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Population dynamics of microorganisms associated with caladium seedpieces

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Title:
Population dynamics of microorganisms associated with caladium seedpieces
Added title page title:
Caladium seedpieces
Creator:
Ferriss, Richard S., 1948-
Copyright Date:
1979
Language:
English
Physical Description:
viii, 138 leaves : graphs ; 28 cm.

Subjects

Subjects / Keywords:
Bacteria ( jstor )
Corms ( jstor )
Fungi ( jstor )
Fusarium ( jstor )
Planting ( jstor )
Population dynamics ( jstor )
Population growth ( jstor )
Pythium ( jstor )
Soil science ( jstor )
Soils ( jstor )
Dissertations, Academic -- Plant Pathology -- UF
Plant Pathology thesis Ph. D
Plants -- Effect of fungicides on ( lcsh )
Soil fungi ( lcsh )
Soil microbiology ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 135-137.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Richard S. Ferriss.

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University of Florida
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University of Florida
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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.
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AAB7113 ( NOTIS )

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POPULATION DYNAMICS OF MICROORGANISMS
ASSOCIATED WITH CALADIUM SEEDPIECES











BY

RICHARD S. FERRISS



















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


1979

















ACKNOWLEDGMENTS


The people and institutions which contributed in some way

to the completion of this dissertation are many and varied.

I would like to thank my major professor, Dr. D. J. Mitchell,

for his suggestions, inspiration, properly conjugated infinitives,

and occasional obstinacy; the other members of my committee,

Dr. D. H. Hubbell, Dr. J. F. Knauss, Dr. D. A. Roberts, and

Dr. R. E. Stall, for their help in many aspects of my

training; Mr. George Richardson and his sons, Mark and Chet,

for their invaluable assistance with my field plots at Bear

Hollow Bulb Farm; the University of Florida for providing

financial support for my studies and a favorable atmosphere

for their completion; dinosaurs, dolphins, and my high school

chemistry teacher, Bob Grove, for nurturing an interest in

the living world during my childhood and adolescence; the

many other teachers I have had over the years for their

gifts of knowledge and sometimes wisdom; the many friends who

have made my stay in Gainesville one of growth and joy; my

mother for her worries and love through the years; and

lastly my daughter, Kate, for her love, enthusiasm and

confusing questions.

















TABLE OF CONTENTS


ACKNOWLEDGMENTS .............................................. ii

ABSTRACT ........................................ ...... .... v

PART 1. THE EFFECT OF FUNGICIDAL SEEDPIECE DUSTS ON THE
POPULATION DYNAMICS OF SOIL MICROORGANISMS
ASSOCIATED WITH GERMINATING OR DECOMPOSING
CALADIUM SEEDPIECES

Introduction ............................................ 1

Materials and Methods ................................... 3

Results .............................................. ... 7

Discussion ........................................ ..... 20

PART 2. THE EFFECT OF SEEDPIECE TREATMENT WITH CAPTAIN
ON THE POPULATION DYNAMICS OF SOIL MICROORGANISMS
ASSOCIATED WITH GERMINATING OR DECOMPOSING
CALADIUM SEEDPIECES IN THE FIELD

Introduction ............................................ 25

Materials and Methods ................................... 26

Results ................................................. 29

Discussion .............................................. 38

PART 3. GROWTH, YIELD, AND EMERGENCE OF CALADIUMS IN
RELATION TO SEEDPIECE WEIGHT

Introduction ......................................... ... 41

Materials and Methods ................................... 42

Results ................................................. 45

Discussion ................ ............................ 54

















APPENDICES

1. REPETITION OF EXPERIMENTS PRESENTED IN PART 1 ............ 56

2. PHYSICAL DISTRIBUTION OF MICROBIAL
POPULATIONS INCREASING ON CALADIUM SEEDPIECES ........ 71

3. THE EFFECT OF FUNGICIDAL SEEDPIECE DUSTS ON
GROWTH, YIELD, EMERGENCE, AND VALUE
OF CALADIUMS ......................................... 87

4. GROWTH OF CALADIUMS IN SOIL INFESTED WITH
FUNGI OBSERVED TO INCREASE POPULATIONS
ON CALADIUM SEEDPIECES .............................. 100

5. TOLERANCE OF FUSARIUM SPP. AND
LASIODIPLODIA SP. TO BENOMYL ......................... 114

LITERATURE CITED ............................................ 135

BIOGRAPHICAL SKETCH .......................................... 138

















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


POPULATION DYNAMICS OF MICROORGANISMS
ASSOCIATED WITH CALADIUM SEEDPIECES

by

Richard S. Ferriss

March, 1979

Chairman: David J. Mitchell
Major Department: Plant Pathology

The short-term population dynamics of fungi and

bacteria associated with caladium (Caladium hortulanum)

seedpieces planted in raw muck soil were investigated.

Sized seedpieces of cultivar Frieda Hemple were planted in

flats of soil and incubated in a growth room at 25-30 C,

or were planted in two areas of a commercial caladium

field with contrasting crop histories. Populations of

Pythium spp., Fusarium spp., other genera of fungi, total

bacteria and fluorescent Pseudomonas spp. were assayed at

0, 2, 4, 8, and 12 weeks after seedpieces were planted.

A suspension of a soil core that contained a seedpiece

and surrounding soil was comminuted in a Waring blender,

and appropriate dilutions were plated on selective media.

The effects of viability of seedpieces (presence or

absence of eyes and epidermis), the use of fungicidal











seedpiece dusts, and planting area in the field on the

population dynamics of the assayed organisms were investigated.

Addition of seedpieces to soil resulted in increase and

subsequent decrease in the populations of several organisms.

For each organism, or group of organisms, the magnitude

of the maximum population was related to seedpiece condition

or treatment. However, regardless of seedpiece condition or

treatment, population maxima occurred in the following

approximate order: fluorescent Pseudomonas spp., Pythium

spp., total bacteria, Mucorales, Fusarium spp., Trichoderma

spp., Verticillium spp., Penicillium spp., and Lasiodiplodia

sp. In general, organisms reached higher populations on

seedpieces which had eyes and epidermis shaved off (shaved)

than on corresponding viable seedpieces (eyed).

Fungicidal dusts used in the experiments were captain; a

mixture of benomyl, thiram, and streptomycin sulfate; and a

mixture of chloroneb, thiram, and streptomycin sulfate.

Effects of the fungicidal dusts on microbial populations

associated with seedpieces were different from effects

observed following incorporation of the fungicidal dusts,

without seedpieces, into soil. Treatment of seedpieces with

any of the fungicidal dusts reduced increases in populations

of fungi on eyed seedpieces, delayed increases in populations

of fungi on shaved seedpieces, and enhanced the magnitude

or duration of increases in populations of both fluorescent

Pseudomonas spp. and total bacteria. The magnitudes of

population increases by different genera of fungi were











greatly altered by the fungicidal dusts; however, the

fungicidal dusts did not foster increases by any fungi

not observed to increase on shaved seedpieces which were

not fungicide-treated. With any particular dust, the fungal

community which increased on eyed seedpieces resembled the

community on shaved seedpieces treated with the same dust

more than it resembled communities on eyed seedpieces

treated with other dusts. In one experiment populations

of Pythium spp. were greater on eyed seedpieces treated

with the dust which included benomyl than on eyed

seedpieces which were not fungicide-treated. Populations of

Pythium spp. increased only slightly on seedpieces treated

with the dusts that included captain or chloroneb. In all

experiments populations of Fusarium spp. were similar on

seedpieces treated with the dust that included chloroneb

and on seedpieces which were not fungicide-treated.

Populations of Fusarium spp. increased only slightly on

eyed seedpieces treated with dusts that included captain or

benomyl, but increased appreciably on shaved seedpieces

treated with these dusts. Increases in populations of

Fusarium spp. on seedpieces treated with the dust that

included benomyl were correlated with the abundance of

benomyl-tolerant isolates.

In the field experiments overall patterns of increase

were similar to those observed in the growth room. Patterns

observed in the field, but not in the growth room, were


vii











an absence of Pythium spp. in one area of the field, a

reduction of increases in populations of Pythium spp. and

Lasiodiolodia sp., and greater involvement of Penicillium

spp. in the succession.


viii
















PART 1


THE EFFECT OF FUNGICIDAL SEEDPIECE DUSTS
ON THE POPULATION DYNAMICS OF SOIL MICROORGANISMS
ASSOCIATED WITH GERMINATING OR DECOMPOSING
CALADIUM SEEDPIECES


Introduction

Treatment of seeds and seedpieces is an important mode of

application of fungicides intended to control soilborne plant

pathogens (10, 16). In spite of the economic importance of

these treatments, information on the ecological operation of

seed treatment fungicides is meager. The effects of fungicides

on populations of soil microorganisms when the fungicide is

incorporated evenly into soil, without the addition of a sub-

strate, have received a good deal of attention (1, 8, 24, 34);

the effects of fungicides on microbial growth associated with

the addition of purified substrates have received a limited

amount of investigation (2). These investigations have not di-

rectly addressed the effects of seed treatment fungicides,

however. Fungicides may alter the generic composition of commu-

nities of fungi which increase populations on substrates in

soil (9), and the persistence of some fungicides is increased

greatly when they are incorporated into soil at high concen-

trations (12, 20). Thus, the presence of a complex substrate

in the form of a seed or seedpiece and the high concentration

of a seed treatment fungicide around a seed or seedpiece










indicate that the effects of fungicides on soil microorganisms

when they are used as seed dressings are not comparable to

effects observed with even incorporation into soil. An under-

standing of the biological effects of seed treatment fungicides

in normal agricultural practice must come from investigations

of effects on communities of microorganisms that are actually

utilizing a seed or seedpiece as a substrate. A model system

appropriate for such research should include a relatively large

seed or seedpiece, a number of available treatment materials,

a method of non-chemically minimizing the introduction of orga-

nisms with the seed or seedpiece, and methods of quantitatively

evaluating the activities of different groups of organisms in

a relatively natural situation.

Caladiums (Caladium hortulanum [Birdsey ) are ornamental

aroids that are grown for corms as a field crop in south-

central Florida. Preparation of propagative material involves

heat treating the corms for control of root-knot nematode, cut-

ting corms into seedpieces with a cutting machine, and dusting

seedpieces with an absorptive dust which may contain fungicides.

Mechanical cutting yields seedpieces that are fairly evenly

distributed in weight between 0.2 and 5.0 g, and emergence and

yield are closely correlated with seedpiece weight (Part 3 of

this dissertation). The low emergence from small seedpieces re-

sults in as little as 25 % of the total seedpiece weight

planted by a grower actually yielding plants. Caladiums are

usually planted by dribbling seedpieces into a furrow, which

places non-germinating seedpieces adjacent to germinating










seedpieces. As soilborne pathogens of caladiums, such as

Pythium myriotylum (26) and Fusarium solani (15), belong to

genera which contain plant-pathogenic members which have been

reported to increase saprophytically on fresh plant material

added to soil (6, 18), this non-germinating seedpiece material

could foster increases in populations of pathogens resulting

in increased disease on germinating plants (3, 33).

This investigation was undertaken to (i) evaluate the

effects of seedpiece treatment fungicides on a community of

microorganisms in a situation similar to that encountered in

normal agricultural practice, (ii) determine whether non-germi-

nating seedpiece material can serve as a substrate for caladium

pathogens in soil, and (iii) determine whether commonly-used

fungicidal seedpiece dusts affect the pathogen-substrate

relationship on non-germinating seedpieces.


Materials and Methods

Palmico muck soil was collected from an area of a commer-

cial caladium field where stunting of plants had been associ-

ated with high populations of Pythium spp. and Fusarium spp.

Analysis of the soil by the University of Florida Soil Science

Department indicated nutrient contents of 840 pg NO3, 104 pg P,

80 pg K, 4720 pg Ca, 488 pg Mg, 376 pg Al, 2.72 pg Cu, and

45.6 pg Fe per gram of soil (dry weight). Soil pH was 4.8

(measurement obtained from a 1:2 suspension of soil in 0.01 M

CaC12). Soil was sifted through a 4-mm sieve and mixed 20 min

in a small cement mixer prior to use in the experiment.










Caladium corms of the cultivar Frieda Hemple were obtained

from a commercial grower. Corms were heat treated in deionized

water at 50 C for 30 min (23). Treated corms were cut into

2.8-3.2 g seedpieces that either had at least one eye present

and were intended to germinate (eyed), or had all eyes and

epidermis shaved off and were intended to decompose (shaved).

Seedpieces were dusted with one of the four following dusts

currently used by growers: diatomaceous earth (Celite 209,

Johns-Manville, Celite Division, Greenwood Plaza, Denver CO

80217); 6.9 % chloroneb, 6.9 % thiram, and 0.6 % streptomycin

sulfate in diatomaceous earth (chloroneb mixture); 3.8 % benomyl,

7.3 % thiram, and 0.6 % streptomycin sulfate in diatomaceous

earth benomyll mixture); and 10 % captain in diatomaceous

earth captain) .

Treated seedpieces were planted in aluminum flats (45 X 28 X

5 cm) which contained 2.5 kg of soil at 42 % water content

(100 X weight of water/ wet weight of soil) and had been

coated with epoxy resin (Resinglas Polyester Resin, Kristal

Kraft Inc., 900 Fourth Street, Palmetto FL 33561). Other flats

were prepared which contained soil without seedpieces (non-amended

soil) or soil into which 0.47 % of one of the four seedpiece

dusts (100 X weight dust/ dry weight of soil) had been incor-

porated evenly. This concentration of seedpiece dust was equal

to the average amount of dust adhering to seedpieces, divided

by the average amount of soil in a sampling core, as described

below. Flats were incubated in a growth room at 25-30 C with

12 hr light cycles (4,000 lx at the level of the plants).










Every two days flats were watered to 53 % water content with

deionized water and weed seedlings were pulled from the soil.

Microbial populations in and around seedpieces were sampled

at 0, 2, 4, 8, and 12 weeks after seedpieces were planted. A

5.6-cm-diameter piece of polyvinylchloride pipe was centered on

the position of a seedpiece, plant, or soil sampling area, and

a core which contained 32.0 1.7 g of soil (dry weight) was

removed. Leaves of plants were cut off at the soil line and

discarded. The soil core was comminuted with 100 ml of auto-

claved, deionized water at low speed in a Waring blender for

1 min, and a diliution series in autoclaved water was prepared

from the initial suspension. Appropriate dilutions were plated

on the following five media for enumeration of microbial pop-

ulations: for Pythium spp., 17 g Difco cornmeal agar, 300 mg

vancomycin (Vancocin, Eli Lilly and Co., Indianapolis IN 46206),

100 mg pentachloronitrobenzene, and 5 mg pimaricin (Delvocid,

Gist-Brocades, Delft, Holland) in 1 liter of water (19); for

Fusarium spp., modified PCNB medium (21); for other fungi,

39 g Difco potato dextrose agar, I ml Turgitol NPX (Union

Carbide Corp., New York NY 10017), 100 mg streptomycin sulfate

(Eli Lilly and Co.), and 40 mg chlortetracycline HC1 (Sigma

Chemical Co., St. Louis MO 63178) in 1 liter water (31); for

bacteria and actinomycetes, 0.3 % tryptic-soy agar (17); and

for fluorescent Pseudomonas spp., King's medium B amended with

cycloheximide, novobiocin, and penicillin (28). For enumeration

of populations of Pythium spp., samples were suspended in

0.3 % agar amended with 3.68 g CaC12*2H20 per liter and then

spread over the surface of solidified medium. For enumeration










of populations of Fusarium spp., samples were suspended in

0.1 % agar amended with 100 mg streptomycin sulfate and 40 mg

chlortetracycline HC1 per liter and then spread over the

surface of solidified medium. For enumeration of populations of

other organisms, cooled medium was mixed with the sample in

the petri dish. Populations of plant-parasitic and free-living

nematodes were assayed in the 12- week samples by the Univer-

sity of Florida Entomology and Nematology Department.

The experiment was performed twice as described herein and

two other times with procedural modifications. In this part of

this dissertation, results are presented for populations of

organisms enumerated in one performance. Results of the other

identical performance are presented in Appendix 1. In all

performances populations of microorganisms were calculated as

the mean of data from three replicate samples. Most population

changes were similar in all performances of the experiment;

however, some differences were observed and are noted in the

results section.











Results

Initial populations of fungi and bacteria in soil are pre-

sented in Table 1. Actinomycetes were recovered only sporadically

after the 0-week sampling in treatments which contained seed-

pieces or plants.

Incorporation of the fungicidal dusts into soil resulted in

relative increases in populations of bacteria and relative de-

creases in populations of total fungi and Fusarium spp., com-

pared with populations in non-amended soil (Fig. i). Incorpora-

tion of diatomaceous earth into soil did not significantly

alter any assayed populations compared with non-amended soil.

The only appreciable differences between the effects of the

fungicidal dusts were a delay in the decrease of populations

of Fusarium spp. with captain and a decrease in the magnitude

of changes in populations of total fungi and bacteria with the

chloroneb mixture. Populations of Pythium spp. were not altered

significantly by any of the dusts when a seedpiece was not

present. Populations of fluorescent Pseudomonas spp. were in-

creased slightly, but nonsignificantly, in soil into which the

fungicidal dusts had been incorporated.

All eyed seedpieces that were assayed at or after the

4-week sampling had emerged and produced leaves. No shaved

seedpieces were observed to produce plants throughout the

course of the experiment.

The effects of the fungicidal dusts on populations of micro-

organisms which were increasing on seedpieces were not related

to changes in populations which followed incorporation of the

fungicidal dusts into soil without the addition of seedpieces.











Populations of Pythium spp. on seedpieces increased to max-

ma at 2 to 4 weeks and then gradually declined (Fig. 2-A,

2-B). Maximum populations were slightly higher on shaved than

eyed seedpieces and were reduced greatly on seedpieces treated

with any of the fungicidal dusts, although populations were

consistently highest on seedpieces dusted with the benomyl mix-

ture. In another repetition of the experiment, populations of

Pythium spp. were higher on eyed seedpieces dusted with the

benomyl mixture than on eyed seedpieces dusted with diatom-

aceous earth. Enumerated populations of Pythium spp. were

predominantly P. spinosum and P. irregulare.

Populations of Fusarium spp. increased more rapidly and

attained higher maxima on shaved than on eyed seedpieces (Fig.

2-C, 2-D). Initial rates of population increase were less, but

maximum populations were similar on seedpieces dusted with the

chloroneb mixture, compared with those dusted with diatomaceous

earth. Populations of Fusarium spp. increased only slightly on

eyed seedpieces dusted with the benomyl mixture or captain. On

shaved seedpieces dusted with the benomyl mixture or captain,

populations increased significantly over those present in non-

amended soil, but were considerably less than populations on

shaved seedpieces dusted with diatomaceous earth. Isolates of

Fusarium spp. from samples which contained shaved seedpieces

dusted with captain, the chloroneb mixture, or diatomaceous earth

were inhibited similarly by benomyl, captain, or thiram in corn-

meal agar; however, isolates from samples which contained seed-

pieces dusted with the benomyl mixture displayed a greater











tolerance to benomyl than isolates from samples which contained

seedpieces treated with the other dusts or non-amended soil.

Investigations of this tolerance are presented in Appendix 5 of

this dissertation. Enumerated populations of Fusarium spp. were

predominantly F. solani with some F. oxysporum and F. roseum.

Fungi other than Pythium spp. and Fusarium spp. increased

to higher populations on shaved than on eyed seedpieces (Fig.

3). Compared with seedpieces dusted with diatomaceous earth,

maximum total populations were decreased on fungicide-treated,

eyed seedpieces and were delayed, but increased in magnitude,

on fungicide-treated, shaved seedpieces. Fungicidal dusts altered

the generic makeup of fungal communities which increased on

seedpieces but did not foster increases by any fungi that

did not increase on shaved seedpieces dusted with diatomaceous

earth (Table 2).

A pathogenicity test of fungi observed to increase popula-

tions on caladium seedpieces indicated that none of the fungi

significantly affected the growth of caladiums when compared to

control treatments (Table 3). However, growth of caladiums was

significantly greater in soil infested with Trichoderma harzianum,

Lasiodiplodia sp., or Verticillium sp. than in soil infested

with Fusarium solani. A more detailed description of this test

is presented in Appendix 4 of this dissertation.

Population increases of total bacteria were greater on

shaved than on eyed seedpieces (Fig. 2-E, 2-F). The fungicidal

dusts did not greatly alter maximum populations on eyed










seedpieces but extended the duration of high populations on

shaved seedpieces.

Populations of fluorescent Pseudomonas spp. increased to

maxima at 2 weeks and then rapidly declined (Fig. 2-G, 2-H).

Maximum populations were greater on shaved than eyed seedpieces,

but in another repetition maxima were greater on eyed than on

shaved seedpieces. In all repetitions 12-week populations were

higher on eyed than on shaved seedpieces. Populations of

fluorescent Pseudomonas spp. were much higher on both eyed

and shaved seedpieces dusted with any of the fungicidal dusts

than on corresponding seedpieces dusted with diatomaceous earth.

Enumerated populations of fluorescent Pseudomonas spp. were

predominantly non-pectolytic isolates of P. fluorescens and

P. putida.

Populations of plant-parasitic nematodes were low and not

significantly different in samples which contained seedpieces

compared with samples which contained non-amended soil. Popula-

tions of free-living nematodes were significantly higher (p=

0.05) by Duncan's multiple range test (30) on shaved seedpieces

dusted with captain or the chloroneb mixture than in non-

amended soil (Table 4).










Table 1. Initial populations of fungi and bacteria in soil




Organism Propagules/g soil


Pythium spp. 3.4 1.3 X 102

Fusarium spp. 2.0 0.2 X 104

Verticillium sp. 4.4 4.6 X 103

Lasiodiplodia sp. 2.2 1.2 X 104

Trichoderma spp. 8.3 2.0 X 104

Penicillium spp. 1.8 0.5 X 105

Total fungi 4.5 0.8 X 105

Fluorescent Pseudomonas spp. 4.8 3.9 X 103

Actinomycetes 7.9 + 2.1 X 106

Bacteria 8.7 1.5 X 107



population standard deviation of the mean. Values are the mean
of 27 samples.










Table 2. Predominant fungi
experiments


increasing on seedpieces in two


Treatment Experiment 1 Experiment 2a


Eyed-benomyl mixture Py L

Eyed-captan T T,V

Eyed-chloroneb mixture F,T F,T

Eyed-diatomaceous earth F,L,Py,T F,L,Py,T

Shaved-benomyl mixture F,L F,L

Shaved-captan F,L,P,T F,L,V

Shaved-chloroneb mixture F,L F,L,V

Shaved-diatomaceous earth F,L,P,Py,T F,L,Py,T,V



aFigures 2 and 3 are for data from experiment 2.

bF=Fusarium spp., L=Lasiodiplodia sp., P=Penicillium spp., Py=Pythium
spp., T=Trichoderma spp., V=Verticillium sp.










Table 3. Effect of planting seedpieces in soil infested with cornmeal-
sand cultures of fungi on growth and emergence of caladium plants




Incorporated % emergence Days to Mean total
organism or amendment at harvest emergence dry weight (g)


Trichoderma harzianum 94 wb 31.0 w 1.72 w

Lasiodiplodia sp. 75 w 28.7 w 1.70 wx

Verticillium sp. 75 w 34.0 w 1.95 w

Pythium irregulare 75 w 33.3 w 1.41 wxy

P. aphanidermatum 75 w 28.7 w 1.57 wxy

Cornmeal-sand medium 88 w 31.7 w 1.16 wxy

Penicillium sp. 69 w 32.8 w 1.27 wxy

Non-amended soil 75 w 30.8 w 1.24 wxy

Pythium spinosum 75 w 35.5 w 0.98 xy

Fusarium solani 75 w 35.1 w 0.71 y


aMean number of days to emergence for harvested plants.

bWithin a column, values followed by the same letter are not
significantly different (p=0.05) by Duncan's multiple range test.










Table 4. Populations of free-living nematodes at 12 weeks after
planting of seedpieces




Treatment Nematodes/g soila


Eyed-benomyl mixture 0.4 wb

Eyed-captan 2.9 wx

Eyed-chloroneb mixture 3.2 wx

Eyed-diatomaceous earth 3.5 wxy

Shaved-benomyl mixture 3.7 wxyz

Shaved-captan 19.5 z

Shaved-chloroneb mixture 17.2 yz

Shaved-diatomaceous earth 9.5 xyz

Soil 1.9 wx



aValues are the mean of three samples.

bMeans followed by the same letter are not significantly different
(p=0.05) by Duncan's multiple range test performed on a square root
+ 2 transformation of data.











A









0 4
0 2 4
WEEKS AFTER MIXING
4
C










o i 4 8 -
0 2 4 8


t D

fs






0
2 0 2 4
WEEKS AFTER


8MI
MIXING


0 2 4 8 12
WEEKS AFTER MIXING

Fig. 1-(A to E). The effects of the incorporation of fungicidal
dusts into soil on the populations of A) total fungi, B) total
bacteria, C) Fusarium spp., D) Pythium spp., and E) fluorescent
Pseudomonas spp. Populations of microorganisms were assayed in
non-amended soil (-- ) and in soil into which the benomyl
mixture (- .--), captain (- ..........), or the chloroneb mixture
(---) had been incorporated. Assays were performed at 0, 2,
4, 8, and 12 weeks after incorporation of the dusts by plating
dilutions on selective media. Each point represents the mean of
three replicates.


WEEKS AFTER MIXING










Fig. 2-(A to H). The effects of treatment of caladium seedpieces
with fungicidal dusts on the population dynamics of Pythium spp. on
A) eyed seedpieces and B) shaved seedpieces, Fusarium spp. on C)
eyed seedpieces and D) shaved seedpieces, total bacteria on E) eyed
seedpieces and F) shaved seedpieces, and fluorescent Pseudomonas spp.
on G) eyed seedpieces and H) shaved seedpieces. Populations of
microorganisms were assayed in non-amended soil (------) and in soil
into which seedpieces dusted with diatomaceous earth (- ), the
benomyl mixture (-..-), captain (......) or the chloroneb mixture
(---) had been planted. Assays were performed at 0, 2, 4, 8,
and 12 weeks after seedpieces were planted by plating dilutions
on selective media. Each point represents the mean of three
replicates.












- A



16

12

.. / '--*------



0 2 4 a
WEEKS AFTER PLANTING
8
C
7
6

5
4

3 -
2



0 2 4 B
WEEKS AFTER PLANTING


4 8
WEEKS AFTER PLANTING


12 0 2 4


WEEKS AFTER PLANTING


14 B

!0

16

12



0 --------


0 2 4 8


0 2 4 8 12
WEEKS AFTER PLANTING

2 G

10

B

6

4

2

( 1 -- ---- "li


0 2


WEEKS AFTER PLANTING


/ \







I-- \, ..-


8


n-










Fig. 3-(A to H). The population dynamics of genera of fungi,
other than Pythium, which had increased populations on eyed
caladium seedpieces dusted with A) diatomaceous earth, C) the
benomyl mixture, E) captain, or G) the chloroneb mixture; and on
shaved caladium seedpieces dusted with B) diatomaceous earth,
D) the benonyl mixture, F) captain, or H) the chloroneb mixture.
Populations of total fungi (-- ), Trichoderma spp. (-----),
Lasiodiplodia sp. (---), Verticillium sp. (-----), and
Fusarium spp. ( ........ ) were assayed in soil samples which each
contained a seedpiece. Assays were performed at 0, 2, 4, 8, and
12 weeks after seedpieces were planted by plating dilutions on
selective media. Each point represents the mean of three
replicates.




























20



0

o10
w




0






15

*
w 10
0


















20






(A
S10
0
0
o


J20


_15
x
-J










0o
0


B
7











0 2 4 8
WEEKS AFTER PLANTING

D
v* ----2




/ D




2 4 8 12
WEEKS AFTER PLANTING


12 0 2 4 8
WEEKS AFTER PLANTING


4 8
WEEKS AFTER PLANTING


2 4 8
WEEKS AFTER PLANTING

C










2 4 B----


8NTIN
PLANTING


2 4 8
WEEKS AFTER PLANTING


2 4 8
WEEKS AFTER PLANTING


--


2 4


F









\__^ '











Discussion

The lack of similarity between the effects of the

fungicidal dusts on microbial populations when the dusts

were incorporated evenly into soil, compared with when they

were used as seedpiece dressings, is indicative of differences

in the processes operating in the two situations. Effects

observed after incorporation of a fungicide into soil without

the addition of a substrate can be interpreted as a killing

of sensitive propagules with concomitant increases of non-

sensitive organisms on the nutrients released (34). This is

illustrated by the relative effects of the three fungicidal

dusts in this experiment. The chloroneb mixture caused less

decrease in fungal populations than did captain or the benomyl

mixture and thus a smaller amount of nutrients was released

for increase of bacteria (Fig. i). When a fungicide is

used as a seedpiece dressing, effects are on actively

growing, interacting populations rather than on non-interacting

resting propagules. Both modification of microbial succession

on seedpieces and initiation of displacement of fungicide-

sensitive organisms by fungicide-tolerant organisms in soil

involve the competitive displacement of some organisms by

others; however, the different natures of the substrates

involved (fresh plant material versus dead microorganisms)

foster the existence of different microbial communities

and thus limit the comparability of the two processes.

This difference is illustrated by the observed behavior

of populations of Pythium spp. and fluorescent Pseudomonas











spp. in the experiment. The fungicidal dusts had little or

no effect on these groups of organisms in soil without

seedpieces, but populations of both were altered significantly

on fungicide-treated seedpieces. These observations emphasize

the necessity of evaluating the biological effects of

pesticides in soil on both populations of resting propagules

and on actively increasing populations.

Although initial populations of microorganisms were

similar to those reported in the literature (7, 17, 18,

21, 27), populations in samples which contained seedpieces

were increased greatly. This association of high populations

with caladium seedpieces is indicative of the niche of the

assayed organisms. The soil used in the experiment was from

a well weeded caladium field; consequently, the pre-

dominant microorganisms were those able to increase pop-

ulations on caladium tissue. This association of high

populations with substrates is probably a major source of

variation in the determination of microbial populations in

randomly-collected soil samples.

Succession on eyed seedpieces was qualitatively similar

to succession on shaved seedpieces. In general, increases

in populations of bacteria, Pythium spp., and Fusarium spp.

were followed by increases in populations of saprophytic

fungi. This placement of potentially parasitic fungi early

in the succession is consistent with previous observations

(11, 14). Quantitatively, almost all organisms reached higher











populations on shaved than eyed seedpieces. This is con-

sistent with consideration that the entire amount of

substrate in a shaved, decomposing seedpiece is available

for the support of microbial growth, while with a germinating

seedpiece, the substrate is partitioned between microorganisms

and the developing plant. Correspondingly, reduced increases

in populations of fungi on eyed seedpieces treated with the

fungicidal dusts, compared with eyed seedpieces dusted with

diatomaceous earth, can be viewed as resulting in a greater

amount of substrate being partitioned to the plant. As

growth of caladiums is proportional to seedpiece weight

(Part 3 of this dissertation), increases in plant growth

with the use of the fungicidal dusts could be attributed

to this increased utilization of seedpiece nutrients by the

plant.

The three fungicidal dusts had different effects on

the two pathogen-containing genera of fungi that were assayed.

On eyed seedpieces, captain and the benomyl mixture gave

good control of Fusarium spp. while captain and the chloroneb

mixture gave good control of Pythium spp. On shaved seed-

pieces the relative activities of the fungicidal dusts

against Pythium spp. and Fusarium spp. were similar to

those on eyed seedpieces. However, all fungicidal dusts

allowed some increase in populations of Pythium spp. and

considerable increase in populations of Fusarium spp. on

shaved seedpieces. This increase on decomposing seedpiece

material, regardless of fungicidal seedpiece treatment,











indicates that planting of small, non-germinating seed-

pieces adjacent to germinating seedpieces may have sig-

nificant epidemiological consequences. Populations of

pathogens could increase on non-germinating seedpiece

material and cause an increased amount of disease because

of higher inoculum density.

Although the fungicidal dusts did not increase total

populations of bacteria on eyed seedpieces, they did

reduce total fungal populations and thus increased the

ratio of bacteria to fungi. As fungicidal dusts increased

the duration of increases in total bacterial populations

on shaved seedpieces, the results of the experiment seem

to indicate a shift in seedpiece colonization away from

fungi and toward bacteria with the use of the fungicidal

dusts.

Increases in populations of fluorescent Pseudomonas spp.

on seedpieces treated with any of the fungicidal dusts,

compared with diatomaceous earth, are of interest in relation

to work on the utilization of these organisms as biological

seed and seedpiece treatments (4, 5). The importance of these

increases in fungicide-related plant growth enhancement is open

to conjecture. It would be of interest to evaluate the

effects of inoculation of caladium seedpieces with fluor-

escent Pseudomonas spp. on plant growth, on the population

dynamics of fluorescent Pseudomonas spp., and on the microbial

succession in general.











The increase in populations of free-living nematodes

on shaved seedpieces dusted with captain or the chloroneb

mixture may indicate either that these dusts reduced com-

petition by fungi with the nematodes or that the dusts

reduced the effects of predacious fungi on the nematodes.

The experimental system used in this study allowed the

discrimination of relatively small differences between treat-

ments in natural soil with a minimal amount of replication.

The system, or one similar to it in which populations of

microorganisms in and around a substrate are assayed over

time, could be used in investigations of a number of

aspects of microbial succession in soil. Mixtures of chemical

and/or biological seedpiece treatment materials could be easily

evaluated for effects on specific target organisms in a

relatively natural situation. In particular, it would be of

interest to determine the effect of initial inoculum density

on the timing and extent of increases by organisms in the

succession. Overall, the system provides the opportunity for

study of a biological succession with a minimal expenditure

of time and materials.

















PART 2


THE EFFECT OF SEEDPIECE TREATMENT WITH CAPTAIN
ON THE POPULATION DYNAMICS OF SOIL MICROORGANISMS
ASSOCIATED WITH GERMINATING OR DECOMPOSING
CALADIUM SEEDPIECES IN THE FIELD


Introduction

Although there has been a good deal of research into

the effects of fungicides on microorganisms in soil under

controlled conditions, attempts to follow the effects of

fungicides on microbial populations in soil under field

conditions have been much less common (13). Since the

behavior of microorganisms may be very different in the

field and under controlled conditions, it is important

that field confirmation be obtained for observations of

chemical influences on microorganisms made under controlled

conditions. In Part 1 of this dissertation the effects of

fungicidal seedpiece dusts on caladium seedpiece succession

were investigated under controlled moisture and temperature

conditions. The research presented in this part of the

dissertation was undertaken to observe the effects

of the fungicidal dusts used in the growth room experiments

on microbial succession on caladium seedpieces in two

contrasting areas of a commercial caladium field.










Materials and Methods

Two locations in a commercial caladium field that had

contrasting crop histories were selected. Location 1 had

supported a cover crop of Japanese barnyard millet

(Echinochloa crugalli var. frumentacea) the previous growing

season and crops of caladiums for 5 years previous to that.

Mid-season stunting of plants associated with high populations

of Pythium spp. and Fusarium spp. and high soil water

content had been observed the last two seasons of caladium

culture. Soil used in the growth room experiments was

obtained from location 1. Location 2 had supported con-

tinuous summer caladium culture and winter weed fallow for

at least the previous 8 years. The grower considered caladium

growth to be above average at location 2.

In each location the experimental plot was prepared by

removing soil from a 1.25 X 2.70 m area to a depth of

15 cm, sifting the soil through an 11 mm screen, and

returning the soil to the excavated area.

Caladium corms of the cultivar Frieda Hemple were obtained

from a commercial grower. Corms were heat treated in de-

ionized water at 50 C for 30 min (23). Treated corms

were cut into 2.8-3.2 g seedpieces that either contained

at least one eye and were intended to germinate (eyed) or

had all eyes and epidermis shaved off and were intended to

decompose (shaved). Seedpieces were dusted with either

diatomaceous earth or 10 % captain in diatomaceous earth.











Treated seedpieces were planted in the experimental plots

on April 21, 1978, during the grower's normal planting

period. Each plot was laid out in a randomized block

design. Each of 17 rows contained one seedpiece in each

of the four seedpiece treatments and an area where no seed-

piece was planted. Seedpieces were planted 4-5 cm deep.

The position of the seedpieces was marked by points of

intersection of nylon string connected to stakes driven

into the ground around the periphery of the plot. Each

plot received 40 liters of water immediately after the

seedpieces were planted and water as provided by the

grower's normal irrigation schedule. Plots were weeded by

hand just before each sampling.

Samples for enumeration of microbial populations in and

around seedpieces were collected from three rows in each

plot at 1 day and 2, 4, 8, and 12 weeks after the

seedpieces were planted. A 5.6-cm diameter piece of poly-

vinylchloride pipe was centered on the position of a

seedpiece, plant, or soil sampling area and a core taken to

a depth of 8 cm. Samples of 64.2 6.8 g of soil (dry

weight) per core were transported to the laboratory in plastic

bags. One day after collection of samples, leaves were

removed from plants and each sample was comminuted with

220 ml of boiled, deionized water in a Waring blender at

low speed for 1 min. A dilution series in autoclaved

water was prepared from the initial suspension. Appropriate

dilutions were plated on the following five selective media










for enumeration of microbial populations: for Pythium spp.,

17 g Difco cornmeal agar, 300 mg vancomycin, 100 mg

pentachloronitrobenzene, and 5 mg pimericin in 1 liter of

water (19); for Fusarium spp., modified PCNB medium (27);

for other fungi, 39 g Difco potato dextrose agar, 1 ml

Turgitol NPX, 100 mg streptomycin sulfate, and 40 mg

chlortetracycline HC1 in 1 liter of water (31); for

bacteria and actinomycetes, 0.3 % tryptic-soy agar (17);

and for fluorescent Pseudomonas spp., King's medium B

amended with cycloheximide, novobiocin, and penicillin (28).

For enumeration of populations of Pythium spp., samples were

suspended in 0.3 % agar amended with 3.68 g CaCl22H20 per

liter and then spread over the surface of solidified medium.

For enumeration of populations of Fusarium spp., samples

were suspended in 0.1 % agar amended with 100 mg strep-

tomycin sulfate and 40 mg chlortetracycline HC1 per liter

and then spread over the surface of solidified medium. For

enumeration of other organisms, cooled medium was mixed with

the sample in the petri dish.











Results

Initial populations of enumerated organisms in non-amended

soil are presented in Table 5. Populations at location 1

were similar to those in the soil used in the growth

room experiments. Populations of all enumerated organisms

except one morphologically identifiable Penicillium biotype

(herein referred to as Penicillium A) and Lasiodiolodia sp.

were lower at location 2 than at location i. Pythium spp.

were not recovered from location 2 at any sampling.

At location 1 Pythium spp. increased to highest

maximum populations on shaved seedpieces dusted with diatom-

aceous earth (Fig. 4-E). Compared with diatomaceous earth,

populations on both eyed and shaved seedpieces treated with

captain were lower at all sampling times except the 12-week

sampling, when average populations of Fythium spp. were

slightly higher on eyed seedpieces dusted with captain than

on either eyed seedpieces dusted with diatomaceous earth or

shaved seedpieces dusted with captain. Populations of Pythium

spp. were lower on captan-dusted seedpieces than in non-

amended soil at the 2-week and 4-week samplings.

Populations of Fusarium spp. attained higher maxima at

location 2 than at location 1 in all treatments except

on shaved seedpieces dusted with diatomaceous earth (Fig. 4-A,

4-B). Maxima were greater on shaved than eyed seedpieces.

On shaved seedpieces captain delayed increases and reduced

maximum populations but allowed considerable increase compared











with non-amended soil. On eyed seedpieces captain greatly

reduced maximum populations of Fusarium spp. compared with

diatomaceous earth but allowed some increase in populations.

Populations of fungi other than Pythiu spp., Fusarium

spp., and Penicillium A reached higher maxima on shaved

seedpieces than on eyed seedpieces at both locations (Fig.

7, 8). Population maxima of these fungi were consistently

higher at location 2 than at location 1. On eyed seed-

pieces at both locations, captain prevented increases in pop-

ulations of Mucorales, fostered increases in populations of

Trichoderma spp., and reduced total fungal populations. On

shaved seedpieces at location 1, captain prevented increases

in populations of Mucorales and allowed only small increases

in populations of other fungi. On shaved seedpieces at

location 2, captain delayed increases in total fungal

populations, prevented increases in populations of Mucorales,

and fostered increases in populations of Lasiodiplodia sp.

and Trichoderma spp.

Penicillium A displayed behavior dissimilar to that of

other fungi. This biotype could be distinguished on the

amended potato dextrose agar used for recovery by a white,

rather than green, colony underside. Populations of Penicillium

A were greater on eyed than on shaved seedpieces at

most samplings and were an order of magnitude higher than

populations of all other fungi combined at both locations

at the 8-week sampling (Fig. 6)











The population dynamics of bacteria were similar at

both locations (Fig. 4-C, 4-D). Maximum populations were

higher on shaved than on eyed seedpieces. Populations of

bacteria were greater on eyed seedpieces treated with captain

than on eyed seedpieces treated with diatomaceous earth at

all samplings except the 12-week sampling at location 1.

On shaved seedpieces captain delayed increases at both

locations. Populations of total bacteria could not be

enumerated at the 2-week sampling because of a procedural

error.

On seedpieces at location 1, populations of fluorescent

Pseudomonas spp. reached maxima at 2 weeks and then

rapidly declined (Fig. 4-F). Populations were similar in

all samples which contained seedpieces at all sampling

times except the 12-week sampling, when populations were

higher on eyed than on shaved seedpieces. At location 2,

populations of fluorescent Pseudomonas spp. could not be

followed due to overgrowth of the selective medium by

non-fluorescent bacteria.









Table 5. Initial populations of fungi and bacteria in soil




Propagules/g soil


Organism Location 1 Location 2


Pythium spp. 5.3 X 102 NRb

Fusarium spp. 37.9 X 103 3.6 X 103

Lasiodiolodia sp. 2.8 X 103 3.2 X 103

Trichoderma spp. 8.8 X 10 3.9 X 10

Penicillium A 4.3 X 104 5.3 X 104

Other Penicillium spp. 14.9 X 10 11.8 X 104

Mucorales 2.6 X 104 0.3 X 104

Total fungi 43.1 X 104 27.7 X 104

Fluorescent Pseudomonas spp. 8.3 X 10 0.1 X 10

Actinomycetes 1.6 X 107 0.9 X 107

Bacteria 20.0 X 107 12.8 X 107



Values are the mean of 15 samples.

b1R=Not recovered.














/'^


2 4 8 12


WEEKS AFTER PLANTING


E -----,- -









0 2 4 8 12
WEEKS AFTER PLANTING


WEEKS AFTER PLANTING
16 .

-12
S F

2"



w iw




0 2 4 8 12
WEEKS AFTER PLANTING


Fig. 4-(A to F). The effects of viability of seedpieces and the
use of captain as a seedpiece dust on the population dynamics of
Fusarium spp. at A) location 1 and B) location 2, total bacteria
at C7 location 1 and D) location 2, E) Pythium spp. at location 1,
and F) fluorescent Pseudomonas spp. at location 1. Populations of
microorganisms were assayed in non-amended soil (--- ), on eyed
seedpieces dusted with diatomaceous earth (-----) or captain
..."-"-), and on shaved seedpieces dusted with diatomaceous earth
----) or captain (-.-). Populations of total bacteria were
assayed at 0, 4, 8, and 12 weeks after seedpieces were planted.
Populations of all other organisms were assayed at 0, 2, 4, 8, and
12 weeks after seedpieces were planted. Each point represents the
mean of three replicates.


6 A


6 B


4



3 /

I ', '.. .

0 2 4 8











24 24
A B



S16 \ 16
x / \
8 I
x j' 'C I




0 - - - -
0 2 4 8 12 O 2 4 8 12
WEEKS AFTER PLANTING WEEKS AFTER PLANTING

Fig. 5-(A,B). The population dynamics of Penicillium A on
caladium seedpieces at A) location 1 and B) location 2. Populations
were assayed in non-amended soil (-- ), on eyed seedpieces
dusted with diatomaceous earth (------) or captain ( ...........), and
on shaved seedpieces dusted with diatomaceous earth (--) or
captain (----). Populations were assayed at 0, 2, 4, 8, and 12
weeks after planting of seedpieces. Each point represents the
mean of three replicates.


_


I






















.- .......- -

0 2 4 8 12
WEEKS AFTER PLANTING





Fig. 6-(A,B). Populations of total fungi (- ), Penicillium
spp. (---), and Trichoderma spp. (------) in non-amended soil
at A) location 1 and B) location 2. Populations were assayed at
0, 2, 4, 8, and 12 weeks after initiation of the experiment. Each
point represents the mean of three replicates.


16
B

12


8


4

0 .....i----T -... .. .. -- ---- -

0 2 4 8
WEEKS AFTER PLANTING











A



8





0 2 4 8 I;
WEEKS AFTER PLANTING


-- .f-l.~ f -

0 2 4 a 12
WEEKS AFTER PLANTING


U Z 4 8 I
WEEKS AFTER PLANTING

D










0 2 4 I;
WEEKS AFTER PLANTING


Fig. 7-(A to D). The population dynamics of genera of fungi other
than Pythium and Penicillium A which had increased populations on
eyed seedpieces dusted with diatomaceous earth at A) location 1 and
B) location 2; and on eyed seedpieces dusted with captain at
C) location 1 or D) location 2. Populations of total fungi
(--- ), Trichoderma spp.(------), Mucorales (-----),
Penicillium spp. (-- ), Lasiodiplodia sp. (--.-- ), and
Fusarium spp. (--.........) were assayed at 0, 2, 4, 8, and 12 weeks
after seedpieces were planted. Each point represents the mean
of three replicates.










16 16



W 0



4 4
^8 --- '' ^9



O 2 4 8 0 2 4 8 12
WEEKS AFTER PLANTING WEEKS AFTER PLANTING
16, 16
D


o a

o 0
x 8





0 0 ..-.-.- .... -.
O 2 4 8 2 0 2 4 8 12
WEEKS AFTER PLANTING WEEKS AFTER PLANTING

Fig. 8-(A to D). The population dynamics of fungi, other than
Pythium spp. and Penicillium A, which had increased populations on
shaved seedpieces dusted with diatomaceous earth at A) location 1
and B) location 2; and on shaved seedpieces dusted with captain at
C) location 1 and D) location 2. Populations of total fungi (- ),
Trichoderma spp. (------), Mucorales (-- ---), Penicillium spp.
(---), Lasiodiplodia sp (------), and Fusarium spp. .......)
were assayed at 0, 2, 4, 8, and 12 weeks after seedpieces were
planted. Each point represents the mean of three replicates.










Discussion

The following trends were observed in both the field

and growth room experiments: (i) succession on eyed seed-

pieces was qualitatively similar to succession on shaved

seedpieces; (ii) increases in populations of Pythium spp.,

Fusarium spp., and bacteria preceded increases in populations

of other fungi; and (iii) almost all organisms reached

higher populations on shaved than on eyed seedpieces. The

much higher populations of Penicillium A on eyed than on

shaved seedpieces represent a significant anomaly. This organism

may have utilized either decaying shoot tissue or corm

epidermis as its substrate; confirmation of any particular

hypothesis would require further experimentation.

A number of aspects of the succession varied with the

plot location in the field. The absence of populations of

Pythium spp. at location 2 illustrates the necessity of

adequate initial populations for the increase of an organism

and may indicate the existence of some natural control

mechanism at that location. Inability to recover populations

of fluorescent Pseudomonas spp. at location 2 is probably

more of a reflection of limitations in the efficiency

of the selective medium than of a lack of increase by

these organisms at that location. The overgrowth of the

medium by non-fluorescent bacteria at location 2 and not

at location 1 does indicate the existence of qualitative

differences in the makeup of bacterial populations at the

two locations, however. Although initial populations of











organisms other than Pythium spp. and fluorescent Pseudo-

monas spp. tended to be higher at location 1, maximum

populations of these organisms were either similar at

both locations or were higher at location 2. This observation

indicates that factors other than initial inoculum density

are important in determining the amount of increase by

these organisms in the observed locations.

As in the growth room experiments, captain, when com-

pared with diatomaceous earth, (i) reduced increases by

Pythium spp. and Fusarium spp. but still allowed considerable

increases in populations of these organisms on shaved seed-

pieces, (ii) reduced increases in populations of total fungi

on eyed seedpieces, (iii) delayed increases in populations

of total fungi on shaved seedpieces, (iv) fostered increases

in populations of Trichoderma spp. and Lasiodiplodia sp.,

(v) tended to foster increases in populations of total

bacteria, and (vi) did not foster increases by any organisms

that did not increase on shaved seedpieces dusted with

diatomaceous earth and planted in the same soil. In contrast

with the growth room experiments, captain did not foster

increases in populations of fluorescent Pseudomonas spp. in

the field experiment. Both this anomaly and the depressed

2-week and 4-week populations of Pythium spp. on captan-

dusted seedpieces may have been due to changes in pop-

ulations during the relatively long period of time between

the collection of soil samples and the population assays.








40


In general, comparison of the results of the field and

growth room experiments indicates that observations of the

behavior of the experimental system under controlled temperature

and moisture conditions may be extrapolated cautiously to

the field. This correlation supports the veracity of

observations made in the growth room experiments and

indicates that the experimental system, or one similar to

it, could be used to evaluate the effects of other para-

meters on microbial populations under controlled conditions

with some confidence in the relevance of observations to

field situations.

















PART 3


GROWTH, YIELD, AND EMERGENCE OF CALADIUMS
IN RELATION TO SEEDPIECE WEIGHT


Introduction

Until recent years caladium seedpieces were cut by

hand, yielding relatively large seedpieces that contained

at least two eyes (29). At the present time, however,

many growers cut seedpieces with locally-manufactured cutting

machines, which cut corms into seedpieces in a random

manner. This method of cutting produces seedpieces of a

range of sizes and yields many seedpieces which do not

contain eyes, and thus do not germinate. Experiments

concerning the fate of this non-germinating seedpiece

material are presented in Parts 1 and 2 of this disser-

tation. The research presented in this part of the disser-

tation was undertaken to provide background information for

the design and interpretation of other experiments and to

provide information on the relationship of seedpiece weight

to emergence, yield, and value of caladiums that would be

of practical use to caladium growers. The investigation

consisted of two experiments conducted in the greenhouse and

one experiment conducted in the field.










Materials and Methods

In all of the experiments, caladium corms were obtained

from a commercial grower. All corms were heat treated at

50 0 for 30 min before being cut into seedpieces. Caladium

corms of cultivars Frieda Hemple and White Wing were used

in the greenhouse and field experiments, respectively.

In greenhouse experiment 1, undersized seedpieces that

were produced in the process of cutting larger seedpieces

for use in another experiment were graded into the following

four weight-classes: 0.2-0.5 g, 0.5-1.25 g, 1.25-2.0 g, and

2.0-3.0 g. All seedpieces contained at least some epidermis.

The seedpieces were planted in aluminum flats (45 X 28 X 5 cm)

which contained Palmico muck soil that had been collected,

sifted, and mixed as described in Part 1 of this disser-

tation. The seedpieces in each weight-class were arranged

evenly over the surface of 730 g of soil (dry weight) that

was evenly distributed over the bottom of a flat and were

covered with 460 g of soil (dry weight). Flats were

incubated in a non-airconditioned greenhouse. Temperatures

varied between approximately 15 C at night and 40 C during

the day. Every 2 to 3 days the flats were watered with

tap water and weed seedlings were pulled from the soil.

Emergence of plants with leaves was recorded weekly. Plants

were harvested and weighed 9 weeks after the seedpieces

were planted.

In greenhouse experiment 2 corms were cut into seedpieces

of the same weight-classes as those used in experiment 1.










All seedpieces were intentionally cut to contain at least

one eye. Palmico muck soil was sifted through a 4-mm

sieve, autoclaved 2 hr on each of two successive days and

aged in a greenhouse for 1 month. Seedpieces were planted

in 26 X 26 X 4 cm plastic flats. In each flat 340 g of

soil (dry weight) were distributed evenly over the bottom

of the flat, nine seedpieces were arranged in the flat in

a 3 X 3 matrix, and the seedpieces were covered with 340 g

of soil (dry weight). Two flats were prepared for each

weight-class of seedpieces as described above, except that

for the 2.0-3.0-g weight-class only three seedpieces were

planted in one of the flats. Flats were incubated in an

airconditioned greenhouse at 30-36 C and were watered daily

with tap water. Emergence of plants with leaves was recorded

weekly. Plants were harvested and weighed 6 weeks after

the seedpieces were planted.

In the field experiment seedpieces that had been cut by

a seedpiece-cutting machine and dusted with 1 % captain in

diatomaceous earth were sorted into the following four

weight-classes: 0.2-0.7 g, 0.7-1.4 g, 1.4-2.1 g, and

2.1-3.0 g. Other samples of seedpieces from the same cutting

run were sorted completely into six weight-classes in order

to determine the weight-class distribution of seedpieces in

the grower's planting material. Seedpieces were planted in

a non-fumigated area of a commercial caladium field that

had been identified by the grower as producing plants of

average to above average yield. Beds which were 1.25 m wide











were prepared by the grower. Seedpieces were planted in five

replicate blocks in each of two beds with weight-class

plots randomized within each block. In each plot four

seedpieces were planted 6 cm deep in each of two rows

running perpendicular to the length of the bed. Rows

within a plot were spaced 20 cm apart and rows in

adjacent plots were spaced 25 cm apart. Within each row

seedpieces were spaced 35 cm apart. The plots received

normal care by the grower, which included overhead irri-

gation, side dressing with fertilizer, and application of

the herbicides paraquat and alachlor. Emergence of plants

identifiable to cultivar was recorded at 4, 8 12, and 30

weeks after seedpieces were planted. Plants were harvested

30 weeks after the seedpieces were planted. The below-ground

portion of each plant was weighed 1 day after harvesting,

and dry weights of all corms produced in each plot were

determined after drying at 15-25 C for 1 month.










Results

Although plants emerged more rapidly and were larger at

harvest in greenhouse experiment 2 than in greenhouse

experiment 1, plant weight was directly related to seedpiece

weight in both experiments (Tables 6, 7). Linear regression

of average fresh weight of plants as a function of average

seedpiece weight indicated: Fresh weight = 0.086 + 2.62 (Seed-

piece weight), with r = 0.9985, for data from experiment 1;

and Fresh weight = 1.29 + 3.23 (Seedpiece weight), with

r = 0.9985, for data from experiment 2. Values of r are

significant at the 1 % level for both experiments.

Value was determined by partitioning dry weight data from

the field experiment into the number of corms in each

plot in each of four weight-classes, multiplying the

number of corms in each weight-class by the current

market price of corms of the corresponding size, and

adding the values of corms in each plot. Parameters used

in the calculations of value are presented in Table 10.

The relationship between size and weight of corms was

determined by linear regression of the average of largest

and smallest corm diameters as a function of the cube root

of corm weight, using measurements of 68 corns as a data

base.

In the field experiment emergence was directly related

to seedpiece weight (Table 8'), and all yield parameters

based on plots or harvested plants were significantly re-

lated to seedpiece weight (Table 9). However, value per







46


total weight of seedpiece material planted was not sig-

nificantly related to seedpiece weight, and value per dry

weight of harvested corms was significantly highest (p = 0.05)

for the lowest seedpiece weight (Table 11). Seedpieces cut

by the grower's seedpiece-cutting machine were fairly

evenly distributed in the weight-classes (Table 12).










Table 6. Emergence and fresh weight of caladium plants from
seedpieces in four weight-classes in greenhouse experiment 1




Seedpiece weight-class


Parameter 0.2-0.5g 0.5-1.25g 1.25-2.0g 2.0-3.0g


Mean seedpiece
weight (g) 0.38 0.73 1.47 2.53

Number of
seedpieces planted 19 35 15 11

Mean fresh weight
of plants (g) 0.92 wa 2.14 x 4.01 y 6.64 z

% emergence
at 5 weeks 15.8 25.7 26.7 27.3

% emergence
at 6 weeks 26.3 45.7 40.0 54.5

% emergence
at 7 weeks 36.8 54.3 46.7 63.6

% emergence
at 8 weeks 47.8 51.4 53.3 63.6

% emergence
at 9 weeks 52.6 51.4 53.3 63.6



aMeans followed by the same letter are not significantly different
(p=0.05) by Duncan's multiple range test performed on a square root
transformation of data.










Table 7. Emergence and fresh weight of caladium plants from
seedpieces in four weight-classes in greenhouse experiment 2




Seedpiece weight-class


Parameter 0.2-0.5g 0.5-1.25g 1.25-2.0g 2.0-3.0g


Seedpiece weight(g)a 0.350.09 0.880.23 1.640.20 2.360.25

Number of
seedpieces planted 18 18 18 12

Mean fresh weight
of plants (g) 2.39 xb 4.27 xy 6.37 yz 9.00 z

% emergence
at 1 week 0 0 0 8.3

% emergence
at 2 weeks 27.8 38.9 27.8 75.0

% emergence
at 3 weeks 61.1 77.8 77.8 91.7

% emergence
at 4 weeks 72.2 94.4 88.9 100

% emergence
at 6 weeks 77.8 94.4 94.4 100



aMean seedpiece weight standard deviation of the mean.

bMeans followed by the same letter are not significantly different
(p=0.05) by Duncan's multiple range test performed on a square root
transformation of data.










Table 8. Emergence of caladium plants from
weight-classes in the field experiment


seedpieces in four


Seedpiece weight-class


Time after planting 0.2-0.7g 0.7-1.4g 1.4-2.1g 2.1-3.0g
(W) (W W


4 weeks

8 weeks

12 weeks

30 weeks


0

1.3

16.3

26.3


0

22.5

36.3

36.3


1.3

43.8

57.5

63.8


3.8

46.3

68.8

75.0










Table 9. Yield of caladium plants from seedpieces in four
weight-classes in the field experiment




Seedpiece weight-class


Parameter 0.2-0.7g 0.7-1.4g 1.4-2.1g 2.1-3.0g


Mean seedpiece
weight (g) 0.43 0.95 1.71 2.77

Fresh weight
per plot (g)a 69.9 xb 164.0 y 327.2 z 408.8 z

Fresh weight
per plant (g)C 34.5 x 50.3 yz 65.4 yz 68.2 z

Corm weight
per plot (g)d 32.9 x 85.8 y 179.2 z 218.6 z

Corm weight
per plant (g)e 15.9 x 26.2 y 35.6 z 36.2 z



aMean fresh weight of corms and root systems per plot.

Within each row, values followed by the same letter are not signif-
icantly different (p=0.05) by Duncan's multiple range test.

CMean fresh weight of corm and root system per harvested plant.

dMean dry weight of corms per plot.

eMean dry weight of corms per harvested plant.































O

0
-t











0


n \
O'





N O


0

C-- C


a




OH *H 0o
*HO H *HO
O Hr Oa

0 .0 0 0

8 Url aP r
O *H 0 Or
U !0 U OR


CM












a









Table 11. Value of corms produced by caladium plants from seedpieces
in four weight-classes in the field experiment




Seedpiece weight-class


Parameter 0,2-0.7g 0.7-1.4g 1.4-2.lg 2.1-3,0g


Value per plot ($)a 0.33 wb 0.68 x 1.29 y 1.65 z

Value per weight
of seedpieces (0/g)0 9.59 w 8.95 w 9.43 w 7.45 w

Value per
yield of corms (0/g) 1.31 w 0.86 x 0.77 x 0.77 x



aMean value of corms produced in each plot.

bWithin a row, means followed by the same letter are not significantly
different (p=0.05) by Duncan's multiple range test.

CMean value of corms per gram of seedpiece material planted.

dMean value per gram of harvested corms.

























co
C)










N













c, o


N
0 L C-
N N











Cr- \o





C -)









1 c



bC 0
C' .-






C) ,a
a) C)

a)


C) H&


C)

r-
0

..
bO






6,r
HH










0 0
H o
cd, P









4 C
o 0
N C)

C oH


H )
*^ I



o o)





C) Ca
fi $H










Discussion

Growth of plants was significantly correlated with

seedpiece weight in both the greenhouse and field experiments,

although growth parameters for the two heaviest seedpiece

weight-classes were not significantly different from each

other in greenhouse experiment 2 and in the field

experiment. This correlation indicates that standardization

of seedpiece weight is an important factor in reducing

variation in experiments involving evaluation of growth of

caladiums.

The low emergence of plants from seedpieces in the

two lightest weight-classes in the field experiment (Table 8),

combined with the distribution of seedpiece weights in the

grower's planting material (Table 12), indicates that small

seedpieces contribute a disproportionately large share of

the amount of non-germinating seedpiece material planted.

The implications of this non-germinating seedpiece material

in the epidemiology of soilborne pathogens of caladiums are

dealt with in Parts 1 and 2 and Appendix 3 of this

dissertation.

The implications of the observations as far as commercial

production of caladiums is concerned are complex. Value was

significantly related to seedpiece weight when the yields

from the same number of seedpieces in each weight-class

were compared; however, value per gram of seedpieces planted

was not related to seedpiece weight, and value per gram of

harvested corm was highest for the lightest seedpieces. This







55


anomaly is the result of the pricing structure of caladiums,

which is based on diameter rather than weight of corms.

This pricing structure has the effect of making smaller

corms more valuable per gram than larger corms (Table 10).

If non-germinating seedpiece material is not a factor in

disease epidemiology, then whether or not it would be

economically advantageous for a grower to plant seedpieces

of a selected weight would depend on costs associated with

planting, harvesting, sorting, and various other procedures

involved in caladium culture. An analysis of such costs is

beyond the scope of this dissertation.

















APPENDIX 1


REPETITION OF EXPERIMENTS PRESENTED IN PART 1


Introduction

Research described in this Appendix is presented in

substantiation of research reported in Part 1 of this

dissertation. Data are presented from the first performance

of the growth room experiment in which the effects of

fungicidal dusts on the population dynamics of microorganisms

associated with caladium seedpieces were evaluated (performance

1) and from a final, partial performance of that experiment

(performance 3). Results from the second performance of the

experiment (performance 2) are presented in Part 1 of this

dissertation.


Materials and Methods

Procedures utilized in the investigation were identical to

those described in Part 1 of this dissertation, except that

soil was collected at different times and, populations of

nematodes were not enumerated. Performance 1 was conducted

exactly as described for performance 2 in Part 1 of this

dissertation. Performance 3 consisted of only the treatments

which involved the dusting of seedpieces with diatomaceous

earth and the control treatment of non-amended soil.










Results

Initial populations of most organisms were similar in

the different performances (Table 13). However, populations of

Trichoderma spp. and Pythium spp. were considerably higher

in performance 1 than in the other performances, populations

of Fusarium spp. were considerably lower in performance 1

than in the other performances, and populations of fluor-

escent Pseudomonas spp. were highest in performance 2,

considerably lower in performance 1, and lowest in per-

formance 3.

In performance 1, 12-week samples of Fusarium spp. and

2-week samples of Pythium spp. were lost due to error,

4-week samples of fungi which were recovered on potato

dextrose agar were altered by inadvertent use of potato

dextrose agar manufactured by Baltimore Biological Laboratories

Cockeysville MD 21030) rather than Difco, and populations of

Pythium spp. in soil amended with the fungicidal dusts without

the addition of seedpieces could not be enumerated with

confidence due to the use of inappropriate dilutions. In

performance 3 12-week samples of bacteria were lost due to

error.

In performance 1 the addition of the fungicidal dusts

to soil without the addition of seedpieces resulted in

changes in microbial populations that were largely similar

to those observed in performance 2 (Fig. 9). Effects observed

in performance 1 that differed from those observed in per-

formance 2 were (i) greater differences between the effects











of the different fungicidal dusts on populations of Fusarium

spp.; (ii) an apparent effect of captain on recovery of

Fusarium spp., as indicated by depression of perceived

0-week populations with that dust; and (iii) a different

ranking of the effects of the fungicidal dusts on populations

of bacteria.

On seedpieces in performance 1 the population dynamics

of Pythium spp. were similar to those observed in performance

2, except that populations were considerably higher on eyed

seedpieces dusted with the benomyl mixture than on eyed seed-

pieces dusted with diatomaceous earth (Fig. 10-A, 10-B). A

slow-growing biotype of P. irregulare accounted for 86 % and

73 % of total populations of Pythium spp. on eyed seedpieces

dusted with the benomyl mixture at the 8-week and 12-week

samplings, respectively. This biotype was not recovered from

any other treatment throughout the course of the experiment.

In performance 3 the population dynamics of Pythium spp.

were similar to those observed on seedpieces dusted with

diatomaceous earth in the other performances (Fig. 12-A).

Populations of Fusarium spp. reached much higher maxima

on shaved than on eyed seedpieces in all performances,

although there was a considerable amount of variation in

actual populations from performance to performance (Fig. 10-C,

10-D, 12-B). On eyed seedpieces the ranking of the effects

of the fungicidal dusts was similar in performances 1 and 2.

On shaved seedpieces the chloroneb mixture gave better control










and the benomyl mixture gave poorer control of increases in

populations of Fusarium spp. in performance 1 than in

performance 2.

Major differences in the behavior of fungi other than

Pythium spp. and Fusarium spp. in the different performances

were (i) a lack of increase in populations of Penicillium

spp. in performance 2, (ii) increases in populations of

Verticillium sp. in performance 2 but not in performances

1 and 3, (iii) increases in populations of Penicillium A

in performance 3 that were similar to those observed in

the field experiment, and (iv) higher initial populations

of Penicillium A on eyed seedpieces than on shaved seedpieces

or in non-amended soil in performance 3 (Fig. 11, 12-E, 13).

The effects of the fungicidal dusts on specific genera of

fungi were similar in performances 1 and 2 (Table 2). In

performance 1 populations of Penicillium spp. increased only

on shaved seedpieces dusted with diatomaceous earth or

captain.

The population dynamics of total bacteria were similar in

all performances, except that the duration of maxima was

extended on eyed seedpieces dusted with the benomyl mixture

or the chloroneb mixture in performance 1 but not in

performance 2 (Fig. 10-E, 10-F).

Population maxima of fluorescent Pseudomonas spp. were

increased on seedpieces dusted with all of the fungicidal

dusts in both performances 1 and 2 (Fig. 10-G, 10-H).

Maxima tended to be greater on eyed seedpieces than on







60


shaved seedpieces in performance 1 and greater on shaved

seedpieces than on eyed seedpieces in performance 2. In

performance 3 the maximum population was attained but was

of greater magnitude or eyed than on shaved seedpieces (Fig.

12-D). In all performances 12-week populations of fluorescent

Pseudomonas spp. were greater on eyed seedpieces than on

shaved seedpieces.









61














a) 0 0 0 0 0 0 0 0 0 0 0
,-H
) t-4 *, 4 ,- ,>- - .- -





o g)




)0 c0




N 0 No -


l C l ,-
( 1
c-



H C












o N
C)
*H C)
C) '-I











CO O ) O 0-
0 s H









0 Hn
C) z) P0

0i P 0 E. -. .)
O C)

' I 0




r' 0 (0 C)



d C)p -H *H a) eC



H1 I H C) H, tO



H i p









3- 4
oE FE W


i- "-"-.- /.
a 2
.. .'.-..... -...-. ....--.....- -.

0 0
0 2 4 12 0 2 4 a
WEEKS AFTER MIXING WEEKS AFTER MIXING

T2 .12
c LD

a. \

"' ''*. .....--. 4



0 0
0 2 4 12 0 2 4 8
WEEKS AFTER MIXING WEEKS AFTER MIXING

Fig. 9-(A to D). The effects, in performance 1, of the incorporation
of fungicidal dusts into soil on populations of A) total fungi,
B) total bacteria, C) Fusarium spp., and D) fluorescent Pseudomonas
spp. Populations of microorganisms were assayed in non-amended
soil (-- ) and in soil into which the benomyl mixture (-----),
captain (.....--...), or the chloroneb mixture (----) had been
incorporated. Assays were performed at 0, 2, 4, 8, and 12 weeks
after incorporation for all organisms, except for Pythium spp. at
2 weeks and Fusarium spp. at 12 weeks. Each point represents the
mean of three replicates.










Fig. 10-(A to H). The effects, in performance 1, of treatment of
caladium seedpieces with fugicidal dusts on the population
dynamics of Pythium spp. on A) eyed seedpieces and B) shaved
seedpieces, Fusarium spp. on C) eyed seedpieces and D) shaved
seedpieces, total bacteria on E) eyed seedpieces and F) shaved
seedpieces, and fluorescent Pseudomonas spp. on G) eyed seedpieces
and H) shaved seedpieces. Populations of microorganisms were
assayed in non-amended soil (------) and in soil into which
seedpieces dusted with diatomaceous earth (-- ), the benomyl
mixture (----.), captain (.........), or the chloroneb mixture
(----) had been planted. Assays were performed at 0, 2, 4, 8,
and 12 weeks after seedpieces were planted by plating dilutions
on selective media. Each point represents the mean of three
replicates.



























O 2 4 8 12
WEEKS AFTER PLANTING


c 8
C


6

x
4



0
3




0 2 4 a 12
WEEKS AFTER PLANTING

____________________I_ I


16 E


'12









0 ,
0 2 4 8
WEEKS AFTER PLANTING
0.


0
WEK FERPATN


0 2 4 8
WEEKS AFTER PLANTING


16 / I
SF

S12







O
0

12 0 2 4 8
WEEKS AFTER PLANTING


S12
0 H


48




S / ...................



12 0 2 4 8
WEEKS AFTER PLANTING


WEEKS AFTER PLANTING


""""~'"'










Fig. 11-(A to H). The population dynamics, in performance 1, of
genera of fungi, other than Pythium, which had increased populations
on eyed caladium seedpieces dusted with A) diatomaceous earth,
C) the benomyl mixture, E) captain, or G) the chloroneb mixture;
and on shaved caladium seedpieces dusted with B) diatonaceous
earth, D) the benomyl mixture, F) captain, or H) the chloroneb
mixture. Populations of total fungi (----), Trichoderma spp.
(------), Mucorales (-.----.), Penicillium spp. (----),
Lasiodiplodia sp. (--.--), and Fusarium spp. (..........) were
assayed in soil samples which each contained a seedpiece. Assays
were performed at 0, 2, 4, 8, and 12 weeks after seedpieces were
planted by plating dilutions on selective media. Each point
represents the mean of three replicates.



























0 2 4 8 12
WEEKS AFTER PLANTING


c












0 2 4 8 12
WEEKS AFTER PLANTING


8a


6 G


x
4


a2


0
0


2


4'











0















0
x
"3
2




0
0



4





,3




o

x
a2
0

0



4








SI
0
0
0


WEEKS AFTER PLANTING


2 4 8
WEEKS AFTER PLANTING


WEEKS AFTER PLANTING


F


E









, -


0 2 4 8 12
WEEKS AFTER PLANTING


2 4 8
WEEKS AFTER PLANTING


-......... ....... - --_'' .


H










............ .,,, .- _ .


--.--.-*"-_________I______,______


t


\











A






0:


0 2 4 8 12
WEEKS AFTER PLANTING


12






0---------- ----- --
0
0 2 4 8 12
WEEKS AFTER PLANTING
24
E /









0 .
0 2 4 8 12
WEEKS AFTER PLANTING


/








0 2 4 8 12
WEEKS AFTER PLANTING

D









o 2 4 8 12
WEEKS AFTER PLANTING


Fig. 12-(A to E). The population dynamics, in performance 3, of
A) Pythium spp., B) Fusarium spp., C) total bacteria,
D) fluorescent Pseudomonas spp., and E) Penicillium A on caladium
seedpieces. Populations were assayed in non-amended soil (-----)
and in soil into which eyed seedpieces (-----) or shaved
seedpieces (----) had been planted. All seedpieces had been
dusted with diatomaceous earth. Assays were performed at 0, 2, 4,
8, and 12 weeks after seedpieces were planted. Each point
represents the mean of three replicates. The 12-week samples
of total bacteria were lost due to error.







68

A B
4








0 0
.. ....................

0 2 4 8 12 2 4 -------- --
WEEKS AFTER PLANTING WEEKS AFTER PLANTING

Fig. 13-(A,B). The population dynamics, in performance 3, of fungi
other than ythium spp. and Penicilliun A which had increased
populations on A) eyed and B) shaved seedpieces dusted with
diatomaceous earth. Populations of total fungi (-- ), Trichoderma
spp. (------), Mucorales (-----), Penicillium spp. (-----),
Lasiodiplodia sp. (---- ), and Fusarium spp. (........... ) were assayed.
Assays were performed at 0, 2, 4, 8, and 12 weeks after seedpieces
were planted. Each point represents the mean of three replicates.










Discussion

The similarity of the repetitions of the growth room

experiments was comparable to the similarity of the field

experiment and the growth room experiments. Certain fungi

increased populations on seedpieces in some performances

but not in others; however, increases in populations of

members of the basic community, which consisted of bacteria,

fluorescent Pseudomonas spp., Pythium spp., Fusarium spp.,

Lasiodiplodia sp., and Trichoderma spp., were observed on

shaved seedpieces dusted with distomaceous earth in all

performances. Observed variation in the genera of fungi

which increased on seedpieces in the different repetitions

of the growth room experiments and in the field experiment

may have been due to differences in initial populations

and/or differences in non-controlled experimental parameters,

such as temperature fluctuations in the growth room,

differences in soil compaction due to differences in soil

moisture content during mixing, and differences in soil

moisture content prior to commencement of the experiments.

The importance of differences in experimental parameters,

rather than initial populations, in causing experiment- to-

experiment variation is supported by the consistent presence

of a relatively high proportion of Penicillium spp. in all

initial populations of fungi but not in populations of fungi

which increased on seedpieces in performance 2. Involvement of

Verticillium sp. in the seedpiece succession in performance 2,

but not in performances I and 3, may indicate that this











organism displaced Penicillium spp. in performance 2. Whatever

the cause of experiment- to- experiment variation, it is

significant that increases on seedpieces by fungi which

increased early in the succession were more consistent than

increases on seedpieces by fungi that increased later in the

succession.

Increases in populations of Penicillium A in performance 3,

although not of the magnitude of those observed in the field

experiment, are of interest in relation to hypotheses

concerning the substrate utilized by this organism. The

observation that populations were highest on eyed seedpieces

at the initial sampling indicates that higher maximum pop-

ulations on eyed seedpieces may have been due to higher

initial inoculum density rather than the utilization of a

particular substrate.

Overall, the similarity of the repetitions substantiates

the veracity of observations made on the behavior of the

experimental system and supports the utilization of the system,

or one similar to it, in further studies of the behavior

of microorganisms in soil.

















APPENDIX 2


PHYSICAL DISTRIBUTION OF MICROBIAL POPULATIONS
INCREASING ON CALADIUM PLANTS


Introduction

Although populations of microorganisms were assayed in

and around caladium plants and seedpieces in Parts 1 and

2 of this dissertation, no attempt was made to determine

the site of observed population increases. The research

presented in this appendix was undertaken to determine

whether increasing populations of microorganisms were

located (i) in soil adjacent to the seedpiece or plant,

(ii) on the surface of the seedpiece or plant, or

(iii) within the tissues of the seedpiece or plant.

Although these three sites could not be differentiated

on shaved seedpieces because of problems in the retrieval

of the disintegrating seedpiece, populations at the sites

were assayed separately on plants growing from eyed seed-

pieces.


Materials and Methods

Soil was collected from a commercial caladium field at

different times and from different areas in the field for

use in three performances of the experiment. In all per-

formances caladium corms were heat treated and cut into










eyed seedpieces as described in Part 1 of this disser-

tation. Seedpieces were dusted with diatomaceous earth, and

dusted seedpieces were planted and incubated as described

in Part 1 of this dissertation.

Populations of microorganisms associated with emerged

plants were assayed at 4, 6, and 9 weeks after seedpieces

were planted in performances 1, 2, and 3, respectively.

Leaves were cut off at the soil line and soil cores were

taken as described in Part 1 of this dissertation. Samples

which contained plants were partitioned by use of the

following sequence of procedures: (i) the complete soil

core was placed in a Waring blender, the plant was

removed from the soil, and 100 ml of autoclaved, deionized

water was added to the soil which remained in the blender

(adjacent soil partition); (ii) the plant was placed in a

beaker which contained 100 ml of autoclaved, deionized

water, the beaker was shaken gently for 3 min, and the

plant was removed (cormsphere partition); and (iii) the

plant was placed in 100 ml of autoclaved, deionized water

(corm partition). Each partition was comminuted at low

speed in the Waring blender for 1 min, A dilution series

was prepared in autoclaved, deionized water from the initial

suspension. Appropriate dilutions were plated on selective

media as described in Part 1 of this dissertation. In

performance 1 samples were plated on all five selective

media. In performances 2 and 3 samples were plated on all

of the media except the medium selective for fluorescent







73


Pseudomonas spp. Populations were determined from three,

two, and three replicate plants in performances 1, 2, and

3, respectively.










Results

In order to calculate total populations in the three

partitions of the plant samples (Tables 14, 15, 16),

populations in the corm and cormsphere partitions were

calculated as if they represented populations in the amount

of soil present in the adjacent soil partition, and then

the populations in the three partitions were added together.

This procedure yielded a population equal to that which

would have been perceived had the plant sample not been

partitioned. Populations are presented only for those

organisms which had a total population in the three par-

titions of the plant sample that was significantly higher

than their population in non-amended soil by comparison

using a one-tailed t-test at p = 0.10 (30). In performance

1 a number of organisms or groups of organisms had

increased populations on plants (Table 14); however, in

performance 2 only Pythium spp., Fusarium spp., Lasiodiplodia

sp., and bacteria had increased populations (Table 15), and

in performance 3 only Pythium spp., Fusarium spp., bacteria,

and an unidentified fungus had higher populations in the

samples which contained plants compared with non-amended soil

(Table 16)

The percentage of the increased population in each

partition was calculated to facilitate comparison of

populations of organisms in the three partitions of the

plant samples in the three performances. For the adjacent










partition, this value was calculated as

% population in adjacent soil =

100 (Population in adjacent soil Population in soil),
Total population in plant samples-- Population in soil

where "total population in plant samples" and "population

in soil" are those values presented in Tables 13, 14, and

15. For the cormsphere and corm partitions, this value

was calculated as

% population in cormsphere or corm =

100 (Population in cormsDhere or corm)
Total population in plant samples Population in soil

The percentages of populations in the three partitions

differed in the three performances of the experiment

(Tables 17, 18, 19).










Table 14. Populations of fungi and bacteria in non-amended soil
and in soil containing caladium plants in performance 1




Propagules/g soil


Organism Soilb Plantc


Pythium spp. 88 270

Fusarium spp. 1.3 X 104 24.2 X 104

Lasiodiplodia sp. 1.2 X 104 18.9 X 10

Trichoderma spp. 2.2 X 10 4.8 X 104

Penicillium A 4.0 X 104 12.3 X 104

Other Penicillium spp. 3.1 X 104 9.0 X 104

Non-identified fungi 6.7 X 104 13.1 X 10

Fluorescent Pseudomonas spp. 0.3 X 103 347 X 103

Bacteria 5.9 X 107 27.4 X 107



aValues are the mean of three samples.

bPopulation in soil without plant.

Mean cumulative population in the soil, cormsphere, and corm
partitions of samples of soil which contained plants.










Table 15. Populations of fungi and bacteria in non-amended soil
and in soil containing caladium plants in performance 2




Propagules/g soil


Organism Soilb Plantc


Pthium spp. 420 800

Fusarium spp. 3.0 X 104 20.2 X 10

Lasiodiolodia sp. 1.5 X 104 63.4 X 104

Bacteria 8.5 X 107 39.3 X 107



aValues are the mean of two samples.

population in soil without plant.

CMean cumulative population in the soil, cormsphere, and corm
partitions of samples of soil which contained plants.









Table 16. Populations of fungi and bacteria in non-amended soil
and in soil containing caladium plants in performance 3




Propagules/g soila


Organism Soilb Plantc


Pythium spp. 11.0 X 102 12.? X 102

Fusarium spp. 2.9 X 10 4.4 X 104

Fungus A 0.8 X 104 8.5 x 104

Bacteria 4.8 X 107 11.3 X 107



aValues are the mean of three samples.

bPopulation in soil without plant.

CMean cumulative population in the soil, cormsphere, and corm
partitions of samples of soil which contained plants.










Table 17. Percentage of increased populations of fungi and
bacteria in plant-sample partitions in performance 1




Partition


Organism Adjacent Cormsphere Corm
soil



Pythium spp. 0 18 82

Fusarium spp. 32 23 46

Lasiodiplodia sp. 34 44 23

Trichoderma spp. 54 35 12

Penicillium A -7 31 76

Other Penicillium spp. 41 24 36

Non-identified fungi 8 44 48

Fluorescent Pseudomonas spp. 0.2 0.7 99.1

Bacteria 59 21 20










Table 18. Percentage of increased populations of fungi and
bacteria in plant-sample partitions in performance 2




Partition


Organism Adjacent Cormsphere Corm
soil



Pythium spp. 41 13 46

Fusarium spp. 12 19 69

Lasiodiplodia sp. 20 56 23

Bacteria 7 20 72










Table 19. Percentage of increased populations of fungi and
bacteria in plant-sample partitions in performance 3




Partition


Organism Adjacent Cormsphere Corm
soil
(%) (%) ()


Pythium spp. 45 28 27

Fusarium spp. 8 31 61

Fungus A 18 15 69

Bacteria 15 15 70










Discussion

Most assayed populations were in the range of those

enumerated in the population dynamics experiments; however,

Pythium spp. populations were considerably lower and higher

than in other experiments in performances 1 and 3, respec-

tively (Tables 13, 14, 15, 16).

The relatively small increases in populations of

organisms in performance 3 may have been the result of

the soil being somewhat dryer in that performance because

of lower bulk density due to initial mixing at a lower

moisture content and/or the result of the sampling being

performed subsequent to increase and decline of populations.

Whatever the reason for the small differences between

populations in the soil and plant samples, data from

performance 3 are less reliable than data from the other

performances.

The percentage of increased populations of Pythium spp.

in the corm decreased in the order of performances 1, 2,

and 3. Although the different performances are not strictly

comparable, the fact that the percentage of the

increased population in the corm was lower in samplings

of older plants may indicate that colonization by Pythium

spp. occurs primarily in tissue exposed at the cut surface

of the corm, and that propagules formed in that tissue are

subsequently shed as the tissue disintegrates and is sloughed

off. This scenario is consistent with the observation that










populations of Pythium spp. tend to reach high levels

early in the succession and then stabilize or decline

slowly (Fig. 2-A, 2-B).

Percentages of increased populations of Fusarium spp.

were fairly evenly distributed among the partitions in

performance 1, but tended to be concentrated in the corm

in performances 2 and 3. This behavior may indicate an

initial increase on substrates diffusing from the cut

surface of the corm, followed by growth into the corm.

Increased populations of Trichoderma spp. were highest

in adjacent soil and the cormsphere in the one performance

in which population increases were observed. This may indicate

that early increase by Trichoderma spp. on seedpieces is on

substrates diffusing from the cut surface of the corm,

rather than on the corm itself. Comparison of the sites

of increase of Trichoderma spp., Pythium spp., and Fusarium

spp. indicates that Trichoderma spp. may be better bio-

logical control agents of Fusarium spp., which have a similar

site of increase, than of Pythium spp., which have a dif-

ferent site of increase.

Increased populations of Penicillium A were highest in

the corm and absent in adjacent soil. If this is the

same organism that was observed in the field experiment

(Fig. 5), then the site of its increase must be assumed

to be the corm itself and/or associated below-ground shoot

tissues.










Increased populations of other Penicillium spp, were

fairly evenly distributed among the partitions. This may

indicate that these organisms have a pattern of increase

similar to that of Fusarium spp., although increases

in populations were small and were observed only in

performance 1.

In both performances 1 and 2, increased populations of

Lasiodiplodia sp. were highest in the cormsphere but of

considerable magnitude in both the corm and adjacent soil.

The hypothesis that this distribution indicates growth into

the corm following that of Fusarium spp. is consistent with

the late increases in populations of Lasiodiplodia sp. that

were observed in the population dynamics experiments (Fig. 3).

Fungus A was observed to increase only in the one

performance of the experiment in which Lasiodiplodia sp.

was not observed to increase. This may indicate that

fungus A displaced Lasiodiplodia sp. under whatever circum-

stances were responsible for the small increases in populations

of Pythium spp. and Fusarium spp. that were observed in

that performance.

Although of small magnitude, increases of a mixture of

non-identified fungi in performance 1 indicate that fungi

other than the enumerated genera may increase on caladium

seedpieces under certain circumstances.

In the one performance of the experiment in which pop-

ulations of fluorescent Pseudomonas spp. were assayed, increased

populations of these organisms were present almost exclusively











in the corm. This site of increase was similar to that

of Pythium spp. Coupled with the observation that these two

groups of organisms increase at the same time in the

succession, this similarity of distribution may indicate

that fluorescent Pseudomonas spp. could be effective bio-

logical control agents of Pythium spp. However, the tight

association of populations of fluorescent Pseudomonas spp.

with the corm may indicate that increased populations are

derived from bacteria carried internally in the seedpiece,

rather than from bacteria present in soil, and thus may

indicate that populations could be difficult to manipulate

by inoculation.

Percentages of increased populations of total bacteria

were highest in adjacent soil in performance 1 and highest

in the corm in performances 2 and 3. This distribution

may indicate that early increases in populations of total

bacteria are on substrates diffusing from the cut surface

of the corm, while later increases are in the corm,

possibly associated with ingressive growth by fungi.

Overall, evaluation of the data indicates early colon-

ization of the seedpiece by Pythium spp. and fluorescent

Pseudomonas spp. with concomitant increases of Trichoderma spp.

and total bacteria on substrates diffusing from the cut

surface. later, Fusarium spp. make intrusive growth into

the seedpiece, accompanied by bacteria and some other fungi

and followed by Lasiodiplodia sp. This scenario is consistent

with observations made in the population dynamics experiments.







86


The most accurate characterization of a microbial

succession, such as the one observed in these experiments,

would probably be derived from a coordinated study of the

physical distribution and population dynamics of organisms

which increase populations on the substrate. Although such

coordination was not present in this research, evaluation of

data on the physical distribution of organisms still served

to substantiate observations on the population dynamics of

those organisms.

















APPENDIX 3


THE EFFECT OF FUNGICIDAL SEEDPIECE DUSTS ON
GROWTH, YIELD, EMERGENCE, AND VALUE OF CALADIUMS


Introduction

Research on the effects of fungicidal seedpiece dusts

on populations of microorganisms associated with caladium

seedpieces is described in Parts 1 and 2 and Appendices

1 and 2 of this dissertation. In this Appendix the effects

of the benomyl mixture, captain, and the chloroneb mixture

on growth of caladiums under a variety of environmental

conditions are reported. Results are presented from (i) a

factorial experiment in which the effects of the seedpiece

dusts and the presence of an adjacent, decomposing seedpiece

on growth of caladiums were evaluated; (ii) a growth room

experiment in which the effects of the seedpiece dusts on

growth of caladiums in autoclaved soil was evaluated; and

(iii) a field experiment in which the effects of the seed-

piece dusts on emergence, yield, and value of caladiums were

evaluated.


Materials and Methods

In all experiments caladium corms were obtained from a

commercial grower. All corms were heat treated at 50 C

for 30 min before being cut into seedpieces. In the










greenhouse and growth room experiments, corms of cultivar

Frieda Hemple were used. In the field experiment corms

of cultivar White Wing were used.

In the greenhouse experiment corms were cut into

2.02 0.13 g seedpieces that each contained at least one

eye (eyed) and 1.03 0.08 g seedpieces that had all

eyes and epidermis shaved off (shaved). Seedpieces were

dusted with the four seedpiece dusts described in Part 1

of this dissertation. Soil was collected, sifted and mixed

as described in Part 1 of this dissertation. Seedpieces

were planted in autoclaved 10- cm clay pots. Materials were

sequentially placed in each pot as follows: 73 g of soil

(dry weight), either an eyed seedpiece or an eyed seedpiece

and a shaved seedpiece spaced 5 mm apart, and 55 g of

soil (dry weight). Ten pots were prepared for each of the

eight combinations of seedpiece dust and seedpiece condition.

Pots were incubated in a non-airconditioned greenhouse in

which temperatures ranged from approximately 15 to 40 C.

Every 2 days pots were watered with tap water and weed

seedlings were pulled from the soil. Emergence of plants with

leaves was recorded daily. Plants were harvested and fresh

weights were determined 12 weeks after seedpieces were

planted. Dry weights of plants were determined after drying

2 weeks at 25-30 C.

In the growth room experiment corms were cut into

1.02 0.6 g seedpieces that each contained at least one

eye. Seedpieces were dusted with the four seedpiece dusts

described in Part 1 of this dissertation. Before use in










the experiment, Palmico muck soil was sifted through a

4-mm sieve, autoclaved 2 hr on each of two successive

days, incubated under irrigation with tap water in a green-

house for 1 week, mixed in a small cement mixer for 20

min, placed in plastic bags, and incubated at 25-30 C for

2 months. One day before seedpieces were planted, soil was

assayed for populations of Pythium spp., Fusarium spp., and

other fungi as described in Part 1 of this dissertation.

A seedpiece was placed on the surface of 55 g soil (dry

weight) in a 10-cm clay pot and covered with 73 g of

soil (dry weight). For treatments with the benomyl mixture,

captain, the chloroneb mixture, and diatomaceous earth seedpiece

dusts, 8, 6, 6, and 8 pots were prepared, respectively.

Pots were incubated in a growth room at 25-30 C with

12 hr of light (4,000 lx at the level of the plants).

Pots were watered and emergence of plants with leaves was

recorded daily. Plants were harvested and weighed 9 weeks

after seedpieces were planted. Dry weights of plants were

determined after drying 6 weeks at 20-30 C.

In the field experiment corms were cut into 2.5-3.5 g

seedpieces and were dusted with the four seedpiece dusts

described in Part 1 of this dissertation. Seedpieces were

planted in a non-fumigated area of a commercial caladium

field that had been identified by the grower as producing

plants of average to above average yield. Plot layout and

care were the same as described for the seedpiece weight

experiment that was conducted in the field (Part 3 of this







90


dissertation). Emergence of plants identifiable to cultivar

was recorded at 4, 8, and 12 weeks after seedpieces were

planted. Plants were harvested 30 weeks after seedpieces

were planted. Dry weights of all corms produced in each

plot were determined after drying at 15-25 C for 1 month.

Value of harvested corms was determined as described in

Part 3 of this dissertation.










Results

Initial populations of microorganisms in the soil used

in the greenhouse experiment were similar to those enumerated

in other experiments (Tables 1, 20 ). Propagules of Pythium

spp. and Fusarium spp. were not recovered from the

autoclaved soil used in the growth room experiment, and

populations of fungi in this soil were predominantly

Penicillium A (Table 20).

The effects of the fungicidal dusts in the green-

house experiment were as follows: (i) time of emergence of

plants from eyed seedpieces planted alone was significantly

decreased by the captain treatment when compared with the

other fungicidal dusts; (ii) time of emergence of plants

from eyed seedpieces planted with an adjacent decomposing

seedpiece was significantly decreased by the captain treatment

compared with diatomaceous earth; (iii) fresh weight of

plants from eyed seedpieces planted alone was significantly

greater for the chloroneb mixture and captain compared with

diatomaceous earth; (iv) fresh weight of plants from eyed

seedpieces planted with an adjacent shaved seedpiece was

significantly greater for the chloroneb mixture compared

with diatomaceous earth or the benomyl mixture; (v) seedpiece

dust did not significantly affect dry weight of plants from

seedpieces planted alone; and (vi) dry weight of plants

from eyed seedpieces planted with an adjacent shaved seed-

piece was significantly lower with diatomaceous earth than

with captain or the chloroneb mixture (Table 20). The only











significant effect of planting a shaved seedpiece adjacent

to an eyed seedpiece was a decrease in fresh weight in

plants from seedpieces dusted with diatomaceous earth.

In the growth room experiment individual seedpiece dusts

did not affect emergence rate, fresh weight, or dry weight

of plants (Table 22). However, when results from the three

treatments with the fungicidal dusts were bulked together

and compared with those from the diatomaceous earth treat-

ment, fungicidal dusts significantly decreased (p = 0.05)

dry weight, but not fresh weight or emergence rate.

In the field experiment emergence was similar with

all af the dusts (Table 23), and neither yield nor value

was affected significantly by seedpiece dust (Table 24).

Bulking of data from the three fungicidal dust treatments

did not affect measures of significance.




Full Text

PAGE 1

POPULATION DYNAMICS OF MICROORGANISMS ASSOCIATED WITH CALADIUM SEEDPIECES BY RICHARD S. FERRISS 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 1979

PAGE 2

ACKNOWLEDGMENTS The people and institutions which contributed in some way to the completion of this dissertation are many and varied. I would like to thank my major professor, Dr. D. J. Mitchell, for his suggestions, inspiration, properly conjugated infinitives, and occasional obstinacy; the other members of my committee, Dr. D. H. Hubbell, Dr. J. F. Knauss, Dr. D. A. Roberts, and Dr. R. E. Stall, for their help in many aspects of my training; Mr. George Richardson and his sons, Mark and Ghet, for their invaluable assistance with my field plots at Bear Hollow Bulb Farm; the University of Florida for providing financial support for my studies and a favorable atmosphere for their completion; dinosaurs, dolphins, and my high school chemistry teacher, Bob Grove, for nurturing an interest in the living world during my childhood and adolescence; the many other teachers I have had over the years for their gifts of knowledge and sometimes wisdom; the many friends who have made my stay in Gainesville one of growth and joy; my mother for her worries and love through the years; and lastly my daughter, Kate, for her love, enthusiasm and confusing questions . ii

PAGE 3

TABLE OF CONTENTS ACKNOWLEDGMENTS ii ABSTRACT v PART 1. THE EFFECT OF FUNGICIDAL SEEDPTECE DUSTS ON THE POPULATION DYNAMICS OF SOIL MICROORGANISMS ASSOCIATED WITH GERMINATING OR DECOMPOSING CALADIUM SEEDPIECES Introduction 1 Materials and Methods 3 Results 7 Discussion 20 PART 2. THE EFFECT OF SEEDPIECE TREATMENT WITH CAPTAN ON THE POPULATION DYNAMICS OF SOIL MICROORGANISMS ASSOCIATED WITH GERMINATING OR DECOMPOSING CALADIUM SEEDPIECES IN THE FIELD Introduction „ 25 Materials and Methods 26 Results 29 Discussion 38 PART 3. GROWTH, YIELD, AND EMERGENCE OF CALADIUMS IN RELATION TO SEEDPIECE WEIGHT Introduction „ 4l Materials and Methods 42 Results 45 Discussion 5^

PAGE 4

APPENDICES 1 . REPETITION OF EXPERIMENTS PRESENTED IN PART 1 % 2. PHYSICAL DISTRIBUTION OF MICROBIAL POPULATIONS INCREASING ON CALADIUM SEEDPIECES 71 3. THE EFFECT OF FUNGICIDAL SEEDPIECE DUSTS ON GROWTH, YIELD, EMERGENCE, AND VALUE OF CALADIUMS 8? k. GROWTH OF CALADIUMS IN SOIL INFESTED WITH FUNGI OBSERVED TO INCREASE POPULATIONS ON CALADIUM SEEDPIECES 100 5. TOLERANCE OF FUSARIUM SPP. AND LASIODIPLODIA SP . TO BENOMYL 114LITERATURE CITED 1 35 BIOGRAPHICAL SKETCH 1 38

PAGE 5

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 POPULATION DYNAMICS OF MICROORGANISMS ASSOCIATED WITH CALADIUM SEEDPIECES by Richard S. Ferriss March, 1979 Chairman: David J. Mitchell Major Department: Plant Pathology The short-term population dynamics of fungi and bacteria associated with caladium ( Caladium hortulanum ) seedpieces planted in raw muck soil were investigated. Sized seedpieces of cultivar Frieda Hemple were planted in flats of soil and incubated in a growth room at 25-30 C, or were planted in two areas of a commercial caladium field with contrasting crop histories. Populations of Pythium spp., Fusarium spp., other genera of fungi, total bacteria and fluorescent Pseudomonas spp. were assayed at 0, 2, k, 8, and 12 weeks after seedpieces were planted. A suspension of a soil core that contained a seedpiece and surrounding soil was comminuted in a Waring blender, and appropriate dilutions were plated on selective media. The effects of viability of seedpieces (presence or absence of eyes and epidermis), the use of fungicidal

PAGE 6

seedpiece dusts, and planting area in the field on the population dynamics of the assayed organisms were investigated. Addition of seedpieces to soil resulted in increase and subsequent decrease in the populations of several organisms . For each organism, or group of organisms, the magnitude of the maximum population was related to seedpiece condition or treatment. However, regardless of seedpiece condition or treatment, population maxima occurred in the following approximate order: fluorescent Pseudomonas spp., Pythium spp. , total bacteria, Mucorales, Fusarium spp., Trichoderma spp . , Yerticillium spp . , Penicillium spp . , and Lasiodiplodia sp. In general, organisms reached higher populations on seedpieces which had eyes and epidermis shaved off (shaved) than on corresponding viable seedpieces (eyed). Fungicidal dusts used in the experiments were captan; a mixture of benomyl, thiram, and streptomycin sulfate; and a mixture of chloroneb, thiram, and streptomycin sulfate. Effects of the fungicidal dusts on microbial populations associated with seedpieces were different from effects observed f ollowing incorporation of the fungicidal dusts , without seedpieces, into soil. Treatment of seedpieces with any of the fungicidal dusts reduced increases in populations of fungi on eyed seedpieces, delayed increases in populations of fungi on shaved seedpieces, and enhanced the magnitude or duration of increases in populations of both fluorescent Pseudomonas spp. and total bacteria. The magnitudes of population increases by different genera of fungi were vi

PAGE 7

greatly altered "by the fungicidal dusts; however, the fungicidal dusts did not foster increases "by any fungi not observed to increase on shaved seedpieces which were not fungicidetreated. With any particular dust, the fungal community which increased on eyed seedpieces resembled the community on shaved seedpieces treated with the same dust more than it resembled communities on eyed seedpieces treated with other dusts . In one experiment populations of Pythium spp. were greater on eyed seedpieces treated with the dust which included benomyl than on eyed seedpieces which were not fungicidetreated. Populations of Pythium spp. increased only slightly on seedpieces treated with the dusts that included captan or chloroneb. In all experiments populations of Fusarium spp. were similar on seedpieces treated with the dust that included chloroneb and on seedpieces which were not fungicide-treated. Populations of Fusarium spp. increased only slightly on eyed seedpieces treated with dusts that included captan or benomyl, but increased appreciably on shaved seedpieces treated with these dusts. Increases in populations of Fusarium spp. on seedpieces treated with the dust that included benomyl were correlated with the abundance of benomyl-tolerant isolates. In the field experiments overall patterns of increase were similar to those observed in the growth room. Patterns observed in the field, but not in the growth room, were

PAGE 8

an absence of Pythium spp. in one area of the field, a reduction of increases in populations of Pythium spp. and Lasiodiplodia sp . , and greater involvement of Penicillium spp. in the succession.

PAGE 9

PART 1 ' THE EFFECT OF FUNGICIDAL SEEDPIECE DUSTS ON THE POPULATION DYNAMICS OF SOIL MICROORGANISMS ASSOCIATED WITH GERMINATING OR DECOMPOSING CALADIUM SEEDPIECES Introduction Treatment of seeds and seedpieces is an important mode of application of fungicides intended to control soilborne plant pathogens (10, l6). In spite of the economic importance of these treatments, information on the ecological operation of seed treatment fungicides is meager. The effects of fungicides on populations of soil microorganisms when the fungicide is incorporated evenly into soil, without the addition of a substrate, have received a good deal of attention (l , 8, 2^, 3^+); the effects of fungicides on microbial growth associated with the addition of purified substrates have received a limited amount of investigation (2). These investigations have not directly addressed the effects of seed treatment fungicides, however. Fungicides may alter the generic composition of communities of fungi which increase populations on substrates in soil (9), and the persistence of some fungicides is increased greatly when they are incorporated into soil at high concentrations (12, 20). Thus, the presence of a complex substrate in the form of a seed or seedpiece and the high concentration of a seed treatment fungicide around a seed or seedpiece

PAGE 10

indicate that the effects of fungicides on soil microorganisms when they are used as seed dressings are not comparable to effects observed with even incorporation into soil. An understanding of the biological effects of seed treatment fungicides in normal agricultural practice must come from investigations of effects on communities of microorganisms that are actually utilizing a seed or seedpiece as a substrate. A model system appropriate for such research should include a relatively large seed or seedpiece, a number of available treatment materials, a method of non-chemically minimizing the introduction of organisms with the seed or seedpiece, and methods of quantitatively evaluating the activities of different groups of organisms in a relatively natural situation. Galadiums ( Caladium hortulanum [Birdsey] ) are ornamental aroids that are grown for corms as a field crop in southcentral Florida. Preparation of propagative material involves heat treating the corms for control of root-knot nematode, cutting corms into seedpieces with a cutting machine, and dusting seedpieces with an absorptive dust which may contain fungicides. Mechanical cutting yields seedpieces that are fairly evenly distributed in weight between 0.2 and 5.0 g, and emergence and yield are closely correlated with seedpiece weight (Part 3 of this dissertation). The low emergence from small seedpieces results in as little as 25 % of the total seedpiece weight planted by a grower actually yielding plants . Galadiums are usually planted by dribbling seedpieces into a furrow, which places non-germinating seedpieces adjacent to germinating

PAGE 11

seedpieces. As soilborne pathogens of caladiums, such as Fythium myriotylum (26) and Fusarium solani (.15) t "belong to genera which contain plant-pathogenic members which have been reported to increase saprophytically on fresh plant material added to soil (6, 18), this non-germinating seedpiece material could foster increases in populations of pathogens resulting in increased disease on germinating plants (3i 33). This investigation was undertaken to (i) evaluate the effects of seedpiece treatment fungicides on a community of microorganisms in a situation similar to that encountered in normal agricultural practice, (ii) determine whether non-germinating seedpiece material can serve as a substrate for caladium pathogens in soil, and (iii) determine whether commonly-used fungicidal seedpiece dusts affect the pathogen-substrate relationship on non-germinating seedpieces. Materials and Methods Palmico muck soil was collected from an area of a commercial caladium field where stunting of plants had been associated with high populations of Pythium spp. and Fusarium spp. Analysis of the soil by the University of Florida Soil Science Department indicated nutrient contents of 840 ug NO-., 104 ug P, 80 ug K, 4?20 ug Ga, 488 ug Mg, 3?6 ug Al, 2.?2 ug Cu, and 45.6 ug Fe per gram of soil (dry weight). Soil pH was 4.8 (measurement obtained from a 1:2 suspension of soil in 0.01 M CaC^). Soil was sifted through a 4-mm sieve and mixed 20 min in a small cement mixer prior to use in the experiment.

PAGE 12

Caladium corms of the cultivar Frieda Hemple were obtained from a commercial grower. Corms were heat treated in deionized water at 50 C for 30 min (23). Treated corms were cut into 2.8-3.2 g seedpieces that either had at least one eye present and were intended to germinate (eyed), or had all eyes and epidermis shaved off and were intended to decompose (shaved). Seedpieces were dusted with one of the four following dusts currently used by growers: diatomaceous earth (Celite 209, Johns-Manville, Celite Division, Greenwood Plaza, Denver CO 80217); 6.9 % chloroneb, 6.9 % thiram, and 0.6 % streptomycin sulfate in diatomaceous earth (chloroneb mixture); 3.8 % benomyl, 7.3 % thiram, and 0.6 ^S streptomycin sulfate in diatomaceous earth (benomyl mixture); and 10 % captan in diatomaceous earth (captan). Treated seedpieces were planted in aluminum flats (45 X 28 X 5 cm) which contained 2.5 kg of soil at 42 % water content (lOO X weight of water/ wet weight of soil) and had been coated with epoxy resin (Resinglas Polyester Resin, Kristal Kraft Inc., 900 Fourth Street, Palmetto FL 33561 ) . Other flats were prepared which contained soil without seedpieces (non-amended soil) or soil into which 0.47 % of one of the four seedpiece dusts (100 X weight dust/ dry weight of soil) had been incorporated evenly. This concentration of seedpiece dust was equal to the average amount of dust adhering to seedpieces, divided by the average amount of soil in a sampling core, as described below. Flats were incubated in a growth room at 25-30 C with 12 hr light cycles (4,000 lx at the level of the plants).

PAGE 13

Every two days flats were watered to 53 % water content with deionized water and weed seedlings were pulled from the soil. Microbial populations in and around seedpieces were sampled at 0, 2, 4, 8, and 12 weeks after seedpieces were planted. A 5 . 6-cm-diameter piece of polyvinylchloride pipe was centered on the position of a seedpiece, plant, or soil sampling area, and a core which contained 32.0 + 1.7 g of soil (dry weight) was removed. Leaves of plants were cut off at the soil line and discarded. The soil core was comminuted with 100 ml of autoclaved, deionized water at low speed in a Waring blender for 1 min, and a diliution series in autoclaved water was prepared from the initial suspension. Appropriate dilutions were plated on the following five media for enumeration of microbial populations: for Fythium spp., 1? g Difco cornmeal agar, 300 mg vancomycin (Vancocin, Eli Lilly and Co., Indianapolis IN 46206), 100 mg pentachloronitrobenzene , and 5 mg pimaricin (Delvocid, Gist-Brocades, Delft, Holland) in 1 liter of water (19); for Fusarium spp., modified PCNB medium (21 ); for other fungi, 39 g Difco potato dextrose agar, 1 ml Turgitol NPX (Union Carbide Corp., New York NY 10017), 100 mg streptomycin sulfate (Eli Lilly and Co.), and 40 mg chlortetracycline HG1 (Sigma Chemical Co., St. Louis MO 63178) in 1 liter water (31); for bacteria and actinomycetes, 0.3% tryptic-soy agar (17); and for fluorescent Pseudomonas spp., King's medium B amended with cycloheximide , novobiocin, and penicillin (28). For enumeration of populations of Pythium spp., samples were suspended in 0.3 % agar amended with 3.68 g CaCl 2 '2H 2 per liter and then spread over the surface of solidified medium. For enumeration

PAGE 14

of populations of Fusarium spp. , samples were suspended in 0.1 % agar amended with 100 mg streptomycin sulfate and 40 mg chlortetracycline HG1 per liter and then spread over the surface of solidified medium. For enumeration of populations of other organisms, cooled medium was mixed with the sample in the petri dish. Populations of plant-parasitic and free-living nematodes were assayed in the 12week samples by the University of Florida Entomology and Nematology Department. The experiment was performed twice as described herein and two other times with procedural modifications. In this part of this dissertation, results are presented for populations of organisms enumerated in one performance. Results of the other identical performance are presented in Appendix 1 . In all performances populations of microorganisms were calculated as the mean of data from three replicate samples. Most population changes were similar in all performances of the experiment; however, some differences were observed and are noted in the results section.

PAGE 15

Results Initial populations of fungi and bacteria in soil are presented in Table 1 . Actinomycetes were recovered only sporadically after the 0-week sampling in treatments which contained seedpieces or plants. Incorporation of the fungicidal dusts into soil resulted in relative increases in populations of bacteria and relative decreases in populations of total fungi and Fusarium spp., compared with populations in non-amended soil (Fig. 1 ) . Incorporation of diatomaceous earth into soil did not significantly alter any assayed populations compared with non-amended soil. The only appreciable differences between the effects of the fungicidal dusts were a delay in the decrease of populations of Fusarium spp. with captan and a decrease in the magnitude of changes in populations of total fungi and bacteria with the chloroneb mixture. Populations of Pythium spp. were not altered significantly by any of the dusts when a seedpiece was not present. Populations of fluorescent Pseudomonas spp. were increased slightly, but nonsignificantly, in soil into which the fungicidal dusts had been incorporated. All eyed seedpieces that were assayed at or after the 4-week sampling had emerged and produced leaves. No shaved seedpieces were observed to produce plants throughout the course of the experiment. The effects of the fungicidal dusts on populations of microorganisms which were increasing on seedpieces were not related to changes in populations which followed incorporation of the fungicidal dusts into soil without the addition of seedpieces.

PAGE 16

Populations of Pythium spp. on seedpieces increased to maxma at 2 to k weeks and then gradually declined (Fig. 2-A, 2-B). Maximum populations were slightly higher on shaved than eyed seedpieces and were reduced greatly on seedpieces treated with any of the fungicidal dusts, although populations were consistantly highest on seedpieces dusted with the benomyl mixture. In another repetition of the experiment, populations of Pythium spp. were higher on eyed seedpieces dusted with the "benomyl mixture than on eyed seedpieces dusted with diatomaceous earth. Enumerated populations of Pythium spp. were predominantly P. spinosum and P. irregulare . Populations of Fusarium spp. increased more rapidly and attained higher maxima on shaved than on eyed seedpieces (Fig. 2-C, 2-D). Initial rates of population increase were less, "but maximum populations were similar on seedpieces dusted with the chloroneb mixture, compared with those dusted with diatomaceous earth. Populations of Fusarium spp. increased only slightly on eyed seedpieces dusted with the benomyl mixture or captan. On shaved seedpieces dusted with the benomyl mixture or captan, populations increased significantly over those present in nonamended soil, but were considerably less than populations on shaved seedpieces dusted with diatomaceous earth. Isolates of Fusarium spp. from samples which contained shaved seedpieces dusted with captan, the chloroneb mixture, or diatomaceous earth were inhibited similarly by benomyl, captan, or thiram in cornmeal agar; however, isolates from samples which contained seedpieces dusted with the benomyl mixture displayed a greater

PAGE 17

tolerance to benomyl than isolates from samples which contained seedpieces treated with the other dusts or non-amended soil. Investigations of this tolerance are presented in Appendix 5 of this dissertation. Enumerated populations of Fusarium spp. were predominantly F. solani with some F. oxysporum and F. roseum . Fungi other than Pythium spp. and Fusarium spp. increased to higher populations on shaved than on eyed seedpieces (Fig. 3). Compared with seedpieces dusted with diatomaceous earth, maximum total populations were decreased on fungicide-treated, eyed seedpieces and were delayed, but increased in magnitude, on fungicide -treated, shaved seedpieces. Fungicidal dusts altered the generic makeup of fungal communities which increased on seedpieces "but did not foster increases by any fungi that did not increase on shaved seedpieces dusted with diatomaceous earth (Table 2). A pathogenicity test of fungi observed to increase populations on caladium seedpieces indicated that none of the fungi significantly affected the growth of caladiums when compared to control treatments (Table 3)> However, growth of caladiums was significantly greater in soil infested with Trichoderma harzianum , Lasiodiplodia sp., or Verticillium sp. than in soil infested with Fusarium solani . A more detailed description of this test is presented in Appendix k of this dissertation. Population increases of total bacteria were greater on shaved than on eyed seedpieces (Fig. 2-E, 2-F). The fungicidal dusts did not greatly alter maximum populations on eyed

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10 seedpieces "but extended the duration of high populations on shaved seedpieces. Populations of fluorescent Pseudomonas spp. increased to maxima at 2 weeks and then rapidly declined (Fig. 2-G, 2-H). Maximum populations were greater on shaved than eyed seedpieces, "but in another repetition maxima were greater on eyed than on shaved seedpieces. In all repetitions 12-week populations were higher on eyed than on shaved seedpieces . Populations of fluorescent Pseudomonas spp. were much higher on both eyed and shaved seedpieces dusted with any of the fungicidal dusts than on corresponding seedpieces dusted with diatomaceous earth. Enumerated populations of fluorescent Pseudomonas spp. were predominantly non-pectolytic isolates of P. fluorescens and P. putida . Populations of plant-parasitic nematodes were low and not significantly different in samples which contained seedpieces compared with samples which contained non-amended soil. Populations of free-living nematodes were significantly higher (p= 0.05) by Duncan's multiple range test (30) on shaved seedpieces dusted with captan or the chloroneb mixture than in nonamended soil (Table 4).

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11 Table 1. Initial populations of fungi and bacteria in soil Organism Propagules/g soil 3Pythium spp. 3.4 ± 1.3 X 10 2 Fusarium spp. 2.0 ± 0.2 X 10^ Verticillium sp. 4.4 ± 4.6 X 10^ Lasiodiplodia sp. 2.2 ± 1.2 X 10^ Trichoderma spp. 8.3 ± 2.0 X 10^ Penicillium spp. 1.8 + 0.5 X 10^ Total fungi 4.5 ± 0.8 X 105 Fluorescent Pseudomonas spp. 4.8 ± 3.9 X 103 Actinomycetes 7.9 + 2.1 X 10 Bacteria 8.7 + 1.5 X 10? a Population ± standard deviation of the mean. Values are the mean of 27 samples .

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12 Ta"ble 2. Predominant fungi increasing on seedpieces in two experiments

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13 Table 3. Effect of planting seedpieces in soil infested with cornmealsand cultures of fungi on growth and emergence of caladium plants Incorporated

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14 Table 4, Populations of free-living nematodes at 12 weeks after planting of seedpieces Treatment Nematodes/g soil a Eyed-benomyl mixture 0,4 w Eyedcap tan 2,9 wx Eyed-chloroneb mixture J. 2 wx Eyed-diatomaceous earth 3-5 wxy Shaved-benomyl mixture 3«7 wxyz Shaved-captan 19.5 z Shaved-chloroneb mixture 17.2 yz Shaved-diatomaceous earth 9-5 xy z Soil 1.9 wx a Values are the mean of three samples. ^Means followed by the same letter are not significantly different (p=0.05) by Duncan's multiple range test performed on a square root + 4 transformation of data.

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15 2 4 8 WEEKS AFTER MIXING 2 4 8 WEEKS AFTER MIXING 8 -I I D

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Fig. 2(A to H). The effects of treatment of caladium seedpieces with fungicidal dusts on the population dynamics of Pythium spp. on A) eyed seedpieces and B) shaved seedpieces, Fusarium spp. on C) eyed seedpieces and D) shaved seedpieces, total bacteria on E) eyed seedpieces and F) shaved seedpieces, and fluorescent Fseudomonas spp. on G) eyed seedpieces and H) shaved seedpieces. Populations of microorganisms were assayed in non-amended soil ( ) and in soil into which seedpieces dusted with diatomaceous earth ( ) , the benomyl mixture ( — . ._ ), captan ( ) or the chloroneb mixture ( ) had been planted. Assays were performed at 0, 2, 4, 8, and 12 weeks after seedpieces were planted by plating dilutions on selective media. Each point represents the mean of three replicates.

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1? ^24 O w 20 IT>

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Fig. J(A to H). The population dynamics of genera of fungi, other than Pythium, which had increased populations on eyed caladium seedpieces dusted with A) diatomaceous earth, C) the be no my 1 mixture, E) captan, or G) the chloroneb mixture; and on shaved caladium seedpieces dusted with B) diatomaceous earth, D) the benomyl mixture, F) captan, or H) the chloroneb mixture. Populations of total fungi ( ), Trichoderma spp. ( ), Lasiodiplodia sp . ( ) , Verticillium sp. (— ) , and Fusarium spp. ( ) were assayed in soil samples which each contained a seedpiece. Assays were performed at 0, 2, k, 8, and 12 weeks after seedpieces were planted by plating dilutions on selective media. Each point represents the mean of three replicates.

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19 _,20 o

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20 Discussion The lack of similarity between the effects of the fungicidal dusts on microbial populations when the dusts were incorporated evenly into soil, compared with when they were used as seedpiece dressings, is indicative of differences in the processes operating in the two situations. Effects observed after incorporation of a fungicide into soil without the addition of a substrate can be interpreted as a killing of sensitive propagules with concomitant increases of nonsensitive organisms on the nutrients released (3^)« This is illustrated by the relative effects of the three fungicidal dusts in this experiment. The chloroneb mixture caused less decrease in fungal populations than did captan or the benomyl mixture and thus a smaller amount of nutrients was released for increase of bacteria (Fig. 1 ) . When a fungicide is used as a seedpiece dressing, effects are on actively growing, interacting populations rather than on noninteracting resting propagules. Both modification of microbial succession on seedpieces and initiation of displacement of fungicidesensitive organisms by fungicidetolerant organisms in soil involve the competitive displacement of some organisms by others; however, the different natures of the substrates involved (fresh plant material versus dead microorganisms) foster the existence of different microbial communities and thus limit the comparability of the two processes. This difference is illustrated by the observed behavior of populations of Fythium spp. and fluorescent Pseudomonas

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21 spp. in the experiment. The fungicidal dusts had little or no effect on these groups of organisms in soil without seedpieces, but populations of both were altered significantly on fungicide-treated seedpieces . These observations emphasize the necessity of evaluating the biological effects of pesticides in soil on both populations of resting propagules and on actively increasing populations . Although initial populations of microorganisms were similar to those reported in the literature (7, 17, 18, 21, 2?), populations in samples which contained seedpieces were increased greatly. This association of high populations with caladium seedpieces is indicative of the niche of the assayed organisms. The soil used in the experiment was from a well weeded caladium field; consequently, the predominant microorganisms were those able to increase populations on caladium tissue . This association of high populations with substrates is probably a major source of variation in the determination of microbial populations in randomly-collected soil samples. Succession on eyed seedpieces was qualitatively similar to succession on shaved seedpieces. In general, increases in populations of bacteria, Pythium spp., and Fusarium spp. were followed by increases in populations of saprophytic fungi. This placement of potentially parasitic fungi early in the succession is consistent with previous observations (ll, 1^-). Quantitatively, almost all organisms reached higher

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22 populations on shaved than eyed seedpieces. This is consistent with consideration that the entire amount of substrate in a shaved, decomposing seedpiece is available for the support of microbial growth, while with a germinating seedpiece, the substrate is partitioned between microorganisms and the developing plant. Correspondingly, reduced increases in populations of fungi on eyed seedpieces treated with the fungicidal dusts, compared with eyed seedpieces dusted with diatomaceous earth, can be viewed as resulting in a greater amount of substrate being partitioned to the plant. As growth of caladiums is proportional to seedpiece weight (Part 3 of this dissertation), increases in plant growth with the use of the fungicidal dusts could be attributed to this increased utilization of seedpiece nutrients by the plant . The three fungicidal dusts had different effects on the two pathogen-containing genera of fungi that were assayed. On eyed seedpieces, captan and the benomyl mixture gave good control of Fusarium spp. while captan and the chloroneb mixture gave good control of Pythium spp. On shaved seedpieces the relative activities of the fungicidal dusts against Pythium spp. and Fusarium spp. were similar to those on eyed seedpieces. However, all fungicidal dusts allowed some increase in populations of Pythium spp. and considerable increase in populations of Fusarium spp. on shaved seedpieces. This increase on decomposing seedpiece material, regardless of fungicidal seedpiece treatment,

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23 indicates that planting of small, non-germinating seedpieces adjacent to germinating seedpieces may have significant epidemiological consequences. Populations of pathogens could increase on non-germinating seedpiece material and cause an increased amount of disease because of higher inoculum density. Although the fungicidal dusts did not increase total populations of bacteria on eyed seedpieces, they did reduce total fungal populations and thus increased the ratio of bacteria to fungi. As fungicidal dusts increased the duration of increases in total bacterial populations on shaved seedpieces, the results of the experiment seem to indicate a shift in seedpiece colonization away from fungi and toward bacteria with the use of the fungicidal dusts . Increases in populations of fluorescent Fseudomonas spp. on seedpieces treated with any of the fungicidal dusts, compared with diatomaceous earth, are of interest in relation to work on the utilization of these organisms as biological seed and seedpiece treatments (k, 5)The importance of these increases in fungicide-related plant growth enhancement is open to conjecture. It would be of interest to evaluate the effects of inoculation of caladium seedpieces with fluorescent Pseudomonas spp. on plant growth, on the population dynamics of fluorescent Pseudomonas spp. , and on the microbial succession in general.

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24 The increase in populations of free-living nematodes on shaved seedpieces dusted with captan or the chloroneb mixture may indicate either that these dusts reduced competition by fungi with the nematodes or that the dusts reduced the effects of predacious fungi on the nematodes. The experimental system used in this study allowed the discrimination of relatively small differences between treatments in natural soil with a minimal amount of replication. The system, or one similar to it in which populations of microorganisms in and around a substrate are assayed over time, could be used in investigations of a number of aspects of microbial succession in soil. Mixtures of chemical and/or biological seedpiece treatment materials could be easily evaluated for effects on specific target organisms in a relatively natural situation. In particular, it would be of interest to determine the effect of initial inoculum density on the timing and extent of increases by organisms in the succession. Overall, the system provides the opportunity for study of a biological succession with a minimal expenditure of time and materials.

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PART 2 THE EFFECT OF SEEDPIECE TREATMENT WITH GAPTAN ON THE POPULATION DYNAMICS OF SOIL MICROORGANISMS ASSOCIATED WITH GERMINATING OR DECOMPOSING CALADIUM SEEDPLECES IN THE FIELD Introduction Although there has been a good deal of research into the effects of fungicides on microorganisms in soil under controlled conditions, attempts to follow the effects of fungicides on microbial populations in soil under field conditions have been much less common (l3)« Since the behavior of microorganisms may be very different in the field and under controlled conditions , it is important that field confirmation be obtained for observations of chemical influences on microorganisms made under controlled conditions. In Part 1 of this dissertation the effects of fungicidal seedpiece dusts on caladium seedpiece succession were investigated under controlled moisture and temperature conditions. The research presented in this part of the dissertation was undertaken to observe the effects of the fungicidal dusts used in the growth room experiments on microbial succession on caladium seedpieces in two contrasting areas of a commercial caladium field. 25

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26 Materials and Methods Two locations in a commercial caladium field that had contrasting crop histories were selected. Location 1 had supported a cover crop of Japanese barnyard millet ( Echinochloa crugalli var. frumentacea ) the previous growing season and crops of caladiums for 5 years previous to that. Mid-season stunting of plants associated with high populations of Pythium spp. and Fusarium spp. and high soil water content had been observed the last two seasons of caladium culture. Soil used in the growth room experiments was obtained from location 1 . Location 2 had supported continuous summer caladium culture and winter weed fallow for at least the previous 8 years. The grower considered caladium growth to be above average at location 2. In each location the experimental plot was prepared by removing soil from a 1.25 X 2.70 m area to a depth of 15 cm, sifting the soil through an 11 mm screen, and returning the soil to the excavated area. Caladium corms of the cultivar Frieda Hemple were obtained from a commercial grower. Corms were heat treated in deionized water at 50 C for 30 min (23). Treated corms were cut into 2.8-3.2 g seedpieces that either contained at least one eye and were intended to germinate (eyed) or had all eyes and epidermis shaved off and were intended to decompose (shaved). Seedpieces were dusted with either diatomaceous earth or 10 % captan in diatomaceous earth.

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27 Treated seedpieces were planted in the experimental plots on April 21, 1978, during the grower's normal planting period. Each plot was laid out in a randomized block design. Each of 17 rows contained one seedpiece in each of the four seedpiece treatments and an area where no seedpiece was planted. Seedpieces were planted 4-5 cm deep. The position of the seedpieces was marked by points of intersection of nylon string connected to stakes driven into the ground around the periphery of the plot. Each plot received 40 liters of water immediately after the seedpieces were planted and water as provided by the grower's normal irrigation schedule. Plots were weeded by hand just before each sampling. Samples for enumeration of microbial populations in and around seedpieces were collected from three rows in each plot at 1 day and 2, 4, 8, and 12 weeks after the seedpieces were planted. A 5.6-cm diameter piece of polyvinylchloride pipe was centered on the position of a seedpiece, plant, or soil sampling area and a core taken to a depth of 8 cm. Samples of 64.2 ± 6.8 g of soil (dry weight) per core were transported to the laboratory in plastic bags. One day after collection of samples, leaves were removed from plants and each sample was comminuted with 220 ml of boiled, deionized water in a Waring blender at low speed for 1 min. A dilution series in autoclaved water was prepared from the initial suspension. Appropriate dilutions were plated on the following five selective media

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28 for enumeration of microbial populations: for Fythium spp. , 17 g Difco cornmeal agar, 300 mg vancomycin, 100 mg pentachloronitrobenzene , and 5 mg pimericin in 1 liter of water (19); for Fusarium spp., modified PCNB medium (27); for other fungi, 39 g Difco potato dextrose agar, 1 ml Turgitol NPX, 100 mg streptomycin sulfate, and 40 mg chlortetracycline HG1 in 1 liter of water (3l); for bacteria and actinomycetes, 0.3 % tryptic-soy agar (17); and for fluorescent Pseudomonas spp., King's medium B amended with cycloheximide , novobiocin, and penicillin (28). For enumeration of populations of Pythium spp. , samples were suspended in 0.3 % agar amended with 3.68 g CaClp^HgO per liter and then spread over the surface of solidified medium. For enumeration of populations of Fusarium spp. , samples were suspended in 0.1 % agar amended with 100 mg streptomycin sulfate and 40 mg chlortetracycline HG1 per liter and then spread over the surface of solidified medium. For enumeration of other organisms , cooled medium was mixed with the sample in the petri dish.

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29 Results Initial populations of enumerated organisms in non-amended soil are presented in Table 5. Populations at location 1 were similar to those in the soil used in the growth room experiments. Populations of all enumerated organisms except one morphologically identifiable Penicillium biotype (herein refered to as Penicillium A) and Lasiodiplodia sp. were lower at location 2 than at location 1. Pythium spp. were not recovered from location 2 at any sampling. At location 1 Pythium spp. increased to highest maximum populations on shaved seedpieces dusted with diatomaceous earth (Fig. k-E) , Compared with diatomaceous earth, populations on both eyed and shaved seedpieces treated with captan were lower at all sampling times except the 12-week sampling, when average populations of Pythium spp. were slightly higher on eyed seedpieces dusted with captan than on either eyed seedpieces dusted with diatomaceous earth or shaved seedpieces dusted with captan. Populations of Pythium spp. were lower on captan-dusted seedpieces than in nonamended soil at the 2-week and 4-week samplings. Populations of Fusarium spp. attained higher maxima at location 2 than at location 1 in all treatments except on shaved seedpieces dusted with diatomaceous earth (Fig. k-A, k— B). Maxima were greater on shaved than eyed seedpieces. On shaved seedpieces captan delayed increases and reduced maximum populations but allowed considerable increase compared

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30 with non-amended soil. On eyed seedpieces captan greatly reduced maximum populations of Fusarium spp. compared with diatomaceous earth but allowed some increase in populations. Populations of fungi other than Pythium spp. , Fusarium spp. , and Penicillium A reached higher maxima on shaved : seedpieces than on eyed seedpieces at both locations (Fig. 7, 8). Population maxima of these fungi were consistently higher at location 2 than at location 1 . On eyed seedpieces at both locations, captan prevented increases in populations of Mucorales, fostered increases in populations of Trichoderma spp., and reduced total fungal populations. On shaved seedpieces at location 1 , captan prevented increases in populations of Mucorales and allowed only small increases in populations of other fungi. On shaved seedpieces at location 2, captan delayed increases in total fungal populations , prevented increases in populations of Mucorales , and fostered increases in populations of Lasiodiplodia sp. and Trichoderma spp . Penicillium A displayed behavior dissimilar to that of other fungi. This biotype could be distinguished on the amended potato dextrose agar used for recovery by a white, rather than green, colony underside. Populations of Penicillium A were greater on eyed than on shaved seedpieces at most samplings and were an order of magnitude higher than populations of all other fungi combined at both locations at the 8-week sampling (Fig. 6)

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31 The population dynamics of bacteria were similar at both locations (Fig. k-C, k-D), Maximum populations were higher on shaved than on eyed seedpieces. Populations of bacteria were greater on eyed seedpieces treated with captan than on eyed seedpieces treated with diatomaceous earth at all samplings except the 12-week sampling at location i. On shaved seedpieces captan delayed increases at both locations. Populations of total bacteria could not be enumerated at the 2-week sampling because of a procedural error. On seedpieces at location 1 , populations of fluorescent Pseudomonas spp. reached maxima at 2 weeks and then rapidly declined (Fig. 4F), Populations were similar in all samples which contained seedpieces at all sampling times except the 12week sampling, when populations were higher on eyed than on shaved seedpieces. At location 2, populations of fluorescent Pseudomonas spp. could not be followed due to overgrowth of the selective medium by non-fluorescent bacteria.

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32 Table 5. Initial populations of fungi and bacteria in soil Propagules/g soil c Organism

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33 2 4 8 WEEKS AFTER PLANTING 2 8 WEEKS AFTER PLANTING 2 4 8 WEEKS AFTER PLANTING Fig. h(A to F). The effects of viability of seedpieces and the use of captan as a seedpiece dust on the population dynamics of Fusarium spp. at A) location 1 and B) location 2, total bacteria at C) location 1 and D) location 2, E) Pythium spp. at location 1, and F) fluorescent Pseudomonas spp. at location 1. Populations of microorganisms were assayed in non-amended soil ( ), on eyed seedpieces dusted with diatomaceous earth ( ) or captan ( )f and on shaved seedpieces dusted with diatomaceous earth ( ) or captan ( ). Populations of total bacteria were assayed at 0, k, 8, and 12 weeks after seedpieces were planted. Populations of all other organisms were assayed at 0, 2, k-, 8, and 12 weeks after seedpieces were planted. Each point represents the mean of three replicates.

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34 Fig. S-vA^). The population dynamics of Penicillium A on caladium seedpieces at A) location 1 and B) location 2. Populations were assayed in non-amended soil ( ), on eyed seedpieces dusted with diatomaceous earth ( ) or captan ( ), and on shaved seedpieces dusted with diatomaceous earth ( ) or captan ( ). Populations were assayed at 0, 2, k, 8, and 12 weeks after planting of seedpieces. Each point represents the mean of three replicates.

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35 WEEKS AFTER PLANTING AFTER PLANTING Fig. 6-(A,B). Populations of total fungi ( ), Penicillium S PP( )i and Trichoderma spp. ( ) in non-amended soil at A) location 1 and B) location 2. Populations were assayed at 0, 2, 4, 8, and 12 weeks after initiation of the experiment. Each point represents the mean of three replicates.

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36

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37 2 4 8 WEEKS AFTER PLANTING AFTER PLANTING .

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Discussion The following trends were observed in both the field and growth room experiments: (i) succession on eyed seedpieces was qualitatively similar to succession on shaved seedpieces; (ii) increases in populations of Pythium spp., Fusarium spp., and bacteria preceded increases in populations of other fungi; and (iii) almost all organisms reached higher populations on shaved than on eyed seedpieces. The much higher populations of Penicillium A on eyed than on shaved seedpieces represent a significant anomaly. This organism may have utilized either decaying shoot tissue or corm epidermis as its substrate; confirmation of any particular hypothesis would require further experimentation. A number of aspects of the succession varied with the plot location in the field. The absence of populations of Pythium spp. at location 2 illustrates the necessity of adequate initial populations for the increase of an organism and may indicate the existence of some natural control mechanism at that location. Inability to recover populations of fluorescent Pseudomonas spp. at location 2 is probably more of a reflection of limitations in the efficiency of the selective medium than of a lack of increase by these organisms at that location. The overgrowth of the medium by non-fluorescent bacteria at location 2 and not at location 1 does indicate the existence of qualitative differences in the makeup of bacterial populations at the two locations, however. Although initial populations of

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39 organisms other than Pythium spp. and fluorescent Pseudo mo nas spp. tended to be higher at location 1, maximum populations of these organisms were either similar at both locations or were higher at location 2. This observation indicates that factors other than initial inoculum density are important in determining the amount of increase by these organisms in the observed locations. As in the growth room experiments, captan, when compared with diatomaceous earth, (i) reduced increases by Pythium spp. and Fusarium spp. but still allowed considerable increases in populations of these organisms on shaved seedpieces, (ii) reduced increases in populations of total fungi on eyed seedpieces, (iii) delayed increases in populations of total fungi on shaved seedpieces, (iv) fostered increases in populations of Trichoderma spp. and Lasiodiplodia sp., (v) tended to foster increases in populations of total bacteria, and (vi) did not foster increases by any organisms that did not increase on shaved seedpieces dusted with diatomaceous earth and planted in the same soil. In contrast with the growth room experiments, captan did not foster increases in populations of fluorescent Pseudomonas spp. in the field experiment. Both this anomaly and the depressed 2-week and 4-week populations of Pythium spp. on captandusted seedpieces may have been due to changes in populations during the relatively long period of time between the collection of soil samples and the population assays.

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In general, comparison of the results of the field and growth room experiments indicates that observations of the behavior of the experimental system under controlled temperature and moisture conditions may be extrapolated cautiously to the field. This correlation supports the veracity of observations made in the growth room experiments and indicates that the experimental system, or one similar to it, could be used to evaluate the effects of other parameters on microbial populations under controlled conditions with some confidence in the relevance of observations to field situations.

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PART 3 GROWTH, YIELD, AND EMERGENCE OF CALADIUMS IN RELATION TO SEEDPIECE WEIGHT Introduction Until recent years caladium seedpieces were cut by hand, yielding relatively large seedpieces that contained at least two eyes (29). At the present time, however, many growers cut seedpieces with locally-manufactured cutting machines, which cut corms into seedpieces in a random manner. This method of cutting produces seedpieces of a range of sizes and yields many seedpieces which do not contain eyes, and thus do not germinate. Experiments concerning the fate of this non-germinating seedpiece material are presented in Parts 1 and 2 of this dissertation. The research presented in this part of the dissertation was undertaken to provide background information for the design and interpretation of other experiments and to provide information on the relationship of seedpiece weight to emergence, yield, and value of caladiums that would be of practical use to caladium growers. The investigation consisted of two experiments conducted in the greenhouse and one experiment conducted in the field. hi

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42 Materials and Methods In all of the experiments, caladium corms were obtained from a commercial grower. All corms were heat treated at 50 G for 30 min before being cut into seedpieces. Caladium corms of cultivars Frieda Hemple and White Wing were used in the greenhouse and field experiments, respectively. In greenhouse experiment 1 , undersized seedpieces that were produced in the process of cutting larger seedpieces for use in another experiment were graded into the following four weight-classes: 0.2-0.5 g, 0.5-1.25 g, 1.25-2.0 g, and 2.0-3.0 g. All seedpieces contained at least some epidermis. The seedpieces were planted in aluminum flats (45 X 28 X 5 cm) which contained Palmico muck soil that had been collected, sifted, and mixed as described in Part 1 of this dissertation. The seedpieces in each weight-class were arranged evenly over the surface of 730 g of soil (dry weight) that was evenly distributed over the bottom of a flat and were covered with 460 g of soil (dry weight). Flats were incubated in a non-airconditioned greenhouse. Temperatures varied between approximately 15 G at night and 40 G during the day. Every 2 to 3 days the flats were watered with tap water and weed seedlings were pulled from the soil. Emergence of plants with leaves was recorded weekly. Plants were harvested and weighed 9 weeks after the seedpieces were planted . In greenhouse experiment 2 corms were cut into seedpieces of the same weight-classes as those used in experiment 1 .

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^3 All seedpieces were intentionally cut to contain at least one eye. Palmico muck soil was sifted through a 4-mm sieve, autoclaved 2 hr on each of two successive days and aged in a greenhouse for 1 month. Seedpieces were planted in 26 X 26 X k cm plastic flats. In each flat 3^0 g of soil (dry weight) were distributed evenly over the bottom of the flat, nine seedpieces were arranged in the flat in a 3X3 matrix, and the seedpieces were covered with 3^0 g of soil (dry weight). Two flats were prepared for each weight-class of seedpieces as described above, except that for the 2.0-3.0-g weight-class only three seedpieces were planted in one of the flats. Flats were incubated in an airconditioned greenhouse at 30-36 C and were watered daily with tap water. Emergence of plants with leaves was recorded weekly. Plants were harvested and weighed 6 weeks after the seedpieces were planted. In the field experiment seedpieces that had been cut by a seedpiece-cutting machine and dusted with 1 % captan in diatomaceous earth were sorted into the following four weight-classes: 0.2-0.? g, 0.7-1.4 g, 1.4-2.1 g, and 2.1-3.0 g. Other samples of seedpieces from the same cutting run were sorted completely into six weight-classes in order to determine the weight-class distribution of seedpieces in the grower's planting material. Seedpieces were planted in a non-fumigated area of a commercial caladium field that had been identified by the grower as producing plants of average to above average yield. Beds which were 1.25 m wide

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1)4 were prepared "by the grower. Seedpieces were planted in five replicate "blocks in each of two beds with weight-class plots randomized within each block. In each plot four seedpieces were planted 6 cm deep in each of two rows running perpendicular to the length of the bed. Rows within a plot were spaced 20 cm apart and rows in adjacent plots were spaced 25 cm apart. Within each row seedpieces were spaced 35 cm apart. The plots received normal care by the grower, which included overhead irrigation, side dressing with fertilizer, and application of the herbicides paraquat and alachlor. Emergence of plants identifiable to cultivar was recorded at 4, 8 12, and 30 weeks after seedpieces were planted. Plants were harvested 30 weeks after the seedpieces were planted. The below-ground portion of each plant was weighed 1 day after harvesting, and dry weights of all corms produced in each plot were determined after drying at 15-25 C for 1 month.

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^5 Results Although plants emerged more rapidly and were larger at harvest in greenhouse experiment 2 than in greenhouse experiment 1 , plant weight was directly related to seedpiece weight in both experiments (Tables 6, 7)Linear regression of average fresh weight of plants as a function of average seedpiece weight indicated: Fresh weight = 0.086 + 2.62 (Seedpiece weight), with r = 0.9985» ^ov data from experiment 1; and Fresh weight =1.29 + 3.23 (Seedpiece weight), with r = 0.9985 1 for data from experiment 2. Values of r are significant at the 1 % level for both experiments. Value was determined by partitioning dry weight data from the field experiment into the number of corms in each plot in each of four weight-classes, multiplying the number of corms in each weight-class by the current market price of corms of the corresponding size, and adding the values of corms in each plot. Parameters used in the calculations of value are presented in Table 10. The relationship between size and weight of corms was determined by linear regression of the average of largest and smallest corm diameters as a function of the cube root of corm weight, using measurements of 68 corms as a data base. In the field experiment emergence was directly related to seedpiece weight (Table 8), and all yield parameters based on plots or harvested plants were significantly related to seedpiece weight (Table 9). However, value per

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46 total weight of seedpiece material planted was not significantly related to seedpiece weight, and value per dry weight of harvested corns was significantly highest (p = 0.05) for the lowest seedpiece weight (Table 11 ). Seedpieces cut by the grower's seedpiece-cutting machine were fairly evenly distributed in the weight-classes (Table 1 2) .

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47 Table 6 . Emergence and fresh weight of caladium plants from seedpieces in four weight-classes in greenhouse experiment 1 Seedpiece weight-class Parameter

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48 Table 7. Emergence and fresh weight of caladium plants from seedpieces In four weight-classes in greenhouse experiment 2 Seedpiece weight-class Parameter

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49 Table 8. Emergence of caladium plants from seedpieces in four weight-classes in the field experiment Seedpiece weight-class Time after planting 0.2-0

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50 Table 9. Yield of caladium plants from seedpieces in four weight-classes in the field experiment Seedpiece weight-class Parameter

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51 a H hi) =tfc c o ft

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52 Table 11 . Value of corms produced by caladium plants from seedpieces in four weight-classes in the field experiment Seedpiece weight-class Parameter 0.2-0.7g o.7-1.4g 1.4-2.ig 2.1-3. 0§ Value per plot ($) a 0.33 w b 0.68 x 1.29 y 1.65 z Value per weight of seedpieces (0/g) 9.59 w Value per yield of corms (0/g) 1.31 w d 8.95 w

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53 3^ =tfc

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54 Discussion Growth of plants was significantly correlated with seedpiece weight in both the greenhouse and field experiments, although growth parameters for the two heaviest seedpiece weight-classes were not significantly different from each other in greenhouse experiment 2 and in the field experiment. This correlation indicates that standardization of seedpiece weight is an important factor in reducing variation in experiments involving evaluation of growth of caladiums . The low emergence of plants from seedpieces in the two lightest weight-classes in the field experiment (Table 8), combined with the distribution of seedpiece weights in the grower's planting material (Table 12), indicates that small seedpieces contribute a disproportionately large share of the amount of non-germinating seedpiece material planted. The implications of this non-germinating seedpiece material in the epidemiology of soil borne pathogens of caladiums are dealt with in Parts 1 and 2 and Appendix 3 of this dissertation. The implications of the observations as far as commercial production of caladiums is concerned are complex. Value was significantly related to seedpiece weight when the yields from the same number of seedpieces in each weight-class were compared; however, value per gram of seedpieces planted was not related to seedpiece weight, and value per gram of harvested corm was highest for the lightest seedpieces. This

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55 anomaly is the result of the pricing structure of caladiums, which is based on diameter rather than weight of corms. This pricing structure has the effect of making smaller corms more valuable per gram than larger corms (Table 10 ). If non-germinating seedpiece material is not a factor in disease epidemiology, then whether or not it would be economically advantageous for a grower to plant seedpieces of a selected weight would depend on costs associated with planting, harvesting, sorting, and various other procedures involved in caladium culture. An analysis of such costs is beyond the scope of this dissertation.

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APPENDIX 1 REPETITION OF EXPERIMENTS PRESENTED IN PART 1 Introduction Research described in this Appendix is presented in substantiation of research reported in Part 1 of this dissertation. Data are presented from the first performance of the growth room experiment in which the effects of fungicidal dusts on the population dynamics of microorganisms associated with caladium seedpieces were evaluated (performance 1 ) and from a final , partial performance of that experiment (performance 3). Results from the second performance of the experiment (performance 2) are presented in Part 1 of this dissertation. Materials and Methods Procedures utilized in the investigation were identical to those described in Part 1 of this dissertation, except that soil was collected at different times and populations of nematodes were not enumerated. Performence 1 was conducted exactly as described for performance 2 in Part 1 of this dissertation. Performance 3 consisted of only the treatments which involved the dusting of seedpieces with diatomaceous earth and the control treatment of non-amended soil. 56

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57 Results Initial populations of most organisms were similar in the different performances (Table 13). However, populations of Trichoderma spp. and Fythium spp. were considerably higher in performance 1 than in the other performances, populations of Fusarium spp. were considerably lower in performance 1 than in the other performances, and populations of fluorescent Pseudomonas spp. were highest in performance 2, considerably lower in performance 1 , and lowest in performance 3In performance 1, 12-week samples of Fusarium spp. and 2-week samples of Pythium spp. were lost due to error, 4week samples of fungi which were recovered on potato dextrose agar were altered by inadvertent use of potato dextrose agar manufactured by Baltimore Biological Laboratories Gockeysville MD 21030) rather than Difco, and populations of Pythium spp. in soil amended with the fungicidal dusts without the addition of seedpieces could not be enumerated with confidence due to the use of inappropriate dilutions. In performance 3 12-week samples of bacteria were lost due to error. In performance 1 the addition of the fungicidal dusts to soil without the addition of seedpieces resulted in changes in microbial populations that were largely similar to those observed in performance 2 (Fig. 9). Effects observed in performance 1 that differed from those observed in performance 2 were (i) greater differences between the effects

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of the different fungicidal dusts on populations of Fusarium spp.; (ii) an apparent effect of captan on recovery of Fusarium spp., as indicated by depression of perceived 0-week populations with that dust; and (iii) a different ranking of the effects of the fungicidal dusts on populations of "bacteria. On seedpieces in performance 1 the population dynamics of Pythium spp. were similar to those observed in performance 2, except that populations were considerably higher on eyed seedpieces dusted with the benomyl mixture than on eyed seedpieces dusted with diatomaceous earth (Fig. 10-A, 10-B). A slow-growing biotype of P. irregulare accounted for 86 % and 73 % of total populations of Pythium spp. on eyed seedpieces dusted with the benomyl mixture at the 8-week and 12-week samplings, respectively. This biotype was not recovered from any other treatment throughout the course of the experiment. In performance 3 the population dynamics of Pythium spp. were similar to those observed on seedpieces dusted with diatomaceous earth in the other performances (Fig. 12-A). Populations of Fusarium spp. reached much higher maxima on shaved than on eyed seedpieces in all performances, although there was a considerable amount of variation in actual populations from performance to performance (Fig. 10-G, 10-D, 12-B). On eyed seedpieces the ranking of the effects of the fungicidal dusts was similar in performances 1 and 2. On shaved seedpieces the chloroneb mixture gave better control

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59 and the benomyl mixture gave poorer control of increases in populations of Fusarium spp. in performance 1 than in performance 2. Major differences in the behavior of fungi other than Pythium spp. and Fusarium spp. in the different performances were (i) a lack of increase in populations of Penicillium spp. in performance 2, (ii) increases in populations of Verticillium sp. in performance 2 "but not in performances 1 and 3, (iii) increases in populations of Penicillium A in performance 3 that were similar to those observed in the field experiment, and (iv) higher initial populations of Penicillium A on eyed seedpieces than on shaved seedpieces or in non-amended soil in performance 3 (Fig. 11, 12-E, 13). The effects of the fungicidal dusts on specific genera of fungi were similar in performances 1 and 2 (Table 2). In performance 1 populations of Penicillium spp. increased only on shaved seedpieces dusted with diatomaceous earth or captan . The population dynamics of total bacteria were similar in all performances, except that the duration of maxima was extended on eyed seedpieces dusted with the benomyl mixture or the chloroneb mixture in performance 1 but not in performance 2 (Fig. 10-E, 10-F). Population maxima of fluorescent Pseudomonas spp. were increased on seedpieces dusted with all of the fungicidal dusts in both performances 1 and 2 (Fig. 10-G, 10-H). Maxima tended to be greater on eyed seedpieces than on

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60 shaved seedpieces in performance 1 and greater on shaved seedpieces than on eyed seedpieces in performance 2. In performance 3 the maximum population was attained but was of greater magnitude on eyed than on shaved seedpieces (Fig. 12-D). In all performances 12-week populations of fluorescent Pseudomonas spp. were greater on eyed seedpieces than on shaved seedpieces.

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61 x 3MO C\i x

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62 AFTER MIXING 24 a WEEKS AFTER MIXING WEEKS AFTER MIXING Fig. 9(A to D). The effects, in performance 1, of the incorporation of fungicidal dusts into soil on populations of A) total fungi, B) total bacteria, C) Fusarium spp., and D) fluorescent Fseudomonas spp. Populations of microorganisms were assayed in non-amended soil ( ) and in soil into which the "benomyl mixture ( ), captan ( ), or the chloroneb mixture ( ) had been incorporated. Assays were performed at 0, 2, h, 8, and 12 weeks after incorporation for all organisms, except for Pythium spp. at 2 weeks and Fusarium spp. at 12 weeks. Each point represents the mean of three replicates.

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Fig. 10(A to H). The effects, in performance 1, of treatment of caladium seedpieces with fugicidal dusts on the population dynamics of Pythium spp. on A) eyed seedpieces and B) shaved seedpieces, Fusarium spp. on C) eyed seedpieces and D) shaved seedpieces, total bacteria on E) eyed seedpieces and F) shaved seedpieces, and fluorescent Pseudomonas spp. on G) eyed seedpieces and H) shaved seedpieces. Populations of microorganisms were assayed in non-amended soil ( ) and in soil into which seedpieces dusted with diatomaceous earth ( ) , the benomyl mixture ( ), captan ( ), or the chloroneb mixture ( ) had been planted. Assays were performed at 0, 2, k, 8, and 12 weeks after seedpieces were planted by plating dilutions on selective media. Each point represents the mean of three replicates.

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Gk 2 4 8 WEEKS AFTER PLANTING D

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Fig. 11(A to H) . The population dynamics, in performance 1, of genera of fungi, other than Pythium , which had increased populations on eyed caladium seedpieces dusted with A) diatomaceous earth, C) the "benomyl mixture, E) captan, or G) the chloroneb mixture; and on shaved caladium seedpieces dusted with B) diatomaceous earth, D) the benomyl mixture, F) captan, or H) the chloroneb mixture. Populations of total fungi ( ), Trichoderma spp. ( ), Mucorales ( ), Penicillium spp. ( ), Lasiodiplodia sp. ( ), and Fusarium spp. ( ) were assayed in soil samples which each contained a seedpiece. Assays were performed at 0, 2, 4, 8, and 12 weeks after seedpieces were planted by plating dilutions on selective media. Each point represents the mean of three replicates.

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WEEKS AFTER PLANTING

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67 24 16 8 n

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WEEKS AFTER PLANTING WEEKS AFTER PLANTING Fig. 13-(A,B). The population dynamics, in performance 3, of fungi other than Pythium spp. and Penicillium A which had increased populations on A) eyed and B) shaved seedpieces dusted with diatomaceous earth. Populations of total fungi ( ), Trichoderma spp. ( ), Mucorales ( ), Penicillium spp. ( ^f, Lasiodiplodia sp. ( ), and Fusarium spp. ( ) were assayed. Assays were performed at 0, 2, 4, 8, and 12 weeks after seedpieces were planted. Each point represents the mean of three replicates.

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69 Discussion The similarity of the repetitions of the growth room experiments was comparahle to the similarity of the field experiment and the growth room experiments. Certain fungi increased populations on seedpieces in some performances but not in others; however, increases in populations of members of the basic community, which consisted of bacteria, fluorescent Pseudomonas spp., Pythium spp., Fusarium spp., Lasiodj-plodia sp. , and Trichoderma spp., were observed on shaved seedpieces dusted with distomaceous earth in all performances. Observed variation in the genera of fungi which increased on seedpieces in the different repetitions of the growth room experiments and in the field experiment may have been due to differences in initial populations and/or differences in non-controlled experimental parameters , such as temperature fluctuations in the growth room, differences in soil compaction due to differences in soil moisture content during mixing, and differences in soil moisture content prior to commencement of the experiments. The importance of differences in experimental parameters, rather than intitial populations, in causing experimenttoexperiment variation is supported by the consistent presence of a relatively high proportion of Peniclllium spp. in all initial populations of fungi but not in populations of fungi which increased on seedpieces in performance 2. Involvement of Verticillium sp. in the seedpiece succession in performance 2, but not in performances \ and J, may indicate that this

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?o organism displaced Peniciliium spp. in performance 2. Whatever the cause of experimenttoexperiment variation, it is significant that increases on seedpieces by fungi which increased early in the succession were more consistent than increases on seedpieces by fungi that increased later in the succession. Increases in populations of Peniciliium A in performance 3> although not of the magnitude of those observed in the field experiment , are of interest in relation to hypotheses concerning the substrate utilized by this organism. The observation that populations were highest on eyed seedpieces at the initial sampling indicates that higher maximum populations on eyed seedpieces may have been due to higher initial inoculum density rather than the utilization of a particular substrate . Overall, the similarity of the repetitions substantiates the veracity of observations made on the behavior of the experimental system and supports the utilization of the system, or one similar to it, in further studies of the behavior of microorganisms in soil.

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APPENDIX 2 PHYSICAL DISTRIBUTION OF MICROBIAL POPULATIONS INCREASING ON CALADIUM PLANTS Introduction Although populations of microorganisms were assayed in and around caladium plants and seedpieces in Parts 1 and 2 of this dissertation, no attempt was made to determine the site of observed population increases. The research presented in this appendix was undertaken to determine whether increasing populations of microorganisms were located (i) in soil adjacent to the seedpiece or plant, (ii) on the surface of the seedpiece or plant, or (iii) within the tissues of the seedpiece or plant. Although these three sites could not "be differentiated on shaved seedpieces because of problems in the retrieval of the disintegrating seedpiece, populations at the sites were assayed separately on plants growing from eyed seedpieces. Materials and Methods Soil was collected from a commercial caladium field at different times and from different areas in the field for use in three performances of the experiment. In all performances caladium corms were heat treated and cut into 71

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72 eyed seedpleces as described in Part 1 of this dissertation. Seedpieces were dusted with diatomaceous earth, and dusted seedpieces were planted and incubated as described in Part 1 of this dissertation. Populations of microorganisms associated with emerged plants were assayed at 4, 6, and 9 weeks after seedpieces were planted in performances 1, 2, and 3» respectively. Leaves were cut off at the soil line and soil cores were taken as described in Part 1 of this dissertation. Samples which contained plants were partitioned by use of the following sequence of procedures: (i) the complete soil core was placed in a Waring blender, the plant was removed from the soil, and 100 ml of autoclaved, deionized water was added to the soil which remained in the blender (adjacent soil partition); (il) the plant was placed in a beaker which contained 100 ml of autoclaved, deionized water, the beaker was shaken gently for 3 min, and the plant was removed (cormsphere partition); and (iii) the plant was placed in 100 ml of autoclaved, deionized water (corm partition). Each partition was comminuted at low speed in the Waring blender for 1 min. A dilution series was prepared in autoclaved, deionized water from the initial suspension. Appropriate dilutions were plated on selective media as described in Part 1 of this dissertation. In performance 1 samples were plated on all five selective media. In performances 2 and 3 samples were plated on all of the media except the medium selective for fluorescent

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73 Pseudomonas spp. Populations were determined from three, two, and three replicate plants in performances 1, 2, and 3. respectively.

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7^ Results In order to calculate total populations in the three partitions of the plant samples (Tables 14, 1 _5 • 16), populations in the corm and cormsphere partitions were calculated as if they represented populations in the amount of soil present in the adjacent soil partition, and then the populations in the three partitions were added together. This procedure yielded a population equal to that which would have been perceived had the plant sample not been partitioned. Populations are presented only for those organisms which had a total population in the three partitions of the plant sample that was significantly higher than their population in non-amended soil by comparison using a one-tailed t-test at p = 0.10 (30) • I n performance 1 a number of organisms or groups of organisms had increased populations on plants (Table 14); however, in performance 2 only Pythium spp. , Fusarium spp., Lasiodiplodia sp. , and bacteria had increased populations (Table 15) » and in performance 3 only Pythium spp . , Fusarium spp . , bacteria , and an unidentified fungus had higher populations in the samples which contained plants compared with non-amended soil (Table 16) The percentage of the increased population in each partition was calculated to facilitate comparison of populations of organisms in the three partitions of the plant samples in the three performances. For the adjacent

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75 partition, this value was calculated as % population in adjacent soil = 100 (Population in adjacent soil Population in soil) , Total population in plant samplesPopulation in soil where "total population in plant samples" and "population in soil" are those values presented in Tables 13, \k, and 15. For the cormsphere and corm partitions, this value was calculated as % population in cormsphere or corm = 100 (Population in cormsphere or corm) . Total population in plant samples Population in soil The percentages of populations in the three partitions differed in the three performances of the experiment (Tables 17, 18, 19).

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Table 14. Populations of fungi and bacteria in non-amended soil and in soil containing caladium plants in performance 1 76 Organism Propagules/g soil c Soil' 4 4 4 4 Plant Pythium spp. 88 Fusarium spp . 1.3X10 Lasiodiplodia sp . 1.2X10' Trichoderma spp. 2.2 X 10' Penicillium A 4.0 X 10' Other Penicillium spp. 3.1 X 10' Nonidentified fungi 6.7 X 10' Fluorescent Pseudomonas spp. 0.3 X 10 Bacteria 5.9 X lo' 270

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7? Table 15. Populations of fungi and "bacteria in non-amended soil and in soil containing caladium plants in performance 2 Propagules/g soil c Organism Soil D Plant c Pythium spp. 420 800 Fusarium spp. 3.0 X 10^ 20.2 X 10^ Lasiodiplodia sp. 1.5 X 10 63.4 X 10 Bacteria 8.5 X 10 7 39.3 X 10 7 a Values are the mean of two samples. ^Population in soil without plant. Mean cumulative population in the soil, cormsphere, and corm partitions of samples of soil which contained plants.

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78 Table l6. Populations of fungi and bacteria in non-amended soil and in soil containing caladium plants in performance 3 Propagules/g soil a Organism Soil° Plant c Pythium spp. Fusarium spp. Fungus A Bacteria a Values are the mean of three samples . '-Population in soil without plant. c Mean cumulative population in the soil, cormsphere, and corm partitions of samples of soil which contained plants. 11.0 X 10 2

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Table 17. Percentage of increased populations of fungi and "bacteria in plant-sample partitions in performance 1 79

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Table 1 8 . Percentage of increased populations of fungi and "bacteria in plant-sample partitions in performance 2 80 Organism

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Table 19. Percentage of increased populations of fungi and bacteria in plant-sample partitions in performance 3 81 Organism Adjacent soil Partition Cormsphere Corm {%) (*) Pythium spp. Fusarium spp, Fungus A Bacteria 45 8 18 15 28 31 15 15 27 61 69 70

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82 Discussion Most assayed populations were in the range of those enumerated in the population dynamics experiments; however, Pythium spp. populations were considerably lower and higher than in other experiments in performances 1 and 3, respectively (Tables 13, i4, 15, 16). The relatively small increases in populations of organisms in performance 3 may have been the result of the soil being somewhat dryer in that performance because of lower bulk density due to initial mixing at a lower moisture content and/or the result of the sampling being performed subsequent to increase and decline of populations. Whatever the reason for the small differences between populations in the soil and plant samples, data from performance 3 are less reliable than data from the other performances. The percentage of increased populations of Pythium spp. in the corm decreased in the order of performances 1, 2, and 3« Although the different performances are not strictly comparable , the fact that the percentage of the increased population in the corm was lower in samplings of older plants may indicate that colonization by Pythium spp. occurs primarily in tissue exposed at the cut surface of the corm, and that propagules formed in that tissue are subsequently shed as the tissue disintegrates and is sloughed off. This scenario is consistent with the observation that

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83 populations of Pythium spp. tend to reach high levels early in the succession and then stabilize or decline slowly (Fig. 2-A, 2-B). Percentages of increased populations of Fusarium spp. were fairly evenly distributed among the partitions in performance 1 , but tended to be concentrated in the corm in performances 2 and J. This behavior may indicate an initial increase on substrates diffusing from the cut surface of the corm, followed by growth into the corm. Increased populations of Trichoderma spp. were highest in adjacent soil and the cormsphere in the one performance in which population increases were observed. This may indicate that early increase by Trichoderma spp. on seedpieces is on substrates diffusing from the cut surface of the corm, rather than on the corm itself. Comparison of the sites of increase of Trichoderma spp . , Pythium spp . , and Fusarium spp. indicates that Trichoderma spp. may be better biological control agents of Fusarium spp., which have a similar site of increase, than of Pythium spp., which have a different site of increase. Increased populations of Penicillium A were highest in the corm and absent in adjacent soil. If this is the same organism that was observed in the field experiment (Fig5), then the site of its increase must be assumed to be the corm itself and/or associated below-ground shoot tissues.

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84 Increased populations of other Penicillium spp. were fairly evenly distributed among the partitions. This may indicate that these organisms have a pattern of increase similar to that of Fusarium spp., although increases in populations were small and were observed only in performance 1 . In both performances 1 and 2, increased populations of Lasiodiplodia sp. were highest in the cormsphere but of considerable magnitude in both the corm and adjacent soil. The hypothesis that this distribution indicates growth into the corm following that of Fusarium spp. is consistent with the late increases in populations of Lasiodiplodia sp. that were observed in the population dynamics experiments (Fig. 3). Fungus A was observed to increase only in the one performance of the experiment in which Lasiodiplodia sp. was not observed to increase. This may indicate that fungus A displaced Lasiodiplodia sp. under whatever circumstances were responsible for the small increases in populations of Pythium spp. and Fusarium spp. that were observed in that performance . Although of small magnitude, increases of a mixture of nonidentified fungi in performance 1 indicate that fungi other than the enumerated genera may increase on caladium seedpieces under certain circumstances. In the one performance of the experiment in which populations of fluorescent Pseudomonas spp. were assayed, increased populations of these organisms were present almost exclusively

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85 in the corm. This site of increase was similar to that of Pythium spp. Coupled with the observation that these two groups of organisms increase at the same time in the succession, this similarity of distribution may indicate that fluorescent Pseudomonas spp. could be effective biological control agents of Pythium spp. However, the tight association of populations of fluorescent Pseudomonas spp. with the corm may indicate that increased populations are derived from bacteria carried internally in the seedpiece, rather than from bacteria present in soil, and thus may indicate that populations could be difficult to manipulate by inoculation. Percentages of increased populations of total bacteria were highest in adjacent soil in performance 1 and highest in the corm in performances 2 and 3. This distribution may indicate that early increases in populations of total bacteria are on substrates diffusing from the cut surface of the corm, while later increases are in the corm, possibly associated with ingressive growth by fungi. Overall, evaluation of the data indicates early colonization of the seedpiece by Pythium spp. and fluorescent Pseudomonas spp. with concomitant increases of Trichoderma spp. and total bacteria on substrates diffusing from the cut surface. Later, Fusarium spp. make intrusive growth into the seedpiece, accompanied by bacteria and some other fungi and followed by Lasiodiplodia sp. This scenario is consistent with observations made in the population dynamics experiments.

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86 The most accurate characterization of a microbial succession, such as the one observed in these experiments, would probably be derived from a coordinated study of the physical distribution and population dynamics of organisms which increase populations on the substrate. Although such coordination was not present in this research, evaluation of data on the physical distribution of organisms still served to substantiate observations on the population dynamics of those organisms.

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APPENDIX 3 THE EFFECT OF FUNGICIDAL SEEDPIECE DUSTS ON GROWTH, YIELD, EMERGENCE, AND VALUE OF CALADIUMS Introduction Research on the effects of fungicidal seedpiece dusts on populations of microorganisms associated with caladium seedpieces is described in Parts 1 and 2 and Appendices 1 and 2 of this dissertation. In this Appendix the effects of the benomyl mixture, captan, and the chloroneh mixture on growth of caladiums under a variety of environmental conditions are reported. Results are presented from (i) a factorial experiment in which the effects of the seedpiece dusts .and the presence of an adjacent, decomposing seedpiece on growth of caladiums were evaluated; (ii) a growth room experiment in which the effects of the seedpiece dusts on growth of caladiums in autoclaved soil was evaluated ; and (iii) a field experiment in which the effects of the seedpiece dusts on emergence , yield , and value of caladiums were evaluated . Materials and Methods In all experiments caladium corms were obtained from a commercial grower. All corms were heat treated at 50 C for 30 min before being cut into seedpieces. In the 87

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greenhouse and growth room experiments, corms of cultivar Frieda Hemple were used. In the field experiment corms of cultivar White Wing were used. In the greenhouse experiment corms were cut into 2.02 ± 0.13 g seedpieces that each contained at least one eye (eyed) and 1.03 ± 0.08 g seedpieces that had all eyes and epidermis shaved off (shaved). Seedpieces were dusted with the four seedpiece dusts described in Part 1 of this dissertation. Soil was collected, sifted and mixed as described in Part 1 of this dissertation. Seedpieces were planted in autoclaved 10cm clay pots. Materials were sequentially placed in each pot as follows: 73 g of soil (dry weight), either an eyed seedpiece or an eyed seedpiece and a shaved seedpiece spaced 5 mm apart, and 55 g of soil (dry weight). Ten pots were prepared for each of the eight combinations of seedpiece dust and seedpiece condition. Pots were incubated in a non-airconditioned greenhouse in which temperatures ranged from approximately 15 to 40 G. Every 2 days pots were watered with tap water and weed seedlings were pulled from the soil. Emergence of plants with leaves was recorded daily. Plants were harvested and fresh weights were determined 12 weeks after seedpieces were planted. Dry weights of plants were determined after drying 2 weeks at 25-30 G. In the growth room experiment corms were cut into 1.02 ± 0.6 g seedpieces that each contained at least one eye. Seedpieces were dusted with the four seedpiece dusts described in Part 1 of this dissertation. Before use in

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the experiment, Palmico muck soil was sifted through a 4mm sieve, autoclaved 2 hr on each of two successive days, incubated under irrigation with tap water in a greenhouse for 1 week, mixed in a small cement mixer for 20 min, placed in plastic bags, and incubated at 25-30 G for 2 months. One day before seedpieces were planted, soil was assayed for populations of Pythium spp., Fusarium spp., and other fungi as described in Part 1 of this dissertation. A seedpiece was placed on the surface of 35 g soil (dry weight) in a 10 -cm clay pot and covered with 73 g of soil (dry weight). For treatments with the benomyl mixture, captan, the chloroneb mixture, and diatomaceous earth seedpiece dusts, 8, 6, 6, and 8 pots were prepared, respectively. Pots were incubated in a growth room at 25-30 G with 12 hr of light (4,000 Ix at the level of the plants). Pots were watered and emergence of plants with leaves was recorded daily. Plants were harvested and weighed 9 weeks after seedpieces were planted. Dry weights of plants were determined after drying 6 weeks at 20-30 C. In the field experiment corms were cut into 2.5-3.5 g seedpieces and were dusted with the four seedpiece dusts described in Part 1 of this dissertation. Seedpieces were planted in a non-fumigated area of a commercial caladium field that had been identified by the grower as producing plants of average to above average yield. Plot layout and care were the same as described for the seedpiece weight experiment that was conducted in the field (Part 3 of this

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90 dissertation). Emergence of plants identifiable to cultivar was recorded at k, 8, and 12 weeks after seedpieces were planted. Plants were harvested 30 weeks after seedpieces were planted. Dry weights of all corms produced in each plot were determined after drying at 15-25 G for 1 month. Value of harvested corms was determined as described in Part 3 of this dissertation.

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91 Results Initial populations of microorganisms in the soil used in the greenhouse experiment were similar to those enumerated in other experiments (Tables 1 , 20 ) . Propagules of Pythium spp. and Fusarium spp. were not recovered from the autoclaved soil used in the growth room experiment, and populations of fungi in this soil were predominantly Penicillium A (Table 20 ). The effects of the fungicidal dusts in the greenhouse experiment were as follows: (i) time of emergence of plants from eyed seedpieces planted alone was significantly decreased by the cap tan treatment when compared with the other fungicidal dusts; (ii) time of emergence of plants from eyed seedpieces planted with an adjacent decomposing seedpiece was significantly decreased by the captan treatment compared with diatomaceous earth; (iii) fresh weight of plants from eyed seedpieces planted alone was significantly greater for the chloroneb mixture and captan compared with diatomaceous earth; (iv) fresh weight of plants from eyed seedpieces planted with an adjacent shaved seedpiece was significantly greater for the chloroneb mixture compared with diatomaceous earth or the benomyl mixture; (v) seedpiece dust did not significantly affect dry weight of plants from seedpieces planted alone; and (vi) dry weight of plants from eyed seedpieces planted with an adjacent shaved seedpiece was significantly lower with diatomaceous earth than with captan or the chloroneb mixture (Table 20). The only

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92 significant effect of planting a shaved seedpiece adjacent to an eyed seedpiece was a decrease in fresh weight in plants from seedpieces dusted with diatomaceous earth. In the growth room experiment individual seedpiece dusts did not affect emergence rate, fresh weight, or dry weight of plants (Table 22). However, when results from the three treatments with the fungicidal dusts were bulked together and compared with those from the diatomaceous earth treatment, fungicidal dusts significantly decreased (p = 0.05) dry weight, but not fresh weight or emergence rate. In the field experiment emergence was similar with all af the dusts (Table 23), and neither yield nor value was affected significantly by seedpiece dust (Table 2+ ) . Bulking of data from the three fungicidal dust treatments did not affect measures of significance.

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93 in the greenhouse

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94 Table 2i . Effect of fungicidal dusts and presence of an adjacent decomposing

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95 Table 22. Effect of fungicidal dusts on caladium emergence and growth in autoclaved soil in the growth room experiment Seedpiece dusi Parameter Benomyl Captan Chloroneb Diatomaceous mixture mixture earth # seedpieces planted

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Ta"ble 23. Effect of fungicidal dusts on emergence of caladiums in the field experiment

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97 Table 24. Effect of fungicidal dusts on yield and value of caladiums in the field experiment Seedpiece dust Parameter Benemy 1 Gaptan Ghloronet DiatomaceouE mixture mixture earth Corm weight per plot (g) a

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Discussion Even though dusting of seedpieces with the fungicidal dusts did not significantly affect yield or value in the field experiment, the observation that the presence of a decomposing seedpiece affected the growth of an adjacent plant only when seedpieces were dusted with diatomaceous earth indicates that the use of a fungicidal seedpiece dust is probably to the advantage of a grower who cuts seedpieces mechanically. In addition, the use of a fungicidal dust may be of benefit in increasing plant growth under more stressful conditions than those present in the field experiment. The small amount of phytotoxicity that the fungicidal dusts exhibited in the growth room experiment in autoclaved soil was apparently balanced by pathogen pressure even under the relatively good growing conditions that were present in the field experiment. Although a decision on the best dust for general usage should be based on the results of a larger number of field tests, the results of these experiments indicate that captan would probably be the best choice as far as price, growth of plants, and minimal phytotoxicity are concerned. The fungicidal dusts apparently nullified the detrimental effect of an adjacent decomposing seedpiece on plant growth even though populations of Pythium spp. and Fusarium spp. increased on decomposing seedpieces treated with the fungicidal dusts. This may have been due to a restriction

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99 of population increases to the interior of the decomposing seedpiece by the fungicidal dusts. However, a decision on the veracity of this or other possible explanations would require further experimentation.

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APPENDIX 4 GROWTH OF CALADIUMS IN SOIL INFESTED WITH FUNGI OBSERVED TO INCREASE POPULATIONS ON CALADIUM SEEDPLEGES Introduction In the course of investigations on microbial succession on caladium seedpieces, a number of fungi that have not been reported previously to be associated with caladiums were observed to increase their populations on seedpieces (Parts 1 and 2 of this dissertation). The research presented in this appendix was undertaken to evaluate the effects of these fungi on growth of caladiums in comparison with a previously reported pathogen of caladiums, Fusarium solani (l5). Results are presented from a single experiment conducted under controlled conditions of light and temperature. A preliminary experiment was initiated but abandoned due to equipment failure. Materials and Methods The following fungi were used in the experiment: four isolates of Fusarium solani (Mart.) Appel and Wr. emend. Snyd. and Hans.; two isolates of a nonidentified species of Lasiodiplodia Griffon and Maubl . ; four isolates of a non-identified species of Penicillium Link; one isolate of 100

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101 Pythium aohanidermatum (Edson) Fitzpatrick; one isolate of Pythium irregulare Buisman; three isolates of Pythium spinosum Sawada and Chen; four isolates of Trichoderma harzianum Rifai; and two isolates of a nonidentified species of Verticillium Nees. One isolate of F, solani , designated F. solani 1 , was obtained from the Florida Type Culture Collection and had been initially isolated from a caladium corm by J. F. Knauss. All other fungi were isolated by mass transfer from isolated colonies on the appropriate selective medium (part 1 of this dissertation). Samples from which the fungi were isolated were of soil and caladium seedpieces in which elevated populations of the appropriate fungus had been observed. Palmico muck soil was sifted through a 4-mm sieve, autoclaved 2 hr on each of two successive days, incubated under irrigation with tap water for 1 week in a greenhouse, mixed in a cement mixer, placed in plastic bags, and incubated 2 months at 25-30 C, Inoculum was grown for 3 weeks in 250 ml flasks which each contained 5 g cornmeal (Dixie Lilly enriched, white, stone ground cornmeal, Martha White Foods Inc. , Nashville TN 37202), 50 g washed sand, and 20 ml deionized water. Each flask of inoculum was used to Infest 510 g of soil (dry weight). Caladium corms of the cultivar Frieda Hemple were heat treated for 30 min at 50 C and cut into 1 . 02 + . 07 g

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102 seedpieces that each contained at least one eye. One seedpiece was placed on the surface of 72 g of infested soil (dry weight) in an autoclaved 10-cm-diameter clay pot, and the seedpiece was covered with ^ g of infested soil (dry weight). Four pots were prepared per isolate, except for two isolates of F. solani for which eight pots were prepared for each isolate. Control pots were prepared which contained soil mixed with sterile cornmeal-sand medium and non-amended soil. Pots were incubated in a growth room at 25-30 G with 12 hr of light (4,000 lx at the level of the plants). Pots were watered daily with tap water. Emergence of plants with visible leaves was recorded daily. Plants were harvested and fresh weights determined 10 weeks after the seedpieces were planted. Dry weights of plants were determined after drying at 20-30 C for 6 weeks. Populations of fungi in the same lot of seedpiece material as that used in the experiment were assayed at k and 24 hr after heat treatment. Ten-gram samples of cut seedpieces that either contained eyes and epidermis or had all eyes and epidermis shaved off were comminuted with 100 ml of autoclaved, deionized water for 1 min at low speed in a Waring blender. Appropriate dilutions were plated on selective media used to assay populations of Pythium spp., Fusarium spp., and other fungi as described in Part 1 of this dissertation.

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103 Populations of fungi in infested soil were determined 1 week after soil was infested and seedpieces were planted. Three grams of soil (dry weight) were removed from the periphery of a pot and comminuted with 100 ml autoclaved, deionized water for 1 min at low speed in a Waring "blender. Appropriate dilutions were plated on the medium selective for the fungus with which the soil in the samule had been infested.

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104 Results Populations of Pythlum spp. were not recovered from assayed seedpiecesj however, seedpieces contained appreciable populations of Fusarium spp., Trichoderma spp., Penicillium spp., Mucorales, and other fungi (Table 25). Populations of fungi appeared to be concentrated in the eyes and epidermis, and populations of Fusarium spp., Trichoderma spp., and Mucorales apparently increased on eyed seedpieces during the 20 hr between samplings. Populations of fungi recovered from autoclaved, nonamended soil were composed of Penicillium spp., Trichoderma spp., and some other fungi (Table 26). Infestation of soil with a fungus greatly increased its population in all cases except that of Pythium aphanidermatum . which was not recovered from soil into which it had been incorporated (Tables 26, 27). Addition of sterile cornmeal-sand medium apparently provided a substrate for those fungi present in non-infested soil. Although no significant alteration of any growth parameter was observed to result from planting of seedpieces in soil infested with any of the fungi compared with non-infested controls; infestation of soil with Trichoderma harzianum , Lasiodiplodia sp. , or Verticillium sp. significantly increased (p = 0.05) total fresh weight, fresh bottom weight, and total dry weight of plants compared with infestation of soil with Fusarium solani (Table 28 ) . The effect on

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105 plant growth of infestation of soil with F. solani 1 was similar to the effect observed with infestation with other F. solani isolates.

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Tahle 25. Populations of fungi in caladium seedpieces at 4 hours and 24 hours after heat treatment 106 Propagules X lO^/g

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107 m a 3 Ch -p id 3 (1) ttf) si q H

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Tahle 2?. Populations of Fythium spp. and Fusarium solani in infested soil Organism Propagules/g soil e Pythium spinosum 12.6 X 10^ P. irregulare 19. 8 X 10-^ P. aphanidermatum Not recovered Fusarium solani isolate 1 1.5 X 10 F. solani isolate 2 1.7 X 10 Population in soil infested with the assayed organism one week after infestation. For Pythium spp. the population is from a single 3-g soil sample. For F. solani the population is the mean of two 3-g soil samples.

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109 Table 28. Effect of planting seedpieces in soil infested with fungi on growth and emergence of caladium plants Incorporated organism or amendment # seedpieces % emergence Days to planted at harvest emergence a Trichoderma harzianum Lasiodiplodia sp. Verticillium sp. Pythium irregulare P. aphan idermatum Cornmeal-sand medium Penicillium sp. Non-amended soil Pythium spinosum Fusarium solani 2 F. solani 1 16 8 8 4 4 8 16 8 12 16 94 w D

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110 Table 28 -extended. Mean fresh Mean fresh Mean total Mean total weight of weight of fresh weight (g) dry weight (g) corm and roots (g) leaves (g) 5.53 v

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Ill Discussion A major problem inherent in the performance of experiments such as this one, in which pathogenicity of a number of different soil fungi is compared, is the diversity of environmental conditions necessary for pathogenesis by soil fungi. Because of this diversity, any conclusive determination of pathogenicity should be based on experiments conducted under conditions of temperature and moisture optimum for disease development and with inoculum densities similar to those present in natural situations. However, the experiment described in this appendix was conducted primarily to provide background information for the interpretation of population dynamics experiments (Parts 1 and 2 of this dissertation); consequently, temperature and moisture regimes that were probably sub-optimal for disease development but which were similar to those used in the population dynamics experiments were used. The lack of a statistically significant effect on plant growth as a result of infestation of soil with any of the fungi compared with non-infested soil may have been due to the inherent non-pathogenicity of the fungi or to aspects of the experimental design such as environmental conditions or the small number of plants used in each treatment. Because of this lack of significance, any conclusions based on the experiment are purely speculative and must await confirmation. However, such speculation may be of use in the identification of areas to be concentrated on in future experiments.

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112 The non-significant increases in plant growth in soil infested with T. harzianum , Lasiodiplodia sp. , or Verticillium sp. may have been due to antagonistic action of these fungi against pathogens present in the experiment. In particular, it could be speculated that these fungi interacted antagonistically with populations of Fusarium spp. present in the seedpieces. This hypothesis is supported by the observation that plant growth increased significantly in soil infested with T. harzianum , lasiodiplodia sp. , or Verticillium sp. compared with soil infested with F. solani. However, the hypothesis is contradicted by the results of the experiment in which the effects of the fungicidal seedpiece dusts on plant growth were investigated in the growth room (Table 21 ) , which was similar to the pathogenicity experiment and was conducted concurrently. If an increase in plant growth in soil infested with T. harzianum , Lasiodiplodia sp. , or Verticillium sp. were due to antagonism to plant pathogens, it would be expected that plant growth would increase in response to the fungicidal dusts in a similar situation. The observation that plant growth decreased in treatments containing fungicidal dusts indicates that the growth increases of plants exposed to the three fungi may have been due to some other factor, such as production of hormones or induction of resistance mechanisms. The observation that the least plant growth occurred in soil infested with F. solani , which has been previously reported to be pathogenic when introduced into caladium

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113 corms (15), my indicate that soilborne populations of this organism are pathogenic. If this is true, then the behavior of populations of Fusarium spp. in the population dynamics experiments may be taken to be that of a pathogen, and the magnitude of increases in populations of Fusarium spp. observed after seedpiece treatment with the different dusts may indicate the effectiveness of the dusts in limiting pathogenesis,

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APPENDIX 5 TOLERANCE OF FUSARIUM SPP. AND IASIODIPLODIA SP. TO BENOMYL Introduction In the course of population dynamics experiments described in Part 1 of this dissertation, it was observed that populations of Fusarium spp. increased to high levels on shaved seedpieces even when the seedpieces had been treated with fungicidal dusts. The experiments described in this appendix were undertaken initially to investigate the possibility that populations which were observed to increase on seedpieces treated with the fungicidal dusts were differentially tolerant to the dust which they had been exposed to in soil. After the initial experiment indicated the existence of tolerance to benomyl in populations of Fusarium spp., further experiments were performed to evaluate this tolerance quantitatively and to investigate tolerance to benomyl in populations of Lasiodiplodia sp. The experiments presented in this appendix were performed in conjunction with other larger experiments and are consequently fragmentary. However, it is hoped that data from them will be of use in the design of future experiments and in the 114

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115 evaluation of the utility of the benomyl mixture as a caladium seedpiece dust. Materials and Methods In all experiments procedures and media used to assay populations of fungi were identical to those described in Part 1 of this dissertation. All fungicides were added to media after autoclaving as suspensions of wettable powder in autoclaved water. In experiment 1 isolates of Fusarium spp. were obtained from dilution plates of modified PGNB medium (22) that had been used in the 4-week sampling of population dynamics experiment treatments which contained shaved seedpieces or non-amended soil (Part 1 of this dissertation). Mycelia and spores were transferred from the center of distinct colonies on dilution plates to sterile plates of modified PGNB medium. Ten isolates were obtained from each of three replicates of each treatment. After 1 week of growth on modified PGNB medium, 8-mm-diameter plugs were transfered from the periphery of each colony to petri dishes which contained non-amended Difco cornmeal agar or cornmeal agar that had been amended with either 1 ug benomyl/imL, 10 ug captan/ml, or 10 ug thiram/ml. The average colony diameter was recorded after incubation at 25 C for 8 days. After measurement of colony diameters, ten isolates that were apparently tolerant to benomyl were transfered from cornmeal agar amended with 1 ug benomyl/ml. to petri dishes which contained non-amended

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116 commeal agar or cornmeal agar amended with 1 jxg, 5 jig, or 50 ug benomyl per ml of medium. The average of two measurements of the distance between the plug and the perimeter of the colony was recorded after incubation for 3 days at 25 G. In experiment 2 appropriate dilutions of three samples of soil in which seedpieces dusted with the benomyl mixture had been planted were plated on modified PCNB medium and modified PGNB medium amended with 5 ug benomyl/ml. Twenty isolates were transfered to sterile plates of modified PGNB medium from each of the six medium-sample combinations. After 1 week of growth, 8-mm-diameter plugs were transfered from the periphery of each colony to petri dishes which contained either non-amended cornmeal agar or cornmeal agar amended with 5 ug benomyl/ml. The average of colony diameters measured in two perpendicular directions was recorded after incubation for 4 days at 25 C. In experiment 3 samples from a single dilution of each of six soil samples in which shaved seedpieces dusted with the benomyl mixture had been planted were plated on modified PGNB medium and modified PCNB medium amended with 2.5i 5, 10, or 20 ug benomyl per ml of medium. In experiment k appropriate dilutions of soil samples which contained seedpieces dusted with the benomyl mixture were plated on modified PCNB medium and on modified PCNB medium amended with 10 ug bemomyl per ml of medium.

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117 Samples were plated from soil which contained either eyed or shaved seedpieces at k, 8, and 12 weeks after the seedpieces were planted. In experiment 5 populations of Fusarium spp. in soil amended with 0.47 % (100 X weight of dust/ dry weight of soil) of the benomyl mixture and in non-amended soil were assayed at 0, 2, 4, 8, and 12 weeks after amendment by plating dilutions on modified PCNB medium and modified PCNB medium amended with 10 ug "benomyl/ml. In experiment 6 isolates of Lasiodiplodia sp. were obtained from dilution plates of potato dextrose agar plus Turgitol and antibiotics that had been used to sample soil which contained seedpieces dusted with either captan or the benomyl mixture. Isolates were transfered to petri dishes which contained non-amended cornmeal agar and cornmeal agar amended with 5 ug benomyl/ml. The diameter of colonies was determined after incubation for k days at 25 C. In experiment 7 appropriate dilutions of soil samples in which seedpieces dusted with the benomyl mixture or diatomaceous earth had been planted were plated on potato dextrose agar plus Turgitol and antibiotics and the same medium amended with 5 >ig benomyl/ml. In experiment 8 growth of caladium plants in soil infested with isolates of F. solani and Lasiodiplodia sp. that were either tolerant or sensitive to benomyl was evaluated. Procedures used were identical to those described in Appendix 4 of this dissertation.

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118 Results In experiment 1 the only Fusarium spp. isolates which displayed a differential sensitivity to a fungicide were recovered from soil which contained shaved seedpieces dusted with the benomyl mixture (Table 29). Nine of the ten isolates transfered to cornmeal agar amended with 0, 1 » 5, or 50 ug benomyl per ml of medium were able to grow in the presence of 50 ug benomyl per ml of medium. The isolate which was inhibited the most in the presence of 1 ug benomyl per ml of medium did not grow in the presence of 50 ug benomyl/ml. In experiment 2 the percentage of benomyl-tolerant Fusarium spp. isolates determined by comparison of recovery on amended and non-amended modified FGNB medium was consistently lower than the percentage determined by transfer of isolates to amended or non-amended cornmeal agar (Table 31 ) . There was no consistent relation between colony diameter on non-amended cornmeal agar and the source or benomyltolerance of isolates. In experiment 3 recovery of populations of Fusarium spp. was not significantly different (p = 0.05) on media amended with 5, 10, or 20 ug benomyl/ml in all samples (Table 32). In samples 3 and 4 recovery was significantly less at 10 ug benomyl per ml of medium than at 2.5 ug per ml of medium.

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119 In experiment k the percentage of "benomyl-tolerant Fusarium spp. isolates on eyed seedpieces apparently increased with time after the seedpieces were planted. On shaved seedpieces there was no apparent effect of time on the percentage of benomyl-tolerant isolates (Table 33). There was a consistent correlation between the total population of Fusarium spp. and the percentage of benomyltolerant isolates among samples of soil which contained the same type of seedpiece and were assayed at the same time (Fig. 14). In experiment 5 there was no significant change in populations of benomyl tolerant Fusarium spp. in either soil amended with the benomyl mixture or non-amended soil over the course of the experiment (Table 34 ) . In experiment 6 all Lasiodiplodia sp. isolates from samples which contained seedpieces dusted with the benomyl mixture were tolerant to 5 ug benomyl per ml of medium, while none of the isolates from the sample which contained a seedpiece dusted with captan was tolerant (Table 35). In experiment 7 benomyl-tolerant Lasiodiplodia sp. isolates were not recovered from samples which contained eyed seedpieces dusted with the benomyl mixture at the 8-week sampling (Table 36). In all other samples of soil which contained a seedpiece dusted with the benomyl mixture, recovery of Lasiodiplodia sp. was higher on medium amended with 5 ug benomyl/ml than on non-amended medium. Some

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120 benomyl-tolerant Lasiodlplodia sp. isolates were recovered from a sample of soil in which a seedpiece dusted with diatomaceous earth had been planted. In experiment 8 the effect on caladium growth of soil infestation with benomyl-tolerant isolates of F. solani or Lasiodiplodia sp. was similar to the effect observed with infestation of soil with benomyl-sensitive isolates of the same fungus (Table 37) .

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Table 29. Relative growth of Fusarium spp. isolates from samples containing shaved seedpieces treated with fungicidal dusts on cornmeal agar amended with fungicides compared with non-amended cornmeal agar 121

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122 Table 30. Growth of Fusarium spp. isolates identified as being tolerant to 1 ug benomyl/ml cornmeal agar on cornmeal agar amended with , 1 , 5 , and 50 ug "benomyl/ml

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123 ft

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124 medium amended with

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125 Table 33. Total and benomyltolerant populations of Fusarium spp, in samples containing seedpieces dusted with the benomyl mixture Propagules X 10^/g soil Week a

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126 -p

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12? Table 35. Relative growth of Lasiodiplodia sp. Isolates on cornmeal agar amended with 5 jig benomyl/ml and non-amended cornmeal agar Sample Seedpiece dust Relative growth (%) 1

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\& 128 3 bD

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129 x: a -P n) •H Is jC -P w

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500 400 130 -J O CO O x 300 CO UJ -j O 2 O cc CL 4 Weeko o 8 Week 4 + 12 Week* • 200 i ..-2 /'"'* •. 5 100 2 3 CC < CO .6 ++• X 20 40 60 80 (%) BEN0MYL TOLERANT Fig. Ik. The relationship between populations of Fusarium spp. and abundance of benomyl-tolerant isolates among samples which' contained seedpieces dusted with the benomyl mixture. Regression lines 1, 2, and 3 are associated with points which represent data from samples which contained shaved seedpieces; and regression lines 4, 5, and 6 are associated with points which represent data from samples which contained eyed seedpieces. Total population of Fusarium spp. in soil was assayed on modified PCNB medium. Percent benomyl-tolerant isolates was calculated as 100 X (population enumerated on modified PGNB medium amended with 10 ug benomyl/ml-fpopulation enumerated on modified PCNB medium) . 100

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131 Discussion The results of the experiments indicate that benomyltolerant isolates of Fusarium spp. comprise a large portion of the total population of Fusarium spp. on shaved seedpieces dusted with the benomyl mixture, that benomyltolerant isolates make up a smaller portion of the total population on eyed seedpieces dusted with the benomyl mixture, and that the proportion of benomyl-tolerant isolates is related to the magnitude of the total population. These observations could indicate that either induction of benomyl-tolerance is dependent on population increases or population increases are dependent on the presence of benomyltolerant isolates. Richardson (25) found that tolerance to benomyl in F. solani was induced rapidly by a single exposure of an actively-growing isolate to concentrations of benomyl in solid media that were greater than about 1 ug/ml. This finding indicates that if F. solani makes up the predominant portion of populations of Fusarium spp. assayed in the caladium experiments, then induction of benomyl-tolerance should be related to increases in total populations, which was the situation observed. However, such induction should not affect the fact that benomyl-tolerant isolates should have a competitive advantage over benomylsensitive isolates in the presence of benomyl. It is possible that on caladium seedpieces the dependence of the rate of induction of benomyl-tolerance on population increases by benomyl-sensitive isolates and the dependence of population increases by benomyl-tolerant isolates on

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132 the rate of induction of benomyl-tolerance could both contribute to the observed relationship between population and the percentage of benomyl-tolerant isolates. Thus, the delay in increases in populations of Fusarium spp. on eyed seedpieces compared with shaved seedpieces could be due to initial growth of benomyl-sensitive isolates and subsequent induction of benomyl-tolerance being fostered by the greater amount of substrate available from a shaved seedpiece. A simple explanation for the observations might be that benomyl-tolerant populations which increase on seedpieces are derived from benomyl-tolerant populations observed to be present in soil to which no seedpiece or benomyl had been added (Table 6). Such an explanation would imply that induction of tolerance such as observed by Richardson (25) is not important on caladium seedpieces. The relationship between population and percentage of tolerant isolates would be due to differential increase by benomyltolerant isolates, and the delay of increases in populations on eyed seedpieces compared with shaved seedpieces would be analogous to the effect of horizontal resistance in delaying the increase of virulent isolates of a pathogen on a vertically resistant plant variety (32). The viability of an eyed seedpiece could thus be considered to reduce the rate of increase of the fungus just as horizontal resistance reduces the rate of increase of a disease.

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133 A major inconsistency between Richardson's (25) findings and the observations made in this investigation is that whereas Richardson reported that benomyl-tolerant isolates grew at only about one-half the rate of benomylsensitive isolates on both non-amended media and media amended with benomyl, in the caladium experiments both benomyl-tolerant and benomyl sensitive isolates grew at similar rates on non-amended cornmeal agar (Table 3). This could indicate that benomyl-tolerant populations of Fusarium spp. observed in this investigation were not P. solani . However, three benomyl-tolerant isolates which were identified as F. solani by spore morphology did not display reduced growth on non-amended medium, and all benomyltolerant isolates observed in the experiments had overall colony morphologies similar to those of isolates positively identified as F. solani . Alternatively, it is possible that the benomyl-tolerant isolates observed in this study possessed a different type of tolerance than that described by Richardson; however, any decision on this possibility must await further experiments. Increases in the proportion of Lasiodiplodia sp. populations that was tolerant to benomyl on seedpieces dusted with the benomyl mixture were similar to those observed with Fusarium spp. Whatever the mechanism of these increases, these observations indicate that populations of all fungi which greatly increase their populations on seedpieces dusted with the benomyl mixture

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134 are comprised of benomyl-tolerant variants of organisms which increase on seedpieces that are not treated with fungicides. Whatever the cause of increased populations of benomyltolerant Fusarium spp. isolates in these experiments, their existence makes the use of the benomyl mixture on caladium seedpieces of questionable advisability. Although the benomyl mixture apparently gave excellent control of increases in populations of Fusarium spp. on eyed seedpieces in the population dynamics experiments, the high populations of benomyl-tolerant Fusarium spp. that occurred on shaved seedpieces could result in a build-up of benomyl-tolerant populations in soil or in corms. A conclusive decision on recommendation of the benomyl mixture for use by growers would require further experimentation; however, on the basis of available data it probably should be recommended that growers alternate the use of the benomyl mixture with the use of other fungicidal dusts.

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LITERATURE CITED 1. AGNIHOTHI, V. P. 1971. Persistence of captan and its effects on microflora, respiration and nitrification of a forest nursery soil. Can. J. Micro "biol. 17:377-383. 2. ATLAS, R. M., D. PRAMER , and R. BARTH. 1978. Assessment of pesticide effects on nontarget soil microorganisms. Soil Biol. Biochem. 10:231-239. 3. BAKER, R. 1971. Analyses involving inoculum density of soil-borne plant pathogens. Phytopathology 61:1280-1292. 4. BROWN, M. E. 1974. Seed and root bacterization. Annu. Rev. Phytopathol. 12:181-197. 5. BURR, T. J., M. N. SGHROTH, and T. SUSLOW. 1978. Increased potato yields "by treatment of seedpieces with specific strains of Pseudomonas fluorescens and P. putida. Phytopathology 68:1377-1383. 6. COOK, R. J. 1970. Factors affecting saprophytic colonization of wheat straw by Fusarium roseum f. sp. cerealis 'culmorum'. Phytopathology 60: 1672-1 676. 7. CURL, E. A. 196l. Influence of sprinkler irrigation and four forage crops on populations of soil microorganisms including those antagonistic to Sclerotium rolfsii. Plant Dis. Rep 45:517-519. 8. FAASSEN, H. G. VAN. 1974. Effect of the fungicide benomyl on some metabolic processes and on numbers of bacteria and actinomycetes in soil. Soil Biol. Biochem. 6:131-133. 9. FARLEY, J. D., and J. L. L0CKW00D. 1969. Reduced nutrient competition by soil microorganisms as a possible mechanism for pentachloronitrobenzene-induced disease accentuation. Phytopathology 59:718-724. 10. FOWLER, D. L. , and J. N. MAHAN. 1976. The pesticide review. U. S. Dept. of Agr. Agr. Stabilization and Conservation Service, Washington. 42 p. 135

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136 11. GARRETT, S. D. 1963. Soil fungi and soil fertility. Pergamon Press, London. 165 p. 12. GRIFFITH, R. L. , and S. MATHEWS. 1969. The persistence in soil of the fungicidal seed dressings captan and thiram. Ann. Appl. Biol. 64:113-118. 13. HELLING, G. S. , P. C. KEARNY, and M. ALEXANDER. 1971. Behavior of pesticides in soils. Adv. Agron. 23:147-240. 14. HUDSON, H. J. 1968. The ecology of fungi on plant remains above the soil. New Phytol. 67:837-874. 15. KNAUSS, J. F. 1975. Description and control of Fusarium tuber rot of caladium. Plant Dis. Rep. 59:975-979. 16. KREUTZER, W. A. i960. Soil treatment. Pages 431-476 in J. G, Horsfall, and A. E. Dimond, eds. Plant pathology: an advanced treatise, Vol. III. Academic Press, New York. 675 p. 17. MARTIN, J. K. 1975. Comparison of agar media for counts of viable soil bacteria. Soil Biol. Biochem. 7:401-402. 18. MLRGETIGH, S. M. 1971 . The role of Pythium in feeder roots of diseased and symptomless peach trees and in orchard soils in peach tree decline. Phytopathology 6l : 357-360. 19. MLRGETICH, S. M. , and J. M. KRAFT. 1973Efficiency of various selective media in determining Pythium populations in soil. Mycopath. Mycol. Appl. 50:15l-l6l. 20. MUNNECKE, D. E. , and K. Y. MIGKAIL. 1967. Thiram persistence in soil and control of damping-off caused by Pythium ultimum. Phytopathology 57:969-971. 21. NASH, S. M. , and W. G. SNYDER. 1965. Quantatative and qualitative comparisons of Fusarium populations in cultivated fields and in no ncultivated parent soils. Can. J. Bot. 43:939-945. 22. PAPAVIZAS, G. G. 1967. Evaluation of various media and antimicrobial agents for isolation of Fusarium from soil. Phytopathology 57:848-852. 23. RHOADES, H. L. 1970. A comparison of chemical treatments with hot water for control of root-knot nematodes in caladium tubers. Plant Dis. Rep. 54:4ll-4l3. 24. RICHARDSON, L. T. 1954. The persistence of thiram in soil and its relationship to the microbial balance and damping-off control. Can J. Bot. 32:335-346.

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137 25. RICHARDSON L. T. 1973Adaptive tolerance of Fusarium solani to benzimidazole derivatives in vitro. Can J. Bot 51:1725-1732. 26. RIDINGS, W. H., and R . D. HARTMAN. 1976. Pathogenicity of Pythium myriotylum and other species of Pythium to caladium derived from shoot-tip culture. Phytopathology 66:?C4~709. 27. ROVIRA, A. D., and D. C. SANDS. 1971. Fluorescent Pseudomonadsa residual component of the soil microflora? J. Appl . Bact. 3^:253-259. 28. SANDS, D. C. , and A. D. ROVIRA. 1970. Isolation of fluorescent Pseudomonads with a selective medium. Appl. Microbiol 20:513-51^. 29. SHEEHAN, T. J. 1967. Caladium production in Florida. Univ. of Fla. Inst, of Food and Agr. Sci. Circ. No. 128B. 7 p. 30. STEEL, R. G. D. ( and J. H. TORRIE. i960. Principles and procedures of statistics. McGraw-Hill, New York. 48 1 p. 31. STEINER, G. W. , and R. D. WATSON. 1965. Use of surfactants in the soil dilution and plate count method. Phytopatholoev 55:728-730. 32. VANDERPLANX, J. E. 1968. Disease resistance In plants. Academic Press, New York. 206 p. 33. VANDERPLANK, J. E. 1975. Principles of plant infection. Academic Press, New York. 21 6 p. 34. WAINWRIGHT, M. , and G. J. F. PUGH. 1975. Effect of fungicides on the numbers of microorganisms and frequency of cellulolytic fungi in soils. Plant Soil 43:561-572.

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BIOGRAPHICAL SKETCH Richard S. Ferriss was born in Orange, New Jersey, on July 7, 1948. He spent his childhood in Madison, New Jersey, and Sarasota, Florida. He is a graduate of Sarasota High School and a veteran of the United States Navy. He received his undergraduate training at Manatee Junior College and the University of South Florida, where he was awarded a Bachelor of Arts in Botany in 1973. He commenced studies at the University of Florida in 1975. Aside from plant pathology and soil microorganisms, his interests include music, camping, and his daughter, Kate. 138

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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. David J. I»?•tchell 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. James F. Knauss Associate Professor of Plant Pathology ;ucuja4 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. /Ci./jJUih Daniel A. Roberts 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. // A^ Dav id H. Hufcbell' Professor of Soil Science

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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. March, 1979 Dean, jCpllege of Agricu Dean, Graduate School

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UNIVERSITY OF FLORIDA 3 1262 08553 1464


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