Group Title: pathogenicity and interrelationship of Pratylenchus coffeae and Phythium splendens on Chinese evergreen /
Title: The Pathogenicity and interrelationship of Pratylenchus coffeae and Phythium splendens on Chinese evergreen
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Title: The Pathogenicity and interrelationship of Pratylenchus coffeae and Phythium splendens on Chinese evergreen
Physical Description: 50 leaves : ill. 28 cm.
Language: English
Creator: Noegel, Kenneth Arlen, 1938-
Publication Date: 1972
Copyright Date: 1972
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Subject: Pratylenchus   ( lcsh )
Pythium   ( lcsh )
Evergreens   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
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non-fiction   ( marcgt )
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Thesis: Thesis (Ph.D.)--University of Florida, 1972.
Bibliography: Includes bibliographical references (leaves 47-49).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Kenneth Arlen Noegel.
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000430713
oclc - 37941135
notis - ACJ0095

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THE PATHOGENICITY AND INTERRELATIONSHIP OF
PERATYLENCHUS COFFLAE AT4O PYTHIUM SPLETDENS
ON CHINESE EVERGREEB








By
KENNETH ARLEN TOEGEL












A DISSERTATION PRESEI ED TO THE CRADUA2T COUNCIL OP
THE UNIVERSITY OF FLORIDA
IN PARTIAL PULFILIZNT OF THE PEQUIREN;2TS F02 TIH
DEGREE Or DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1972














ACKIO'WLEDGLIENTS


I wish to thank my supervisory committee members, Dr. V. G. Perry,

chairman; Dr. H. N. Miller, cochairman; Dr. G. C. Smart, Jr. and

Dr. P. Decker for their assistance in the preparation of this manuscript.

I wish to express my appreciation to Dr. V. G. Perry and

Dr. H. N. Miller for their guidance, encouragement, and generous use of

their laboratory facilities and photographic equipment.

I particularly wish to express sincerest thanks to my wife, Claire,

for her patience, understanding, and assistance during the course of

this graduate study.















TABLE OF CONTENTS


Acknowledgments ................................................ i

List of Tables ................................................... vi

List of Figures .................................................. vii

Abstract ......................................................... viii

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

Literature Review ................................................ 3

Materials and Methods

General ..................................................... 7

Laboratory Experiments ...................................... 8

1. Colonization of Pratvlenchus coffee on sterile ex-
cised roots ........................................ 8

2. Isolation of Pvthiunm slndens from diseased Chinese
evergreen .......................... ............... 9

3. Growth of Pythlum splandens on selected artificial
media ........ .................................... 11

4. Growth of Pythiu splendens at selected temperatures 11

Greenhouse Experiments ...................................... 12

1. Comparison of the reproductive potential of specimens
of Pratvlenchus coffee reared on Chinese evergreen
and excised corn roots ............................. 12

2. Host preference of Pratylenchus coffee from Chinese
evergreen ........................................ 12

3. The pathogenicity of Pratylenchus coffee to Chinese
evergreen ......................................... 13

4. The pathogenicity of six fungi to rooted tip cuttings
of Chinese evergreen ............................... 13
































Results

Labor,


Green


5a. Pathogenicity of Pythium spendens to mechanically
damaged roots of Chinese evergreen ................ 14

5b. Pathogenicity of Pythium splendens to unrooted tip
cuttings of Chinese evergreen ...................... 14

5c. Pathogenicity of ythium solendens to unrooted tip
cuttings of Chinese evergreen under cool tempera-
ture conditions .................................... 15

Ga. Inoculation of Fratylenchus coffee and Pythium
splendens to Chinese evergreen ..................... 16

6b. Pathogenicity and interaction of ytium splendens
and Pratylenchus coffee on Chinese evergreen ...... 16

7a. Pathogenicity of Pythium splendens to cuttings of
coleus, geranium, and begonia ...................... 18

7b. Pathogenicity of Pvthiu splendens to seedlings of
radish, coleus, geranium, and cucumber ............ 18



atory Erperiments ........................................ 20

1. Colonization of Pratylenohus coffee on sterile ex-
cised roots ........................................ 20

2. Isolation of Pythium splendens from diseased Chinese
evergreen .......................................... 21

3. Growth of Pythium splendens on selected artificial
media .............................................. 21

4. Growth of Pythium solendens at selected temperatures 21

house Experiments ........................................ 22

1. Comparison of the reproductive potential of speci-
mens of Pratylenchus coffee reared on Chinese
evergreen and excised corn roots ................... 22

2. Host preference of Pratylenchus coffee from Chinese
evergreen .......................................... 22

3. The pathogenicity of Pratylenchus coffee to Chinese
evergreen .......................................... 23

4. The pathogenicity of six fungi to rooted tip cuttings
of Chinese evergreen ................................ 23








5a. Pathogenicity of thium splendens to mechanically
damaged roots of Chinese evergreen ................ 24

5b. Pathogenicity of Pytiu plendens to unrooted tip
cuttings of Chinese evergreen ...................... 24

5c. Pathogenicity of Pythiu splendens to unrooted tip
cuttings of Chinese evergreen under cool tempera-
ture conditions .................................... 24

6a. Inoculation of Pratylenchus coffee and Pythium
Llendens to Chinese evergreen ..................... 31

6b. Pathogenicity and interaction of Pythium solendens
and Pratylenchus coffee on Chinese evergreen ...... 31

7a. Pathogenicity of Pythium splendens to coleus,
geranium and begonia................................ 37

7b. Pathogenicity of Pythium _pendens to radish, coleus
geranium, and cucumber seedlings.................... 37

Discussion

Laboratory Experiments ........................................ 42

Greenhouse Experiments ....................................... 42

Summary .......................................................... 45

Literature Cited .................................................. 47

Biographical Sketch ............................................... 50















LIST OF TABLES


1. The effect of Pratylenchus coffee and Pvthium slndens,
singly and in combination, on growth of Chinese evergreen,
Aglaonema modestum ........................................... 32

2. The pathogenicity and interaction of Pythium solendens and
Pratylenchus coffee on Chinese evergreen, Allaonema modestum. 36













LIST OF FIGURES


1. Typical root rot of Chinese evergreen incited by Pratylenchus
coffee in peat ............................................ 26

2. The effect of Pythium splendens on mechanically wounded roots
of Chinese evergreen ........................................ 28

3. Pathogenicity of Pythium solendens to unrooted tip cuttings
of Chinese evergreen ........................................ 30

4. The effect of Pythium splendens and Pratylenchus coffeae,
singly and together, on Chinese evergreen ................... 35

5. Pathogenicity of Pythium splendens to unrooted tip cuttings
of geranium and begonia ..................................... 39

6. Pathogenicity of Pythium splendens to selected vegetable end
ornamental seedlings ........................................ 41









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


THE PATHOGENICITY AND INTERRELATIONSHIP OF
PRATYLENCIIUS COPFEAN ANFD PYTHIUMA SPLENDE!13
ON CHINESE EVERGROIEl


By


Kenneth Arlen Noegel

August, 1972


Chairman: Dr. V. G. Perry
Cochairman: Dr. H. IT. Miller
Major Department: Entomology and Nematology


A research program was initiated to study the roles of the fungus

Pythium splendens and the nematode Pratylenchus coffee in the etiology

of the root rot of Chinese evergreens grown in central Florida nurseries.

A technique was devised whereby Pythium splendens was isolated con-

sistently from diseased Chinese evergreens with a mininmr. of contamina-

tion by other organisms associated with the root rot.

Pratylenchus coffee was surface sterilized in 0.1% streptomycin

sulphate aid successfully colonized on sterile excised corn roots growing

on Tiner's medium Many P. coffee were obtained from the excised corn

roots for inoculum purposes.

Pythium solendens produced maximum vegetative growth on V-8 agar,

but also grew well on potato dextrose and corn meal agars. The optimum

temperature for growth was between 25 and 50 C, with maximum growth

occurring at 30 C. The fungus failed .to grow at 40 C and produced sparse

growth at 15 and 20 C.








Pratylenchus coffee, from Chinese evergreens, reproduced on straw-

berry, coffee, and rough lemon, but not on tea. _Pthiun splendens was

pathogenic to rooted and unrooted cuttings of geranium, and to unrooted

cuttings of begonia, but not to rooted cuttings of begonia. The fungus

did not damage coleus cuttings. Seedlings of coleus, radish, geranium,

and cucumber were severely damaged by the fungus.

Pratylenchus coffee was capable of destroying the root systems of

Chinese evergreens within four months after inoculation, but only when

the plants were growing in soil with a high organic matter content such

as peat.

Pythium splendens was highly pathogenic to Chinese evergreen in

sandy and in peat soils. The fungus was capable of destroying estab-

lished root systems and preventing formation of new roots, and did not

require mechanical wounds for entry into host tissues. P. Bolendens

was more pathogenic at 22 C than at higher temperatures.

No synergistic activity was noted when Pratylenchus coffee and

Pythiun splendens were inoculated to Chinese evergreen simultaneously.

Idore damage occurred when the fungus was inoculated to plants three

weeks after the nematode.

Pratylenchus coffee populations were reduced in the presence of

Pythium splendens on Chinese evergreen. Plants growing in sandy soil

and inoculated vi.th the nematode alone increased in total plant and root

weights more than did uninoculated plants.














INTRODUCTION


The production of ornamental plants in Florida for sale on local,

national and international markets has increased significantly during

the last 25 years. Those sold as foliage ornamentals (as opposed to

flowering ornamentals) constitute a major portion of ornamental plant

production. Many of these are tropical plants grown in nurseries and

used by consumers primarily as container-grown house plants or indoor

landscape materials. Several thousand types and species of plants are

included. The groups that are produced most abundantly include Chinese

evergreen, Aglaonema simplex (Blume), which has been grown commercially

in Florida since 1934 (32).

Chinese evergreens and many of the other foliage plants are propa-

gated from tip cuttings. The cuttings are planted in organic mixes,

usually peat and muck soils, for rooting. After the cuttings have

rooted they are transplanted and grown to sizes desired by the markets.

High soil moisture and air humidity are maintained throughout the pro-

duction period. Chinese evergreens are sensitive to cold, thus they are

usually grown in glasshouses or plastic houses wherein the temperature

is controlled. In some cases various types of cooling devices are used

during hot weather.

The environment necessary for propagation and growth of Chinese

evergreen is favorable to the growth of pathogens such as fungi and

nematodes. In 1943 Tisdale (31) reported a severe root rot of plants

collected in Orange County. He isolated the fungus Pythium splendens








Braun from diseased tissues and was able to reproduce the disease

symptoms under experimental conditions. Tisdale and others have also

reported the findings of various other fungi from Chinese evergreen.

Root rot diseases are spread throughout the Florida foliage nurseries

and are so severe that, unless control measures are utilized, commercial

production is limited.

Chemical control of Chinese evergreen root rot has been attempted

(15, 32). Soil sterilants, such as fumigants, were found to be more

effective than fungicides.

Various nematodes such as Rj2dpholus similis Thorne, Helicotylenchus

spp., and Pratylenchus spp. are common parasites of various ornamental

plants, including Chinese evergreen, grown in Florida. Pratylenchus

coffee (Zimmerman) Filipjev and Schuurmans Stekhoven is consistently

associated with the root rot of Chinese evergreen. Perry and Miller (22)

were able to control the root rot conditions through the application of

nematicides to established plants. Since the root rot of Chinese

evergreen was first reported to be incited by the fungus Pythium splendens,

control of the disease by the application of nematicides raised questions

as to the nature of the root rot, especially to the roles played by

Pythinm splendens and the nematode Pratylenchus coffeae.

A research program was initiated to determine more definitely the

types and extent of diseases caused by Pythiu splenens and Pratylenchus

coffee, singly and in combination. Such data are necessary for judicious

uses of control procedures such as sanitation and chemical pesticides.












LITERATURE REVIEW


The literature on the interrelationships between nematodes and other

organisms in producing plant diseases is voluminous and has been reviewed

by Miller (16), Pitcher (23), and Slack (27). It is apparent from the

literature that many diseases are true complexes involving two or more

organisms. Nematodes play important roles in plant diseases by vectoring

bacterial, fungal, and viral diseases; by mechanically wounding hosts and

thereby providing a means of ingress; by providing necrotic host tissue

for weak pathogens; and by altering the physiology of a plant in a manner

which makes it more suitable for a pathogen or less resistant to invasion

by a pathogen.

Members of the nematode genus Pratylenchus, the lesion nematodes, are

involved in disease complexes to varying degrees. One of the earliest re-

ports of a lesion nematode-fungus interaction was that of Hastings & Bosher

(8) in which they found that Cylindrocarpon radicicola caused greater

damage when rat9 inchus enetrans was present. Slootweg (29) postulated

that Pratylchu penetrans created wounds which allowed the entry of

Cylindrocarpon radicicola into bulbs. Norton (20) found that Macrophomina

phaseolj and Pratylenchus hexincisus contributed to charcoal rot of sorghum

independently.

McKeen & Mountain (13) reported synergism between Pratylenchus

ponetrans and Verticillium albo-atrum (identified as V. dahlia in a sub-

sequent publication (18)) in causing eggplant wilt. The nematode increased

wilt at low and intermediate levels of Verticillium inoculum. Nematode







populations were greater at these inoculum levels than in the absence of

the fungus. The nematode alone did not affect eggplant. Mountain & McKeen

(18) found that on eggplant and tomato a relationship exists between the

incidence of Verticillium wilt and the increase in numbers of Pratylenchus

penetrans in the presence of the fungus. The reason for the increased rate

of reproduction was not established. In further work on the Verticillium

wilt-Pratylenchus penetrans disease complex of eggplant, Mountain & McKeen

(19) indicated that the nematode's role in the wilt might be that of a

wounding agent which provides a necrotic avenue to the vascular elements

of the host. Faulkner & Skotland (6) reported a synergistic interaction

between Verticillium dahliae and Pratylenchus mina in producing wilt of

peppermint. They also found that the nematode's rate of reproduction was

greater in the presence of the fungus, and concluded that the fungus

causes changes in the host physiology that are favorable to the nematode.

Benedict & Mountain (1) theorized that Pratylenchus nin-'s aids the

development of Rhizoctonia solani in root rot of winter wheat.

Conroy et al. (4) found that Pratylenchus penetrans caused an increase

in infection incidence of Verticillium albo-atrum on tomato. Infection by

the fungus increased in relation to increases in the nematode inoculum

level. They did not find, as did other workers, that the nematode popula-

tion increased in the presence of the fungus. Their work indicated that

the nematode's role in this complex is to provide infection courts or

similar changes in the roots which favor fungus development.

O'Bannon and Tomerlin (21) found that Pratylenchus coffee, inocula-

ted to rough lemon seedlings, reduced shoot growth by 22% after 16 months.

The nematode reached a population peak of 10,000 nematodes per gram of

root 9 months after inoculation at ambient greenhouse temperatures.

Radewald et al. (24) reported that Pratylenchus coffee, feeding on







rough lemon, increased at temperatures from 26 C to 32 C with the greatest

increase occurring at 29.5 C. P. coffee reduced total plant weight 38%

at 26 C. The nematode reduced root weight 4T7 at 26 C and 29.5 C. P.

coffee survived in stored, excised rough lemon roots and were infective

after 4 months at temperatures of 4.5 C to 32 C. Survival was greatest

when the roots were stored in sand with a moisture content near field

capacity, and lowest in oven-dried soil.

Feldnesser and Hannon (7) working with mixed populations of Pratylenchus

spp. which included P. coffeae, demonstrated that Pratvlenchu spp. were

capable of stunting the growth of rough lemon and Milam lemon rootstocks.

Their work indicates that Pratylenchus spp., which are found frequently

in citrus roots, pose a serious threat to the Florida citrus industry.

Schieber and Grullon (25) reported that Pratylenchus coffee caused

damage to the absorbent rootlets of coffee in the Dominican Republic.

Safflower var. Ute was highly susceptible to root rot caused by

Pythium splendens, particularly under conditions of flooding (34).

Hendrix and Powell (9) reported that isolates of Pythiun splendens

from pecan roots and soil reduced the root weights of inoculated seedlings

by 41 to 48V.

Summirs et al. (29) consistently isolated Pythium splendens from

diseased -renaf plants in Florida.

The following plants were reported as hosts for Pythium splendens by

Middleton (14); Ananas comosus, Begonia spp., Cajanus caan, Canavalia

ensiformis, Carica pap Chrysanthemum sp., Citrus aurantium, Coleus

sp., Oucumis sativus, Geranium sp., Helianthus annuus, Hordeum vulgare,

Ipomea betatas, Linum usitatissimum, Medicago sativa, Nicotiana tabacum,

Pelargonium spp., Phaseolus aureus, P. vulgaris, Piper betle, Raphanus

sativus, Saccharum officinarum, Triticum aestivum, Vicia faba, and Vigna




6

sinensis. Other workers have reported many additional plants as hosts for

P. splendeno.














MATERIALS AND METHODS


General

Root rot-diseased Chinese evergreen plants were obtained from com-

mercial nurseries in the Apopka, Florida, area and maintained in green-

houses at the University of Florida Horticultural Farm near Gainesville

as a continual source of material for study. I also collected additional

diseased plants from the Apopka area from time to time.

Several methods were compared for the extraction of the nematode

Pratylenchus coffee from nematode-infected roots. The methods discussed

below gave the most consistent results and therefore were utilized during

the course of this research. When nematodes were needed for inoculum,

infected roots were placed in large beakers filled with water, and air

was bubbled through the water. After 24-48 hr the nematodes were col-

lected on a 325-mesh sieve. Recovery was very good, and the nematodes

apparently were not harmed by this method. When it was necessary to de-

termine the effect of a treatment on nematode populations, the roots were

cut into small pieces, placed in a food blender with sufficient water to

cover, and comminuted for 30 sec. The roots were collected on a 325-mesh

sieve and washed onto a facial tissue resting on hardware cloth in the

top of a 6-inch plastic pot filled with water. After 24-48 hr the nema-

todes were collected on a 325-mesh sieve and washed into Syracuse watch

glasses for counting with the aid of a dissecting microscope. This technique

usually gave better results than the technique described by Young (33).







Laboratory Exoeriments

1. Colonization of Pratylenchus coffee on sterile excised roots.

Techniques for surface sterilization of plant parasitic nematodes

have been described (2, 3, 11, 12, 17, 30). Several of these methods

were tried before adopting the procedure described below. In an effort

to remove Pratylenchus coffee, as much as possible, from the organisms

associated with the root rot of Chinese evergreen, the nematode was

reared in a greenhouse on corn, Zea mays L. 'Iona', growing in 6-inch

clay pots containing sterile soil. Nematode-infected corn roots were

washed and placed in 2000-ml beakers containing 0.2 streptomycin sulphate

in sterile water and air was bubbled through the water for 12-24 hr. The

nematodes were collected on a 325-mesh sieve and washed into sterile 4-oz

screwcap bottles with the aid of a plastic wash bottle containing 0.1%

streptomycin sulfate in sterile distilled water. The nematodes were

stored in a refrigerator at 5 C, used immediately, or pipetted directly

onto plates of sterile 1.0% water agar containing 0.1% streptomycin

sulphate.

Corn was chosen as a suitable host for rearing Pratylenchus coffeae

on excised roots. Iona corn seeds were placed in a 4-inch plastic pot

and a circular piece of hardware cloth was placed in the top of the pot

to retain the seeds. Water from a cold water tap was directed into the

pot through rubber tubing to remove the Arasan coating from the seeds.

The seeds then were placed in a 150-ml beaker. A double thickness of

cheesecloth was fastened across the top of the beaker with a rubber band.

Mercuric chloride at 0.1% concentration was poured through the cheese-

cloth until it covered the seeds. The seeds were swirled for 60 sec,

then rinsed in sterile distilled water, followed by a 60 see period of








exposure to 0.5% sodium hypochlorite, and two rinses of sterile distilled

water. All rinses were poured directly through the cheesecloth covering

the beaker, and the cheesecolth was not removed during the sterilization

process. The seeds were transferred from the beaker, with forceps dipped

in 95% ethyl alcohol and flamed, onto sterile 1.5% water agar in petri

dishes in order to promote germination and to observe for contaminants.

In order to study the effects of Pratylenchus coffee on Chinese

evergreen under sterile conditions, healthy excised roots of the plant

were treated with 0.5% sodium hypochlorite, 0.1% mercuric chloride, 0.1%

Roccal, or 0.1% streptomycin sulfate, and combinations of these for

exposure intervals of 30 sec followed by sterile water rinses. The

treated roots were placed on Tiner's nutrient medium (30) and observed

for contaminants.


2. Isolation of Pythium plendens from diseased Chinese evergreen.

Diseased plants were examined in the laboratory and subjected to the

methods described below in an attempt to isolate Pythiun slendens. Root

and stem sections, from within diseased portions and from the periphery

of diseased portions, were included in the tests.

A. Soil and comminuted root tissue were processed according to the

dilution technique described by Johnson et al. (10). Dilutions were

plated in 1.5% water agar, corn meal agar, lima bean agar, and potato

dextrose agar.

B. Stem and root sections were surface sterilized in 0.1% mercuric

chloride, 0.1% Roccal, or 95% ethyl alcohol and plated on water agar,

potato dextrose agar, lima bean agar, and corn meal agar. Plant sections,

untreated except for brief washing in tap water, were also plated on

these agars.








C. Peptone dextrose agar as described by Johnson et al. (10) as

well as other selective antibiotic agars were tried (5, 26).

D. Diseased tissue was inoculated into apple fruits and isolations

were made from within and around necrotic lesions which developed. The

isolations were plated on selected agar media.

E. Another technique was devised by the author. Roots and stems

were washed in running water to remove soil and adhering debris. Sections

0.5 to 1.0 cm in length were removed from small lateral roots having

necrotic and water-soaked root tips. Similar sized sections were removed

from both sides of necrotic areas on main roots. Stem sections were cut

from canes on which the roots had been destroyed and the infection had

advanced into the stem base. A 6-inch diameter glass funnel was placed

in a ring stand in a sink. A piece of rubber tubing was fastened on one

end to a faucet and on the other end to the stem of the funnel. A circle

of window screen was cemented into the funnel at the top of the stem. All

plant sections were placed in the funnel and a circular piece of window

screen, held in place by a weight, was placed inside the top of the funnel.

Running cool tap water was used to constantly agitate the root and stem

sections. This washing was maintained for 6 hr after which the sections

were placed with the aid of flamed forceps into sterile petri dishes con-

taining sterile distilled water. The petri dishes then were placed in

temperature cabinets at 15 C for 48 hr. Root and stem sections next

were plated on corn meal agar, under sterile conditions, and returned to

the 15 C cabinet. When fungi began to grow from the sections into the

agar, hyphal tip isolations were made onto corn meal agar.








3. Growth of Pythium splendens on selected artificial media.

Six-mo-diameter plugs were cut with a cork borer from potato dextrose

agar cultures of P. splendens. The plugs were transferred to the center

of 100-mm-diameter petri dishes containing potato dextrose agar, corn

meal agar, V-8 agar, or lima bean agar. The fungus was inoculated onto

five plates of each type of agar and incubated in temperature cabinets

at 30 C. Measurements of the radial growth of the fungus were made 24

hr after inoculation. Two measurements were made on each plate and

averaged.

The experiment was repeated with measurements taken 20 hr after

inoculation.


4. Growth of Pythium snlendens at selected temperatures.

Six-mm-diameter plugs of a potato dextrose agar culture of P. solendens

were removed with a cork borer and placed in the center of 100-mm-diameter

petri dishes containing potato dextrose agar. Five petri dishes each had

been held in temperature cabinets at 10, 20, 30, and 40 C for 24 hr prior

to inoculation. Each group of five dishes was returned to the appropriate

temperature cabinet immediately after inoculation with P. solendens.

Measurements of radial growth of the fungus were made 30 hr after inocu-

lation. Two measurements were made on each plate and averaged.

A second experiment was conducted at temperatures of 15, 20, 30, and

35 C using the procedures described for the first test. Measurements of

fungus growth were made 24 and 48 hr after inoculation.

A third experiment was conducted at temperatures of 15 and 35 C.

Measurements of fungus growth were made 24 and 48 hr after inoculation.

A fourth experiment was conducted at temperatures of 20, 25, and 30 C.

Measurements of fungus growth were made 24 hr after inoculation.








Greenhouse Exneriments

1. Comnarison of the reproductive potential of specimens of Pratylenchus
coffee reared on Chinese evergreen and excised corn roots.

Pratylenchus coffee was obtained from Chinese evergreen by comminu-

ting the roots in a food blender and processing them with the plastic pot

technique described earlier. P. coffee was also obtained from excised

corn roots growing on Tiner's medium, by flooding the agar with sterile

water. The nematodes were collected from both sources after 12 hr of

incubation. Groups of 200 nematodes were hand picked, using a nylon

bristle pick, into dishes of water. The water, containing the nematodes,

was poured into the sterile field soil around the roots of young, rooted

Chinese evergreens growing in 4-inch clay pots. Each plant was inocu-

lated with 200 nematodes. Ten plants received specimens of P. coffee

reared on Chinese evergreens, and 10 plants received specimens of the

Chinese evergreen population which had been reared for several genera-

tions on excised corn roots. The plants were maintained in a greenhouse.

Forty-five days after inoculation, plants were harvested, and the roots

comminuted and incubated for 24 hr for nematode extraction.


2. Host preference of Pratylenchus coffee from Chinese evergreen.

An experiment was conducted to compare the host preference of

specimens of Pratylenchus coffee extracted from Chinese evergreen with

the host range reported for P. coffeae in the literature.

Five plants each of strawberry, coffee, tea, rough lemon and Chinese

evergreen were planted in sterile soil in 4-inch plastic pots. One

hundred Pratylenehus coffee in 25 ml of water were poured into the soil

around the roots of each test plant.' The plants were placed in a green-

house, and received normal maintenance during the course of the experiment.







Forty-two days after inoculation, the roots were removed from the

plants and processed by the plastic pot technique described earlier.

The nematodes were extracted and counted to study the suitability of the

plant varieties tested as possible hosts.


3. The pathogenicity of Pratylenchus coffee to Chinese evergreen.

Healthy, 1-year-old Chinese evergreens, growing in methyl bromide-

sterilized peat contained in 6-inch clay pots, were inoculated with

Pratylenchus coffee. Two hundred and fifty P. coffee, extracted from

Chinese evergreen, were inoculated to each of 10 plants by pouring the

nematodes, in 50 ml of water, into holes made in the potted peat at 3

points around the base of each plant. Fifty ml of water, collected during

the extraction of the nematodes and passed through a 325-mesh sieve, were

inoculated to each of 10 plants in an identical manner. An additional

10 plants received 50 ml of distilled water each, but no nematodes. The

plants were placed in a growth room at 29.4 C where they received 16 hr

of artificial light in each 24 hr cycle. The plants were examined and

data obtained 4 months after inoculation.


4. The pathoenicity of six fungi to rooted tip cuttings of Chinese
evergreen.

Pythium splendens, Pythium acanthicum, Rhizoctonia sp., and 3

species of Fusarium were consistently isolated from diseased roots of

Chinese evergreen during preliminary trials. A test was designed to

study the effect of these fungi on Chinese evergreen with healthy roots

and mechanically wounded roots. Tip cuttings of Chinese evergreen,

rooted in flats of sterile soil, were removed from the flats and the

roots washed free of soil. Half of the plants were wounded by

pruning the tips of main and lateral roots with scissors. The roots of

the other plants were not wounded. A single, 2-week-old agar plate









culture of each fungus was placed individually in a food blender with

150 ml of water and comminuted for 15 sec and then poured into a 400-ml

beaker. The blender container was rinsed with 0.5% sodium hypochlorite

and hot tap water after treatment. Each fungus was inoculated to 10

plants, 5 with healthy roots and 5 with wounded roots, by dipping the

roots in the inoculum solution for 60 sec. Control plants with healthy

roots and wounded roots were dipped in a solution prepared with a sterile

plate of potato dextrose. The plants immediately were placed in methyl

bromide-sterilized soil contained in 4-inch plastic pots. They were

randomized on a bench in a growth room having a photoperiod of 16 hr and

a temperature of 29.4 C.


5a. Pathoenicity of Pythiu splendens to mechanically damaged roots of
Chinese evergreen.

The roots of 10 Chinese evergreen were wounded by pruning the root

tips with scissors. Five plants were placed in steam-sterilized sand

and peat mix in 6-inch plastic pots. Two hundred and fifty grams of a

3-week-old culture of P. solendens was mixed with enough sterile soil

mix to fill 5 additional 6-inch plastic pots. Five plants were placed

in the fungus-inoculated soil. The plants were placed randomly on a

greenhouse bench.

One hundred and eleven days after inoculation, the plants were

removed from the pots, and the roots were washed and examined. Whole

plant and root weights were recorded.


5b. Pathogenicity of Pythium splendens to unrooted tic cuttings of
Chinese evergreen.

Five fresh tip cuttings, taken from mature Chinese evergreens, were

planted in sterile soil contained in 4-inch plastic pots. The freshly








cut bases of an additional 5 tip cuttings were dipped in an inoculum

solution prepared by comminuting two 17-day old potato dextrose agar

cultures of Pthium splendens in 100 ml of distilled water in a food

blender. The inoculum solution was poured around the base of each

cutting as it was planted in sterile soil contained in a 4-inch plastic

pot. The pots then were placed in a greenhouse in which the ambient

temperature was above 32 C during the daytime.

Two months after inoculation, the plants were removed from the pots,

washed and examined. Weights of plant, roots, and lateral roots were

recorded.


5c. Pathogenicity of Pythium solendens to unrooted tip cuttings of
Chinese evergreen under cool temperature conditions.

Tisdale (32) reported that Pythiua solendens was more pathogenic at

moderate temperatures than at high temperatures. An experiment was set

up to observe the severity and rapidity with which the root rot incited

by P. splendena progressed under cool temperature conditions.

Two hundred and fifty grams of a 16-day-old oat seed culture of the

fungus was mixed with enough sterile field sand to fill ten 4-inch

plastic pots. Ten fresh, unrooted tip cuttings of Chinese evergreen

were planted in the pots of inoculated soil. Ten similar cuttings were

planted in pots containing only sterile soil. The plants were placed in

a growth room at 22 C and a 16 hr photoperiod.

The plants were examined periodically for evidence *of root develop-

ment and disease development. Thirty-five days after inoculation, the

plants were removed from the pots and the roots examined for disease

symptoms.








6a. Inoculation of Pratvlenchus coffeae and Pythium solendens to Chinese
evergreen.

Healthy tip cuttings of Chinese evergreen, rooted in flats of methyl

bromide-sterilized peat, were planted in 6-inch clay pots of steam-

sterilized field soil. Five plants were inoculated with 100 P. coffee

each. The nematodes, in 50 ml of water, were poured into holes made in

the soil at the base of each plant. Each of 5 plants was inoculated in

a similar manner with 50 ml of inoculum prepared by comminuting 3

potato dextrose agar cultures of P. splendens in 150 ml of water in a

food blender. The inoculum volume was adjusted to 500 ml. Five plants

were inoculated with P. coffee and P. splendens together, following the

procedures described above. Five untreated plants were held as controls.

The plants were placed in a greenhouse and maintained with normal watering

and fertilization.

All plants were examined 4 months after inoculation. Weights of the

entire plant and of the roots were recorded. The roots of Pratylenchus

coffeae-inoculated plants were processed using the food blender-plastic

pot technique, and nematode counts were recorded.


6b. Pathogenicity and interaction of Pythium solendens and Pratylenchus
coffee on Chinese evergreen.

An experiment was designed to study the pathogenicity of P. splendens

and P. coffee, singly and in combination, to Chinese evergreen, and

possible interactions between the two organisms. This experiment included

the following treatments of 10 plants each:

1. Unrooted tip cuttings inoculated with the fungus.

2. Mechanically pruned rooted tip cuttings inoculated with the

fungus.









5. Healthy rooted tip cuttings inoculated with the fungus.

4. Healthy rooted tip cuttings inoculated with 500 nematodes per pot.

5. Healthy rooted tip cuttings planted in soil inoculated with the

fungus and then inoculated with 500 nematodes per pot.

6. Healthy rooted tip cuttings inoculated with 500 nematodes per

pot; the soil was inoculated with the fungus 3 weeks later.

7. Unrooted tip cuttings, uninoculated.

8. Mechanically pruned tip cuttings, uninoculated.

9. Healthy rooted tip cuttings, uninoculated.

Oat cultures of P. solendens were prepared by autoclaving 250 g of

oat seeds and 250 ml of distilled water in 1000-ml flasks. The sterile

oats were inoculated with 6-irm plugs taken from a potato dextrose agar

culture of P. splendens. The fungus grew throughout the oat medium in

about 7 deas. One flask of inoculum was added to each volume of sterile

field soil sufficient to fill ten 6-inch pots.

Pralylenchus coffee was obtained by incubating roots of nematode-

infected Chinese evergreen in water through which air was bubbled. The

nematodes were collected on a 325-mesh sieve and condensed in a small

amount of water. The volume was adjusted so that each 10 ml of the

water contained 500 P. coffee. The treatments were randomized on a

greenhouse bench. Normal cultural practices were followed.

Seventy days after the initial inoculations took place, the plants

were removed from the pots for examination. Disease ratings, total

fresh plant weight, total fresh root weight, and nematode counts were

recorded. Nematodes were extracted by placing a 10 g subsample from

chopped-up roots of each replicate in a food blender and comminuting for

30 sec. The root debris and nematodes were condensed on a 325-mesh









sieve and washed onto facial tissues supported by hardware cloth in the

tops of water-filled 6-inch plastic pots. The nematodes were identified

and counted after 48 hr.


7a. Pathogenicity of Pvthium splendens to cuttings of coleus, geranium,
and begonia.

The ornamental plants coleus, geranium, and begonia are listed as

hosts for ythium splendens (14). In an effort to further define the

nature of the P. splendens isolated from Chinese evergreen, an experiment

was conducted to ascertain the pathogenicity of the fungus to cuttings of

coleus, geranium, and begonia.

Five hundred grams of a 14-day-old oat seed culture of Pythium

splendens was mixed in enough soil to fill thirty 4-inch plastic pots.

Five rooted cuttings and 5 unrooted tip cuttings of each type of plant

were placed in the fungus-infested soil. A like number of cuttings were

placed in sterile soil in 4--inch plastic pots to serve as uninoculated

controls. The plants were placed in a greenhouse and observed daily for

disease symptoms.


7b. Pathogenicity of Pythium splendens to seedlings of radish, coleus,
geranium, and cucumnber.

Seven hundred and fifty grams of an oat seed culture of P. splendens

was mixed with enough sterile soil to fill fifteen 90-cubic-inch plastic

trays. Fifteen trays were filled with sterile soil. Seeds of radish,

Raphanus sativus L. 'Long Scarlet Short Top' and 'Cherry Belle'; coleus,

Coleus sp. 'Rainbow Mixed'; geranium, Geranium sp. 'Zonale Mixed'; and

cucumber, Cucumis sativus L. 'Improved Long Green' were each planted in

3 flats of fungus-infested soil and 3 flats of sterile soil. Thirty

seeds of each crop, except coleus, were planted per tray. One hundred





19



and fifty coleus seeds were planted per tray. The trays were placed in

an air conditioned greenhouse.

Emergence counts and observations on damping-off were made.














RESULTS


Laboratory ExDeriments

1. Colonization of Pratylenchus coffee on sterile excised roots.

The sterilization method was successful and no contaminants were

noted on the surface-sterilized corn seeds germinated on sterile 1.5%

water agar. One- to 2-inch-long roots from the corn seedlings were

excised with flamed scissors and transferred to 4-oz screwcap bottles

containing Tiner's medium (30). The technique described earlier for

surface sterilizing nematodes was successful on Pratylenchus coffee.

Surface-sterilized nematodes were pipetted from solutions, or picked

from agar plates onto small blocks of sterile agar, and transferred to

bottles containing excised corn roots growing on Tiner's medium. The

nematode reproduced well on the sterile excised roots. Over 2000 nematodes

were obtained from each excised root about 4 months after inoculation.

The nematodes fed, almost without exception, inside the corn roots, and

migrated throughout the roots. No lesions were noted; however, heavily

parasitized roots turned a brownish-yellow color. As the nematode popu-

lation increased there was a steady migration by the nematodes out of the

roots. Females were observed laying eggs in the agar adjacent to excised

roots as they browsed along the sides of the roots. Colonies of

Pratylenchus coffee were thus maintained on sterile excised corn roots

and used as inoculum as needed.

Efforts to sterilize excised roots of Chinese evergreen were unsuccess-

ful. When sterilizing agents were used in sufficient strength to eradicate









contaminants, the roots were killed.


2. Isolation of Pythium splendens from diseased Chinese evergreen.

Isolation techniques A, B, C, and D were not successful. Antibiotics

and sterilizing agents often killed all fungi in diseased material. The

more vigorous growing organisms usually overran the agar plates within

hours after plating plant material.

Technique E, devised by the author after much trial and error, was

successful in isolating the fungus. This technique proved to be highly

successful and reliable for isolating Pythium from root rot-diseased

Chinese evergreens. It was also used for isolating Phytophthora from

diseased petunias and other ornamental bedding plants.


3. Growth of Pythium splendens on selected artificial media.

V-8 agar provided the maximum growth of Py thium splerden when

measured 24 hr after inoculation with the growth averaging 72 mm in

diameter. The growth on potato dextrose agar (PDA), corn meal agar (CMA)

and lima bean agar (LBA) averaged 62, 59.8 and 47.8 mm in diameter

respectively.

The second experiment, in which measurements were made 20 hr after

inoculation, gave basically the same results as the first experiment.

The growth of P. slendens on V-8 agar, PDA, CMA, and LBA was 63.8, 55.9,

52.7, end 44.1 mm in diameter respectively.


4. Growth of Pythium splendens at selected temperatures.

In the first experiment Pythium splendens produced maximum growth at

30 C, 30 hr after inoculation. The fungus growth measured 0, 76.7, 38.3,

and 0 mm in diameter at 40, 30, 20, and 10 C respectively.








Growth of P. splendens in the second experiment, recorded 24 hr after

inoculation, was 0, 24.1, 55, and 13.4 mm in diameter at 15, 20, 30, and

35 C respectively. The growth measured at 48 hr was 15.8, 90, 90, and

24.1 mm in diameter at 15, 20, 30, and 35 C respectively.

The grov-th of P. splendens in the third experiment, recorded at 24

hr, was O and 9 mm at 15 and 35 C respectively. The 48-hr measurements

were 36.4 and 41.2 mm at 15 and 35 C respectively.

The measurements in the fourth experiment, recorded at 25 hr, were

20.7, 44.1 and 53.0 mm at 20, 25, and 30 C respectively.


Greenhouse Experiments

1.-


The roots of Chinese evergreens inoculated with the Chinese evergreen

population of Pratylenchus coffee yielded an average of 159 nematodes

per 10 g of roots. The plants inoculated with the Chinese evergreen

population which had been reared for several generations on excised corn

roots yielded 167 nematodes per 10 g of roots. The two populations did

not differ significantly in reproductive potential. The nematodes reared

for several generations on excised corn roots were capable of feeding and

reproducing on Chinese evergreen as well as the Chinese evergreen popula-

tion.


2. Host or-ference of Pratylenchus coffee from Chinese evergreen.

Nematode counts made after processing the roots of strawberry,

coffee, tea, rough lemon, and Chinese evergreen, previously inoculated

with Pratylenchus coffee, revealed that the nematode did not reproduce

on tea. The roots of rough lemon and coffee yielded an average of 49 and

98 nematodes per 10 g of roots. Strawberry and Chinese evergreen were









better hosts; their roots yielded an average of 189 and 197 nematodes

respectively per 10 g of roots.


3. The pathogenicity of Pratylenchus coffee to Chinese evergreen.

Four months after inoculation, the plants were removed from the pots

and the roots washed clean for examination.

The plants which received 250 P. coffee each were stunted in

comparison to the growth of control plants. The lower leaves of the

nematode-infected plants were chlorotic and drooping. Root damage was

similar to that seen on root rot-diseased Chinese evergreens in commer-

cial nurseries (Pig. 1). Roots were generally rotted-off at the base

of the cane and those remaining displayed numerous dark, necrotic areas.

Pratylenchus coffee were extracted from root pieces of nematode-

inoculated plants.

One-cm-thick serial sections were removed from the bases of the

nematode-infected canes, comminuted in a food blender, and placed on

facial tissues in the tops of water-filled 6-inch plastic pots for 48

hr. The 0-1 cm, 1-2 cm. and 2-3 cm sections yielded averages of 211.1,

156.3 and 30.3 Pratylenchus coffee respectively. Sections taken up to

10 cm from the bases of the canes did not yield nematodes.

All plants in both sets of controls remained healthy.


4. The pathogenicity of six fungi to rooted tip cuttings of Chinese
evergreen.

Twenty days after the Chinese evergreens with healthy and wounded

roots were inoculated with the 6 fungi, the plants were removed from the

pots and the soil washed from their roots. Root systems were examined

for symptoms of root rot. Only Pythium splendens-inoculated plants,

showed symptoms which were typical of early Chinese evergreen root rot;









i.e. root tips were turning tan in color, necrotic, or sloughing. Healthy

roots and wounded roots were damaged to the same extent. No evidence of

root rot was found on the roots of control plants.


5a. Pathogenicity of Pythium splendens to mechanically damaged roots of
Chinese evergreen.

Chinese evergreens on which the roots had been mechanically wounded

and inoculated with P. splendens developed typical root rot symptoms

(Pig. 2). The few new roots produced were much smaller than those of

the uninoculated controls. Both old and new roots were necrotic. The

control plants initiated vigorous root growth which replaced those

pruned off.

The fungus damage reduced plant weight by 17.6% and root weight by

31.0% in comparison to the controls.


5b. PathoPenicity of Pythium splendens to unrooted tip cuttings of
Chinese evergreen.

Unrooted tip cuttings of Chinese evergreen, inoculated with

P. splenden3, were limited in new root development because of the

severity of the fungus infection. Roots were rotted-off at the base of

the cane in some instances. Uninoculated tip cuttings developed healthy

roots (Fig. 3).

The total fresh plant weights, total fresh root weights, and lateral

root weights of fungus-infected plants were reduced 16.3, 25.4 and 89.1%,

respectively, in comparison to the control plants.


5c. Pathogenicity of Pythium plendens to unrooted tip cuttings of
Chinese evergreen under cool temperature conditions.

The roots of the test plants were examined for evidence of the root

rot incited by Pythiu splendens on Chinese evergreen. Disease severity



























Fig. 1. Typical root rot of Chinese evergreen incited by
Pratylenchus coffee in peat.

a. Roots before removal of potting soil. Left, untreated
control; right,inoculated with Pratylenchus coffee.

b. Roots after removal of soil. Left, untreated control;
right, inoculated with Pratylenchus coffee. Note that
many of the roots sloughed off infected plants during soil
removal.




26

















..























b
I BD J

,,".`: ('i t
,' i 4 I1


































Fig. 2. The effect of Pthium splendens on mechanically
wounded roots of Chinese evergreen. Left, un-
inoculated control; right, inoculated with
Pythium splendens.





28































Fig. 3. Pathogenicity of Pythium splendens to unrooted tip
cuttings of Chinese evergreen. Left, uninoculated
control; right, inoculated with Pythium slendens.





30








was rated using a 1 to 5 scale in which 1 = no disease, 2 = very light,

3 = light, 4 = moderate, and 5 = severe. The plants from uninoculated

sterile soil had produced small healthy roots, and all received a rating

of 1. The tip cuttings from P. splendens-infested soil received ratings

of 5. No healthy roots were found, and the basal portion of each cutting

was severely rotted. The disease caused by Pythium splendens did appear

to be slightly more pathogenic at 22 C than at the higher temperatures of

the preceding experiment in which the test plants were held in a green-

house where ambient temperatures were often higher than 32 C.


6a. Inoculation of Pratylenchus coffee and Pythium splendens to
Chinese evergreen.

Pratylenchus coffee was not as pathogenic to Chinese evergreen in

this experiment as in others and had little effect on whole plant and

root weights (Table i). Neither necrosis nor lesions were noted on

nematode-infected roots.

Although Pthium splendens also was not as pathogenic in this ex-

periment as in others, it did cause necrosis and sloughing of root tips

and lateral roots.

The combination of Pratylenchus coffee and Pthium splendens was

only slightly more pathogenic than the fungus alone. Root rot symptoms

and degree of damage were similar to that caused by P. splendens alone.

The nematode reached higher population levels in plants inoculated

with the nematode alone than in plants inoculated with the nematode and

fungus.


6b. Pathogenicity and interaction of Pythium slenns and Pratylenchus
coffeae on Chinese evergreen.

Unrooted tip cuttings inoculated with P. splendens were severely









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damaged by the fungus and very few roots were found on the plants (Pig. 4).

Total plant and root weights were greatly reduced in comparison to the

control plants (Table 2).

The fungus caused about the same degree of damage to wounded rooted

cuttings as to healthy rooted cuttings. Total plant and root weights

were reduced. Root necrosis and sloughing were evident. Wounded cuttings

did not maintain new root growth because of the root rot incited by

Pythium splendens. The wounds created with scissors provided infection

courts for the fungus. The severity of the damage to the healthy cuttings

indicates that Pythium solendens does not require wounds to gain ingress

into the host.

Plants inoculated with Pratylenchus coffee alone showed an increase

in total plant and root weights, and a proliferation of lateral roots.

Healthy plants, inoculated with both pathogens simultaneously,

showed less damage than healthy plants which received the fungus alone.

Both treatments greatly reduced root weights.

Plants inoculated with the fungus 3 weeks after being inoculated

with the nematode had a slight reduction in total plant weight. Total

root weight was reduced to the same extent as that of healthy plants

inoculated with the fungus alone. Root damage was slightly greater with

the delayed fungus inoculation than when both pathogens were inoculated

simultaneously.

All control plants exhibited vigorous, healthy, root growth.

Root samples of plants inoculated with P. coffee alone, P. coffee

and P. splendens simultaneously, and P. splendens 3 weeks after P. coffee

yielded 422.8, 132.3, and 249.5 nematodes, respectively.





















Fig. 4. The effect of Pythium splendens and Pratylenchus
coffee, singly and together, on Chinese evergreen
growing in sandy soil.

Top row. Pythium solendens on: left, unrooted tip
cutting; center, wounded rooted cutting; right,
healthy rooted cutting.

Center row. Left, Pratylenchus coffee on healthy
rooted cutting; center, Pythium solendens inoculated
to healthy rooted cutting; right, Pythium solendens
inoculated to healthy rooted cutting 3 weeks after
Pratylenchus coffee.

Bottom row. Uninoculated controls: Left, unrooted
tip cutting; center, wounded rooted cutting; right,
healthy rooted cutting.

Note proliferation of lateral roots caused by
Pratylenchus coffee (center row, left).










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7a. Pathogenicity of Pythium splendens to coleus, geranium and begonia.

Geranium was the most susceptible to Pythium splendens of the three

ornamental plants tested. Blackening of the stems of unrooted tip

cuttings and some rooted cuttings of geranium was evident 3 days after

planting (Fig. 5). Eighteen days after planting, all geranium unrooted

cuttings and 2 rooted cuttings were dead.

Begonia unrooted tip cuttings also displayed blackening of the stem

(Fig. 5). Two of the unrooted cuttings died within 18 days after

planting. The rooted cuttings of begonia and rooted and unrooted cuttings

of coleus were not damaged by the fungus.


7b. Pathogenicity of Pythium snlendens to radish, coleus, geranium, and
cucumber seedlings.

Final stand counts of seedlings emerged in trays of sterile soil

and trays of Pythium splendens-infested soil were compared. The final

stand count was made 14 days after planting. The fungus killed 10% of

the seedlings of radish var. Long Scarlet Short Top, geranium var. Zonale

Mixed, and cucumber var. Improved Long Green. The stands of radish var.

Cherry Belle and coleus var. Rainbow Mixed were reduced by 90% and 98.7

respectively (Fig. 6).

Pythium splenden,isolated from Chinese evergreen, was extremely

pathogenic to seedlings of radish, coleus, geranium, and cucumber, and

appears to have a host range similar to that reported in the literature.




























Fig. 5. Pathogenicity of Pythiu lendens to unrooted tip
cuttings of geranium (left) and begonia (right).














~fi~
s :C+






























Fig. 6. Pathogenicity of Pythium splendens to selected vegetable
and ornamental seedlings.

a. Uninoculated controls. Reading clockwise from upper
left tray; coleus var. Rainbow Mixed, geranium var. Zonale
Iixed, radish var. Long Scarlet Short Top, radish var.
Cherry Belle, and cucumber var. Improved Long Green.

b. Inoculated with Pythium splendens.





_1_













DISCUSSION


Laboratory Experiments

Surface-sterilized specimens of Pratylenchus coffee did not cause

lesions when feeding in sterile, excised corn roots growing on an arti-

ficial medium. This indicates the possibility that secondary parasites

invading the nematode's feeding sites are responsible for the lesions

associated with Pratylenchus spp. under nonsterile conditions.

V-8 agar produced the greatest amount of growth by the fungus

Pythium splerdens. The fungus also grew well on potato dextrose and

corn meal agars. The optimum temperature for growth was between 25 and

30 C, with maximum growth occurring at 30 C. The fungus failed to grow

at 40 C and produced sparse growth at 15 and 20 C.


Greenhouse Experiments

Pratylenchus coffee reproduced on strawberry, coffee and rough

lemon, but not on tea, in the host preference test. It would be of

interest to determine if the Pratylenchus coffee found naturally

occurring on citrus in Florida would become established on and pathogenic

to Chinese evergreen. Conversely, it would also be of interest to

determine if the P. coffee from Chinese evergreen is capable of causing

severe damage to citrus. According to Dr. J. H. O'Bannon, USDA

Nematology Investigations, Orlando, Florida, P. coffee has the potential

to be a severe pathogen on citrus (personal communication, 1971). If

P. coffee from Chinese evergreen is pathogenic to citrus, the movement

of infested ornamental nursery stock may require regulation within the








state of Florida to prevent the infestation of citrus nurseries.

Soil type evidently plays an important role in the severity of the

damage caused by Pratylenchus coffee to Chinese evergreen. The nematode

seems to be more pathogenic in peat or high organic matter content soils

such as are found in the commercial nurseries of central Florida. P.

coffee was capable of destroying the root system of Chinese evergreen

growing in peat within 4 months after inoculation. The nematode's

pathogenicity to Chinese evergreen was greatly reduced in sandy soils.

The fungus ythium sendens was highly pathogenic to Chinese

evergreen, and was capable of destroying established root systems or

preventing the formation of roots on tip cuttings. It did not require

mechanical wounds for entry into the host's tissues. The fungus was

more pathogenic at 22 C than at higher greenhouse temperatures. This

correlates with Tisdale's findings (32).

No synergistic activity was noted in experiments 6a and 6b when the

nematode and fungus were inoculated to Chinese evergreen simultaneously.

The two organisms inoculated together caused only slightly more damage

than the fungus alone in experiment 6a and less damage in experiment 6b.

More damage occurred when the fungus was inoculated to plants 3 weeks

after the nematode than when they were inoculated to plants together.

It is possible that the nematode created wounds which provided infection

courts or aided in the ingress of the fungus.

The presence of the fungus in the same roots as the nematode did

not cause an increase in the population of the nematode in these tests

as was reported in a Verticillium dahliae-Pratylenchus penetrans complex

on eggplant and tomato (18). Nematode populations were probably lower

in fungus-inoculated treatments because of the reduction in feeding area








created by the severity of Pythium solenden's pathogenicity.

Plants inoculated with Pratylenchus coffee alone displayed an

increase in total plant and root weights in some tests. This phenome-

non, although not new in host-parasitic nematode relationships, was

surprising in view of the nematode's pathogenicity to Chinese evergreen

in other tests. The only explanation in this instance would appear to

be the influence of soil type. P. coffee feeding caused healthy rooted

cuttings of Chinese evergreen to produce many fine lateral roots. This

observation is supported by the data presented in Table 2. Fresh root

weights of P. coffeae-infected Chinese evergreens increased by 18.3%

over comparable uninoculated check plants. The difference in root

growth can be seen clearly when Figures 4D and 41 are compared.

Pythium splendens was pathogenic to rooted and unrooted cuttings of

geranium, and to unrooted cuttings of begonia, but not to rooted cuttings

of begonia. Apparently the fungus functioned as a wound parasite in

rotting only the unrooted begonias. It was not damaging to coleus

cuttings.

Seedlings of coleus, radish, geranium, and cucumber were severely

damaged by the fungus.














SUMMARY


The roles of the fungus, Pythium solendens, and the nematode,

Pratylenchus coffee, in the etiology of the root rot of the Chinese

evergreen, APlaonema modestum, were studied.

A technique was devised whereby Pythium splendens was isolated

consistently from diseased Chinese evergreen.

The nematode, Pratylenchus coffee, was surface sterilized in 0.1%

streptomycin sulphate and successfully colonized on sterile excised corn

roots growing on Tiner's medium. Numerous nematodes, free of root rot

associated organisms, were obtained from the excised corn roots for

inoculum purposes.

Pythium splendens exhibited optimum growth on V-8 agar. Potato

dextrose agar and corn meal agar provided slightly less growth, and lima

bean agar the poorest. The optimum temperature for growth of the fungus

was 30 C.

Only _Pythium splendens, from among several fungi consistently found

in association with Chinese evergreen root rot, proved to be pathogenic

to Chinese evergreen. The fungus was extremely pathogenic to healthy

rooted plants, unrooted tip cuttings, and plants with wounded roots.

The root rot incited by P. splendens was slightly more damaging at 22 C

than at higher temperatures.

Pratylenchus coffee was capable of inducing a root rot of Chinese

evergreen within 4 months after inoculation. However, the nematode was

highly pathogenic only in a high organic content soil such as peat.








Pratylenchus coffee and Pythium splendens inoculated together

caused only slightly greater damage to Chinese evergreen than the fungus

alone in one experiment. The fungus inoculated alone, and the fungus

inoculated 3 weeks after the nematode, proved to be equally damaging to

the roots of Chinese evergreen. Both treatments were more severe than

when the fungus and nematode were inoculated simultaneously. The fungus

inoculated alone greatly reduced total fresh plant weight as compared to

the nematode-fungus combinations. The nematode inoculated alone increased

total fresh plant and total root weights in sandy soil. Pratylenchus

coffee populations did not increase in number in the presence of Pythium

splendens. The nematode populations probably decreased because of the

rapidity and severity of the root rot incited by P. splendens.

Pratylenchus coffee reproduced on rough lemon, coffee, and straw-

berry, but not on tea.

The reproductive potential of specimens of Pratylenchus coffee

grown on either Chinese evergreen or excised corn roots for several

generations was virtually identical when both populations were inoculated

to Chinese evergreen.

Pythium splendens was pathogenic to unrooted and rooted cuttings of

geranium, unrooted cuttings of begonia, and seedlings of radish, geranium,

cucumber, and coleus. Rooted begonia cuttings and rooted and unrooted

coleus cuttings were not damaged. The isolate of Pythium splendens

obtained from Chinese evergreen, Arlaonema modestum, has a host range

similar to that described in the literature.













LITERATURE CITED


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3. Chen, T. A., J. Kiernan, & W. F. Mai. 1965. A technique for
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4. Conroy, J. J., R. J. Green, Jr., & J. M. Ferris. 1972. Interaction
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49


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BIOGRAPHICAL SKETCH


Kenneth Arlen IHoegel was born January 13, 1938, in Starke, Florida.

He attended public schools in Starke, Florida, and was graduated from

Bradford County High School in 1956.

He enrolled in the University of Florida in 1956 and received a

Bachelor of Science in Agriculture degree with a major in entomology in

1960. He enrolled in graduate work in nematology and received a Master

of Science in Agriculture degree in 1963 while working as a graduate

assistant in the Florida State Collection of Arthropods, Florida Depart-

ment of Agriculture. Following completion of formal course requirements

for the Ph. D. program, he took a position in the Department of Plant

Pathology as research assistant to Drs. V. G. Perry and H. N. Miller.

In October, 1968, he joined the Research Department of Chemagro,

Division of Baychem Corporation, at Stanley, Kansas. In April, 1971, he

was transferred to Chemagro's Vero Beach Laboratories research farm in

Florida, and placed in charge of the Nematicide-Fungicide Section.

He is a member of the Society of Nematologists, the Organization of

Tropical American Nematologists, and the American Phytopathological

Society.

He is married to the former Carolyn Claire Douglas, and they have

an adopted son, Jonathan Kenneth.








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



.IG. Perry, Coirman
Professor of Nmatology

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



H. N. Miller, Cochairman
Professor of Plant Pathology

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



G. C. Smart, Jr.
Associate Professor of Nematolo

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



P. Decker
Professor of Plant Pathology







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

August, 1972


Dean, Collee of Agriculture



Dean, Graduate School




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