Title: Population dynamics of microorganisms associated with caladium seedpieces
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 Material Information
Title: Population dynamics of microorganisms associated with caladium seedpieces
Alternate Title: Caladium seedpieces
Physical Description: viii, 138 leaves : graphs ; 28 cm.
Language: English
Creator: Ferriss, Richard S., 1948-
Copyright Date: 1979
 Subjects
Subject: Soil microbiology   ( lcsh )
Soil fungi   ( lcsh )
Plants -- Effect of fungicides on   ( lcsh )
Plant Pathology thesis Ph. D
Dissertations, Academic -- Plant Pathology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Richard S. Ferriss.
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 135-137.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099112
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000013939
oclc - 05982479
notis - AAB7113

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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.




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