts 21+ Sectioned material 2k Light Microscopy 27 Purification of Virus and Virus-induced Cytoplasmic Inclusions 27 Serology J>1+ Distribution and Effects of Dasheen Mosaic Virus 37 Surveys 37 Symptom Expression 37 Quantitative Effects 1+5 vi

PAGE 7

Page Elimination of Dasheen Mosaic Virus ^9 Seed Propagation h9 Shoot-tip Culture 55 DISCUSSION. . ' 63 LITERATURE CITED 7k BIOGRAPHICAL SKETCH 80 vii

PAGE 8

LIST OF TABLES TABLE Page 1 Araceous plants susceptible to dasheen mosaic virus , . „ 3.8 2 Plants not infected with dasheen mosaic virus following manual inoculations .... £1 3 Systemic symptoms expressed on cocoyam leaves formed after inoculation of plants with dasheen mosaic virus 44 1+ Effects of dasheen mosaic virus on fresh weights and leaf sizes of caladium, dieffenbachia, Fhilodendror. selloum and Zantedeschia elliottiana plants 43 viii

PAGE 9

LIST OF FIGURES FIGURE Page 1 In vitro culture of 'Candidum* caladiura and fully developed plant transferred from culture to soil "Frontispiece' 2 Thin sections of DMV-infected caladium leaf tissue showing pinwheel, circular, bundle and laminated aggregate inclusions 26 3 Stained epidermal leaf cells of caladium containing cytoplasmic inclusions and negatively stained particles of dasheen mosaic virus ... 29 if Ultraviolet absorption spectrum of a partially purified preparation of dasheen mosaic virus from dief fenbachia 31 5 Laminated aggregate and tubular inclusions and particles of dasheen mosaic virus purified from infected caladium and dief fenbachia leaves 33 6 Serological reactions of dasheen mosaic virus to potato virus Y and tobacco etch antisera . • 36 7 Dasheen mosaic virus symptoms in dieffenbachia and Philodendron selloum ifO 8 Dasheen mosaic virus symptoms in cocoyam and 'Candidum' caladium leaves if2 9 Foliar symptoms of dasheen mosaic virus expressed by dieffenbachia plants during a 12 month study period under two temperature regimes if7 10 Dieffenbachia spadix during pollen shed and at various stages after pollination 51 11 Caladium spadix prior to pollen shed, at pollen shed, and at various stages after pollination 33 12 Dieffenbachia seedlings 12 months after germination 57

PAGE 10

FIGURE Page 13 In vitro culture of shoot-tips used to eliminate dasheen mosaic virus and other phytopathogens from infected aroids 60

PAGE 11

By Robert Dale Kartman June, 1974 Cliairman: Dr. F. '.V. Zettler Major Department: Plant Pathology Dasheen mosaic virus (DMV) was recovered from species representing 13 genera in the Araceae, but did not infect seedlings of « species in 1 7 other families. The following results of this investigation support earlier reports that DMV is a member of the "potato virus Y" group of plant viruses: 1) . mea n particle length of 782 nm was recorded, 2) characteristic cylindrical inclusions were observed and 3) the virus proved serologically related to potato r and tobacco etch' viruses. Surveys indicate that DMV is prevalent in commercial plantings of aroids and induces serio, losses. Infected plants were encountered in all Caladiu ° rtUlanU ' m (cal ^->> Colocasia esculenta (taro) and Kanil-soma sagittifolnnm (cocoya*) fields surveyed in Florida and in all stock beds of Dieffenbachia. nicta (dieffenbachia) xn ornamental foliage plant nurseries visited. Based on these surveys, it appears that DMV has become uniformly established in certain cultivars of caladiu. and dieffenbachia )US Lum

PAGE 12

The deleterious effects of DMV on yields of caladium, dieffenbachia, Philodendron selloum and Zantedeschia elliottiana were assessed in controlled experiments. Weight losses of ZfO, 63, 30 and 59% and reductions in leaf area of 33, 66, 30 and 51% v/ere recorded, respectively. Virus-free plants of caladium, dief fenbachia, and Zantedeschia spp. were obtained through zee J. propagations, although caladium and dief fenbachia progeny differed from parental plants. Excised shoot-tips of caladium, cocoyam and taro v/ere successfully cultured aseptically and free of DMV on the revised medium of Murashige and Skoog. Callus of all three species proliferated into numerous plantlets on this medium and plantlets survived transplanting from culture to soil. Caladium plantlets assumed characteristic foliar variegation within three months after transplanting and attained maturity within 6-7 months. xii

PAGE 13

INTRODUCTION The family Araceae includes about 105 genera and 1Z+001500 species of plants which are primarily of tropical or subtropical distribution, although several species occur in temperate climates. Species in eight genera are indigenous to the United States. The typical aroid inflorescence is a spadix subtended by a large, often showy spathe. These plants are mostly monoecious and dichogamous. Many aroids are economically important. In Florida, species of Aglaonema, Dief fenbachia . Philodendron . Scindan sus and Syngonium account for over half of a 525,700,000/ year foliage industry (16). Approximately 9 6% of the world's commercially produced caladiums are also grown in Florida (39). Cryptocoryne spp. are of considerable significance to the aquarium plant industry of Florida, and another aquatic aroid, Pistia stratiotes, ranks as one of the world's most noxious waterweeds. On an international basis, perhaps the most significant aroid genera are those containing edible species. The cocoyams ( Xanthosoma spp.) and taros (Colocasia spp.) are carbohydrate staples in many tropical and subtropical areas throughout the world. In Florida approximately 3000 acres of the former are produced for the Cuban populace (^,54). Of lesser

PAGE 14

importance as food crops are species of Alocasia, Amor'oho phallus , and Cyrtosperma . These are grown principally in Japan, parts of Southeast Asia, and in the South Pacific. Most horticulturally important aroids are propagated exclusively by vegetative means. Many aroid species are sterile (51) and Hill described taro as having "been cultivated for no long that it never flowers" (38). A few aroids, however, are readily seed propagated including some species of Agalonema , Anthurium and Philodendron . For many aroids, special techniques must be employed to achieve fertilization (46). Since, however, most ornamental aroids are heterozygous cultivars, progeny do not necessarily reflect parental types (46,71). Continued vegetative propagation has enabled certain phytopathogens to become prevalent in stock plantings of many aroids. Viruses can become especially serious under these circumstances and indeed infections of tomato spotted wilt virus in Zantedeschia spp. and cucumber mosaic virus in Arum italicum are well documented (22,45*66,67). Virus-like diseases have also been reported in species of Amorrhophallus (10,12), Anthurium (34,35,70), Philodendron (65) and P. stratiotes (53). In addition, a virus causing the "chirke disease" of large cardamon was found to be sap-transmissible to the aroid Acorus calamus (56). In 1970, Zettler et al. (74>75) reported a filamentous virus infecting Colocasia esculenta in Florida which proved mechanically transmissible to seedlings of Philodendron

PAGE 15

3 selloum and Zantedeschia elliottiana . Unlike the filamentous anthurium virus described by Herold, it failed to infect tobacco. This virus was designated as dasheen mosaic virus (DMV) and was characterized as belonging to the Potato Y virus group of Brandes (7) and Brandes and Bercks (8) in: (i) being aphid transmitted in a stylet-borne manner by aphids, (ii) having a mean particle lenglh of 730 nm, and (iii) inducing characteristic cylindrical inclusions. An isolate of DMV was later characterized by Alconero (1) as having a (i) dilution end point between 1/100 and 1/1000, (ii) thermal inactivation point in 10 minutes between 60 and 65 C and (iii) longivity in vitro of 75 hours at 26 C. Since its discovery in 1970, DMV has been reported from Zantedeschia spp. in the Netherlands (69) and from taro and/or cocoyam in the British Solomon Islands (23,40,41,42), Trinidad (42), Venezuela (17), Puerto Rico (2), Hawaii (2), the Fiji Islands (J>2) and Japan (3). In addition, a mosaic disease of Philodendron selloum in California (65) is assumed to be induced by DMV (H. H. Hill, personal communication). Two bacilliform viruses of taro have been described in the British Solomon Islands (23,40,42). These viruses in combination with DMV reportedly kill infected plants causing a condition referred to locally as "alomae". Unlike DMV, however, neither bacilliform virus has been detected outside the island of Malaita. Alomae-infected plants were not detected elsewhere in the Protectorate, nor were they observed in material from Trinidad or Ghana.

PAGE 16

The purpose of this study was to further characterize DMV, assess its distribution and importance among aroids in Florida and to investigate practical control measures for this virus. Attempts were also made to identify other viruses of aroids in Florida. Portions of this dissertation have been published elsewhere (26,27,28,29,30,31).

PAGE 17

MATERIALS AND METHODS Characterization of Dasheen Mosaic Virus Host-range Determinations All host range determinations were based upon manual transmission trials using inoculum expressed from symptomatic leaves and diluted with deionized water. Six hundred mesh carborundum was used as an abrasive. Unless otherwise noted, all test plants were grown from seed. Two or more leaves were inoculated per test plant. At least 4 P. sel loum seedlings were inoculated after all host range trials to insure that the inoculum used was infectious, except in one instance when two caladium seedlings were used subsequent to inoculation of Rhoeo discolor . Test plants were maintained for at least 14 days following inoculations. Regardless of symptom expression, attempts were made to recover virus from test plants. In recovery trials, at least 3 P. selloum seedlings were rubbed with extracts from inoculated leaves and with extracts from leaves which developed subsequent to inoculation. Whereas aroids were tested at various times throughout the year, all non-aroids were inoculated August-October when greenhouse temperatures were 20-30 C. At least 5 individuals of each plant species were inoculated except that only 4, 4

PAGE 18

and 2 plants of Nicotiana sylvestrus , N. glutinosa and N. clevelandii were used, respectively. Transmission Electron Microscopy All plant material was examined using a Philips Model 200 electron microscope operating with an accelerating voltage oT 60 KV. Size determinations were made by comparing projected electron micrographs with projected micrographs of a,5^,86if line/inch diffraction grating. Leaf extracts All material used for examining virus particle morphology was obtained from negatively stained leaf extract preparations. Mounts were prepared by dicing a small piece of symptomatic tissue (ca. 3nim ) in several drops of 1-2% phosphotungstic acid (PTA), pH 6.8, contained on a glass slide. The diced tissue was mixed briefly with the PTA and a droplet of the mixture was placed on a specimen grid coated with Formvar and strengthened with arc-vaporized carbon. After one minute, excess fluid was absorbed with filter paper. Specimens were then examined with the electron microscope. A similar procedure was used for preparing virus-induced cytoplasmic inclusions for examination except that leaf extracts were stained in 1% ammonium molybdate, pH 6.5> rather than PTA (55). Sectioned material For electron microscopic observations of virus-induced

PAGE 19

cytoplasmic inclusions, 2mm sections of leaf tissue were cut from Caladium hortulanum 'Candidum' and Dief fenbachia picta •Exotica 1 leaves with symptoms. The leaf sections were placed in a 6% solution of glutaraldehyde in cacodylate buffer at pH 7.2. The tissue was placed under vacuum for 5 minutes and fixed for 1-3 hours at if-5 0. The tissue was then washed twice for 15 minutes each with cacodylate buffer and post fixed for 1.5 hours at 23 C with 2% osmium tetroxide. The tissue was v/ashed twice for ten minutes each with buffer and deionized water, respectively. The tissues were progressively dehydrated in a series of increasing concentrations of ethanol to 100%. At 70% ethanol they were stained for 5-10 hours in 2% uranyl acetate at J>-k C. The tissue was subsequently transferred through two changes of 100% acetone for 15 and 30 minutes, respectively, at 23 C The embedding plastic was an Epon 812, Araldite "M" mixture as described by Mollenhauer (if7 ) The embedding procedure was done in three steps of 30, 70 and 100% plastic contained in acetone for 1 hour each after which it was placed in a vacuum oven at 60 C to remove the acetone. The tissue was placed in a drying oven overnight at 60 C and further polymerized in an oven at 80 C for 1-2 days. Tissues were sectioned with glass knives mounted on a Sorvall Model MT-2 ultramicrotome. They were transferred to acid-cleaned, uncoated specimen grids and stained for 15 minutes in 0.5/= aqueous uranyl acetate, rinsed in water, and subsequently stained for 5 minutes in lead citrate (58). The

PAGE 20

sections were then rinsed in deionized water and allowed to dry prior to examination with the electron microscope. Scanning Electron Microscopy Apical shoots of 'Candidum' caladium were prepared for examination with a Cambridge Mark II stereoscanning electron microscope operating with an accelerating voltage of 10-20 KV. Shoot tissue comprising the apical dome and 1-2 leaf primordia were dissected and mounted on a 12mm diameter aluminum specimen stub covered with a thin layer of silver base paint. The specimens were examined as fresh material rather than being coated with metal. Light Microscopy Epidermal strips were removed from the abaxial surfaces of DMV-infected and healthy leaves of caladium, dieffenbachia and Colocasia esculenta (taro) and prepared for examination with a light microscope according to the procedures described by Christie (13,14) for staining virus-induced inclusions. Epidermal strips were placed in 5% Triton X-100 for 5 minutes and subsequently stained for 10 minutes in calcomine orange and "Luxol" brilliant green. The strips were rinsed briefly in 95% ethanol and mounted in Euparal on a glass slide. Purification of Virus and Virus-induced Cytoplasmic Inclusions Attempts were made to purify virus particles and cytoplasmic inclusions from leaves of 'Candidum 1 caladium and 'Exotica' dieffenbachia with symptoms and from entire symp-

PAGE 21

tornatic shoots of the latter. All DMV-infected plants were obtained from commercial propagators in Orange County. All purification procedures were conducted at 4 C and 500 g of tissue were used in each purification run. The virus purification procedure was as described by Hiebert and McDonald (36) except that the final step involved tfiree rather than one cycle of differential centrifugation. The procedure used to purify the cytoplasmic inclusions induced by DMV was as described by Hiebert et al. (37) and modified by Hiebert and McDonald (36). Serology Attempts were made to obtain antiserum specific to DMV. Virus purified from 'Exotica 1 dieffenbachia leaves was emulsified (1:1, v/v) with Freund's incomplete adjuvant and injected intramuscularly into a rabbit. Only one injection was made. The blood was collected 26 days after injection, incubated at 37 C in a water bath, and allowed to clot. The serum fraction was collected, placed in glass ampules, and frozen. Antigens were prepared by triturating 2 g of symptomatic or healthy »Candidum' caladium leaf tissue with a mortar and pestle in 2 ml of deionized water. The triturated sap was then expressed through cheesecloth and centrifuged at approximately 2600 £ for 30 minutes. The supernatant was then discarded and the pellet resuspended in 1 ml of water. In this trial, the gels consisted of 0.8% Noble agar,

PAGE 22

10 0.5% sodium dodecyl sulfate (SDS) , and 1% sodium azide in water (2if). The Ouchterlony agar double-diffusion technique was used in all serological studies. The respective antisera were placed in center wells and the antigens in peripheral wells. Test plates were incubated 1-k days at 25 C and observed periodically. In all tests, normal serum and leaf extracts from healthy plants were used as controls. In other trials, attempts were made to determine the serological relationships of DMV to other viruses of the potato Y virus group. These tests were conducted with antisera to pepper mottle virus, potato virus Y, tobacco etch virus and turnip mosaic virus, obtained from D. 2. Purcifull (Department of Plant Pathology, University of Florida, Gainesville). Antigens of DMV were prepared by triturating k g of symptomatic or healthy leaf tissue of P. selloum with a mortar and pestle in 3 ml of water. The triturated sap was then expressed through cheesecloth and centrifuged at approximately 2600 £ for 30 minutes. The supernatant was discarded and the pellet was resuspended in 8 ml of a 3% aqueous pyrrolidine solution. Antigens were either derived from P. selloum seedlings which had been inoculated 8 or % days previously with a DMV isolate originally from 'Candidum' caladiums or from pooled leaf tissue from P. selloum seedlings which had been inoculated 13 days previously with DMV isolates from various naturally infected caladium varieties 'Itocapus', •John Peed 1 , 'Dr. Groover 1 , 'Avalon Pose 1 , 'W. B. Halderman *

PAGE 23

and 'Crimson 7/ave ' . Antigens to the other PVY-type viruses were obtained from D. Batchelor (Department of Plant Pathology 3 University of Florida). These antigens were prepared by triturating 2 g of leaf tissue in l+ ml of water. The sap was then expressed through cheesecloth, pipetted into vials and freeze dried. When virus samples were needed, 1+ ml/vial of a J>% pyrrolidine solution was added. The solution was stirred and allowed to stand for 30 minutes prior to use. The gels used in these tests consisted of 0.6% Noble agar, 0.05M Borate buffer at pH 8.2 and 0.02% sodium aside. Distribution and Effects of Dasheen Mosaic Virus Surveys Surveys were conducted from 1969-1971 in an attempt to assess the prevalence of DMV in Florida. These surveys included foliage nurseries in Orange and Dade counties, caladium fields in Highlands and Orange counties, Xanthosoma sagittifolium (cocoyam) fields in Dade County, an aquarium plant nursery in Palm Beach County and experimental and ornamental plantings of taro at the University of Florida, Gainesville. In addition, aroid samples were solicited from areas outside continental United States, and assessed for DMV infection. All plants received were maintained in isolation and destroyed upon completion of tests. Infections of DMV were assessed on the basis of symptoms expressed and recovery of virus, to at least 1 of k or more manually inoculated seedlings of P. selloum . Certain samples

PAGE 24

12 were further checked by electron microscopic examinations of negatively stained leaf extracts. Symptom Expression Throughout the course of this study, various aroids infected with DMV were maintained in greenhouses and periodically examined for symptoms expressed. In one experiment, 70 rooted cane pieces of 'Exotica' dieffenbachia were obtained from a foliage nursery in Orange County, Florida. All were cuttings of the same developmental stage when collected, and all except /f exhibited foliar distortion and mosaic. Half of the plants were placed in a greenhouse where the temperature varied from 20-ifO C, whereas the others were maintained in a different greenhouse at 2^-30 C. The light intensities of both greenhouses were equivalent and plants in both greenhouses were routinely watered and fertilized. Each plant was observed routinely from April 20, 1971 to March 1, 1972 and the symptoms expressed on each leaf recorded. On July 19, half the plants in each greenhouse were transplanted from if to 6 inch pots and examined for any resultant effects on symptom expression. In a similar experiment, 6 cocoyam plants rendered free of DMV through tissue culture were selected and manually inoculated with DM 1 /. Thereafter, these plants were maintained for 5 months in a greenhouse at 20-/i0 C. Throughout this time, these plants were checked periodically for foliar symptoms.

PAGE 25

Quantitative 5f f a r.t.w The effects of DMV on growth of caladium, dieffenbachia, P. selloum and Z. elliottiana were assessed after manual inoculation. All planting stock used was propagated from seed and maintained in isolation until inoculated. Plants of P. ^llomn and Z. elliottiana were inoculated as seedlings and an equal number of non-inoculated seedlings served as controls. Seedlings of caladium and dieffenbachia were maintained in isolation until they attained a size suitable for vegetative propagation. They were then vegetatively propagated and genotypically identical pairs were selected for comparisons between inoculated and non-inoculated plants. Inoculated and non-inoculated plants were maintained in pots on greenhouse benches for observation. All data except for Z. elliottiana were recorded within a 12 month period. Results for Z. elliottiana were recorded 2.5 years after inoculation during which time the corms were harvested, stored, and replanted twice. Fresh corm weights, leaf areas (maximum length X maximum width) and petiole lengths were recorded for caladium and Z. elliottiana . Fresh shoot weights, leaf areas, and shoot heights were recorded for dieffenbachia and total fresh plant weights and leaf areas were recorded for P. selloum . Elimination of ^ she en Mosaic Virus Seed Propagation Seed of caladium, dieffenbachia, and Zantedeschia spp.

PAGE 26

Ik were produced by intraspecific crosses. These crosses were made between plants of 'Candidum' caladium, 'Exotica' dieffenbachia, Z. albo-maculata , Z. elliottiana , or Z. rehmannii . All plants used in the crosses were shown to be infected with DMV based on symptoms and transmission to seedlings of P, selloum . Dieffenbachia plants were grown in a stock bed in a fiberglass house at Apopka and pollinated MayJune, 1971. The caladiums were grown in Gainesville either in a greenhouse or outdoors and pollinated May-June, 1972. The Zantedeschip spp. were grown in a greenhouse at Gainesville maintained at 2^-30 C and pollinated May-June, 1971. •The pollination procedures vrere similar to those described by McColley and Miller (46) for philodendron. All crosses were made between 6-10 a.m. or 5-8 p.m. Pollen was collected daily with a camel-hair brush and transferred within i+8 hours after shedding to neighboring receptive blooms of different plants. Bloom receptivity was indicated by the increased stickiness of the stigmatic surfaces of the spadix. Bloom receptivity of caladium and dieffenbachia was further indicated when the spathe began to unfurl revealing the distal portion of the spadix. Prior to pollination, the spathe was cut away from the spadix, discarded, and the pollen was gently applied to the proximal ovulate portion of the spadix with a brush. Shoot-tip Culture Attempts were made to culture the following aroids in vitro : 'Candidum 1 and 'Frieda Hemple • caladium, taro, cocoyam, 'Exotica* dieffenbachia, Asrlaonema modes turn ( Chinese evergreen),

PAGE 27

15 P. selloun, Cryntocory-e cordate and C. nevillii. In this study, the "revised" median of Kurashige and Skoog (50) was used exoept that (i) Hesc-ino^^ (1 . lnositol dehydrate) was substituted for .ffio-inositol, (ii) Edaaln was omitted and (iii) each liter of medium contained 8 rather than 10 g of agar. The kinetin level employed was 1.0 m g /l of medium and the indole-acetic acid level was 15.0 m g /l. Shoot-tips from all plants were excised and treated sequentially as follows: shoots were (i) trimmed to <1. 5 cm^, (ii) rinsed in flowing tap water, (iii) submerged for 10 -nutes in 0.52% sodium hypochlorite, (iv) trimmed further, (v) submerged for 5 minutes in 0.26* sodium hypochlorite and (VI) rinsed briefly „ sterile distilled water, with the aid of a dissecting microscope, each shoot was then trimmed until only the apical dome and one or two leaf primordia remained (»«. 13A.B). Finally, each shoot was transferred, cut surface down, to the surface of 1 5 ml of solidified medium contained in a 30 ml screw-cap vial. All cultures were maintained at 21-25 c and provided 12 hours of daily l ight (ca. ?50 ft-c) from incandescent and fluorescent bulbs. Oaladium, cocoyam and taro plantlets which had differentiated in culture were separated from undifferentiated callus and transplanted individually to sterili zed sand/peat (1 . 1} contained in 7 . 5 cn clay pots. AH potted ^^ ^ Placed in insect-proof cages in a growth room and observed ^r virus symptoms before being transplanted to larger pots and transferred to greenhouses. Differentiated Cryntocorvr.

PAGE 28

16 spp. plantlets were transferred to sterilized sand contained in 3.8 1 wide-mouth glass jars. Special precautions were taken to ascertain the presence or absence of infected material. Plants were maintained for at least 3 months in observation cages before being transferred to greenhouses. Samples were further checked for DMV infection by electron microscopic examination of leaf dip preparations and/or through manual transmission attempts to seedlings of P. selloum.

PAGE 29

RESULTS Characterization of Dasheen Mosaic Virus Host-range Determinations Dasheen mosaic virus proved to have a wide host range within the Araceae and was recovered from members of 9 of the 12 tribes and 13 of the 16 genera represented (Table 1). The following aroids did not become infected when manually inoculated as seedlings: Aglaonema commutatum , Anthurium bakerii, A. cris-oimar~inatum, Peltandra sp., P. stratiotes and Snathiphyllum floribundum . None of the non-araceous plant species including Tetragonia ex pans a proved susceptible to DMV when manually inoculated (Table 2). Neither host range differences nor differences in symptom expression were noted when single isolates from 'Candidum' caladium, 'Exotica' dieffenbachia and taro were compared. All three isolates infected manually inoculated seedlings of Anthurium scandens , caladium, dieffenbachia and P. selloum, and all three isolates induced identical symptoms in P. sel loum seedlings inoculated in recovery trials. 17

PAGE 30

18 <; < CQC3CQ<<«<<«3:< <<<<<<<

PAGE 31

j-y O o PQ < < <
PAGE 32

20

PAGE 33

21 Table 2. Plants not infected with dasheen mosaic virus following manual inoculations a Family Species Cultivar Aizoaceae Amaranthaceae Amaxyllidaceae Bromeliaceae Chenopodiaceae Commeliniaceae Compositae Convolvulaceae Cruciferae Curcurbitaceae Gramineae Tetragonia exnansa Amaranthus tricolor Gomphrena globosa Amaryllis sp. Aechmea pubescens Beta vulgaris Chenopodium amaranticolor Chenonodium quinoa Rhoeo discolor Zinnia elegans Ipomoea purpurea Brassica perviridis Cucurbita neno Curcurbita vevo var. melopepo Avena sativa Lolium sp. Pennisetum typhoideum Sorghum vulgare Triticum aestivum Zea mays Scarlet Tender green Small sugar Early Prolific Straightneck Red Rustproof Ryegrass Pearl Georgia 615 Michigan Amber Golden Cross Bantam

PAGE 34

Table 2 continued 22 Family Species Cultivar Iridaceae Labiatae Leguminosae Liliaceae Phytolaccaceae Solanaceae Gladiolus hortulanus Ocimum basilicum Arachis hypogaea Cassia occidentalis Cassia tora Cyamo-psis tetragonoloba Desnodiun canum Glycine max Phaseolus vulgaris Pisum sativum Vicia faba Vigna unguiculata Allium cepa Lilium longiflorum Phytolacca americana Capsicum frutescens Datura stramonium Lyconersicon esculentum Nicotiana clevelandii Florunner Early Runner Dixie Runner Bragg Red Kidney Michelite 62 Alaska Little Marvel Long pod Early Ramshorn Blackeye Yellow Bermuda California Wonder Manalucie Nicotiana glutinosa

PAGE 35

Table 2 continued 23 Family Species Nicotians hybrid b Nlcotiana sylvestris Nicotiana tabacum Petunia hybrida Physalis floridana Cultivar Samsun M All plants tested were inoculated as wHii^o t instance attempts were made to reefer lm to P lliV** 7 2-6 weeks after inoculation. g ellou q § £ ir§fch h ?is r L d e ( fi 5 ' ;! eVeland11 X * Siutinosa, developed

PAGE 36

2Af Transmission Electron Microscopy Leaf extracts Electron microscopic examinations of negatively stained leaf dip preparations of various aroids exhibiting mosaic symptoms revealed the presence of filamentous rods (Fig. 3B) similar to those reported by Zettler _et _al. (7A-j75). Measurements of virus particles from cocoyam plants infected with a DMV isolate originally from 'Candidum 1 caladium were similar to those previously reported for DMV in taro (7^*75); i.e., 77% of 95 particles measured were 726 to 815 nm long with a main maximum at 801 nm and a mean length of 782 nm. Such particles were not seen in extracts from healthy cocoyam plants. Striated inclusions were seen in leaf extracts of 'Candidum' caladium and 'Exotica' dieffenbachia leaves stained with ammonium molybdate. These inclusions resembled those reported by others for viruses of the potato Y virus group (20). Tubes and laminated aggregates were observed in leaf extracts from both caladium and dieffenbachia. No apparent differences in specific inclusion morphology were noted between these species. Sectioned material Ultrathin sections of 'Candidum' caladium infected with DMV revealed the presence of pinwheel, circle, bundle and laminated aggregate inclusions (Fig. 2) similar to those described by Zettler et al. (7^,75) for DMV-infected taro plants. Of the sections examined, the relative numbers of tubes seemed to be much greater than the number of pinwheels or laminated aggregates. Virus particles were commonly found in close association

PAGE 37

Figure 2. A,E) Thin sections of a caladium leaf infected with dasheen mosaic virus showing pinwheel (PV/), circle (C), bundle (B) and laminated aggregate (LA) inclusions. Scale line of A is 900 nm and B is 450 nm. Note close association of virus particles with inclusions.

PAGE 38

26 *

PAGE 39

27 with these cytoplasmic inclusions (Fig. 2). Light Microscopy Amorphous cytoplasmic inclusions were seen in stained epidermal strips taken from 'Candidum' caladium, 'Exotica 1 dieffenbachia and taro when examined with a light microscope (Fig. 3). Neither nuclear nor nucleolar inclusions were observed in any of the epidermal strips. No amorphous cytoplasmic inclusions were observed in epidermal strips from healthy plants of caladium, dieffenbachia and taro. Purification of Virus and Virus-induced Cytoplasmic Inclusions DMV particles were partially purified from dieffenbachia shoot and leaf tissue (Fig. 5C). This material had a maximum ultraviolet absorbtion at 260 nm in aqueous 0.02K Tris buffer, pH 7»kt and 10 M Cleland's reagent. The UV absorption spectrum from 230-360 nm was very similar to that reported for other PVY type viruses (Fig. /f). The 260/280 ratio of ca. 1.507 (not corrected for light scattering), however, is a value higher then that recorded for other flexous-rod shaped viruses (personal communication, E. Hiebert). Attempts to purify particles of DMV from caladium were unsuccessful. A green mucilaginous pellet resulted from the first high speed centrifugation. The purification procedures were discontinued at this step since the pellet proved difficult to resuspend and contained a relatively low concentration of virus particles based upon electron microscopic examination. DMV-induced cytoplasmic inclusions were partially purified

PAGE 40

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PAGE 41

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PAGE 42

Figure l+. Ultraviolet absorption spectrum (not corrected for light scattering) of dasheen mosaic virus, purified from 'Exotica' dieffenbachia and contained in 0.02 M Tris buffer and 10~->M Cleland's reagent.

PAGE 43

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PAGE 44

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3k from symptomatic caladium and dieffenbachia leaves and dieffenbachia shoots. Striated plate-like inclusions as well as striated tubular inclusions were observed iron; both dieffenbachia and caladium tissue. 7/hereas plates predominated in dieffenbachia extracts (Fig. 5A), tubes predominated in extracts of caladium (Fig. 53). Serology No reactions were noted between center wells containing serum prepared from DMV and peripheral wells with extracts from healthy or DMV-infected caladium leaves. Precipitin reactions formed betv/een peripheral v/ells with extracts of DMV-infected P. selloum leaves and center wells containing antisera to TEV and PVT (Fig. 6). Nevertheless, the homologous reactions v/ere much stronger and spurred over the heterologous reactions. The heterologous reactions were stronger when DMV antigens were prepared from P. selloum plants inoculated 10-13 days prior to extraction than from plants inoculated 56 days earlier. No reactions were observed between antisera of pepper mottle or turnip mosaic viruses and extracts from DMV-infected P. selloum leaves. Nor were any reactions observed between any of the antisera wells and peripheral wells containing extracts of healthy tobacco or P. selloum leaves.

PAGE 47

Figure 6. Serological relationship of dasheen mosaic virus (DMV) to potato Y (PVY) and tobacco etch (TEV) viruses. The center wells contain: (A) PVY antiserum, (B) TEV antiserum, (C) Normal serum. The peripheral wells in each pattern were filled with pyrrolidine treated tissue extracts from: (1) healthy tobacco, (2) PVY-infected tobacco, (3) and (5) DMV-infected Philodendron selloum , (4) TEV-infected tobacco, and (6) healthy P. selloum.

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36

PAGE 49

Distribution and Effects of Dasheen Mosaic Virus Surveys Symptoms of DMV infection were observed in all commercial, ornamental and experimental aroid plantings surveyed in Florida. Areas surveyed included: if and 1 caladium fields in Highlands and Orange counties, respectively, if cocoyam fields in Dade County, 1 experimental and 2 ornamental taro plantings in Alachua County, 1 aquarium plant .nursery in Palm Beach County, and 3 and 6 foliage nurseries in Dade and Orange counties, respectively. Dasheen mosaic virus was recovered to seedlings of P. selloum , and filamentous particles were observed in leaf dip preparations from the following aroias obtained in these surveys: A. modestum , C. cor data , caladium ( 'Candidum' , 'Caroline V/horton 1 , 'Crimson V/ave ' , 'Dr. Groover', 'Exposition, 'Frieda Hemple ' , "Miss Chicago 1 , 'White Christmas'), taro, an ornamental Colocasia sp., Dieffenbachia amoena and dieffenbachia ( 'Exotica' , 'Perfection') and cocoyam. Dasheen mosaic virus was also recovered to P. selloum and virus particles observed in taro samples received from India, Hawaii, and the Fiji Islands, and in cocoyam samples from Trinidad. A sample of Xanthosoma brasilensis from Guadeloupe likewise proved infected with DMV. Symptom Expression Foliar mosaic symptoms were observed in all plants from which DMV was recovered (Table 1). Dasheen mosaic virus, however, was rarely isolated from tissues without such symptoms.

PAGE 50

38 The foliar mosaic pattern of taro and cocoyam was typically associated with the major veins and resembled a "feathery" appearance (Fig. 8B), a characteristic noted by others (2,17, 2.2,25). A "vein mosaic" pattern (6) was typically expressed on the first leaves of P. selloum seedlings to show symptoms after inoculation with DMV (Fig. 7C). On subsequently formed leaves, however, this veinal pattern was not apparent (Fig. 7D). Prominent foliar distortion symptoms were frequently noted for certain aroids, including 'Exotica' and 'Perfection* dieffenbachia (Fig. 7A), Philodendron nittieri , P. selloum , Zantedeschia aethiorlca , Z. albo-maculata and Z. elliottiana . Distortion symptoms induced by DMV in dieffenbachia included epinasty and, in many instances, a marked disruption of the vertical symmetry of the unfurled leaf (Fig. 7A). As such leaves became fully expanded, torn regions resulted due to uneven expansion rates of maturing lamellar tissues. Whereas DMV symptoms of some aroids were not conspicuous and did not markedly deter from their attractiveness, other aroids were severely affected, particularly 'Exotica' and 'Perfection' dieffenbachia and Z. elliottiana . Among variegated aroids such as caladium and dieffenbachia, DMV-induced mosaic symptoms supplanted inherited patterns, which detracted from their attractiveness (Fig. 73, 8A). Several authors have observed that leaves with symptoms appear intermittently among infected plants (2,75). This phenomenon was apparent for most aroids observed in this study,

PAGE 51

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« "eluding caladium, cocoyam, diefferbachia, P. selloum and tare infected plants of certain species, however, such as P. pittieri expressed syaptoas continually throughout the observation period. In one experiment involving cocoyam plants inoculated with PHV, successively produced leaves .ere examined for expressed symptoms (Table 3). For all six plants, mosaic symptoms were apparent on the first leaf to appear following inoculation, hereafter, leaves v,ith a P d without symptoms were produced on each of the plants, and, with one exception, leaves with symptoms appeared in succession following the emergence of symptomless leaves. The duration of this intermittent expression of symptoms among aroids was not assessed in this study, although it was noted that plants of P. selloum inoculated with DM, a= seedlings in October, logo, still lnteraittently ^^ leaves with and without mosaic symptoms 4.5 years later. An unsuccessful attempt was made during this investigation to correlate certain environmental conditions with the appearance of symptoms using DKV-infected -Exotica, dieffen»achia plants. Both temperature and pot size, as it relates to growth rate, were studied. The periodicity of symotom expression was identical at both temperature regimes tested, although Plants growing in the greenhouse where temperature fluctuated between 2 and ,0 C averaged 16. 2 leaves per plant compared to only 12 . 6 leaTC s per plant for the plants growing where the temperature was controlled between 24-50 C (557 and 429 leaves per 33 and 54 plants, respectively). At both

PAGE 56

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45 regimes, the lowest proportion of leaves with symptoms emerged June-September whereas leaves with the highest proportions emerged October-December and April-May (Fig. 9). This symptom periodicity data generally correspond with observations of 'Exotica' dieffenbachia plants grown commercially at Apopka, Florida (Knauss, personal communication). Similarly, no effect on symptom expression was noted in this experiment as a result of transplanting plants from 4 to 6" pots, although plants transferred to larger pots grew more vigorously than their counterparts in smaller containers: at 20-40 C and 2430 C, an average of 16.8 and 13.2 leaves per plant were produced in 6" pots compared to only 13.6 and 11.9 leaves per plant in 4" pots, respectively (303 and 238 leaves for 18 and 18 plants and 234 and 191 leaves for 15 and 16 plants, respectively) . Quantitative Effects Plants infected with DMV proved stunted in comparison to their healthy counterparts (Table 4) regardless of whether or not mosaic symptoms were evident on plants when recorded. Fresh corm weights and leaf areas (maximum length x maximum width) of 69 caladium plants were 40 and 33% less than healthy plants, respectively. Corm weights and leaf areas of 8 Z. elliottiana plants were likewise reduced 59 and 51%, respectively. Similarly, fresh shoot weights and leaf areas of 27 infected dieffenbachia plants were 63 and 66;o less than controls; and total fresh plant weights and leaf areas of 36 P. selloum plants were 30 and 30/o less, respectively (Table 4).

PAGE 58

Figure 9. Symptoms of dasheen mosaic virus expressed in newly emerged leaves of 'Exotica* dief fenbachia plants in relation to date of emergence and temperature regime at which plants were maintained. Thirty-four and 33 plants were maintained at 24-30 and 2O-/1O C, respectively. Bars indicate the percentage of leaves with symptoms according to the order of foliar succession.

PAGE 59

100. 80604020< w > s 100' »-3 20-40 C 80< 604020 24-30 C JUN AUG OCT DEC FEB MONTH

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All these results were highly significant at the 1% level. Elimination of Dasheen Mosaic Virus Seed Prot)a-Tation Dieffenbachia fruits, although cream colored during most Of their development, became red when ripe approximately 1012 weeks after pollination. Dieffenbachia fruits did not dehisee but remained loosely attached to the spadix (Fig. 10). A spadix bore 15-30 ovaries, each containing a single round geed 5"6 mm in diameter which was green when mature. Seeds germinated within 20 days after they were removed from the fruit and planted in moist peat. Unlike dieffenbachia, caladium fruits ripened 5-6 weeks after pollination and abscissed from the spadix (Fig. 11). The exposed fruit surfaces remained green throughout their development without apparent color change at the time of abscission. The exposed surfaces of the ovary walls were creamcolored at maturity. Ovulate portions of the spadix contained approximately 200 seed-bearing ovaries, each containing 1-14 oval seeds which were 1-1.5 mm in length and light tan in color. As many as 1500 seeds were obtained from a single spadix. Seeds were removed from the fruit and planted in moist peat. Seeds germinated readily 8-10 days after planting. Seed germination rate decreased markedly, however, when seeds were dried and stored 2 weeks at 25 C and 53/^ relative humidity. Fruits of Zantedeschia spp. also remained green throughout

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u a c o O-H S-.-H O O ft co +> -P -H CO -rj -^ ft* 1 -' a5 aS c; -p •H CO CO H 3 .3 H " © o © ft C 5 o «M -H vO O-P 1 © o S ftC ShS rt co aS -H -H X © ^ O CO ft as ^t3 3 i © h CQ 9"* H C © t3 © -£ RSH CO HH O ftO

PAGE 65

53

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5k their development, but did not abscise from the spadix at maturity (if— 6 weeks after pollination). Each fruit contained one seed. Seedlings were maintained in a greenhouse at about 60% shade in isolation from commercial aroid stock. Plants were transferred from peat to a steam-sterilized soil/perlite mix contained in clay pots and treated routinely with a liquid formulation of 20-20-20 fertilizer. Although parental 'Exotica' dieffenbachia, 'Candidum' caladium, and Zantedeschia spp., plants used in this study were virus-infected as evidenced by symptoms observed at the time of pollination, none of the more than 500 resulting dieffenbachia, 1000 caladium and 100 Zantedeschia progeny displayed virus symptoms, indicating that the seedlings were virus-free. A small percentage of the dieffenbachia seedlings exhibited albinistic tendencies and died shortly after germination. The juvenile foliage of the surviving dieffenbachia progeny was non-variegated and variegated leaves did not develop until i+8 months after germination. Similarly, juvenile leaves of caladium were homogeneously green and variegated leaves did not develop until about 3 months after germination. A year after germination a marked variation was noted among the 207 randomly selected dieffenbachia seedlings which had grown to 30-34 cm in height. Fifty-three of the progeny exhibited none of the white variegation typical of the 'Exotica' parents. The foliar patterns of the remaining progeny varied from those having relatively localized areas of white to those

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55 having white patterns exceeding in area that of the original P-ents (Fig. 1 2A) . A few plants exhiblted & ^^ ^ ^^ coloration unlike the .^oticaparents. Marked differences in leaf shape and apical dominance were also noted among the progeny. Whereas some of the leaves had dimensions similar to those of the parents, others were somewhat more lanceolate. Axillary buds of some progeny grew only after the apical meristem was removed whereas the axillary buds of other progeny proliferated regardless nf +ho ™~^ c less of the Presence or absence of the apical shoot (Fig. 12B). little variation was noted among the 100 randomly selected caladium seedlings 8 months after germination. Fifty-one percent of the progeny had predominately white leaves with 22 and 27% having predominately green or intermediately white leaves, respectively. Forty-one percent had striped petioles with 55 and « having predominately green or purple petioles, respectively. The ^t . CandidM , plaQts by compar . sM haye pre _ dominately white leaves with striped petioles. Minor differences were noted in leaf shape or apical dominance between the •Candidua' parents and the progeny. All seedlings of Zantedeschi, spp. resembled tbg Darents from which they were derived. These data correspond closely to those obtained by Eyohitsu (60) for the self-pollinated progeny of yellow, white and pink ZantedescM, S pp. Shoot-tin Culture Attempts to culture pathogen-free plants of caladiu*, tare and cocoya* were apparently successful. Forty of 50,

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Figure 12. Dief fenbachia seedlings 12 months after germination. Note differences in foliar variegation patterns (A) and in degree of shoot . proliferation (B).

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57

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58 3 of 6, 4 of 6 and 6 of 6 shoots of 'Candidum' and 'Frieda Heraple ' caladium, taro and cocoyam, respectively, developed in vitro without apparent microbial contamination . Shoot tips, originally cream colored, became green within 2 weeks after excision. Within 8 weeks, callus masses developed and differentiated numerous shoot buds. Within 18 weeks, many plantlets with roots and shoots developed from these buds (Fig. 13C). Plantlets of all three species survived transfer to soil and developed rapidly into vigorously growing plants (Fig. 13D). Every 3 months each caladium culture generated 10-20 plantlets suitable for transplanting and enough callus for an additional 10-20 cultures. 'Candidum' caladium callus has been maintained in culture for at least 1.5 years without apparent adverse affects expressed by plantlets after transfer to soil. All attempts to culture dieffenbachia, Chinese evergreen and philodendron were unsuccessful either because microbe-free shoot tips could not be obtained or because they failed to grow in vitro . Six of 60, 2 of 2 and 2 of 3 shoot-tips of dieffenbachia, Chinese evergreen and philodendron, respectively, were without apparent microbial contamination. However, none of these developed callus or differentiated into plantlets. Shoottips, originally cream colored, became green 1-2 weeks after excision, but eventually became necrotic and died. Similar attempts with C. cordata and C_. nevillii were successful; however, both of these species failed tc proliferate in culture.

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Figure 13. (A,B) Scanning electron micrograph of caladium apical shoot showing first (1) and second (2) leaf primordia and anical dome (a). • Scale line is 0.166 mm. C) Six-week-old culture of 'Frieda Hemple ' caladiums on Kurashige-Skoog medium. D) Cocoyam plant 6 weeks after transfer to soil from culture. Scale line is 5.4 cm. E) Mature pathogen-free Candidum' caladiums grown under greenhouse conditions.

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60

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Three of 10 and 2 of 5 shoot tips of C. cordata and C. nevillii , respectively, were without microbial contamination and became green 1-2 weeks after excision but differentiated only one plantlet per shoot tip. Attempts to culture dieffenbachia on other media were likewise unsuccessful. Three of 20 and 2 of 20 apical shoots proved microbe free when placed on Vacin-Went (68) and Knudson C (43) medium, respectively. However, as with the MurashigeSkoog medium, none of the 5 shoots survived in culture. Plantlets of 'Candidum' caladium and taro obtained from some shoot tips in culture proved to be infected with DMV. Virus-infected plantlets were readily detected in vitro and as plants growing in soil. These observations were confirmed through inoculations of P. selloum seedlings and electron microscopy. Plants of both caladium cultivars, taro and cocoyam derived from other shoot-tips, however, proved free of virus and were used in continued propagations. Virus-free plants of 'Candidum' and 'Frieda Hemple ' caladiums developed typically variegated leaves 10-12 weeks after transplanting into soil. Some of the 'Candidum' caladiums flowered 6-7 months after transplanting and produced corms l+5 cm in diameter with leaves 20-27 cm wide and 25-32 cm long (measured from lamella tip to point of petiole attachment) (Fig. 13E). After 1 year, these plants had corms ca. 9.5 cm in diameter with a fresh harvest weight of ca. 200 g. Mature plants were identical to their commercial counterparts, except that they appeared to be more vigorous. In one experiment,

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62 lZf DMV-free and 11+ DMV-infected plantlets were transplanted from culture to soil and maintained under identical growing conditions. After 6 months, corm weights of healthy plants were significantly greater (t value = 2.6; P = < 5%) than those from diseased plants (56.9 and l±0.2 g/corm, respectively). Approximately 350 virus-free 'Candidum' caladium plantlets have been grown to maturity since this study was initiated. These plants were propagated in a screened greenhouse on raised asbestos-cement benches previously sterilized with a 3% commercial formulation of sodium hypochlorite. The plants were routinely treated with either nicotine sulfate or malathion for insect control and twice with Truban (5-ethoxy-3-trichloromethyll,2,Jf-thiadiazole) for the control of pythiaceous fungi. During their development, none of the plants ever had symptoms of DMV. At harvest (ca. 6 months after planting), none of the plants had apparent discolored or necrotic roots or corms.

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DISCUSSION This study provides additional evidence that DMV is a member of the potato Y group of viruses as originally described by Brandes (7), supplemented by Edwards on (18), and recognized by' the International Committee on Nomenclature of Viruses (25, 72.). The mean length of 782 nm for particles in leaf extracts from DKV-infected cocoyam plants corresponds closely to the values of 759 nm for particles in cocoyam by Debrot and Ordosgoitti (17) and 750 and 751 nm for particles in taro by Zettler et al. (74,75) and Debrot and Ordosgoitti (17), respectively. Similar flexous rod particles were found in leaf extracts of DKV-infected plants representing 12 other aroid genera investigated in this study. Similarly, cylindrical inclusions as described by Edwards on et al. (20) were observed in extracts and/or thin sections of leaves from DMV-infected plants of caladium, dieffenbachia and Z. elliottiana . These inclusions closely resembled those described by Zettler e_t al. (74,75) for DMV-infected taro except that, in dieffenbachia, laminated aggregate inclusions were more prevalent than tubular inclusions. The relatedness of DMV to potato Y and tobacco etch viruses as indicated by serological data further supports the contention that DMV is a member of this virus group. These serological 63

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b^ results were confirmed by other workers who found that DMV and 7 other viruses considered to be members of this group reacted positively to antiserum of tobacco etch D-protein (62). Attempts to obtain an antiserum specific to DMV were not successful in this study. This is presumably due to relatively low virus titers in the leaf tissues used for purification and to a relatively low purity of the final injected viral preparation, as indicated by the relatively high 260/280 value of 1.59 recorded. Herold (34,35) reported a mean particle length of 754 nm for a virus of Anthurium andraeanum which was reported to be transmitted by white flies and manually transmissible from tobacco to other solanaceous plant species. Although DMV was recovered from and inoculated to Anthurium spp. in this study, transmission to tobacco was never accomplished. In addition, transmissibility by white flies is not considered to be a property typical of other members of the potato Y virus group. Hence, it is concluded to be unlikely that Herold *s virus and DMV are synonymous. The relation of DMV to other aroid viruses is difficult to judge considering the general paucity of information provided by these previous studies. Saychaudhuri and Ganguly (56) for example re-ported that the aphid-transmissible "Chirke" disease of cardamon was manually transmissible to the aroid Acorus calamus . However, further information that would enable a correlation to be made between this virus and DMV was not provided. Also, in this study, neither cardamon

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65 nor Acorus sp. were available for inoculation with DMV. Dasheen mosaic virus was shown to have a wide host range within the Araceae. Although DMV infected species in 13 of the 16 aroid genera tested none of the inoculated k5 species of 17 other plant families became infected. These results, however, do not necessarily indicate that DMV is not synonymous with any other previously described members of the potato Y virus group. The plant species included in the host range were not all-inclusive with respect to all 103 members of the group listed by Edwards on (19). James et al. (l+O) reported that DMV "occasionally" infected plants of T. ex-pans a , although they apparently did not effect recovery of DMV back to any seedling aroid. Repeated efforts to infect T. exnansa with DMV in this study were unsuccessful despite precautions taken to insure inoculum viability. If susceptible, T. expansa must be considered a marginal host, and certainly less satisfactory than seedlings of P. selloum as indicators for DMV. No evidence for virus inactivators or inhibitors among aroids was noted in this study, as DMV was readily recovered to P. selloum seedlings. In addition to DMV, an isolate of cucumber mosaic virus from Commelina sp. manually inoculated to caladium seedlings was readily recovered to tobacco indicator plants (unpublished data of the author). The apparent lack of inhibitory substances is not surprising considering the study of Simons et al. (63) who showed that extracts of neither Dief fenbachia seguine nor Monster a deliciosa

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66 contained substances inhibitory to the transmission of tobacco mosaic virus. Since its recognition in 1969 > DMV has been recovered from various locations including the United States (75) > Europe (69, 75), the South Pacific (23,32,40,42), the Caribbean (2,42), South America (17) and Japan (3). In this study, specimens from India, Hawaii, the Fiji Islands, Trinidad and Guadeloupe were received, in addition to samples from Florida, and determined to be infected with DMV, thus confirming the international distribution of this virus. This widespread occurrence of DMV presumably reflects: 1) the antiquity of aroids as cultivated plants, 2) their obligatory means of vegetative propagation, 3) their extensive dissemination as popular cultivated plants and 4) the facility of DMV to be transmitted by aphids. The widespread occurrence of DMV in commercial caladiums is further abetted by certain circumstances peculiar to this industry. According to grower estimates, 96% of the world's caladiums are from Florida, 76?o of which are produced in Highlands County on less than 600 acres (39). This crop is restrictively cultivated on acidic muck soil and with a minimum of crop rotation or isolation. Considering that the variety 'Candidum' comprises over 30% of the total production and that all these plants are derivatives of a single plant developed before the turn of the century (33), the ubiquity of DMV infections noted in this study for caladiums was seemingly inevitable. This research shows DMV to be a serious pathogen of aroids.

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67 Foliar symptoms are narked in some aroids, particularly 'Exotica' and 'Perfection' dieffenbachia. These symptoms in dieffenbachia merit grower concern, and, in some instances, entire beds have been abandoned due to foliar disfigurement. Symptoms in most aroids, however, are relatively inconspicuous -or absent altogetherand incited a minimum of concern, likely accounting for the relatively little attention devoted to aroid viruses. Of particular interest in this study was the, intermittency of expressed symptoms for most aroids, but absence of apparent symptoms does not indicate recovery from virus. This fact was indicated in prolonged and controlled observations of several aroids, in particular cocoyam, 'Exotica' dieffenbachia, and P. selloun . In every instance, leaves with pronounced mosaic symptoms were expressed following periods when only symptomless leaves had developed. For this reason, it is concluded that most aroids naturally infected with DMV are unreliable indicators of infection. Several authors (10,12,23*40,41,42) have used vegetatively propagated aroids in virus studies, and concluded that symptoms expressed on inoculated plants indicated successful transmission. Such results should be viewed with caution unless workers can assure that plants were not already infected v/hen inoculated. This investigation did not divulge reasons for the intermittency of symptom expression, although it was concluded that temperature was not likely to be a major contributing factor since leaves of 'Exotica' dieffenbachia expressed symptoms synchronously at two different temperature regimes. A

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more likely factor in effecting symptom expression might be photoperiodic response. The author is not aware of published reports citing such a factor being responsible for intermittent symptom expression, although Reiner t and Kasperbauer (57) have shown that the phytochrome system affects the multiplication of tobacco ringspot virus; and as reviewed by Butler and Downs (9)> the phytochrome system has been implicated as having a major role in the development of photoperiodically sensitive plants. Accordingly, studies should be undertaken to ascertain the role of light in masking symptoms; the possibility exists that for certain foliage plants, symptoms can be masked by artificially manipulating day length. Similarly, such manipulation could also be employed on a practical basis to subdue virus multiplication prior to dissection of shoot tips for tissue culture, or, conversely, to augment virus multiplication to increase the reliability of DMV assays. Quantitative losses due to DMV are more substantial than qualitative losses although they are probably far less apparent, when plants are ubiquitously infected. In controlled experiments with plants of caladium, dieffenbachia, P. selloun and Z. elliottiana , fresh plant weights were reduced as much as 63% and no less than 30/o« Similarly, leaf areas were reduced 30 to 66% even when expressed foliar symptoms were not apparent. These studies show conclusively that practical control of DMV is contigent upon the elimination of virus from propagating

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stock. Two approaches for obtaining virus-free plants were given serious consideration in this study: seed propagation and shoot-tip culture. Alconero (1) attempted heat elimination of DMV from cocoyaa but failed to inactivate the virus at temperatures the plant would withstand. Success with modification of this technique may be successful with other aroids, although relatively few viruses of the potato Y group have been controlled in this manner (52). Moreover, heat therapy is unlikely to completely eliminate other pathogens, including species of Pythium , Sclerotinia , Fusarium and Rhizoctonia known to be pathogenic to certain aroids. Seed propagation is an effective =eans of eliminating most phytopathogers (L). Seed of many aroids were collected during this study (Table 1) and in no instance was there any evidence for seed transmission, even when seed was collected from plants known to be infected with DMV". Certain aroids, particularly P. selloun , are normally seed propagated, and resultant progeny closely resemble parental plants. As this study shows, however, progeny of some aroids, such as caladium and dieffenbachia, differ markedly from parental plants. For such aroids, therefore, this technique cannot be used on a practical scale to eliminate DMV from commercially desirable cultivars such as 'Candidum' caladium or 'Exotica' dieffenbachia. Nevertheless, results with caladium and dieffenbachia suggests a rich -but ignoredgenetic potential for deriving improved horticultural varieties. For example, a need exists for "color" among foliage plants, such as that provided by the

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70 golden green variegated pothos ( Scindansus aureus). A concerted breeding program with Dief fenbachia spp., could enrich the industry considerably by infusing it with color, as this study suggests. Similarly, horticulturalists have expressed interest in the possibility of developing less poisonous variants of dieffenbachia than are currently available (5). The genetic potential of caladiums has previously been demonstrated as evidenced by the 2000 or more named varieties developed by such workers as Nehrling, Mead and Leitze (33) • Whereas these evaluations were based principally upon foliage characteristics, incorporation of other factors such as coldhardiness and disease resistance could be considered under the auspices of a future concerted breeding program. Theoretically, shoot-tip culture affords the most ideal means of eliminating DMV and other phytopathogens while retaining the genetic integrity of the parental stock. In certain instances, this technique has the considerable added advantage as a means to mass propagate plants under aseptic conditions (k8) • This research resulted in successfully culturing in vitro caladium, cocoyam, _C. cordata, C. nevillii and taro aseptically. This appears to be the first report of the in vitro culture of aroids. Interestingly, with the exception of the Cryotocoryne spp., the success reported herein involved plants of the tribe Colocasieae whereas failures involved other tribes in the Araceae. Although shoot-tips of both Crypto coryne spp. differentiated into single plants which survived transfer to sand, neither species formed callus or proliferated

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in culture indicating that the tissue culture techniques used in this study, while ideally suited for the Colocasieae, will have to be modified for members of other tribes. Attempts to culture dieffenbachia, A. modes turn and P. selloum v/ere not successful either because of contamination, or because explants failed to survive in culture. It is possible that proliferation for these plants could be effected through the use of growth regulators such as 2,4-D, as shown by Carter e_t al. (ll) to be essential for the proliferation of oat callus; Pathogen-free plants derived from tissue culture may effectively be used in pathogenicity trials with specific disease agents. Plants of ' Candidum' caladiums were used, for example, in determining that Pythium rayriotylum v/as a significant root pathogen whereas three other species of Pythium were not (59). Tissue culture may also be used as a valuable precautionary tool for those endeavoring to introduce exotic species or cultivars of caladium, cocoyam or taro into new areas. Leon (44) emphasised the dangers of inadvertently importing pathogens in vegetative plant parts and announced the need for defining areas where pathogen-free material could be obtained. Although some pathogens, such as DMV, may already be distributed world-wide, others may have a restricted range. Thus, culturing new accessions of these aroids in vitro , and screening plantlets before their release, would greatly minimize importing dangerous nev; pathogens accidentally. The rapid proliferation of caladium, cocoyam and taro in

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72 culture indicates that these plants can be propagated at an unprecidented rate. A newly developed caladium cultivar, for example, cannot be made available commercially until sufficient saleable stock can be produced which, according to grower estimates requires ca. 6-9 years during which time enough material has been accumulated to plant 0.5 acres with ca. 20,000 seed pieces. This study shows that at least 1020 plantlets and 10-20 new cultures can be generated in vitro every three months. Realizing that each plantlet can attain maturity 6 months later, it would be theoretically possible to produce sufficient pathogen-free stock within three years to supplant the entire caladium industry of Florida consisting of an annual production of 40-50 million corms. A similar approach was considered for narcissus by Stone (6/+) who divulged the feasibility of replacing the entire narcissus stock of the Isles of Scilly with virus-free material. Regardless of technique used to eliminate DMV and other pathogens from aroid planting stock, procedures should be adapted to prevent the re-establishment of pathogens. Presumably certification programs similar to those established for numerous other crops, such as potatoes and chrysanthemums, could be developed and standards appropriate for specific aroids established. Regretably, no such programs for aroids have been developed in Florida or elsewhere despite their economic importance. Ironically, DMV is firmly established in the cocoyam and taro accessions of the Federal Experiment Station at Mayaguez, Puerto Rico (2), and at the Saman Mocho Experiment

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Station in Venezuela (17). Presumably higher standards of disease freedom should be initiated at research centers. Once released, it is logical to assume that pathogenfree plants will become re-infected. This factor does not negate the wisdom of certification programs, however, and many, such as the bean seed program of New York, have withstood the test of time. Indeed, although DMV is aphidtransmitted, rates appear to be low and appro:rimate the findings of Saladini and Zettler (61) for their observations of unexpectedly low incidences of sugarcane mosaic virus infections in St. Augustinegrass plantings in Florida. Zettler (personal communication) noted that only 2 of 112 and 34 of 157 Philodendron sellourn seedlings became infected with DMV during test feedings (involving five aphids per test plant) by individuals of Aphis craccivora and Hyzus uersicae allowed single acquisition probes, respectively. These results are in marked contrast to the results with other potato Y type viruses of such plants as cowpea (73) and pepper (76), which have relatively high rates of aphid transmission.

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LITERATURE CITED 1. Alconero, R. 1972. Hot v/ater treatments of corms of Xanthosoma snp. infected with dasheen mosaic virus. Plant Dis. Rep. 56:320-321. 2. Alconero, R., and F. '.V. Zettler. 1971. Virus infections of Colocasia and Xanthosoma in Puerto Rico. Plant Dis. Rep. 53:306-308. 3. Arai, K., Y. Doi, and K. Yora. 1970. Notes on the filamentous virus found from mosaic of taro. Ann. Phytopathol. Soc . Japan 36:373 (Abstr.). 4. Baker, K. F. 1957. The U. C. system for producing healthy container grown plants. Calif. Agr. Exp. Sta. Ext. Service Manual 23. 552. p. 5. Barnes, B. A., and Lauretta S. Fox. 1955. Poisoning with Die ffenbachia . J. Hist. Med. Allied Sci. 10: 173 -ihi: 6. Bos, L. 1970. Symptoms of virus diseases in plants. Cent. Agr. Pub. Doc, V/ageningen. 206 p. 7. Brandes, J. I964. Identifizierung von gestreckten pflanzenpathogenen viren auf morphologischer Grundlage, Mit. Biol. Bundesanstalt Land-Forstwirtsch. BerlinDahlem 110.5-130. 8. Brandes, J., and R. Bercks. 1965 . Gross morphology and serology as a basis for classification of elongated plant viruses. Adv. Virus Res. 11:1-24.. 9. Butler, W.L., and R. J. Downs. I960. Light and plant development. Sci. Amer. 203:56-63. 10. Capoor, S. P., and D. G. Rao. 1969. Observations on a mosaic disease of Amorpho-challus camnanulatus . Indian Phytopathol. 22:433-440. 11. Carter, 0., Y. Yamada, and E. Takahashi. 1967Tissue culture of oats. Nature 214:1029-1030. 74

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12. Chatter jee, S. N., S. P. Capoor, R. D. Ram, and M. R. Nimbalkar . 1971. Effect of mosaic virus on production of corms of Amorrho-phallus camnanulatus . Indian Phytopathol. 24: 821-023. 13. Christie, R. G. 1967. Rapid staining procedures for differentiating riant virus inclusions in enidermal strips. Virology 31:268-271. 14. Christie, R. G. 1971. A rapid diagnostic technique for plant viruses. Proc. Pest Control Conf., Univ. Fla., Gainesville 5:65-68. 15. Christie, S. R. 1969. Nicotiana hybrid developed as a host for plant viruses. Plant Dis. Rep. 53:939-941. 16. Conover, C. A., R. T. Pool, J. F. Knauss, R. A. Hamlen, and R. '
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25. Harrison, 3. D., J. T. Finch, A. J. Gibbs, M. Hollings, R. J. Shepherd, V. Valenta, and C. Wetter. 1971. Sixteen groups of plant viruses. Virology 45:356-36326. Hartman, R. D. 1973. Pathogen-free caladiums obtained through shoot-meristem tip culture. Phytopathology 63: 442 (Abstr.). 27. Hartman, R. D. 1974. Dasheen mosaic virus and other phytopathogens eliminated from caladium, taro and cocoyam by culture of shoot-tips. Phytopathology 64: 237-240. 28. Hartman, R. D., and F. 7/. Zettler. 1972. Dasheen mosaic virus infections in commercial plantings of aroids in Florida. Phytopathology 62:804 (Abstr.). 29.' Hartman, R. D., and F. ff. Zettler. 1972. Mericloning as a potential means for obtaining virus free plants from aroids commercially produced in Florida. Proc. 4th Organic Soil Vegetable Crops (Horticulture) Workshop, Belle Glade, Fla., Feb. 22-24. 60-62. 30. Hartman, R. D., and F. '.V. Zettler. 1974. Effects of dasheen mosaic virus on yields of caladium, dieffenbachia and philodendron. Phytopathology 64: (in press). 31. Hartman, R. D., F. 7/. Zettler, J. F. Knauss, and Eleanor M. Hawkins. 1972. Seed propagation of caladium and dieffenbachia. Proc. Fla. State Hort. Soc. 85:404-409. 32. Haynes, P. H., C. 7/. Meister, L. D. Butler, and F. 7/. Zettler. 1974. The occurrence of dasheen mosaic virus in taro (Colocasia esculenta) in Fiji. Proc. 3rd Int. Symp. Trop. Root Crops, Ibadan, Nigeria (in press). 33. Hayward, 7/. 1950. Fancy-leaved caladiums. Plant Life 6:131-142. 34. Herold, F. 1967. Investigaciones sobre virus de plantas en Venezuela. Acta Cient. Venezolana Supl. 3:381-386. 35. Herold, F. 1967. Investigation of a virus disease of Anthurium andraeanum . Phytopathology 57:8 (Abstr.). 36. Hiebert, S., and J. G. McDonald. 1975. Characterization of some proteins associated with viruses in the potato Y group. Virology 56:349-361. 37. Hiebert, E., D. E. Purcifull, R. G. Christie, and S. R. Christie. 1971. Partial purification of inclusions induced by tobacco etch virus and potato virus Y. Virology 43:638-646.

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77 38 * *£*• JF. 1937. Economic Botany. 1st ed., McGrawHill Book Co., Inc. New York. 592 p. r.cbrawSrI Rep. ^f;. ^ ladlUm bulbs Highlands County h °' J V^i i^f 1 ' H \?% Kenten » a nd R. D. Woods. 1973. Virus-like particles associated with two diseases of ^fn^iflf^ 4 ^! Ch0tt ^ the ^^IklLs. 41. Kenten, R. H., and R. D. Wood«? 107? t» 42. Kenten, R. H., and R. D. '/foods. 1973. Viruses '"\£Hia and Xanthosoma News Summ. 19:38-41 *«w«« f a , n ., ana «. d. Woods. I973. Viru«?P» ancient root andTelf-vSVe-

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78 51. Nicolson, D. H. 1969. A revision of the genus Aglaonema (Araceae). Smithsonian Contributions Botany. No. 1. 69 P. 52. Nyland, 6., and A. C. Goheen. 1969. Heat therapy of virus diseases of -perennial plants. Ann. Rev. Phytopathol. 7:331-35^/ 53. Pettet, A., and S. J. Pettet. 1970. Biological control of Pistia stratiotes L. in '.Yes tern State, Nigeria. Nature 226:282. 54. Plucknett, D. L., R. S. De La Pena, and F. Obrero. 1970. Taro (Colocasia esculenta ). Field Crop Abstr. 23:*fl3426. 55. Purcifull, D. E. 1968. Disruption of watermelon mosaic virus induced inclusions by phosphotungstate. Virology 36:690-693. 56. Raychaudhuri, S. P., and B. Ganguly. 1965. Further studies on chirke disease of large cardamon ( Amomum subulatum Roxb.). Indian Phytopathol. 18:373-377. 57. Reinert, R. A., and M. J. Kasperbauer. 1966. Influence of red and far red light on virus content of callus tissues cultured from Nicotiana tabacum . Phytopathology 56:1108-1109. 58. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaoue stain in electron microscopy. J. Cell. Biol. 17:208-212. 59. Ridings, Y/. H., and R. D. Hartman. 1973. Pathogenicity of Pythium spp. to pathogen-free caladiums derived from shoo t-mer is terntip cultures. Abstr. No. 0112 in Abstracts of Papers. 2nd Int. Congr. Plant Pathol. 60. Ryohitsu, S. 1956. Intercrossing among pink calla, white spotted calla and yellow calla. Kasai Publishing and Printing Co. Tokyo. 57 p. 61. Saladini, J. L. , and F. Y/. Zettler. 1973. Resistance of St. Augustinegrass to infection by sugarcane mosaic virus. Phytopathology 63:162-166. 62. Shepard, J. F. , G. A. Secor, and D. E. Purcifull. 1974-. Immunochemical cross-reactivity between the dissociated capsid proteins of PVY group plant viruses. Virology (in press). 63. Simons, J. N., R. Swidler, and L. M. Moss. 1963 . Succulent-type plants as sources of plant virus inhibitors. Phytopathology 53:677-683.

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79 64. Stone, 0. M. 1973. T he elimination of viruses from Narcissus tazetta cv. Grand Soleil d'Or, and ravl° multxplxcation of virus-free clones. Inn^plfSiol. 65. Stoner, M. ?. 1964. A aosaic disease of Philodendron Belloum. Pnytopathology 54:1437 (Abstr.) . 66. Tompkins, C. M., and M. 7/. Gardner. 193^. Spotted wilt of head lettuce. Phytopathology 24:1135.Ill36. 67. Tompkins, C. M., and H. H. P. Severin. 1 95 Snottpd git of white, yellow, and pink calli,. ^Jg^, 68, 70 'sas&i. ^.^rts: Soif^fii^i: 71 • West, E., and H. T J. r T illp-r incr c ~ dend.on'hybrids/'Pr^^. 1 !^, fe^! g s g^ ?3 * Z ?oS er, A ? •''-•! R * G * C1 ^istie, and J. R. Edwardson correla&vit" a ? r S ? iSSi ?? ? f V±rUS ^°» leafsec?Srs 33^549-552. ^tracellular inclusions. Virology Z A^ir ? V' #9 M ' J ' Foxe ' and K0. Hartman. 1970 A mosaic disease of dash^en cavern ^ = -pTi f y/u virus. Phytopathology c5:^h! (ISstr.f. fllanentous Zettler, F. W., M. J. Foj:e B D «-_+_„ . „ .._. pathololf g^9W? ° ther araCe ° US «"»*•• ^ ? 76 z fgha!fcc?B\JS^&riE g,SS? 74 75.

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BIOGRAPHICAL SKETCH Robert Dale Hartman was born in Miami, Florida on March 31, 1%8. In June, I966 he was graduated from Hialeah High School located in Hialeah, Florida. In September, 1966 he entered Miami Dade Junior College where he was awarded an A. A. degree in June, I968. He subsequently entered the University of Florida where he received his B.S. degree in agriculture with majors in plant pathology and entomology and graduated with High Honors in June, 1970. He v/as supported in his upper division work by an Academic Scholarship and a National Defense Loan. He enrolled in the Graduate School of the University of Florida in the Department of Plant Pathology in January, 1971 and began work toward his Ph.D. degree in plant pathology with a minor in Ornamental Horticulture. His Ph.D. work was supported by a NDEA Title IV Fellowship and a departmental assistantship. He is a member of Phi Kappa Phi, Gamma Sigma Delta, The American Phytopathological Society and The Florida State Horticultural Society. He was married to the former Linda Diane Rockwell on October 25» 1969 and has a three year old daughter, Sandee Lynn. He is currently in the Army Reserve serving as a pay specialist with the rank of Sp. 5. His term of enlistment will terminate April, 1976. 80

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OHininn f V ^ "** thiS StUd ^ and that ^ my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doct^Jf Philosophy F. William Zettler, Chairman Associate Professor of Plant Pathology I certify that I have read this study and that in my op^on it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy 9 M ^L-^u^. $. R. Edwardson Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy £ E. Hiebert Assistant Professor of Plant Pathology on . .J ce ^ify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy J. F. Knauss Assistant Professor of Plant Pathology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. D. E. Purcifull Associate Professor of Plant Pathology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. 0.d&~. J2 T. J. Sheehan Professor of Ornamental Horticulture This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. June, 1974 Dean, College of Agriculture Dean, Graduate School