Biological control of water weeds with plant pathogens

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Biological control of water weeds with plant pathogens
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Florida Water Resources Research Center Publication Number 45
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Freeman, T. E.
Charudattan, R.
Conway, K. E.
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University of Florida
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VATER RESOURCES

research center

Publication No. 45
Biological Control of Water Weeds With Plant Pathogens
By
T.E. Freeman
(Principal Investigator)
Plant Pathology Department
University of Florida
Gainesville













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Biological Control of Water Weeds With Plant Pathogens



By


T. E. Freeman
R. Charudattan
K. E. Conway


PUBLICATION NO. 45


FOIRIDA WATER RESOURCES RESEARCH CENTER


RESEARCH PROJECT TECHNICAL COMPLETION REPORT



OWRP Project Number A-033-FLA



Annual Allotment Agreement Numbers


14-34-0001-7019
14-34-0001-7020
14-34-0001-8010


Report Submitted October, 1978


The work upon which this report is based was supported in part
by funds provided by the United States Department of the
Interior, Office of Water Research and Technology
as Authorized under the Water Resources
Research Act of 1964 as amended.










TABLE OF CONTENTS


BIOLOGICAL CONTROL OF WATERWEEDS WITH
PLANT PATHOGENS 1

Introduction 1
Relevence of Research
Research Approach

DEVELOPMENT OF CERCOSPORA RODMANII AS A
BIOLOGICAL CONTROL FOR EICHHORNIA CRASSIPES 7

Introduction 7
Pathogenicity Evaluation 8
References 14

DISEASE RESISTANCE MECHANISMS IN WATERHYACINTH
AND THEIR SIGNIFICANCE IN BIOCONTROL PROGRAMS
WITH PHYTOPATHOGENS 16

A DUTCH ISOLATE OF FUSARIUM ROSEUM 'CULMORUM'
MAY CONTROL HYDRILLA IN FLORIDA 23

Introduction 23
Experimental 24
Conclusion 29
References 30

GERMINATION AND STORAGE OF AND INFECTION BY
UREDOSPORES OF UROMYCES PONTEDERIAE 31


OTHER STUDIES 34

Survey for C. rodmanii in Florida and
Louisiana 34
Rating scale for C. rodmanii 36
Pathogenicity of Phycomycetes to
Hydrilla 38
Other Pungi Isolated from Hydrilla 41
Effect of C. rodmanii on Fish 41

Survey of Hydrilla Undergoing Annual
Decline for Pathogenic Bacteria 43

Introduction 44
Materials and Methods 46
Results 48
Discussion 49
Literature Cited 53







Comparison of Three Nozzle Systems
for Spraying Cercospora rodmanii. 55

Materials and Methods 56
Results 57
Conclusion and Recommendations 60

SUMMARY AND CONCLUSION 61

PROJECT PUBLICATIONS 63


im














BIOLOGICAL CONTROL OF WATER WEEDS WITH PLANT PATHOGENS






Introduction


Plant pathogens have many characteristics that make
them ideal candidates as biocontrols for aquatic weeds. They
are: (1) numerous and diverse; (2) frequently host specific;

(3) easily disseminated and self-perpetuating; (4) will not
completely eliminate a host species; and (5) do not normally

affect man or other animals. With these points in mind, a

modest program was begun at the University of Florida in 1970,
with the object of the evaluation and subsequent use of plant i

pathogens as biocontrol agents for aquatic weeds. The program

was expanded with the aid of a matching grant from the U.S.
Department of Interior's Office of Water Resources Research,
subsequent support from the Florida Department of Natural

Resources, the U. S. Army Corps of Engineers and from the
annual allotment program of the Florida Office of Water
Resources Research.

The program progressed rapidly considering the lack of
initial background information. We have developed a


'*







considerable backlog of information (see list of project

publications) about diseases affecting aquatic plants. The

objective of the utilization of plant pathogens in biocontrol

programs for at least one noxious aquatic plant is nearing

fruitition. We have reached the stage where large-scale

field evaluation of the fungus Cercospora rodmanii for control

of waterhyacinth is warranted. An additional three or four

organisms also should soon reach this point. We have also

attempted to find and research diseases with biocontrol

potential for other aquatic weeds.


Relevance of Research

The aquatic weed problem is one of considerable pro-

portion that appears to be growing in magnitude rather than

diminishing or even stabilizing. This is occurring despite

the expenditure of considerable sums of money and human

energy in the application of conventional methods of mechani-

cal and chemical control.

The Florida Department of Natural Resources estimates

over 20 million dollars are expended annually in Florida for

aquatic weed control. These control efforts are concentrated

primarily on the estimated 100 thousand hectares of water-

hyacinth (Eichhornia crassipes) and 40 thousand hectares of

hydrilla (Hydrilla verticillata) that occur in the state.

Lesser attention is given to the approximately 20 thousand

hectares of other aquatic weeds, such as Eurasian watermilfoil








(Myriophyllum spicatum) and alligatorweed (Alternathera
philoxeroides) (Burkhalter, personal communication.) Despite

these efforts, aquatic weed infestations have increased

steadily in the years since these plants were introduced. The
range of these plants has also expanded to include virtually

all of Florida. Within the last 5 years, Eurasian watermil-

foil was found in the St. Johns River watershed and hydrilla

was found to infest Rodman Reservoir on the Cross Florida
Barge Canal, Okeechobee, Orange, and Lochlossa Lakes.

Florida is by no means unique in having a tremendous

aquatic weed problem. Proliferating water weed populations
are of concern in the rest of the United States, Middle

Europe, Africa, Asia, and South and Central America. Indeed

the problem is world-wide, but is more acute in the warmer
latitudes where waterhyacinth, hydrilla, watermilfoil,

alligator weed, salvinia (Salvinia spp.) and water lettuce
(Pistia stratioties), are the major offenders. Reasons for
the increasing aquatic weed problem are complex, but are

definitely related to man and his activities. With the

increase in population and the accompanying environmental

problems, it has become apparent that new methods of aquatic

weed control must be found. Conventional methods have not
been entirely satisfactory either because of cost, overall

ineffectiveness, or environmental pollution. The energy

problem as it relates to fossil fuel supply has also served
to emphasize the need for low-energy methods of control.


I




-4-



In recent years, biological control methods have received

considerable attention. Various species of herbivorous .

insects, fish, snails, and even the manatee have been, or are

being, investigated for their ability to exert some control

pressure on noxious aquatic plants. Some of them, such as

the alligatorweed flea beetle, have been reasonably effective,

especially in.an integrated control program. Surprisingly,

until our program was initiated, plant pathogens had been

rarely considered as biocontrol agents. They have all the

prerequisites of a biocontrol agent and thus offer an untapped

reservoir of potential usefulness, either alone or in an inte-

grated control program with insects and perhaps, chemicals.

Our research efforts are aimed at bringing to fruitition this

use of plant pathogens in control programs for aquatic weeds.


Research Approach

We are using two approaches in our efforts to utilize

plant pathogens to control aquatic weeds. They are:

1) The use of endemic or native plant pathogens as a type

of "biological herbicide" through the artificial

induction of epiphytotics. We consider this to be

the most rapid approach from an operational stand-

point.

2) The search for and ultimate utilization of exotic

plant pathogens. This has been the classical

approach successfully used by entomologist in their


1





-5-


biological control efforts toward imported weeds.

This facet involves the search for pathogens near

the center of origin of the noxious species, in an

area where climatic conditions are similar to those

where the pest is a problem in this country. This is

slower of the two approaches from an operational

standpoint.

Our research efforts since the inception of this project

are indicated by the titles on the list of publications.

Publications Number 23 and 30 of the Florida Office of Water

Resources Research summarizes the first six years of our

research work.

During the past two and one-half years, our efforts have

been directed primarily toward those pathogens with definite

biocontrol potential. These are: the endemic pathogens of

waterhyacinth, Acremonium zonatum and Cercospora rodmanii

and two exotic ones, Uredo eichhorniae and Fusarium culmorum

for hydrilla control. We have carried out extensive cultural

studies in the laboratory, greenhouse studies and,:in the case

of the endemic pathogens, various small scale field tests.

These latter tests have shown A. zonatum and C. rodmanii to

have considerable potential as biocontrols. We have tested

both of these at locations in Florida and in Lake Concordia

in Louisiana. In this latter test, the two pathogens were.

combined with two insects (Neochetina eichhorniae and Arzama

densa) in all possible combinations. This test was conducted




-6--


in cooperation with the U.S. Army Corps of Engineers, Water-

ways Experiment Station and the U.S. Department of Agriculture

with the approval of the Louisiana Department of Agriculture

and the Louisiana Fish and Game Commission. We believe C.

rodmanii to have been the cause of a spectacular decline of

waterhyacinth in Rodman Reservoir in 1971. This natural

decline saved the Army Corps of Engineers approximately

$35,000 in spray cost in that body of water (Zeiger, personal

communication). Laboratory and greenhouse studies with A.

Zonatum on waterhyacinth have elucidated a general resistant

mechanism in this plant that accounts for its disease

reaction.

Work with the two exotic pathogens is being done in our

quarantine facility, which is limited in size. Therefore, the

work is progressing at a slower pace than with the endemic

pathogens.

This report summarizes the research conducted under

project A-033-Fla. For more detail on specific points, the

reader is referred to published articles. The project is

being continued.


m














DEVELOPMENT OF CERCOSPORA RODMANII AS A BIOLOGICAL

CONTROL FOR EICHHORNIA CRASSIPES

K. E. Conway, T. E. Freeman, and R. Charudattan




Introduction

A decline of populations of waterhyacinth was first
noticed in 1970 in Rodman Reservoir, a large impoundment of
water (3,491 ha) near Orange Springs, Florida. Symptoms

associated with waterhyacinth during this decline were a
yellowing of the plant, formation of spindly petioles and a
rot of the root portion of the plant. It was estimated that
this decline saved approximately $20,000 for weed control in
this reservoir for one season. Unfortunately, this decline
was less severe in each succeeding year and allowed the
waterhyacinth to almost completely reestablish in the reser-
voir. -A survey of fungi associated with waterhyacinth in
this reservoir (Conway, et al. 1974) resulted in the isola-
tion of a Cercospora that was later determined to be a new
species, Cercospora rodmanii Conway (Conway 1976a).



From Proc. EWRS 5th symposium on aquatic weeds. 1978.


I.-



j




-8-


Pathogenicity Evaluations

The pathogenicity of the fungus was evaluated in

several preliminary tests (Conway 1975) which indicated that

the fungus was capable of producing lesions and chlorosis on

leaves and petioles of waterhyacinth. This testing culmi-

nated in a small-scale field test (Conway 1976b) in a small

isolated pool of Lake Alice, located on the campus of the

University of Florida. Procedures for the preparation and

application of the fungus have been published (Conway 1976b).

Approximately 1 Kg of a mycelial suspension was inoculated

onto waterhyacinth in an area of 65 M2. Infection was

evident on the inoculated plants within two weeks. Although

the fungus was applied to a small area of waterhyacinth, the

results from this test indicated that once the disease was

established on plants it was capable of spreading via wind-

borne conidia to infect most of the waterhyacinths in the

pool (1.7 ha). Even though the populations of waterhyacinth

regrew in the spring, there were indications that the new

populations had been severely stressed, based on their

slower growth rates when compared to noninoculated plants on

the other side of the lake. In addition, the fungus was

capable of overwintering on the plants and initiated the

disease cycle again in the spring.

Host specificity of C. rodmanii was tested (Conway and

Freeman 1977) on over 85 economically and ecologically impor-

tant plants that are either grown commercially or occur


I








naturally in Florida. A modified centrifugal (related

plants) and varietal (economic plants) strategy was used to

determine which plants would be included for testing.

Plants tested represented 22 families of higher plants and

were evaluated in both the greenhouse and the field. The

results indicated that C. rodmanii was pathogenic only to

waterhyacinth and was safe to use as a biological control

in Florida. In a recent publication of preliminary results

concerning an attempt to control waterhyacinth with an inte-

grated pathogen-insect combination (Addor 1977a), it was

erroneously stated that C. rodmanii had a wide host range.

Thus, this publication should be disregarded in favor of I'

Addor's (1977b) later publication of the completed results

in which C. rodmanii was correctly noted as being host

specific for waterhyacinth.

As integrated pathogen-insect field test which include

the pathogens, C. rodmanii and Acremonium zonatum (Sawada)

Gams, and two insects, Neochetina eichhorniae Warner and

Arzama densa Walker, was conduced in Lake Concordia,

Louisiana (Addor 1977). These organisms were evaluated

alone and in combination on waterhyacinths confined in

frame's enclosed in an area of approximately 3.25 M 2. C.

rodmanii was applied as a mycelial suspension at a rate of

48 gm/M2 and A. zonatum was applied at a rate of 96 gm/M2.

The stocking rates for the insects N. eichhorniae and A. ':

densa were 150 and 50 insects per frame, respectively. The




-10-'

FIGURE 1


160


140


120


100


80


60

40

20



160


140


120


100


80

60

40

20


174 194 214 234 254
TIME, DAYS


254 274


174 194 214 234
TIME, DAYS


160


140


120


100


80

60

40

20


274


254


174 194 214 234 254
TIME, DAYS


274


274


THE EFFECT OF COMBINATIONS OF PLANT PATHOGENS AND INSECTS
ON THE WEIGHT OF WATERHYACINTH

INTEGRATED CONTROL OF WATERHYACINTH
LAKE CONCORDIA, LOUISIANA, USA
23 JUNE 30 SEPTEMBER


CONTROL







ARZAMA


160


140


120


100


80

60

40

20


174 194 214 234
TIME, DAYS


CONTROL








ARZAMA
NEOCHETINA
CERCOSPORA


CONTROL








AR7
NEdA TINA
CERCOSPORA
ACREMNITM


CONTROL







ARZAMA
NEOCHETINA





-11-


frames were weighed at two week intervals from 19 June to I

30 September 1975. The criterion used for damage to these

populations of waterhyacinth was the difference in weight

of the treated plants vs. the untreated plants. The impor-

tant result of this study was in indication that damage to

waterhyacinth increased with the number of organisms used to

stress the plants. The additive effect of combinations of

plant pathogens and insects is illustrated in Figure 1. The

reduction in the weights of the waterhyacinth in the frames

by each organism as well as in various combinations of each

organism were determined. Acremonium zonatum caused the

least reduction in weight of the waterhyacinths when compared

to each of the other organisms. However, greater reductions

in the weight of waterhyacinths were achieved as more organisms

were applied to these plots. The greatest reduction of weight

was achieved at the end of the test period when all four

organisms were used to stress the plants.

Following the Lake Alice study, the ability of C.

rodmanii to stress waterhyacinth populations was tested

further in Florida. The fungus was re-introduced onto water-

hyacinths in the area of Rodman Reservoir where the disease

had been isolated originally. Five applications, each con-

sisting of approximately 1 kg of mycelial inoculum, were
2 J
applied every two weeks to a small area (65 M2) of water-

hyacinth along the shoreline beginning in February 1975. The

disease became established in this area within two weeks of

the first application. During the next six months the
,iii




-12-


disease spread to infecL most of the waterhyacinth in the

area (approximately 15.0 ha). At that time (July), some of

the individual waterhyacinth plants had died and sunk

beneath the surface of the water. Waterlettuce (Pistia

stratiotes L.) and yellow cow lily (Nuphar luteum (L.)

Sibthorp and Smith) had replaced these dead plants in the

waterhyacinth mats. By August, the enitre population of

waterhyacinth was under severe stress from the pathogen and

there was approximately 7.0 ha of open water where origin-

ally there had been complete coverage of waterhyacinth.

During the Fall of 1975, the area of open water reached a

maximum of 10.0 ha before cold winter temperatures limited

the spread of the disease. No additional inoculations of

the fungus have been applied to waterhyacinth in this area

and each year since the original inoculation, the disease

has overwintered and initiated epidemics the following

spring. The disease spread to infect waterhyacinth in most

of the reservoir and as a result of the stress placed on

the plants by the disease it has not been necessary to

expend either large sums of money or energy for water-

hyacinth control in the reservoir during 1975-77. A com-

plete elimination of waterhyacinth is not possible using

the disease alone and fluctuations in the waterhyacinth

populations have occurred which are influenced by environ-

mental factors which condition the host-pathogen balance.

During the Rodman study it was determined that under





-13-


optimal conditions waterhyacinths were capable of producing

one new leaf every 5-6 days. This rate will vary depending

on environmental conditions and during unfavorable periods

this rate may decline to less than one leaf produced over a

three week period. Therefore, the success of the epidemic

will depend upon the rate at which the pathogen can infect

and kill these new leaves. In order to determine if there

was an optimal concentration of inoculum necessary to

initiate disease, an inoculum rate experiment was begun in

a small lake southeast of Gainesville, Florida. Water-

hyacinths were confined in 9 M2 frames and treated at three

mycelial inoculum concentrations: 48 gm/M2, 96 gm/M2, and

192 gm/M2. Results showed that regardless of the initial

inoculum level, the rate of disease spread became equalized

after a period of time due to inoculum buildup on the

inoculated plants and cross infectivity between plots. The

maximum rate of damage produced by C. rodmanii was assessed

at the 192 gm/M2 inoculum level and this rate was not I'

exceeded even with an additional application of inoculum

later in the year. The maximum rate of damage caused by C.

rodmanii during this experiment corresponded to the death-

of 1.0-1.3 leaves of the waterhyacinth every three weeks.

Therefore, when conditions exist which favor disease deve- VA

lopment and which limit leaf production to less than one

leaf per three weeks, C. rodmanii can infect and kill leaves

faster than the plant can produce new leaves. The plant




-14-


becomes debilitated and over a period of time will die unless

conditions change to favor its regrowth.

The use of C. rodmanii as a biological control for

waterhyacinth has been patented by the University of Florida.

The University is working with Abbott Laboratories, Chicago,

Illinois, U.S.A., to produce a commercial product of the

fungus. Evaluation of a product has already begun.

References

ADDOR, E. E. (1977a) A field test of selected insects and

pathogens for control of waterhyacinths. Report I.

Preliminary results for the 1975-76 season. Tech. Rpt.

A-77-2, U.S. Army Corps of Engineers, Vicksburg, MS,

USA 54 pp.

ADDOR, E. E. (1977b) Controlled field tests of selected

insects and pathogens in combination on waterhyacinth.

Proc. Research Planning Conf. Aquatic Plant Control

Program. Misc. Paper A-77-3, U.S. Army Corps of

Engineers, Vicksburg, MS pp. 236-268.

CONWAY, K. E. (1975) Procedures used to test endemic plant

pathogens for biological control of waterhyacinth.

Proc. Amer. Phytopath. Soc. 2:31 (Abstr.).

(1976a) Cercospora rodmanii, a new pathogen of

waterhyacinth with biological control potential. Can.

J. Bot. 54:1079-1083.

(1976b) Evaluation of Cercospora rodmanii as a

biological control of waterhyacinth. Phytopath. 66:914-916.





-15-


CONWAY, K. E. and T. E. FREEMAN (1977) Host specificity of
Cercospora rodmanii, a potential biological control of
waterhyacinth. Plant Dis. Rptr. 61:262-266.
CONWAY, K. E., T. E. FREEMAN and R. CHARUDATTAN (1974) The
fungal flora of waterhyacinths in Florida. Part I.
Water Resources Research Center, Univ. Florida, Publ.
No. 30, Gainesville, Florida, 11 pp.
I I





-16-


DISEASE RESISTANCE MECHANISMS IN WATERHYACINTHS AND THEIR
SIGNIFICANCE IN BIOCONTROL PROGRAMS WITH PHYTOPATHOGENS 1
R. D. Martyn



The waterhyacinth [Eichhornia crassipes (Mart.) Solms.]
is a free-floating vascular hydrophyte that has colonized
much of Florida's inland waters. In 1970, a program was
initiated at the University of Florida to study biological
control of this noxious plant with phytopathogens. One of
the pathogens currently being studied is the fungus Acremo-
nium zonatum (Sawada) Gams. It causes severe spotting on
both leaves and petioles of this plant under conditions of
high humidity.
During field trials with this fungus, it was observed
that small, young, plants displayed fewer symptoms after
infection than did larger plants in the same plots. It also
appeared that large plants infected with A. zonatum exhibited
a faster rate of leaf regeneration than did smaller plants.
The present study was initiated to determine if small plants '
I were in fact more resistant to A. zopatum than large plants; __

if meristematic activity in the plants was altered after
infection; and, if so, to what extent host phenolic compounds
and their oxidizing enzymes, namely polyphenoloxidase (PPO),
were responsible.


1 From Ph.D. Dissertation, University of Florida.




I




-1 /-


Waterhyacinths displayed various degrees of resistance

to A. zonatum depending on their morphotypic state of deve-

lopment. Results of this study indicate that these differ-

ences in resistance are due to the variations in phenol

chemistry among plants of different sizes and to subsequent

changes induced by infection (Table S-1).

Small plants are more resistant to fungal attack than

are medium or large plants, based upon the number of lesions/

leaf after infection. It appears that the presence of high

concentrations of phenolic compounds does not itself impart

resistance to the pathogen. Rather it is the oxidation of

these compounds by enzymes, such as polyphenoloxidase (PPO),

which is responsible for the resistance. This view is

supported by qualitative and quantitative data on the

phenols in plant morphotypes and is coincident with the

observed differences in resistance.

Small plants, by virtue of having fewer phenol cells/

mm2 leaf area, have less total phenol content/leafthan

larger plants. If phenol content alone, was responsible

for disease resistance, then small plants would be more

susceptible than large plants but they were not. In this

case PPO activity is apparently the mediating factor. The

rate of enzyme activity in small plants is three-fold that

in large plants; presumably therefore, oxidation of poly-

phenols to quinones is much greater in small plants. Thus,

small plants are initially more resistant to pathogenic





-18-


attack than are larger plants.

After the disease has progressed for several weeks the

differences in resistance among the morphotypes is no longer

evident. Each plant size exhibits a percent-total-diseased

leaf area which is statistically the same (approximately

40%). It is believed that this equalization of disease

severity results from a gradual loss in resistance by small

plants while at the same time there is a gradual increase in

resistance by large plants. Again, quantitative data of the

phenol metabolism can be correlated with this change.

The total phenol content decreased significantly after

infection in small and medium-sized plants. This is coinci-

dent with a reduction in PPO activity. The coupling of

these two phenomena may account for the decrease in resis-

tance of small plants. Large plants, on the other hand,

retain their total phenol content and at the same time

exhibit a three-fold increase in PPO activity. Therefore,

an increase in polyphenol oxidation would be expected to

occur and could account for the increase in resistance in

large plants.

In essence, then, the point being made is: if infected H

small plants retained the phenol content and PPO activity of

healthy plants, then disease severity would probably be

limited to much less than 40%. Similarly, if infected large

plants retained the PPO activity of healthy plants, disease

would progress to a much higher percentage, perhaps 60-70%.










TABLE 1. DIFFERENCES AND SIMILARITIES AMONG HEALTHY AND

A. ZONATUM-INFECTED WATERHYACINTH MORPHOTYPES.


MORPHOTYPE
ASSESSMENT CRITERIA
SMALL MEDIUM LARGE

MEAN # LESIONS/LEAF 3.7 12.8 18.3
% TOTAL DISEASE 41,3 37.0 39.5
MEAN # PHENOL CELLS/MM2 33.6 41.8 48.7
PPO RATE (HEALTHY) 1.53 0.80 0.47
PPO RATE (DISEASED) 0.90 0.70 1.36
PPO LOCALIZATION (HEALTHY) 3 CELL TYPES 3 CELL TYPESA 3 CELL TYPESA
PPO LOCALIZATION (DISEASED) ALL CELLSB ALL CELLS ALL CELLSB
TYPE OF PHENOLIC ACIDS (HEALTHY) 6 6 9
TYPE OF PHENOLIC ACIDS (DISEASED) 7 7 9
TOTAL PHENOLS (HEALTHY) 92 vG/G 106 pG/G 104 yG/G
TOTAL PHENOLS (DISEASED) 80 pG/G 96 vG/G 105 pG/G
FUNGAL GROWTH (HEALTHY) STIMULATIVEC STIMULATIVEC STIMULATIVEC
FUNGAL GROWTH (DISEASED) STIMULATIVED STIMULATIVED STIMULATIVED
LEAF REGENERATION (HEALTHY) 27.3% 28.5% 46.1%
LEAF REGENERATION (DISEASED) 21.6% 33.9% 93.3%


AVASCULAR PARENCHYMA, BUNDLE SHEATH, AND PHENOL CELLS; BALL CELLS WHICH CONTAIN CHLORO-

PLASTS; Cp=0.05; GROWTH INCREASED OVER CONTROLS: Dp=0.05; GROWTH INCREASED OVER HEALTHY

f fe





-20-


However, because each morphotype responds to infection

differently (in most cases in contrast to each other) disease

severity balances among the plant sizes at approximately 40%

of the leaf-surface area.

If disease severity is viewed, not from a percentage

of leaf-area infected, but as a reduction in plant growth,

then data on leaf regeneration rates among the morphotypes

becomes of prime importance. It has been observed that

infected large plants regenerate two to three times as many

new leaves as do infected small plants. This too, is cor-

related with the plant's phenol chemistry.

It has been shown that A. zonatum is capable of synthe-

sizing indoleacetic acid in vitro and that this is one

explanation for the increased growth observed in large plants.

More important, however, is the fact that phenols are known

inhibitors of IAA oxidase, the enzyme responsible for con-

trolling the IAA level in the plant. It has already been

pointed out that the different waterhyacinth morphotypes

vary in phenol content, both prior to and after infection.

The higher phenol content in large plants could account for

the increased growth observed in large plants by inhibition

of the IAA oxidase system.

Perhaps the most significant data supporting a positive

role for phenols in disease resistance comes from the loca-

lization studies of PPO in healthy and diseased plants.

Enzyme activity is localized in the thylakoids of chloroplasts




-21-


in only three cell types in healthy plants. After infection

there is a "turn on" in PPO activity in all cells which con-

tain chloroplasts. This turn on in PPO activity is highly

suggestive of a vital role for enzymatic oxidation of poly-

phenols during disease.

Before disease can ensue, the pathogen must come into

contact with and penetrate its host. In this regard, A.

zonatum can enter the waterhyacinth by either of two ways:

through open stomata or by directly penetrating the unbroken

cuticle of the leaf. Intracellular colonization is enhanced

by the diffuse secretion of cellulolytic enzymes and perhaps

by the localized secretion of pectolytic enzyems.

Growth of A. zonatum was either unaffected or stimu-

lated by seven different phenolic acids in concentrations up

to 1000 ppm in minimal media. When yeast extract was added

to the media as a growth supplement, one phenolic acid, p-

coumaric, was found to be inhibitory. In addition, fungal

growth was enhanced on media containing yeast extract and

extracts from diseased leaves over that on media containing

healthy-leaf extracts.

Several cytological changes were observed in the cells

from infected waterhyacinth leaves. First, chloroplasts in -

cells of healthy leaves have an abundance of starch granules

which disappear after infection. Second, there are only a

few plastoglobuli in chloroplasts in healthy cells, but

after infection, they increase both in size and in number.




-22-


Third, there is a noticeable increase in microbodies in the

cytosol of infected cells. It is believed that each of

these cytological changes is the result of a shift in host

metabolism induced by infection.

It is concluded that waterhyacinths have at least two

distinct biochemical defense mechanisms that are related to

phenol metabolism and plant size. The first is the presence

of high concentrations of polyphenols in specialized phenol-

cells which, under the proper conditions, can serve as

toxicants to potential pathogens. The second proposed

defense mechanism of waterhyacinths is an acceleration of

its growth rate brought about by the inhibition of IAA

oxidase by the phenolic compounds.

Which of the above mechanisms is operational is depen-

dent upon the plant's morphotypic stage of development. It

is believed that initially small plants defend against

pathogenic attack by virture of their high PPO activity

whereas large plants respond by increased leaf production.

Medium-sized plants appear to have a combination of both
I,
mechanisms.

In consideration of A. zonatum as a potential biocon-

trol agent for waterhyacinths, it is concluded that best

control would be achieved with small, young, plants rather ,

than with larger, more mature plants. In this regard,

control procedures should be initiated early in the spring

when new plants start to grow and colonize the body of water.




-23-


A DUTCH ISOLATE OF FJSARIUM ROSEUM 'CULMORUM' MAY

CONTROL HYDRILLA VERTICILLATA IN FLORIDA1

R. Charudattan and D. E. McKinney


Introduction


It is estimated that a fifth of all fresh water ponds,

lakes and rivers in Florida is infested with Hydrilla verti-

cillata L. F. Royle (Hydrocharitaceae), and the weed is

spreading rapidly. Since its introduction into Florida

waters around 1960, this weed has moved to several other

states in the U.S.A. Serious economic losses and ecologic

damages resulting from this submerged weed have spurred

research on biological, chemical, and mechanical controls.

Among biological agents researched are plant pathogens

(Charudattan, et al., 1974; Freeman, 1977); however, very

few disease of submerged weeds are known (Zettler and Freeman,

1972) and those found on Hydrilla (Charudattan, 1973; Charu-

dattan and Lin, 1974; Freeman, 1977) have not been suffi-

ciently damaging or specific to this host to promote their

use in the field.

In 1974, a disease of Stratiotes aloides L. (Hydrocha-

ritaceae) was discovered near Wageningen by Dr. J. C. J. van

Zon who brought it to our attention. Mature plants had


I
From Proc. EWRS 5th Symposium on aquatic weeds.





r-24-


symptoms of root- and crown-rots and severely diseased
plants appeared to sink gradually as a consequence of tissue
decay. A few infected plant parts were taken to Gainesville,
where a group of fungi were cultured from them including
predominantly a Fusarium roseum 'Culmorum' (Lk. ex Fr.)
Synd. & Hans. In view of the close taxonomic relationship
between S. aloides and H. verticillata, the pathogenic poten-
tial of these fungi to the latter was of obvious interest to
us. Among the fungal isolates obtained from S. aloides, I 1
only 'Culmorum' was capable of killing Hydrilla (Charudattan
and McKinney, 1977). Results presented here will prove that
the Dutch 'Culmorum' is a virulent pathogen of Hydrilla unlike
most other fusaria tested on this host, and that it may help
control of Hydrilla in Florida.

Experimental .
The effects of the 'Culmorum' isolate on Hydrilla were
determined in three test systems. The first one consisted

of incubating 8 to 10 cm long terminal portions of Hydrilla
shoots in 3 X 15 cm glass tubes with 40 ml of sterile water

to which were added dense macroconidial suspensions. Control
- tubes were without conidia. Fungal inocula, consisting of

filtered macroconidial suspension obtained from potato dext-
rose agar cultures, were quantitated with a hemacytometer.
Inoculum levels between 2500 and 250,000 conidia per ml (100,
000 and 10 million conidia per tube containing 40 ml of water)
were set up my mixing suitable concentrations of conidial

suspensions. Inoculated and control Hydrilla tubes

i *





-25-


were incubated under diffuse light at 22 + 2 C for several

weeks. Damage to Hydrilla from the Dutch 'Culmorum' was

usually evident as chlorosis and discoloration of inoculated

shoots 10 to 14 days after inoculation. In 3 weeks, death

and lysis or regrowth of partially damaged Hydrilla were

observed. The threshold of inoculum needed to damage

Hydrilla was found to be 1 million conidia per tube or

25,000 per ml. A dose and effect relationship was seen on

inoculated Hydrilla; at lower inoculum levels the shoots

were only partially damaged or killed while at higher inocu-

lum levels the effects were drastic and lethal.

In the second system, 20 liter aquarium tanks were

layered with river sand, filled with 14 liters of water,and

planted with 100 terminal ends of Hydrilla shoots, each with

an active growing bud. After two days, the tanks were inocu-

lated with conidial suspensions of 'Culmorum' at approxi-

mately 80,000 or 90,000 conidia per ml of water in tanks.

Three weeks after inoculation, Hydrilla shoots started to

discolor and developed signs of rotting. In about 5 weeks,

the shoots broke down completely, and some that were still

green were defoliated and uprooted, and floated to the water

surface.

In the third system, the fungus was grown for two

weeks on a sterilized mixture of 9 parts sand, 1 part oat

meal and 3 parts water, and mixed with the bottom sand in

Hydrilla tubes at 1:1 and 1:10 proportions (w/w) of inoculum




-26-


and sand. Controls had sand-oat-water mixture without the

fungus, mixed with an equal weight of sand. A Hydrilla

plant with shoots, roots, and at least one tuber was planted

per inoculated and control tubes. After a week, the inocu-

lated plants turned pale and were dead by the end of 14

days.

In all these systems, the inoculated fungus could be

reisolated from inoculated, dead, dying, or green Hydrilla

shoots after surface sterilization and plating on potato

dextrose agar. Controls did not yield the fungus. In

addition, the conidia were observed to germinate on, and

penetrate into Hydrilla tissue which confirmed the pathogenic

capability of the fungus.

In order to decide that the effects of the 'Culmorum'

isolate on Hydrilla were specifically due to its infectivity

and not due merely to massive numbers of fungal spores in

water, a comparative inoculation test was set up. In this

test, three unidentified Fusarium spp., isolated from

Hydrilla in Florida, a F. roseum from Ficus elastica Roxb.

and a F. roseumi 'Graminearum' from Eichhornia crassipes

(Mart.) Solms in Florida were included. The test tube

procedure described first was used, with inoculum densities.

between 2500 and 250,000 conidia per ml of treated water.

The results confirmed that the Dutch 'Culmorum' was

indeed unique in its effects on Hydrilla. The three

Fusarium spp. from Hydrilla and the Ficus isolate of F.




-27-


roseum did not damage Hydrilla even at higher levels of I

inoculum. The 'Graminearum' from E. crassipes was capable

of damaging Hydrilla, inciting similar symptoms as the Dutch

'Culmorum'. However, the threshold of inoculum needed to

cause damage by this isolate was approximately 60,000

conidia per ml, or 2.4 times higher than that of 'Culmorum'.

The Dutch 'Culmorum' isolate hence was not only pathogenic

to Hydrilla but also was more virulent than any Fusarium

tested.

In another experiment, conidia and mycelial fragments

of the Florida isolate of 'Graminearum' from E. crassipes

were applied either as suspension or was injected into bottom

sand around 25 rooted Hydrilla shoots maintained in 4 liter i

glass jars under 2.5 liters of water. For inoculum, the

fungus was grown on potato dextrose broth for a week. About

30 g of wet, filtered mycelium and conidia were blended in

125 ml of sterile water. The resulting slurry was applied

with an 100 ml hypodermic syringe, fitted with a blunt

needle, at 10, 20, and 40 ml portions consisting of 0.96 g,

1.92 g and 3.84 g of conidia and mycelium per liter. The

inoculum was suspended over Hydrilla in water or injected

into the soil. Control plants received equal amounts of

sterile water. Inoculum applied as suspension caused consi-

derable turbidity to water but also was effective in killing

most of the Hydrilla by 3 weeks. In jars with soil-injected

inoculum, some damage and death of Hydrilla shoots were

visible, but mostly the plants were healthy, similar to the
I .




-28-


controls.

Since the Dutch isolate is still maintained under

quarantine due to its foreign origin, the effects of the

local 'Graminearum' isolate was tested in an outdoor, large

scale test. Plastic swimming pools of 3.04 m diameter and

0.76 m height were layered with river sand, each was planted

with 45 kg of fresh Hydrilla, and filled with irrigation

water. After five weeks, pools were inoculated with mycelial

homogenates. One pool was inoculated with a suspension of

approximately 0.18 g/liter of conidia and mycelium and a

second pool at 1 g/liter. Control pools were maintained.

There were isolated patches of dead Hydrilla a month follow-

ing inoculation, but no appreciable control of this plant was

achieved in pools. This lack of field efficacy may be due to

insufficient levels of inoculum used or poor virulence of

'Graminearum' or both. Test with higher inoculum levels of

'Graminearum' as well as with other 'Culmorum' isolated from

U.S.A. are in progress.

Host range of the Dutch 'Culmorum' to a few common

aquatic plants of Florida and a limited number of crop hosts

has been tested. Rooted aquatic plants in glass containers

were screened, using inoculum of 125,000 conidia per ml. At

this level, the isolate was lethal to Ceratophyllum demersum

L. (Ceratophyllaceae); Egeria densa Planchon, and Vallisneria

americana Michx. (both of Hydrocharitaceae) and Najas quada-

lupensis (Spreng.) Magnus (Najadaceae). On E. crassipes, it





-29-


caused severe root rot. Alternanthera philoxeroides (Mart.)
Griseb. (Amaranthaceae); Nuphar luteum (L.) Sibthorp. and
Smith (Nymphaeaceae); and Ruppia maritima L. (Ruppiaceae)
were not affected by this isolate.
In preemergence infectivity trials using ca. 38,000
conidia/g of soil, the 'Culmorum' did not depress germination
of seeds or cause seedling blights on bean (Phaseolus
vulgaris L. var. Pole, Blue Lake.); celery (Apium graveolens
L., var. dulce DC., var. Pascal); corn (Zea mays L., var.
Silver Queen); lettuce (Lactuca sativa L., var. Bibb);
pepper (Capsicum annuum var. Yolo); sorghum Sorghum vulgare
Pers., var. unknown); and soybean (Glycine max Merr., var.
Forrest). Other crop hosts are under testing. When com-
plete, the host range test will have included most of the
economic crop plants grown in Florida and several ecologi-
cally important plants.

Since 1971 several hundred fungi and bacteria have
been tested for pathogenicity to Hydrilla (Charudattan,
Freeman, unpublished). To date no other F. roseum 'Culmorum'
or another pathogen possessing virulence comparable to
'Culmorum' has been discovered in the U.S.A. or elsewhere.
The Dutch 'Culmorum' appears to be a significant pathogen
of Hydrilla.

Conclusions
Results of our tests with a Dutch isolate of R. roseum

'Culmorum' from S. aloides up to now have been encouraging

i ''i
I, *[I




-30-


with regard to its potential as a biological control. The

outcome of studies on its field efficacy and safety based on

host range testing will determine if this foreign pathogen

could be released for control of Hydrilla in Florida and

elsewhere.


References

CHARUDATTAN, R. (1973) Pathogenicity of fungi and bacteria

from India to hydrilla and waterhyacinth. Hyacinth

Control J. 11:44-48.

CHARUDATTAN, R., T. E. FREEMAN, K. E. CONWAY & F. W. ZETTLER

(1974) Studies on the use of plant pathogens in bio-

logical control of aquatic weeds in Florida. Proc.

EWRC 4th Intern. Symp. Aquatic Weeds, Vienna. 144-151.

CHARUDATTAN, R. & C. Y. LIN (1974) Isolates of Penicillium,

Aspergillus, and Trichoderma toxic to aquatic plants.

Hyacinth Control J. 12:70-73.

CHARUDATTAN, R. & D. E. McKINNEY (1977) A Fusarium disease

of the submerged aquatic weed Hydrilla verticillata.

Proc. Am. Phytopathol. Soc. 4:222 (Abstr. S-5).

FREEMAN, T. E. (1977) Biological control of aquatic weeds

-with plant pathogens. Aquatic Botany 3:175-184.

ZETTLER, F. W. & T. E. FREEMAN (1972) Plant pathogens as

biocontrols of aquatic weeds. Annu. Rev. Phytopathol.

10:455-470.







h-





-31-



GERMINATION AND STORAGE OF AND INFECTION BY

UREDOSPORES OF UROMYCES PONTEDERIAE1

D. E. McKinney




Collection. Uredospores of Uromyces pontederiae may

be obtained from field-collected detached leaves of Pontederia

cordata or from infected plants maintained in the greenhouse

using a cyclone spore collector. Spores produced under con-

ditions of 100% RH and temperatures above 30 C will have

reduced germination. A three-day interval is necessary

between spore collections from infected plants in the green-

house, to obtain uredospores with good germinability.

Germination. Both U. pontederiae and Uredo eichhorniae

uredospores were stimulated to germinate by -- and 8-ionone,

2-heptanone, 5-methyl-2-hexanone and 2-hexanone, better than

by 1-octanol, 1-nonanol and n-nonanal. Spores of U. ponte-

deriae were best germinated under stimulation by chemicals

in vapor or liquid phase in Conway diffusion cells. Stimu-

lation was less effective when spores were seeded on 2.0%

water agar containing concentrations of these chemicals:

Chemical pretreatment of U. pontederiae uredospores prior to

their contact with H20 was best accomplished by 20 min.

exposure of spores to chemicals in a hydrated atmosphere.

An apparent source of stimulus for germination of U.



1From M.S. Thesis, University of Florida.




22-



.. edcriae uredospores the host plant, P. cordata.

Germination of U. pontederiae uredospores may be

inIuited in dense concentrations by the presence of an endo-

genous, water soluble, self-inhibitor. A second endogenous

metabolite may be involved in stimulation of germ tube

growth. Uromyces pontederiae spores seemed to be inhibited

by light, but not irreversibly so. Uromyces pontederiae

spores germinated between 10 and 30 C when stimulated, and

if not, between 15 and 30 C.

Storage. Uredospores of U. pontederiae are best

stored at -196 C in liquid nitrogen, which stops all metabolic

processes and freezes residual spore water. Storage under

temperatures of -12 C is best when spores have been vacuum-

dried for three hours, which decreases damage by residual

liquid solutes. Rapid viability losses were observed with

U. pontederiae spores at temperatures of -12 C or less under

conditions of no treatment, or storage over desiccants.

Humidities of 35, 52, and 61% were beneficial to spore sur-

vival at 5 C. Spores did not exhibit cold-dormancy.

Inoculation. Of the inoculation media tested, best

results were obtained with spore-talc mixtures and 0.27%

water agar'. nt-mlton iocuoT ted Asp s oft

was best accomplished by vapor stimulation with 2-heptanone

in a dew chamber. Incorporation of a stimulant in 0.27%

water agar containing U. pontederiae uredospores was less

efficient in stimulation of germination on laminae.


I.





-33-


Pretreatment of uredospores of U. pontederiae by vapors of

stimulants prior to inoculation was not successful in

stimulation of spores on inoculated laminae, although this

technique worked in Conway diffusion cells.

Infection. Infection of P. cordata by U. pontederiae

occurs at temperatures of 15, 20, and 25 C. Apparently due I

to static growth rate of the host, uredosori were not pro-

duced on P. cordata at 15 C, even after 5 wk incubation, but

were produced 1 wk after plants were transferred to warmer

temperatures in the greenhouse. Uredosori were produced at

16 and 17 days at 20 and 25 C, respectively. Spores germi-

nated poorly and under stress on leaves at 30 C and did not

infect. No spores germinated at 25 C. Uredospores of U. &

pontederiae germinated equally at 25 C on leaves of pickerel-

weed and waterhyacinth, but penetrated and formed uredosori

only on the former host.

I









1 ]I ,




-34- -



OTHER STUDIES

In addition to those summarized in the previous

sections, several additional short-term studies have been

conducted. These studies are summarized in this section.

Two of these concerning a comparison of spray nozzles for

application of C. rodmanii and an evaluation of the role of

bacteria in the decline of hydrilla were special projects

assigned to and conducted by students. Their final reports

are included as the final portion of this section.


Survey for C. rodmanii in Florida and Louisiana:

During July and August of 1978, M. T. Olexa surveyed

several locations in the state of Florida for the presence

of C. rodmanii. The survey was prompted by two factors; 1)

there had been several unconfirmed reports of the disease at

various locations and 2) we needed a more complete record

of the distribution of the fungus in state prior to the

establishment of large scale field tests. During the survey,

numerous sites ranging from near Tallahassee in the north to

Ft. Lauderdale in the South were visited (see Figure 2).

Suspected diseased plants were collected and transported to

G-ain-esv-illewh1ere-.thepresence or. absence -of C.' rodminii wais

verified by R. Cullen. He based his verification on both

microscopic examination and isolations of the fungus from

diseased tissue. The fungus was found to be present in

6 of 22 sites surveyed. These six coupled with the six


I M




- --- --- _- .-- j, -- _.j .i Ij I 1U I %II IIJ u n uLL_


LEGEND


Lakes and waterways
surveyed.


Areas surveyed
presence of Cercospora
not confirmed.


in which the
rodmanii was


u / sl h ^ '}"

_- / /,,,,.4
;' ; \>-ae i i\
v- **' -*z(' / I~



.-V ,: -: < zr "^.
^-^^ / ^~ \ /



.^ i w l~t -
C/4


Areas surveyed
presence of Cercospora
confirmed.


which
rnanii


in which the
rodmanii was


0 Areas previously surveyed in
the presence of Cercospora rod-
has been confirmed.


Fig. 2


- czZ~-'-~ --~


'Wnt'u O1 GltOGV





-36-


prior known locations bring to 12 sites in which the
fungus is now known to occur. See Figure 2 for present
distribution of C. rodmanii in Florida.
In attempts to evaluate the potential of C. rodmanii
for waterhyacinth control in Louisiana the, Army Corps of
Engineers has transferred diseased plants from the Lake
Concordia experiment to locations in South Louisiana. In
September of 1978 we accompanied Mr. E. Addor of the Corps
on a survey of several of these sites. Plants were collect
and returned to Gainesville for examination. Cercospora
rodmanii was present on plants from4 of 9 locations visited.
The fungus was present on plants from a site North of Hayes
LA; sites I and II on Pecan Island, LA; and site I in Bayou
Manchac. It was not present at either Sorrento II, Hayes
West, Centerville C, Bayou Louise or Manchac IV sites.
Results of these surveys indicate that C. rodmanii is
becoming widespread in both Florida and Louisiana. There
are indications based on observations that in several of
these areas it is beginning to exert a degree of control.

Rating scale for C. rodmanii damage.
One of the problems encountered in plant disease worK
is that of evaluation of disease damage on a quantitative
basis. Based on his years of experience with the disease,
Dr. Conway was able to devise a rating scale system for C.
rodmanii damage based on individual leaf damage. This
system is shown in Figure 3. The system worked exceptional


!I





r, :2.


&


Figure 3
RATING SCALE SYSTEM FOR DAMAGE TO LEAVES OF WATERHPYACINTH BY CERCOSPORA RODMANII.


NUMERICAL
RATING
SYMPYTOrS


0
No spots on
leaf of
petiole.


1
1-41 spots on leaf,
no petiolar spotting.


2 3
Less than 25% of leaf Less than 50%
surface with spots, no .:- of leaf surface
coalescence or-petiolar with spots, some
spotting. coalescence, no"
_____ petiolar spotting..
'0 .. e


Less than 25% of
leaf surface with
spots, coalescence,
some tip-dieback
and petiolar spottin


5 6 7 8
Less than.50% of Loss than 75% spots,- Greater than 75% spots, Dead leaf Blade,
leaf surface with spots, coalescence,(30%)tip- coalescence,(60%) tip- Petiole green,but
coalescence, 10% tip- dieback, increasing dieback, coalescing heavily spotted.
dieback, petiole spotting. _petiolar spotting,___.. spots on petiole ,. ... .....
J~afite^"""" ~--"'' ""..rflifirt, -iiMteZ'' ..7r~1e


9
Dead leaf blade
and petiole
(submerged).



.4




-38-


we in the Fish Prairie studies where inoculum concentration

and subsequent spread were variable being evaluated. It is,

therefore, recommended that this system be used in all

future work with C. rodmanii. It is readily adaptable to

rating entire plant damage by simply rating all the leaves

on a given plant and dividing the total rating by number of

leaves per plant.


Pathogenicity of Phycomycetes to Hydrilla.

Phycomycetes are fungi frequently referred to as

water molds. As the name implies they are adapted to sur-

vival in an aquatic environment. Several species are para-

sitic on higher plants which they attack under conditions

of high moisture. Many of these pathogens have a wide host

range and will attack several plant species. Most of these

latter are soil inhabiting. We tested the susceptibility

of hydrilla to 25 such pathogens belonging to three genera.

Out of these, 3 would consistently attack hydrilla under

conditions of this test (sprigs of hydrilla in test tubes

of distilled water). Results are shown in Table 2. However,

larger scale tests in gallon jugs and 5 gallon aquaria

yeilded unconisstant .patlhogenicity results. Similar results

(inconsistent) were also obtained with unidentified species

of Pythium and Phytophthora isolated from declining hydrilla

in Orange Lake, FL. Despite this inconsistency, the search

for pathogens of hydrilla in the Phycomycete group should

be continued because of the adaption of this group of fungi

to an aquatic mode of existence.





-39-


Table 2. Reaction of Hydrilla following inoculation with

various Phycomycetes.


Phycomycete Original host Hydrilla reaction


Aphanomyces cochliodes

A. euteiches

Phytophthora cinnamoni

P. citrophthora

P. cryptogea

P. dreschleri

P. erythroseptica

P. palmivora

P. parasitica

P. parasitica

P. stellata

Pythium acanthicum

P. aphanidermatum

P. carolinianum

P. deboryanum

P. graminicolum

P. herbicoides .

P. irregulare

P. irregular

P. myriotylum

P. paroecandrum

P. polytylum


Beta vulgaris

Pisum sp.

Persea americana

Citrus sp.

Aster sp.

Citrus sp.

Solanum tuberosum

Ficus sp.

Lycopersicon esculentum

Nicotiana tabacum

unknown

unknown

Chrysanthemum sp.

unknown

unknown

unknown


unknown


unknown

Caladium sp.

unknown

Zea mays

unknown


-(?)




-40-



P. splendens unknown

P. ultimum unknown

P. vexans unknown


a+, ++, +++ = slight, moderate, and high degree of sus-
ceptibility indicated, = not susceptible, -(?) =
indefinite.









Other fungi isolated from Hydrilla.

During the summer of 1977, Dr. Olexa isolated over 175

cultures of fungi from declining hydrilla primarily from

Remuda Canal in Southwest Florida and Orange Lake and Rodman

Reservoir in North Central Florida. Only one of these,

tentatively identified as a microconidial Fusarium sp. was

consistently pathogenic on hydrilla. However, pathogenicity

level was considered too low to be of value in a biological

control program. Therefore, the search for hydrilla patho-

gens with biocontrol potential must be continued.


Effect of C. rodmanii on fish.

One of the major concerns in the use of plant pathogens

for biological control of aquatic weeds is that they will

harm fish. To determine if C. rodmanii was detrimental, it

was tested against the fish Gambusia affinis in a standard

96 hr. bioassy. Ground up mycelium and spores of C. rodmanii

was placed in the fish containers at rates ranging from

0.4 gm/liter to 6.34 gl liter. The lowest rate corresponded

to an inoculum level of 48 gm/M2 which was the inoculation

r -ate-used in Lake Concordia.,aiidthel-owest- rateused in Fish
Prair e. The highest rate is equivalent to a surface area

rate of 800 g/M2 which is 4 times higher than the highest

rate we have ever used to inoculated waterhyacinth with C.

rodmanii. None of the fish in any of the treatments were

adversely affected. In fact at the highest rate, the fish

ate the fungus which was subsequently isolated from their



V^




-42-


feces. Therefore based on this limited test, we feel that

C. rodmanii poses no threat to fish but other species need

to be tested.





-43-


SURVEY OF HYDRILLA UNDERGOING ANNUAL

DECLINE FOR PATHOGENIC BACTERIA


Daryl E. McKinney















PT 621

RESEARCH PROJECT




-44-


Intro actionn

Hydrilla verticillata (L. f.) Royle is a submersed

vau~alar aquatic macrophyte. It is a monocot and belongs

to the Hydrocharitaceae family. The plant is thought to

have been introduced to Florida from South America around

1940 (4). Since its introduction, it has spread to many

major freshwater lakes and streams and is threatening all

those not currently infested. It grows in dense mats that

may quickly destroy the public usefulness of any body of

water so infested.

Florida may have been the first site of introduction,

but the plant has now become a problem in many southeastern

and southwestern states. In these areas and in northern

Florida it grows similar to an annual plant.

In early spring, as the water temperature increases

and days become longer, new shoots arise from stem frag-

ments, tubers, and turions that have survived the winter in

the hydrosoil. Hydrilla soon outgrows native aquatic plants

since it utilizes light more efficiently (6). It grows

towards the surface of the water from depths as great as 40

to 50 feet. The hydrilla may then form dense, entangled mats

that can reduce light penet ration ro less than. 3~ a. 3 c ...R-.

depth (3). Throughout the summer, hydrilla stores starch in

its stems, stolons, and underground rhizomes. By late

summer the mat is at a maximum density. An increase in

epiphytic growth is commonly observed at this time (2). As

the summer passes into fall, hydrilla begins to undergo what





-45-


is known as annual decline. At this time of the year the

weather is the warmest, with the growing season and light

intensity near maximum. Plants undergoing annual decline

exhibit chlorotic leaves and stems that may become trans-

parent. Leaf abscission is common and stems fragment

easily. These symptoms are always associated with the sur-

face growth of the hydrilla.

There are several possible explanations for annual

decline. It is possible that hydrilla loses an excessive

amount of photosynthate, as dissolved organic matter (DOM),

through its leaves and epiphytes then use the DOM. This
would correlate with the observed growth increase of epip-

hytes (1). These epiphytes (bacteria and algae) may then

interfere with C02 diffusion or photosynthesis by the

hydrilla (5). When photosynthetic processes are reduced

below equilibrium with the respiration processes the plant

would begin to die. The epiphytes may produce toxic meta-

bolites that damage hydrilla tissue. Another theory is

that plant pathogens are involved in annual decline (2).

This research project was initiated to study the

possibility that annual decline of hydrilla is-cauAed.by a-

plant pathogenic bacterium. The criteria of the study are

that (a) the pathogen will be isolated from diseased

hydrilla, (b) it will be grown in pure culture, (c) inocu-

lated on healthy hydrilla where it would have to cause

symptoms associated with annual decline, and (d) the




-46-


bacterium would have to be reisolated from the test plants.


Materials and Methods

Isolation Bacterial isolations were made from samples

of hydrilla expressing symptoms of annual decline. The

samples were collected on October 14, 1977, near the middle

of Orange Lake. Isolations were made at the site of collec-

tion and also in the lab.

At the site isolations were made by cutting necrotic

hydrilla stem and leaf tissue into approximately pieces

and surface sterilizing 20 of these pieces. Sterilization

was accomplished by rinsing the hydrilla pieces with a 10%

clorox solution in a petri plate for one minute followed by

two rinses of sterile deionized water. The surface steri-

lized pieces were then plated on hydrilla infusion agar

(10 g crushed hydrilla and 15 g Bacto agar in one liter of

deionized water), nutrient agar, and potato dextrose agar.

Plates were then incubated at 25 C for three days.

Lab isolations were made from hydrilla spigs maintained

in sterile deionized water. The sprigs were shaken vigor-

ously in three sterile water rinses to remove most of the

ep. Z --piphytic :-ba-terie-a.-. :-Spr i s ,-rs r then cr ? er d wvi th -a -.-ar

rod in small tubes containing 2 ml of sterile saline solu-

tion. Loopfuls of the resulting suspensions were streaked

on hydrilla infusion agar, NA and PDA. Plates were incu-

bated at 25 C in the dark for three days.

Seven apparently different bacteria were then selected





-47-


from the site and lab isolations based on colony morphology

and color. Stock cultures of these seven bacteria were

maintained on NA slant tubes under paraffin oil in the

refrigerator.


Inoculum preparation Two liter flasks containing 500 ml of

nutrient broth were inoculated from stock cultures and shake

cultures at approximately 60 strokes per minute for 24 hours.

Isolates six and seven grew extremely slow at these condi-

tions. They were examined microscopically and found to be

myceloid, apparently actinomycetes. They were not tested

for pathogenicity dueto their slow growth and the fact that

few actinomycetes have been found to be plant pathogens.

The remaining five bacterial isolates were prepared

for inoculum by first sedimenting them from the nutrient

broth by centrifugation (10,000 K for 10 minutes). The

supernatant was decanted and the pellet was resuspended in

50 ml of sterile saline (0.85%) solution. Each isolate was

then adjusted to 0.25 transmittance with a colorimeter.

This equalled approximately a 108 cells/ml concentration.


noculation Healthy hydrilla, was collected from

Rodman Reservoir on October 18, 1977. Sprigs of hydrilla

were washed thoroughly with running tapwater before being

rinsed twice with sterile deionized water.

Two inoculum systems were used. The first consisted

of incubating an 80 to 100 mm long growing hydrilla shoot




-48-


in a 30 X 150 mm glass tube in a bacterial suspension. The

suspension was prepared by adding 4 ml of a 108 cells/ml

bacterial concentration to 36 ml of sterile water. This

resulted in a 107 cells/ml concentration around each

hydrilla sprig. Three replications were made of each treat-

ment. A 4 ml saline solution was added to each of three

control tubes. All the tubes were mixed with a Vortex

mixer.

The second inoculation method was similar to the first

except tubes were vacuum infiltrated two mintues at 25 mm

Hg vacuum after inoculation.

All tubes were incubated at approximately 22 C on the

lab windowsill for three weeks after inoculation.

Disease assessment after three weeks was made by

visually comparing inoculated tubes with the control tubes.

Hydrilla sprigs were rated as either healthy (H), chlorotic

(C) or necrotic (N), (see Table 1).

Besides visually assessing the inoculated hydrilla,

reisolations were made from all inoculated and control

hydrilla sprigs. A central piece of each stem containing

one node and three leaves was surface sterilized and rinsed

twice with sterile water before being crushed with a glass

rod in a 2 ml saline solution.


Results

The seven colony types are listed in Table 1. The

majority (4/7) of the colonies were gray or white. This




-49-


agrees with Berg's (2) finding of three white colony types

commonly isolated from hydrilla undergoing annual decline.

Colony types 2 and 3 may be the same bacterium, with 3 being

a rough mutant. All bacterial types grew similarly on NA as

they did on hydrilla infusion agar. This indicates a lack i;

of specific growth requirements. I!

Results from inoculated hydrilla sprigs show that the

bacteria tested were not pathogenic to hydrilla (Table 2).

The chlorosis observed was probably due to a nutrient

deficiency and not a pathogenic response.

Reisolations (Table 3) from the inoculated and control

hydrilla resulted in a random pattern of bacteria reisolated.

Many of the reisolations contained bacteria that had not

been inocualted on that particular hydrilla sprig. These

bacteria were compared to stock tubes and related visually

on the basis of their simularities in gross morphology.


Discussion

Bacterial diseases are associated with enormous concen-

trations (10 cells/ml) of bacteria in diseased tissue.

In concentrations of this magnitude it is seldom that they

are not present in reisolations from such tissue especially

if more than one reisolation is made. There was no pre-

ponderance of any one bacterial type found in any of the

reisolations and none of the colony-types tested produced

symptoms of annual decline in test inocualtions.

Berg (2) previously isolated three white bacterial




-50-


TABLE 1. Description of the seven isolated bacteria

grown on NA plates.


Isolate


1 White, gummy, some slime production

2 Gray, some slime, smooth colony

3 Gray, some slime, rough colony

4 Yellow, some slime (nonfluorescent
on KMB)

5 Yellow, copious slime, myceloid

6 White, very slow growth, myceloid

7 Pink, some slime, gummy, slow
growth, myceloid





-51-


TABLE 2. Visual comparison of hydrilla sprigs

three weeds after inoculation.


Inoculation Isolate Inoculated Control
method Tube 1 2 3 4 5 (saline only)


Method 1 1 H* H H H H H

2 H H H H H H

3 H H C* H C H


Method 2 1 H C H C H H

2 H H H H H C

3 H H H H H H


*H= Healthy; C= Chlorotic; N= Necrotic




- ~


TABLE 3. Reisolations (2-3 days on NA).


TREATMENT


Inoculation


Tube


Method 1


Control


(5)


2 (2) (3) (4)


Method 2


2 (7) (6)
3 --


3


(3)


(4) (5)


(1)


(4) (5)


(2) (7)
(2) (5)


(2)


(2) (5)


(3)
(4)


(4) (2)


(5) (7)

(2) (5)


(4) (7)


(1)


( ) contain bacterial type reisolated, compared with stock cultures.






-53-


types from hydrilla expressing annual decline and inoculated

unknown concentrations of these on healthy hydrilla. He

found symptoms similar to annual decline only to occur when

he mixed the three different bacteria and again inoculated

unknown concentrations on hydrilla. He reasoned that annual

decline was due in part to toxins (or toxin) produced by

the three bacteria. It is more probable that he used

excessive numbers of bacteria in his inoculations and that

normally non-toxic metabolites produced by the bacteria

became concentrated to such an extent that they were toxie.

His tests with the toxins found them to be non-specific on

other aquatic plants, this supports the idea that they are

super-concentrated metabolites and not toxins per se.

It is probable that the bacteria found in this survey

were epiphytic and not associated directly with the symptoms

of annual decline of hydrilla. This view is supported by

the results of reisolations (Table 3) of inoculated and non-

inoculated hydrilla which indicate the presence of these

bacteria on healthy hydrilla.


Literature Cited .

1. ALLEN, H. L. 1971. Primary productivity, chemoorgan-

otrophy and nutritional interactions of epiphytic

algae and bacteria on macrophytes in the littoral of

a lake. Ecol. Monogr. 41:97-127.

2. BERG, R. H. 1977. Annual decline of the aquatic

macrophyte Hydrilla verticillata (L. f.) Royle. Ph.D.





-54-


Dissertation, Univ. of FL.

4. HALLER, W. T. 1976. Hydrilla, a new and rapidly spread-

ing aquatic weed problem. Fla. Ag. Expt. Station

Circular S-245.

5. SAND-JENSEN, K. 1977. Effect of epiphytes on eelgrass

photosynthesis. Aquat. Bot. 3:55-63.

6. VAN, T. K., W. T. HALLER and G. BOWES. 1976. Comparison

of the photosynthetic characteristics of three sub-

mersed aquatic plants. Plant Physiol. 58:761-768.

7. WAITE, T. D. and R. MITCHELL. 1976. Some benevolent

and antagonistic relationships between Ulva lactuca

and its microflors. Aquat. Bot. 2:13-22.





I-55-











COMPARISON OF THREE NOZZLE SYSTEMS FOR SPRAYING

CERCOSPORA RODMANII

by

Deborah F. Reese







As a senior resear-ch project I undertook the assignment

of determining if nozzle systems have any effect on the

efficacy of biological control organisms. Under the direc-

tion of Dr. Kenneth E. Conway and with the technical assis-

tance of Richard Cullen, a field test was carried out on

plots in Thrasher Pond on Fish Prairie, south of Micanopy,

FL.

The objectives of this experiment were to see if

nozzel types have any affect on the size of inoculum par-

ticles and pathogenicity of Cercospora rodmanii, Conway, a

biological control organism currently being experimented

with for waterhyacinth control.


Materials and Methods

Nozzle The following nozzle types selected for

testing: Delevan RD-10, #45 core (raindrop); Delevan WR-

25 (mister): and Delevan hollow cone, #10 disc (hollow cone).

Each nozzle was used in conjunction with a Spray

Systems gun jet #12GH, adapted with a Delevan gun #3160 and

two 14 inch extensions for ease in covering the plots from

a boat. A portable spray power rotary pump with a modified

even flow tank was used to deliver the spray.. E-ach nozzle

was calibrated to determine the amount of flow/second. The

raindrop nozzle delivered 300 ml/sec., the mister 200 ml/

sec.,


Inoculum. amount used and method of application: Isolate


I,


-bb-




-57-


WH 9 of C. rodmanii, was grown on potato-dextrose broth with

0.5 % yeast extract (PDBY) for approximately two and one-

half weeks. A concentration of 48 b/M2 wet weight mycelium

was used. The mycelium was diluted with water to give the

proper spray volume.

Each plot received the same amount of inoculum, 6000

ml. To insure an even inoculation, the plots were sprayed

in the following series; 3000 ml of inoculum was applied to

waterhyacinths in plot #33, 34, and 35. Then the remaining

3000 ml of the total 6000 ml was applied in series to plots

#35, 34, and 33.


Plots Frames (made from PVC pipe) 9 m2 were floated

on the pond. Each frame contained approximately the same

number, age, and degree of previous infection (this variable

was unavoidable as field test with C. rodmanii for rate and

effect is an ongoing experiment at this pond). The frames

were numbered #33, 34, and 35. The inoculum was applied to

waterhyacinths in frame #33 using the raindrop nozzle.

Waterhyacinths in frame #34 were inoculated using the mister

nozzle. And waterhyacinths in frame #35 were inoculated

using the-hOTow tone nozzle. Each frame was rated-for-

damage before inoculation and at two and seven weeks after

inoculation.


Results

Immediately after spraying, ten leaves per plot were




-58-


removed. Each leaf was examined for average size of

inoculum particles and average number of particles/unit area

2.33 cm2) using a binocular microscope. Dimensions of ten

particles per leaf were recorded and the area of an elispe

formula was used to calculate the area of each particle.

The unit area was chosen at random on each leaf. The follow-

ing is a breakdown of data on particle size and number of

particles/unit area:





Average Area Range Particles/
Nozzle (mm2) (mm) Unit area

raindrop 0.99 0.62-2.2 8.3

mister 1.6 0.61-5.6 13.1

hollow cone 1.3 0.46-2.8 10.2


The leaves of ten waterhyacinth plants from each of

the three plots were rated using a system of numbers 0.9.

Zero being no infection and nine being death and/or sub-

mersion of the leaf. For the purpose of this experiment,

we limited the data collected to inoculum sprayed out for

this test. Therefore, only the leaves with damage of 0-6

were counted. The following is a breakdown of damage

caused by inoculum sprayed using the three nozzle types:


*' I 1 -I,





-59-


March April May
31 26 31


Raindrop: (Plot #33)
Total Damage 106 170 158
Average damage/plant 10.6 17.0 15.8
Average damage/leaf .2.0 2.2 2.2

Mister: (Plot #34)
Total Damage 102 165 162
Average damage/plant 10.2 16.5 16.2
Average damage/leaf 1.8 2.3 2.3

Hollow cone: (Plot #35)
Total Damage 107 146 122
Average damage/plant 10.7 14.6 12.2
Average damage/leaf 1.9 2.2 2.0


Following inoculation plants in plot #33 had the

greatest amount of total damage and average damage/plant.

Plants in plot #34 had intermediate amount of total damage

and average damage/plant. Plants in #35 had the least total

damage and average damage/plant. But plants in plot #34

had the greatest amount of average damage/leaf, plot #33

intermediate and plot #35 the least. The average damage/

leaf was studied because each plant did not have the same

amount of leaves.

Visually on April 26, the plants -in pl-ot 33 had. the-

most spotting. Plants in plot #35 ahd intermediate spotting

and plants in plot #34 had the least spotting.

On May 31, the plots were rated again to see if nozzle

type had any affect on the long-term pathogenicity of the

inoculum. The plants in plot #34 had the greatest total

damage, average damage/plant, and average damage/leaf. The




-60-


plants in plot #33 had intermediate total damage, average

damage/plant and average damage/leaf. The plants in plot

#35 had the least amount of total damage, average damage/

plant and average damage/leaf. Visually it was hard to

tell which plots had been sprayed with inoculum from

different nozzles.


Conclusions and Recommendations

Using the premise that the larger particle size of

inoculum and the more inoculum particles/leaf is best, I

concluded that the mister nozzle would be the best nozzle

for spraying C. rodmanii. The nozzle delivered the largest

particle and the most particles/leaf, although infection

got a slower start. During the course of this test,

inoculum sprayed by the mister nozzle produced the most

damage on the plants.

I believe the calculation of data concerning total

damage had too many variables. Not knowing exactly which

leaves were inoculated and which leaves were already

infected with C. rodmanii statistically affected the data

results. We should have tagged the oldest living leaf and

the - 1, r- e s:r -1 ea f --recammend that t Ti-- st e e's -Ve-d

tagging the youngest and oldest leaf before inoculation.

Then we will rate those leaves between the tags at the

beginning and end of the test to insure that only those

leaves inoculated directly with C. rodmanii are rated for

statistical analysis.





-61-


SUMMARY AND CONCLUSIONS

During the past two and one-half years, considerable

additional progress has been made in reaching our goal of

the utilization of plant pathogens in biological control

programs for aquatic weeds.

The pathogen Cercospora rodmanii has been shown to

be effective against waterhyacinth in tests in both

Louisiana and Florida. Methods of culturing and dissemina-

tion of this fungus for biocontrol purposes have been

developed and considerable basic information concerning the

host parasite relationship has been elucidated. This fungus

shows so much promise that the University has decided to

apply for a patent for its use in biocontrol programs and

Abbott Laboratories has entered into an agreement with the

University to develop it into a marketable product form for

possible worldwide distribution.

Two exotic pathogens also show biocontrol potential

in our tests. The rust fungus, Uredo eichhorniae, from

Argentina appears to have potential in biocontrol programs

for waterhyacinths. A Dutch fungus, Fusarium roseum
'Culmorum' show.s; promise for hydrilla control. Research

with both of these fungi has been slowed because of the

necessity of conducting research on them in quarantine.

In addition to the above studies, several other

investigations have been conducted with other pathogens and

potential pathogens on various weed host. As a result, we


i'




-62-


feel our program has advanced faster than anticipated and

is nearing our goal, at least in some areas. Based on

this work, we can conclude that our original proposition

is correct plant pathogens are viable candidates as

biocontrol agents for aquatic weeds.





-63-


PROJECT PUBLICATIONS

1. CHARUDATTAN, R. 1973. Pathogenicity of fungi and

bacteria from India to hydrilla and waterhyacinth.

Hyacinth Contr. J. 11:44-48.

2. CHARUDATTAN, R. and C. Y. LIN. 1973. Penicillium,

Aspergillus, and Trichoderma isolates toxic to -

hydrilla and other aquatic plants. Hyacinth Contr.

J. 12:70-73.

3. CHARUDATTAN, R. 1973. Evaluation of foreign pathogens

as biocontrols of hydrilla and waterhyacinth in the

U.S.A. Second International Congress of Plant

Pathology, Proc. (Abstr. 0390).

4. CHARUDATTAN, R. 1974. Evaluation of foreign pathogens

as biocontrols of hydrilla and waterhyacinth in the

U.S.A. WSSA Newsletter. 2:11 (Repring of No. 3).

5. CHARUDATTAN, R., T. E. FREEMAN, K. E. CONWAY, and F. W.

ZETTLER. 1974. Studies on the use of plant pathogens

in biological control of aquatic weeds in Florida.

Proc. EWRC 4th International Symposium on Aquatic

Weeds, Vienna, 144-151.

6. CHARUDATTAN, R. and C. Y. LIN. :1974.-; slates of

Penicillium, Aspergillus, and Trichdderma toxic to

aquatic plants. Proc. EWRC 4th International Sym-

posium of Aquatic Weeds, Vienna. 142-143. (Abstr.).

7. CHARUDATTAN, R. 1975. Use of plant pathogens to

control aquatic weeds. In Impact of the use of

microorganisms on the aquatic environment. Ecological
'




-64-


Res. Series, U.S. Environmental Protection Agency,

Corvallis, OR. 259 p.

8. CHARUDATTAN, R. 1975. Weed control with plant

pathogens. Agrichemical Age. Jan.-Feb. 1975.

9. CHARUDATTAN, R. and K. E. CONWAY. 1975. Comparison

of Uredo eichhorniae, the waterhyacinth rust with

Uromyces pontederiae. Mycologia. 67:653-657.

10. CHARUDATTAN, R. and K. E. CONWAY. 1975. Mycolep-

todiscus terrestris leaf-spot on waterhyacinth.

Plant Dis. Reptr. 66:77-80.

11. CHARUDATTAN, R., K. E. CONWAY and T. E. FREEMAN. 1975.

A blight of waterhyacinth, Eichhorinia crassipes

caused by Bipolaris stenospila (Helminthosporium

stenospilum). Proc. Phytopathology Soc. 2:65 (Abstr.).

12. CHARUDATTAN, R., K. E. CONWAY and T. E. FREEMAN. 1976.

A blight of waterhyacinth, Eichhorniae crassipes,

caused by Bipolaris stenospila (Helminthosporium

stenospilum). Proc. Am. Phytopathol. Soc. 2:65

(Abstr.).

13. CHARUDATTAN, R., D. E. McKINNEY, H. A. CORDO and A.

SILVEIRA-GUIDO. 1976. Uredo eichhorniae, a potential

biocontrol agent for waterhyacinth. Proc. IV Inter- -

national Sym. on Biol. Contr. Weeds. 210-213.

14. CHARUDATTAN. R. and D. E. McKINNEY. 1978. A Dutch

isolate of Fusarium roseum 'Culmorum' may control

Hydrilla verticillata in Florida. Proc. EWRs Sym.

on Aquatic Weeds. 5:219-224.





-65-


15. CHARUDATTAN, R., B. D. PERKINS, and R. C. LITTRELL.

1978. Effects of fungi and bacteria on the decline

of arthropod-damaged waterhyacinth (Eichhorniae

crassipes) in Florida. Weed Science 26:101-107.

16. CONWAY, K. E., T. E. FREEMAN and R. CHARUDATTAN.

1974. The fungal flora of waterhyacinths in Florida,

Part I. Water Resources Research Center, Univ. of

Florida Publication No. 30, Gainesville, FL.

17. CONWAY, K. E. and J. W. KIMBROUGH. 1975. A new

Doratomyces from waterhyacinth. Mycotaxon. 2:127-13L

18. CONWAY, K. E. 1975. Procedures used to test endemic

plant pathogens for biological control of water-

hyacinth. Proc. Phytopathology Soc. 2:31 (Abstr.).

19. CONWAY, K. E. 1976. Cercospora rodmanii, a new

pathogen of waterhyacinth with biological control

potential. Canad. J. Bot 54:1079-1083.

20. CONWAY, K. E. 1976. Evaluation of Cercospora rodmanii

as a biological control of waterhyacinth. Phyto7

pathology 66:914-917.

21. CONWAY, K. E. and T. E. FREEMAN. 1976. The potential

of Cercogpora rodmanii as a biological controIlQo a --Z -

waterhyacinths. Proc. IV International Sym. on Biol.

Contr. of Weeds. 207-209.

22. CONWAY, K. E. and T. E. FREEMAN. 1977. Host speci-

ficity of Cercospora rodmanii a potential biological

control of waterhyacinth. Plant Dis. Reptr. 61:262-

266.




4L,




-66-


23. CONWAY, K. E., T. E. FREEMAN and R. CHARUDATTAN. 1978.

Development of Cercospora rodmanii as a biological

control for Eichhorniae crassipes. Proc. EWRS Sym.

on Aquatic Weeds. 5:225-230.

24. FREEMAN, T. E. and F. W. ZETTLER. 1971. Rhizoctonia

blight of waterhyacinth. Phytopathology 61:892

(Abstr.).

25. FREEMAN, T. E. and F. W. ZETTLER. 1972. A disease

of waterhyacinth with biological control potential.

Abstr. of 1972 meeting of Weed Sci. Soc. of America

61.

26. FREEMAN, T. E. 1973. Survival of sclerotia of

Rhizoctonia solani in lake water. Plant Dis. Reptr.

57:601-602.

27. FREEMAN, T. E., F. W. ZETTLER, and R. CHARUDATTAN.

1973. Utilization of phytopathogens as biocontrols

for aquatic weeds. III International Sym. on Biol.

Contr. of Aquatic Weeds. Montpellier, France.

28. FREEMAN, T. E., R. CHARUDATTAN, and F. W. ZETTLER.

1973. Biological control of water weeds with plant

pathogens. Univ. of Florida Water Resources Research

Center Publication No. 23.--

29. FREEMAN, T. E. and R. CHARUDATTAN. 1974. Occurrence

of Cercospora piaropi on waterhyacinth in Florida.

Plant Disease Reptr. 58:277-278.

30. FREEMAN, T. E., F. W. ZETTLER and R. CHARUDATTAN.





-67-


1974. Phytopathogens as biocontrols for aquatic

weeds. PANS. 20:181-184.

31. FREEMAN, T. E., F. W. ZETTLER and R. CHARUDATTAN.

1974. Utilization of phytopathogens as biocontrols

for aquatic weeds. Proc. Conf. on Intergrated

Systems of Aquatic Plant Control. U.S. Army Engineer

97-102.

32. FREEMAN, T. E. 1975. Rhizoctoniosis of aquatic plants

McGraw-Hill Encyclopedia of Science and Technology

Yearbook. 327-328.

33. FREEMAN, T. E., R. CHARUDATTAN and K. E. CONWAY. 1975.

Use of plant pathogens for bioregulation of aquatic

macrophytes.

34. FREEMAN, T. E., R. CHARUDATTAN and K. E. CONWAY. 1976.

Status of the use of plant pathogens in the biological

control of weeds. Proc. IV International Sym. on Biol.

Contr. of Weeds. 201-206.

35. FREEMAN, T. E., R. CHARUDATTAN, K. E. CONWAY, F. W.

ZETTLER AND R. D. MARTYN. 1976. Biological control

of water weeds with plant pathogens. Fla. Water

Resources Res. Center Pub. 36.- 39 p. ----

36. FREEMAN, T. E. 1977. Biological control of aquatic

weeds with plant pathogens. Aquatic Bot. 3:175-184.

37. FREEMAN, T. E. 1978. Biological control of aquatic

weeds with plant pathogens. In E. 0. Ganstad

"Aquatic plant control in river basin management".





-68-



CRC publishers. West Palm Beach.

38. Ganstad, E.O., T. E. FREEMAN, F. W. ZETTLER, R. E.

RINTZ, R. CHARUDATTAN, K. E. CONWAY and H.-E. HILL.

1974. Aquatic weed control with plant pathogens.

U.S. Army Corps of Engineers. Waterways Experiment

Station. Vicksburg, MS 62 p.

39. GOEDEN, R. E., L. A. ANDRES, T. E. FREEMAN, P. HARRIS,

R. L. PIENKOWSKI, and C. R. WALKER. 1974. Present

status of projects on the biocontrol of weeds with

insects and plant pathogens in the United States and

Canada. Weed Science. 22:490-495.

40. HAYSLIP, H. F. 1972. Evaluation of Plant Pathogens as

biocontrols of Eurasian watermilfoil (Myriophyllum

spicatum L.) M.S. Thesis, Univ. of Florida, Gainesville.

41. HAYSLIP, H. F. and F W. ZETTLER. 1973. Past and current

research on diseases of Eurasian watermilfoil

(Myriophyllum spicatum L.) Hyacinth Contr. J. 11:38-40.

42. HILL, H. R. 1972. Survey and evaluation of plant

pathogens of alligatorweed (Alternanthera philoxeroides

(Mart.) Griseb.). M.S. Thesis, Univ. of Florida,

Gainesville.

43. HILL, H. R. and R. E. RINTZ. 1972. Observations of

declining water lettuce populations in Lake Izabel,

Guatemala. Proc. Southern Weed Sci. Soc. 25:374-380.

44. HILL, H. R. and F. W. ZETTLER. 1973. A virus-like

stunting disease of alligatorweed from Florida.




-69-


Phytopathology. 63:443 (Abstr.).

45. HILL, H. R., F. W. ZETTLER and T. E. FREEMAN. 1972.

Plant pathogens with potential for biological control

of aquatic weeds. Proc. Southern Weed Sci. Soc.

25:388 (Abstr.).

46. JOYNER, B. G. 1972. Characterization of a Rhizoctonia

sp. pathogenic to aquatic plants. M.S. Thesis, Univ.

of Florida, Gainesville.

47. JOYNER, B. G. and T. E. FREEMAN. 1973. Pathogenicity

of Rhizoctonia solani to aquatic plants. Phytopathol.

63:681-685.

48. MARTYN, R. D. 1977. Disease resistance mechanisms in

waterhyacinths and their significance in biocontrol

programs with phytopathogens. Ph.D. Dissertation,

Univ. of Florida 204 p.

49. MARTYN, R. D. and T. E. FREEMAN. 1978. Evaluation of

Acremonium zonatum as a potential biocontrol agent

of waterhyacinth. Plant Dis. Reptr. 62:604-608.

50. McKINNEY, D. E. 1978. Germination and storage of an

infection by uredeospores of Uromyces pontederiae

.. -.M;S- Thesis, Univ. of-Florida. 114 p-. -=

51. RIDINGS, W. H. and F. W. ZETTLER. 1972. Aphanomyces

blight of amazon sword plant. Phytopathology. 62:

806 (Abstr.).

52. RIDINGS, W. H. and F. W. ZETTLER. 1973. Aphanomyces

blight of amazon sword plant. Phytopathol. 62:289-295.




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53. RINTZ, R. E. 1973. Zonal leafspot of waterhyacinths.

Hyacinth Contr. J. 11:41-44.

54. RINTZ, R. E. 1973. Location, identification and

characterization of pathogens of the waterhyacinth.

Ph.D. Dissertation, Univ. of Florida, Gainesville.

55. RINTZ, R. E. and T. E. FREEMAN. 1972. Fusarium

roseum pathogenic to waterhyacinth in Florida.

Phytopathology. 62:806 (Abstr.).

56. ZETTLER, F. W. and T. E. FREEMAN. 1972. Plant pathogens

as biocontrols of aquatic weeds. Annu. Rev. Phyto-

pathology. 10:455-470.

57. ZETTLER, F. W. and T. E. FREEMAN. 1973. Potential for

the use of plant pathogens as biocontrol agents of

weeds. Proceedings 2nd International Congress of

Plant Pathology. St. Paul, Minn.










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Full Text

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JER RESOURCE researcli center Publication No. 45 Biological Control of Water Weeds With Plant Pathogens I By T.E. Freeman (Principal Investigator) Plant Pathology Department University of Florida Gainesville E SITY OF FLORID

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Biological Control of Water Weeds With .Plant Pathogens By T. E. Freeman R. Cllarudattan K. E. CorMay PUBLICATICN 00.45 a-mr Project Nurrber A-033-FIA Annual Allobnent Agreenent Nu1rbers 14-34-0001-7019 14-34-0001-7020 14-34-0001-8010 Report Sulinitted October, 1978 The upon which this report is based was supported in part by funds provided by the United States Depart::nent of the Interior, Office of Water Research and Technology as Authorized tmder the Water Resources Research Act of 1964 as amended.

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TABLE OF CONTENTS BIOLOGICAL CONTROL OF WATERWEEDS WITH PLANT PATHOGENS 1 Introduction 1 Re1evence of Research Research Approach DEVELOPMENT OF CERCOSPORA RODMANII AS A BIOLOGICAL CONTROL FOR EICHHORNIA'CRASSIPES 7 Introduction 7 Pathogenicity Evaluation 8 References 14 DISEASE RESISTANCE IN WATERHYACINTH AND THEIR SIGNIFICANCE IN BIOCONTROL PROGRAMS WITH PHYTOPATHOGENS 16 A DUTCH ISOLATE OF FUSARIUM ROSEUM 'CULMORUMt MAY CONTROL HYDRILLA IN FLORIDA 23 Introduction 23 Experimental 24 Conclusion 29 References 30 GERMINATION AND STORAGE OF AND INFECTION BY UREDOSPORES OF UROMYCES PONTEDERIAE 31 OTHER STUDIES 34 Survey for c. rodmanii in Florida and Louisiana Rating scale for C. rodmanii Pathogenicity of Phycomycetes to Hydri11a Other Fungi Isolated from Hydri11a Effect of C. rodmanii on Fish Survey of Hydri1la Undergoing Annual 34 36 38 41 41 Decline for Pathogenic Bacteria 43 Introduction 44 Materials and Methods 46 Results 48 Discussion 49 Literature Cited 53

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Comparison of Three Nozzle Systems for Spraying Cercospora rodmanii. Materials and Methods Results Conclusion and Recommendations SUMMARY AND CONCLUSION PROjECT PUBLICATIONS 55 56 57 60 61 63 I

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-'"-:.....-'---, -1 BIOLOGICAL CONTROL OF WATER WEEDS WITH PLANT PATHOGENS Introduction Plant pathogens have many characteristics that make, them ideal candidates as biocontro1s for aquatic weeds. They, are: (1) numerous and diverse; (2) frequently host specific; (3) easily disseminated and self-perpetuating; (4) will not completely eliminate a host species; and (5) do not normally affect man or other animals. With these points in a modest program was begun at the University of Florida in 1970, wi th the obj E:ct of the evaluation and subsequent use of plant pathogens as biocontrol agents for aquatic weeds. The program was expanded with the aid of a matching grant from the U. s. Department of Interior's Office of Water Resources Research, subsequent support from the 'Florida Department of Natural the U. S. Army Corps of Engineers and from the annual allotment program of the Florida Office of Water Resources Research. The program progressed rapidly considering the lack of initial background information. We have developed a

PAGE 6

-2-considerable backlog of information (see list of project publications) about diseases affecting aquatic plants. The objective of the utilization of plant pathogens in biocontrol programs for at least one noxious aquatic plant is nearing fruitition. We have reached the stage where large-scale field evaluation of the fungus Cercospora rodmanii for control of waterhyacinth is warranted. An additional three or four organisms also should soon reach this point. We have also attempted to find and research diseases with biocontrol potential for other aquatic weeds. Relevance of Research The aquatic weed. problem is one of considerable proportion that appears to be growing in magnitude rather than diminishing or even stabilizing. This is occuring despite the expenditure ofconsider
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I -3-spicatum) and alligatorweed (Alternathera philoxeroides) (Burkhalter, personal communication.) Despite these efforts, aquatic weed infestations have increased steadily in the years since these plants were introduced. The range of these plants has also expanded to include virtually all of Florida. Within the last 5 years, Eurasian watermilfoil was found in the St. Johns River watershed and hydrilla was found to infest Rodman Reservoir on the Cross Florida Barge Canal, Okeechobee, Orange, and Lochlossa Lakes. Florida is by no means unique in having a tremendous aquatic weed problem. Proliferating water weed populatiOns are of concern in the rest of the United States, Middle Europe, Africa, ASia, and South and Central America. Indeed the problem is world-wide, but is more acute in the warmer latitudes where waterhyacinth, hydrilla, watennilfoil, alligator weed, salvinia(SaTvTnia and water lettuce (Pistia are the maj or offenders. Reasons for the increasing aquatic weed problem are complex, but are definitely related to man and his activities. With the increase in population and the accompanying environmental problems it has bec01ne appa.rentthat new methods of aquatic weed control must be found. Conventional methods have not been entirely satisfactory either because of cost, overall ineffectiveness, or environmental pollution. The energy problem as it relates to fossil fuel supply has also served to emphasize the need for low-energy methods of control.

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.... : ...... .. -4-In recent years, biological control methods have considerable attention. Various species of herbivorous insects, fish, snails, and even the manatee have been, or are being, investigated for their ability to exert some control p.re-ssure on noxious aquatic plants. Some of them, such as thealligatol'weed flea beetle, have beenreaso-nahly'effective, especially in" an integrated control program. Surprisingly, until our program was initiated, plant pathogens had been rarely considered asbiocontrol agents. They have all the prerequisites of a biocontrol agent and thus offer an untapped reservoir of potential usefulness, either alone or in an inte-.:-'.' grated, wi '. and chemicals. Our research efforts are aimed at bringing. to Jruitition this 'f ";./ ""-,, '.... use of plant in cont:rol programs for, aqllatic weeds. Approach .;-.' We are using"tw6 toutilize"C plant pathogensto control aquatic weeds. They are: 1) The use of or native plant pathogens' as a type {)f "biologicai herbicide" tbroughthe'a-ttificl.al induction of epiphytotics:' We' consider this ,to be the most rapHFa_pproach from an: point. 2) The 6f plant pathogens. This has been the classical 'approach used by entomologist in their 3M'fiiJ5A! &tiWlkCt LI

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I I i I -5-biological control efforts toward imported weeds. This facet involves the search for pathogens near the center of origin of the noxious species, in an area where climatic conditions are similar to those where the pest is a problem in this country. This is slower of the two approaches from an operational standpoint. Our research since the inception of this project are indicated by the titles on the list of publications. Publications Number 23 and 30 of the Florida Office of Water Resources Research summarizes the first six years of our research work. During the past two and years, our efforts have been directed primarily toward those pathogens with definite biocontrol potential. These are: the endemic pathogens of waterhyacinth, Acremonium zonatum and Cercospora rodmanii and two exotic ones, Uredo eichhorniae and Fusarium culmorum for hydrilla control. We have carried out extensive cultural studies in the laboratory, greenhouse studies and, :in the case of the endemic pathogens, various small scale field tests. These latter tests have shown A.zonatum and C. rodmaniito have considerable potential as biocontrols. We have tested both of these at locations in Florida and in Lake Concordia in Louisiana. In this latter test, the two pathogens were--combined with two insects (Neochetina eichhorniae and Arzama densa) in all possible combinati6ns. This test was conducted

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-6--ln cooperation with the U.S. Army Corps of Engineers, Water\vays Experiment Station and the U.S. Department of Agriculture with the approval of the Louisiana Department of Agriculture and the Louisiana Fi$h and Game Commission. We belj.eve C. rodmanii to have been the cause of a spectacular decline of waterhyacinth in Rodman Reservoir in 1971. This natural decline saved the Army Corps of Engineers approximately $35,000 in spray. cost in that body of water (Zeiger, personal communication). Laboratory and greenhouse studies withA. Zonatum on waterhyacinth have elucidated a general resistant mechanism in this plant that accounts for its disease reaction. Work with the two exotic pathogens is being done in our quarantine facility J which: is limited in size. Therefore, the work is progressing at a slower pace than with the endemic pathogens. This report summarizes the research conducted under proj ect A-033-.Fla; For more detail on specific points J the reader is referred to published articles. The is being continued.

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I t I -7-DEVE.LOPMENT OF,CERCOSPORA RODMANII AS A BIOLOGICAL CONTROL FOR EICHHORNIA CRASSIPES 1 K. E. Conway, T. E. Freeman, and R. Olarudattan Introduction A decline of populations of waterhyacinth was first noticed in 1970 in Rodman Reservoir, a large impoundment of water (3,491 ha) near Orange Springs, Florida. Symptoms associated with waterhyacinth during this decline were a yellowing of the plant, formation of spindly petioles and a rot of the root portion of the plant. It was estimated that this decline saved approximately $20,000 for weed control in this reservoir for one season. Unfortunately, this decline was less in each succeeding year and allowed the waterhyacinth to almost completely reestablish in the reser-voi r; ,A survey of -fungi---as50clatedwlthwaterhyacintli in this reservoir (Conway, etal. 1974) resulted in the isolation of a Cercospora that was later determined to be a new species-, CercQspora rodmanii Conway (Conway 1 From Proc. EWRS 5th symposium on aquatic weeds. 1978.

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-8-. Pathogenicity Evaluations The pathogenicity of the fungus was evaluated in several preliminary tests (Conway 1975) which indicated that the fungus was capable of producing lesions and chlorosis on leaves and.petioles of waterhyacinth. This testing culmi-nated in a small-scale field test (Conway 1976b) in a small isolated pool of Lake Alice, located on the campus of the University of Florida. Procedures for the preparation and application of the fungus have been published (Conway 1976b). Approximately I Kg of a mycelial suspension was inoculated onto waterhyacinth in an area of 65 M2. Infection was evident on the inoculated plants within two weeks. Although the fungus was applied to a small area of waterhyacinth, the results from this test indicated that once the disease was established on plants it was capable of spreading via wind-borne conidia to infect most of the waterhyacinths in the pool (1.7 hal. Even though the populations of waterhyacinth regrew in the spring, there were indications that the new populations had been severely stressed, based on their slower growth rates when compared to noninoculated plants on the other side of the lake. In addition, the fungus was capable oibverwinterirtg ihe plants initiated;the disease cycle again in the spring. Host specificity of C. rodmanii was tested (Conway and Freeman 1977) on over 85 economically and ecologically impor-tant plants that are either grown commercially or occur

PAGE 13

-9-naturally in Florida. A modified centrifugal (related plants) and varietal (economic plants) strategy was used to determine which plants would be included for testing. Plants tested represented 22 families of higher plants and were evaluated in both the greenhouse and field. The results indicated that C. rodmanii was pathdgenic only to waterhyacinth and was safe to use as a biological control in Florida. In a recent publication of preliminary results concerning an attempt to control waterhyacinth with an inte-grated pathogen-insect combination (Addor 1977a), it was erroneously stated that rodmanii had a wide host range. Thus, this publication should be disregarded in favor of Addor's (1977b) later publication of the completed results in which C. rodmanii was correctly noted as being host specific for waterhyacinth. As integrated pathogen-insect field test which include the pathogens, C .. rodmanii and Acremonium zonatum (Sawada) Gams, and two insects, Neochetina eichhorniae Warnei and Arzama densa Walker, was conduced in Concordia, Louisiana (Addor 1977). These organisms were evaluated alone and in combina.tion on waterhyacinths confined in frames enclosed in an area of approximately 3.25 MZ. C. rodmanii was applied as a mycelial suspension at a rate of 48 gm/M2 and A.zonatum was applied at a rate of 96 gm/M2. The stocking rates for the insects N. eichhorniae and A. densa were 150 and 50 insects per frame, respectively. The

PAGE 14

160 140 120 100 80 60 40 20 160 140 120 100 80 60 40 20 -10--FIGURE 1 CONTROL ARZAMA 174 194 214 234 254 274 TIME, DAYS CONTROL ARZAMA NEOCHETINA CERCOSPORA ---174 194 214 234 254 274 TIME, DAYS 160 140 120 100 80 -60 40 20 CONTROL ARZAMA :NEOCHETINA __ -4 174 194 214 234 254 274 TIME, DAYS 160r-__________________ 140 120 100 80 60 40 CONTROL ARlJ\MA NEOO:ffiTINA CERCOSPQRA, ACREMJNIUM 20 174 194 214 234 254 274 TIME, DAYS 1HE EFFECT OF CCNBINATIONS OF PLANT PA1HOGENS AND INSECTS ON '!HE WEIGHT OF WATERHYACINTII INTEGRATED CONTROL OF WATERHYACINIH LAKE CONCORDIA, LOUISIANA, USA 2.3 JUN"E -30 SEPTEMBER I I I I I I

PAGE 15

-11-frames were weighed at two week intervals from 19 June to 30 September 1975. The criterion used for damage to these populations of waterhyacinth was the difference in weight of the treated plants vs.the untreated plants. The important result of tnis study was in indication that damage to waterhyacinth increased with the number of organisms used to stress the plants. The additive effect of combinations of plant pathogens and insects is illustrated in Figure 1. The reduction in the weights of the waterhyacinth in the frames by each organism as well as in various combinations of each organism were determined. Acremonium zonatum caused the least reduction in weight of the waterhyacinths when compared to of the other organisms. However, greater reductions in the weight of waterhyacinths were achieved as more organisms were applied to these plots. The greatest reduction of weight was achieved at the end of the test period when all four organisms were used to stress the plants. Following the Lake Alice study, the ability of c. rodmanii to stress waterhyacinth populations was tested further in Florida. The fungus was re-introduced onto water,hya,ci:rJ.ths in the where the disease > ...... -- -. -. had been isolated originally. Five applications, each consisting of approximately 1 kg of mycelial inoculum, were applied every two weeks to a small area (65 M2) of wat,er-hyacinth along the shoreline beginning in February 1975. The disease became established in this area within two weeks of the first application. During the next six months the

PAGE 16

-12-disease spread to infect most of the waterhyacinth in the area (approximately 15.0 hal. At that time (July), some of the, individual waterhyacinth plants ,had died and sunk beneath the surface of the water. (Pistia stratiotes L. .. >c) and yellow cow lily (Nuphar 1uteum (L.) Sibthorp and Smith) had replaced these dead plants in the waterhyacinth mats. By August, the enitre population of waterhyacinth was under severe stress from the pathogen and there was approximately 7.0 ha of open water where originally there had been complete coverage of waterhyacinth. Dur.ing the Fall of 1975, the area of open water reached a maximum of 10.0 ha before cold winter temperatures limited the spread of the disease. No additional inoculations of the fungus have been applied to waterhyacinth in this area and each year since the original inocuiation, the disease has overwintered and initiated epidemics the following spring. The disease spread to infect w,terhyacinth in most of the reservoir and as a result of the stress placed on the plants by the disease it has not been necessary to expend either large sums of money or energy for waterhyacinth control in the reservoir during 1975-77. A com-""'pfeteelimination: is not "possihle-'uslpg the disease alone and fluctuations in the' waterhyacinth populations have occurred which are influenced by environmental factors which condition the During the Rodman study it was determined that under "'.'.. .. ---...

PAGE 17

-13-optimal conditions waterhyacinths were capable of producing one new leaf every 5-6 days. This rate will vary depending on environmental conditions and during unfavorable periods this rate may decline to less than one leaf produced over a three week period. Therefore, the success of the epidemic will depend upon the rate at which the pathogen can infect and kill these new leaves. In order to determine if there was an optimal concentration of inoculum necessary to initiate disease, an inoculum rate experiment was begun in a small lake southeast of Gainesville, Florida. Waterhyacinths were confined in 9 M2 frames and treated at three mycelial inoculum concentrations: 48 gm/M2 96 gm/M2, and 192 gm/M2. Results showed that regardless of the initial inoculum level, the rate of disease spread became equalized -after a periodof time due to inoculum buildup on the inoculated plants and cross infectivity between plots. The maximum rate of damage produced by c. rodmanii was assessed at the 192 gm/M2 inoculum level and this rate was not exceeded even with an additional application of inoculum later in the year. The maximum rate of damage caused byC. ,. this experTment"cOrresponaed of 1.0-1.3 leaves of the waterhyacinth every three weeks. Therefore,. when conditions exist which favor disease deve-. lopment and which limit leaf production to less than one leaf per thre.e weeks, C. rodmanii can infect and kill leaves faster than the plant can produce new leaves. The plant

PAGE 18

-14-. becomes debilitated and over a period of time will die unless conditions change to favor its regrowth. The use of C. rodmanii as a biological control for waterhyacinth has been patented by the University of Florida. The University is working with Abbott Laboratories, Illinois,. to produce a commercial product of the ;: fungus. Evaluation of a product has already begun. References ADDOR, E. E. (1977a) A field test of selected insects and pathogens for control of waterhyacinths.Report I. Preliminary results for the 1975-76 Tech. Rpt. A-77-2, U.S. Army Corps of Engineers, Vicksburg, MS, USA 54 pp. ADDOR, E. E. (1977b) Controlled field tests of selected insects and pathogens in combination on wa"terhyacinth; Proc. Research Planning Coni. Aquatic Plant Gontro1 Program. Misc. Paper A.,.77-"3, U.S. Army Corps of Engineers, Vicksburg, MS pp.236-268. CONWAY, K. E. (1975) ,Procedures used to test endemic plant "pathogens for biological control of waterhyacinth. Proc. Amer. Phytopath. Soc. 2:31 (Abstr.). rodmanii, a new o( waterhyacinth wi thbiologica1 control potential.' Can. J. Bot. 54:1079-1083. (1976b) Evaluation of as a biological control of waterhyacinth. Phytopath. 66:914-916. ----------------------------................. ...

PAGE 19

-15-CONWAY, K. E. and T. E. FREEMAN (1977) Host specificity of Cercospora rodmanii, a potential biological control of waterhyacinth. Plant Dis. Rptr. 6-1:262-266. CONWAY, K. E., T. E. FREEMAN and R. CHARUDATTAN (1974) The fungal flora of waterhyacinths in-Florida. Part I. Water Resources Research Center, Univ. Florida, Publ. No. 30, Gainesville, Florida, 11 pp. ,--..

PAGE 20

" -16-DISEASE RESISTANCE MECHANISMS IN WATERHYACINTHS AND THEIR SIGNIFICANCE IN BIOCONTROL WITH PHYTOPATHOGENS 1 R. D. Martyn The waterhyacinth [Eichhornia crassipes (Mart.) is a free-floating vascular hydrophyte that has colonized much of Florida's inland waters. In 1970, a program was initiated at the University of Florida to study biological, control of this noxious plant with phytopathogens. One of the pathogens currently being studied is the fungus Acremo nium zonatum (Sawada) Gams. It causes severe spotting on both leaves and petioles of this plant under conditions of high humidity. During field trials with this fungus, it was observed that small, young, plants displayed fewer symptoms after infection than did larger plants in the same plots. It also appeared that large plants infected with A. zonatum exhibited a faster rate of leaf regeneration than did smaller plants. The present study was initiated to determine if small plants were :in fact more resistant to A. than l.arge plants; -,:-;. -: --/ ;: ..:.-. -if meristematic activity in the plants was altered after infection; and, if so, to what extent host phenolic compounds' and their oxidizing enzymes, namely polyphenoloxi
PAGE 21

-1/-Waterhyacinths displayed various degrees of resistance to A. zonatum depending on their morphotypic state of deve-lopment. Results of this study indicate that these differ-ences in resistance are due to the variations in phenol chemistry among plants of different sizes and to subsequent changes inauced by infection (Table 5-1). Small plants are more resistant to fungal attack than are medium or large plants, based upon the number of ,lesionsl leaf after infection. It appears that the presence of. high concentrations of phenolic compounds does not itself impart resistance to the pathogen. Rather it is the oxidation of these compounds by enzymes, such as polyphenoloxidase which is responsible for the resistance. This view.'is supported by qualitative and quantitative data on the phenols in plant morphbtypes and is coincident with the observed differences in resistance. Small plants, by virtue of having fewer phenol cells! mm2 leaf area, have less total phenol content/leaf,than larger plants. If phenol content alone, was responsible for disease resistance, then small plants would be more susceptible than large plants but they were not. In this case PPO activity isa.pparently the mediating factor. The rate of enzyme activity in small plants is three-fola in large plants; presumably therefore, oxidation of poly-phenols to quinones is much greater in small plants. small plants are initially more resistant to pathogenic t I I

PAGE 22

-18-attack than are larger plants. After the disease has progressed for several weeks the differences in resistance among the morpho types is no longer evident. Each plant size exhibits a percent-total-diseased leaf area which is statistically the same (approximately 40%). It is believed that this equalization of disease severity results from a gradual loss in resistance by small plants while at the same time there is a gradual increase in resistance by large plants. Again, quantitative data of the phenol metabolism can be correlated with this change The total phenol content decreased significantly after infection in small and medium-sized plants. This is coincident with a reduction in PPO activity. The coupling of these two phenomena may account for the decrease in resistance of small plants. Large plants, on the other hand, retain their total phenol content and at the same time exhib ita three -fold increase "in PPO acti vi ty. Therefore, an increase in polyphenol oxidation would be expected to occur and could account for the increase in resistance in large plants. In essence, then, the point being made is: if infected ,-.:;'-'-'-.. --"':7 .--. ',"-_ ; ."" small plants retained the phenol content and PPO activity of healthy plants, then disease severity would probably be limited to much less than 40%. Similarly, if infected large plants retained the PPO activity of healthy plants, disease would progress to a much higher percentage, perhaps 60-70%.

PAGE 23

( TABLE L DIFFERENCES AND SIMILARITIES AMONG HEALTHY AND A.ZONATUM-INFECTED WATERHYACINTH MORPHOTYPES. ---, ---."'---------" ASSESSMENT CRITERIA SMALL MEAN # LESIONS/LEAF % TOTAL DISEASE MEAN # PHENOL CpLLS/MM2 PPO RATE (HEALTHY) PPO RATE (DISEASED) PPO (HEALTHY) PPO LOCALIZATION (DISEASED) TYPE OF PHENOLIC ACIDS (HEALTHY) TYPE OF PHENOLIC ACIDS (DISEASED)' TOTAL PHENOLS (HEALTHY) TOTAL PHENOLS (DISEASED) FUNGAL GROWTH (HEALTHY) FUNGAL GROWTH (DISEASED) LEAF REGENERATION (HEALTHY) LEAF REGENERATION (DISEASED) ".,' i 3.7 41.3 33.6 1. 53 0.90 3 CELL TYPESA ALL CELLSB 6 7 92 llG/G 80 llG/G STIMULATIVEC STIMULATIVED 27.3% 21. 6% MORPHOTYPE MEDIUM 12.8 37.0 41. 8 0.80 0.70 3 CELL TYPESA ALL CELLSB 6 7 106 llG/G 96 llG/G STlMULATIVEC STIMULATIVED 28.5% 33.9% LARGE 18.3 39.5 48.7 0.47 1. 36 3 CELL TYPESA ALL CELLSB 9 9 104 llG/G 105 llG/G STIMULATIVEC STIMULATIVED 46.1% 93.3% AVASCULAR PARENCHYMA, BUNDLE SHEATH, AND PHENOL CELLS: BALL CELLS WHICH CONTAIN CHLOROPLASTS; Cp=0.05; GROWTH INCREASED OVER CONTROLS: Dp=O.05; GROWTH INCREASED OVER HEALTHY I I-' to .... "-',--'= .,r. ." _'T'. --i>:: ______ ''''''' ____ ''''_'''' ____ ';''' __ '' __ ''(U .. ... "' ... i4"' ..... "" .. ''''I111 .. I\Ij '"i",111 Hn

PAGE 24

-20-However, because each morphotype responds to infection differently (in most cases in contrast to each other) severity balances among the plant sizes at approximately 40% of the leaf-surface area. If disease severity is viewed, not from a percentage of leaf-area infected, but as a reduction in plant growth, then data on leaf regeneration rates among the morphotypes becomes of prime importance. It has been observed that infected large plants regenerate two to three times as many new leaves as do infected small plants. This too, is correlated with the plantts phenol chemistry. It has been shown that A. zonatum is capable of synthe-sizing indoleacetic acid in vitro and that this is one explanation for the increased growth observed in large More important, however, is the fact that phenols are known inhibitors of IAA oxidase, the enzyme responsible for controlling the IM level in the plant. It has already been pointed out that the different waterhyacinth morphotypes vary in phenol content, both prior to and after infection. The higher phenol content in large plants could account for increased growth obser:v.ed in large plants by inhibition of the IAA oxidase system. Perhaps the most significant data supporting a positive role for phenols in disease resistance comes from the loca lization studies of PPO in healthy and diseased plants. Enzyme activity is localized in the thylakoids of chloroplasts

PAGE 25

-21-in only three cell types in healthy plants. After infection there is a "turn on" in PPO activity in all cells which con-tain chloroplasts. This turn on in PPO activity is highly suggestive of a vital role for enzymatic oxidation of polyphenols during disease. Before disease can ensue, the pathogen must come into contact with and penetrate its host. In this regard, A. zonatum can enter the waterhyacinth by either of two ways: through open stomata or by directly penetrating the unbroken cuticle of the leaf. Intracellular colonization is enhanced by the diffuse secretion of cellulolytic enzymes and perhaps by the localized secretion of pectolytic enzyems. Growth of A. zonatum was either unaffected or stimu-lated by seven different phenolic acids in concentrations up to 1000 ppm in minimal media. When yeast extract was added to the media as a growth supplement, one phenolic acid, coumaric, was found to be inhibitory. In addition, fungal growth was enhanced on media containing yeast extract and extracts from diseased leaves over that on media containing healthy.leaf extracts. Several cytological changes were observed in the cells from infected waterhyacinth leaves. First, in cells of healthy leaves have an abundance of starch granules which disappear after infection. Second, there are only a -few plastoglobuli in chloroplasts in healthy cells, but after infection, they increase both in size and in number.

PAGE 26

-22-Third, there is a noticeable increase in microbodies in the cytosol of infected cells. It is believed that each of these cytological changes is the result of a shift in host metabolism induced by infection. It is-concluded thatwaterhyacinths have at least two distinct biochemical defense mechanisms that are related to phenol metabolism and plant size. The first is the preselice of high concentrations of polyphenols in specialized phenolcells which, under the proper conditions, can serve as toxicants to potential pathogens. The second proposed defense mechanism of waterhyacinths is an acceleration of its growth rate brought about by the inhibition of lAA oxidase by the phenolic compounds. Which of the-above mechanisms is operational is dependent upon the plantls morphotypic stage of development. It is believed that initially small plants defend against pathogenic attack by virture of their highPPO activity whereas large plants respond by increased leaf production. Medium-sized plants appear to have a combination of both mechanisms. In consideration "of A. zonatum as a. potential biocon-" ,. _.', .'." ,-'-.-trol agent for waterhyacinths, it is concluded that best control would be achieved with small, young, plants rather than with larger, more mature plants. In this regard, control procedures should be initiated early in the spring when new plants start to grow and colonize the body of

PAGE 27

-23--A DUTCH ISOLATE OF FUSARIUM ROSEUM 'CULMORUM' MAY CONTROL HYDRILLA VERTICILLATA IN FLORIDAI R. Charudattan and D. E. McKinney Introduction It is estimated that a fifth of all fresh water ponds, lakes and rivers in Florida is infested with Hydrilla verticilIata L. F. Royle (Hydrocharitaceae), and the weed is spreading rapidly. Since its introduction into Florida waters around 1960, this weed has moved to several other states in the U;S.A. Serious economic losses and ecologic damages resulting from this submerged weed have spurred research on biological, chemical, and mechanical controls. Among biological agents researched are plant pathogens (Charudattan, et 1974; Freeman, 1977); however, very few disease of submerged weeds are known (Zettler and 1972) and those found on Hydrilla (Charudattan, 1973; Charu-dattan and Lin, 1974; Freeman, 1977) have not been suffi-ciently damaging or specific to this host to promote their use in the field. In 1974, a disease of Stratiotes aloides L. (Hydrocharitaceae) was near Wageningen by Dr. J. C. J. van Zan who brought it to our attention. Mature plants had 1 -From Proc. EWRS 5th Symposium on aquatic weeds.

PAGE 28

,;.' -24-symptoms of root-and crown-rots and severoly diseased plants appeared to sink gradually as a consequence of tissue decay. A 'few infected plant parts were taken to Gainesville, where a group of fungi were cultured from them including predominantly a Ftisarium roseum 'Culmorum' eLk. ex Fr.) Synd. & Hans. In view of the close taxonomic relationship between S. aloides and H. verticillata, the pathogenicpotential of these fungi to the latter was of obviousinterest'to us. Among the fungal isolates obtained from S. aloides, only 'Culmorum' was capable of killing Hydrilla (Charudattan and McKinney, 1977). Results presented here will prove that the Dutch 'Culmorum' is a virulent pathogen of Hydrilla unlike fusaria tested on this host, and that it may help control of Hydrilla in Florida. Experimental The effects of the 'Culmorum' isolate on Hydrilla w.are determined in three test systems. The first one consisted of incubating 8 to 10 cm long terminal portions of Hydrilla shoots in 3 X 15 cm glass tubes with 40 ml of sterile water to which were added dense macroconidial suspensions. Control t.ubes were without cbnidia.Fungal inocula, consisting .of filtered macroconidial suspension obtained from potato dext-rose agar cultures, were quantitated with a hemacytometer. Inoculum levels between 2500 and 250,000 conidia per ml (100, 000 and 10 million conidia per tube containing 40 ml of water) were set up my mixing suitable concentrations of conidial sUspensions. Inoculated and control.Hydrilla tubes

PAGE 29

-2 5-were incubated under diffuse light at 22 + 2 C for several weeks. Damage to Hydrilla from the Dutch 'Culmorum' was usually evident as chlorosis and discoloration of inoculated shoots 10 to 14 days after inoculation. In 3 weeks, death and lysis or regrowth of partially damaged Hydrilla were observed. The threshold of inoculum needed to damage Hydrilla was found to be 1 million conidia per tube or 25,000 per mI. A dose and effect relationship was seen on inoculated Hydrilla; at lower inoculum levels the shoots were only partially damaged or killed while at higher inoculum levels the effects were drastic and lethal. In the second system, 20 liter aquarium tanks were layered with river sand, filled with 14. liters of water, and planted with 100 terminal ends of Hydrilla shoots, each with an active growing bud. After two days, the tanks were inocu-lated with conidial suspensions of 'Culmorum' at approximately 80,000 or 90,000 conidia per ml of water in tanks. Three weeks after inoculation, Hydrilla shoots started to discolor and developed signs of rotting. In about 5 weeks, the shoots broke down completely, and some that were still green were defoliated and uprooted, and floated to the water surface. In the third system, the fungus was grown for two weeks on a sterilized mixture of 9 parts sand) 1 part oat meal and 3 parts water, and mixed with the bottom sand in Hydrilla tubes at 1:1 and 1:10 proportions (w/w) of inoculum

PAGE 30

i l I I l l -26-anci sand. Controls had sand-oat-water mixture without the fungus, mixed with an equal weight of sand. A Hydrilla plant with shoots, roots, and at least one tuber was planted per inoculated and control tubes. After a week, the inocula-ted plants turned pale and were dead by the end of 14 days. In all these systems, the inoculated fungus could be reisolated from inoculated, dead, dying, or green Hydrilla shoots after surface sterilization and plating on potato dextrose agar. Controls did not yield the fungus. In additio_n, the conidia "rere observed to germinate on, and penetrate into Hydrilla tissue which confirmed the pathogenic capability of the fungus. In order to decide that the effects of the 'Culmorum.' isolate on Hydrilla were specifically due to its infectivity and not due merely to massive numbers"of fungal spores in water, a comparative inoculation test was set up. In this test, three unidentified Fusarium spp., isolated from Hydrilla in Florida, a F. roseum from Ficus elast'ica Roxb. and a F. roseu'm 'Graminearum' from Eichhornia c'rassipes (Mart.) Solmsin Florida were included. The test tube" procedure described fi;'-5't: th inoc.ulum den.si between 2500 and 250,000 conidia per ml of treated water. The results confirmed that the Dutch tCulmorum' was indeed unique in its effects on Hydrilla. The three Fusarium spp. from Hrdrilla and the Ficus isolate of F.

PAGE 31

J -27-roseum did not damage Hydri11a ev'n nt higher of inoculum. The 'Graminearum' from E. crassipes was capnble of damaging Hydrilla, inciting similar symptoms as the Dutch 'Gulmorum'. However, the threshold of inoculum needed to cause damage by this isolate was approximately 60,000 conidia per m1, or 2.4 times higher than that of 'Culmorum'. The Dutch 'Culmorum' isolate hence was not only pathogenic to Hydrilla but also was more virulent than any Fusarium tested. In another experiment, conidia and mycelial fragments of the Florida isolate of 'Graminearum' fromE. crassipes were applied either as suspension or was injected into bottom sand around 25 rooted Hydril1a shoots maintained in 4 liter glass jars under 2.5 liters of water. For inoculum, the fungus was grown on potato dextrose broth for a week. About 30 g of lvet, filtered mycelium and conidia were blended in 125 ml of sterile water. The resulting slurry was applied with an 100 ml hypodermic syringe, fitted with a blunt needle, at 10, 20, and 40 rnl portions consisting of 0.96 g, 1.92 g and 3.84 g of conidia and mycelium per liter. The inoculum was Hydrilla in water?r injected .. into the soil. Control plants received equal amounts of sterile water. Inoculum applied as suspension caused considerable turbidity to water but also was effective in killing most of the Hydrilla by 3 weeks. In jars with soil-injected inoculum, some damage and death of Hydrilla shoots were visible, but mostly the plants were healthy, similar to the

PAGE 32

-28-controls. Since the Dutch isolate is still maintained under quarantine due to its foreign origin, the effects of the local 'Graminearum' isolate was tested in an outdoor, large scale test. Plastic swimming pools of 3.04 m diameter and 0.76 m height were layered with river sand, each was planted with 45 kg of fresh Hydrilla, and filled with irrigation water. After five weeks, pools were inoculated with mycelial homogenates. One pool was inoculated with.a suspension of approximately 0.18 g/liter of conidia and mycelium and a second pool at I g/liter. Control pools were maintained. There were isolated patches of dead Hydrilla a month ing inoculation, but no appreciable control of this plant was achieved in pools. This lack of field efficacy may be due to insufficient levels of inoculum used or poor virulence of 'Graminearum' or both. Test with higher inoculum levels of 'Graminearum' as well as with other 'Culmorum' isolated from U.S.A. are in progress. Host range of the Dutch 'Culmorum' to a few common aquatic plants of Florida and a limited number of crop hosts has been tested. Rooted aquatic plants in glass containers were screened, using inoculum of 125,000 conidia per ml. At this level, the isolate was lethal to Ceratophyllum demersum L. (Ceratophyllaceae); Egeria densa Planchon, and Vallisneria americana Michx. (both of Hydrocharitaceae) and Najas quadalupensis (Spreng.) Magnus (Najadaceae). On E. crassipes, it I. IF I Jj 7 Lll SEW II ZUWi db ..

PAGE 33

.1 I 1 -29-caused severe root rot. Alternanthera (Mart.) Griseb. (Amaranthaceae); Nuphar luteurn (L.) Sibthorp. and Smith (Nymphaeaceae); and maritima L. (Ruppiaceae) were not affected by this isolate. In preemergence infectivity trials using ca. 38,000 conidia/g of soil, the 'Culmorum' did not depress germination of seeds or cause seedling blights on bean (Phaseolus vulgaris L. var. Pole, Blue Lake.); celery (Apium graveolens L., var. dulce DC., var. Pascal); corn (Zea mays L., var. Silver Queen); lettuce (Lactuca sativa L., var. Bibb); pepper (Capsicum annuum var. Yolo); sorghum Sorghum vulgare Pers., var. unknown); and soybean (Glycine max Merr., var. Forrest). Other crop hosts are under testing. When complete, the host range test will have included most of the economic crop plants grown in Florida and several ecologi:ocally important plants. Since 1971 several hundred fungi and bacteria have been tested for pathogenicity to Hydrilla (Charudattan, Freeman, unpublished). To date no other F. roseum I Culmorum' or another pathogen possessing virulence comparable to 'CulmoTum' has been discovered in the U. S or eIsel-There. The Dutch lCulmorum' appears to be a significant pathogen of Hydrilla. Conclusions Results of our tests with a Dutch isolate of R. roseum I Culmorum" from S. aloides up to now have been encouraging

PAGE 34

-30-with to its as a biological control. The outcome of studies on its field efficacy and safety based on host range testing will determine if this foreign pathogen could be released for control of Hydri11a in Florida and elsewhere. References CHARUDATTAN, R. (1973) Pathogenicity of fungi and bacteria from India to hydri11a and waterhyacinth. Hyacinth Control J. 11:44-48. CHARUDATTAN, R., T. E. FREEMAN, K. E. CONWAY & F. W. ZETTLER (1974) Studies on the use of plant pathogens in bio-logical control of aquatic weeds in Florida. Proc. EWRC 4th Intern. Symp. Aquatic Weeds, Vienna. 144-151. CHARUDATTAN, R. & C. Y. LIN (1974) Isolates of Penicillium, Aspergillus, and Trichoderma toxic to aquatic plants. Hyacinth Control J. 12:70-73. C HARUDAT TAN R. & D. E. McKINNEY (1977) A Fusarium disease of the submerged aquatic weed Hydri11a verticil1ata. Proc. Am. Phytopathol. Soc. 4:222 (Abstr. 8-5). FREEMAN, T. E. (1977) Biological control of aquatic weeds with plant pathogens. Aquatic Botany 3:175-184. ZETTLER, F. W. & T. E. FREEMAN (1972) Plant pathogens as biocontro1s of aquatic weeds. Annu. Rev. Phytopathol. 10:455-470.

PAGE 35

-31-GERMINATION AND STORAGE OF NID INFECTION BY UREDOSPORES OF UROMYCES PONTEDERIAE1 D. E. McKinney Collection. -Uredospores of Uromyces pontederiae may be obtained from field-collected detached leaves of Pontederia cordata or from infected plants maintained in the greenhouse using a cyclone spore collector. Spores produced under conditions of 100% RH and temperatures above 30 C will have reduced germination. A three-day interval is necessary between spore collections from infected plants in the greenhouse, to obtain uredospores with good germinability. Germination. -Both U. pontederiae and Uredo eichhorniae uredospores were stimulated to germinate by -and a-ionone, 2-heptanone, 5-methyl-2-hexanone and 2-hexanone, better than by l-octanol, 1-nonanol and n-nonanal. Spores of U. pontederiae were best germinated under stimulation by chemicals in vapor -or liquid phase in Conway diffusion cells. Stimulation was less effective when spores were seeded on 2.0% water agar containing concentrations of these Chemical pretreatment of U. pontederiae uredospores prior to their contact with H20 was best accomplished by 20 min. exposure of spores to chemicals in a hydrated atmosphere. An apparent source of stimulus for germination of U. lFrom M.S. Thesis, University of Florida.

PAGE 36

-32-edcriae urcdospores _, t.hc host plant, P. cordata. Germination of U. pontederiae uredospores may be bi ted in dense concentrations by the presence of an endo-genous, water soluble, self-inhibitor. A second endogenous metabolite may be involved in stimulation of germ tube growth. Uromyces pontederiae spores seemed to be inhibited by light, but not irreversibly so. Uromyces pontederiae spores germinated between 10 and 30 C when stimulated, and if not, between 15 and 30 C. Storage. -Uredospores of U. pontederiae are best stored at -196 C in liquid nitrogen, which stops all metabolic processes and freezes residual spore water. Storage under temperatures of -12 C is best when spores have been vacuumdried for three hours, which decreases damage by residual liquid solutes. Rapid viability losses were observed with g.pontederiae spores at temperatures of -12 C or less under conditions of no treatment, or storage 9ver desiccants. Humidities of 35, 52, and 61% were beneficial to spore sur-vival at 5 C. Spores did not exhibit cold-dormancy. Inoculation. Of the inoculation media best results were obtained with mixtures and 0.27% ",," was best accomplished by vapor stimulation with 2-heptanone in a dew chamber. Incorporation of a stimulant in 0.27% water agar containing U. pontederiae uredospores was less efficient in stimulation of germination on laminae.

PAGE 37

-33-Pretreatment of uredospores of Q. pontederiae by vapors of stimulants prior to inoculation was not successful in stimulation of spores on inoculated laminae, although this technique worked in Conway diffusion cells. Infection. -Infection of P. cordata by U. pontederiae occurs at temperatures of 15, 20, and 25 C. Apparently due to static growth rate of the host, uredosori were not produced on P. cordata at 15 C, even after 5 wk incubation, but were produced 1 wk after plants were transferred to warmer temperatures in the greenhouse. Uredosori were produced at 16 and 17 days at 20 and 25 C, respectively. Spores germinated poorly and under stress on leaves at 30 C and did not infect. No spores germinated at 25 C. Uredospores of U. pontederiae germinated equally at 25 C on leaves of pickerelweed and waterhyacinth, but penetrated and formed uredosori only on the former host.

PAGE 38

-34-OTHER STUDIES In addition to those summarized in the previous sections, several auditional short-term studies have been conducted. These studies are summarized in this section. Two. of these concerning a comparison of spray nozzles for application of C. rodmanii and an evaluation of the role of bacteria in the decline of hydrilla were special projects assigned to and conducted by students. Their final reports are included as the final portion of this section. Survey for G.rodmanii in F lor ida: a"n"d Louisiana: During July and August of 1978, M. T. Olexa surveyed several locations in the state of Florida for the presence of C. rodmanii. The survey was prompted by two factors; 1) there had been several unconfirmed reports of the disease at various locations and 2) we needed a more complete record of the distribution of the fungus in state prior to the establishment of large scale field tests. During the survey, numerous sites ranging from near Tallahassee in the north to Ft. Lauderdale in the South were visited (see Figure 2). Suspected diseased plants were collected and transported to .. .---i 1 I I I verified by R. Cullen. He based his verification on both microscopic examination and isolations of the fungus from diseased tissue. The fungus was found to be present in 6 of 22 sites surveyed. These six coupled with the six w:

PAGE 39

i' c LIDEND ----Lakes and waterways surveyed. Areas surveyed presence 01 Cercospora not confirmed. in which the' rodrnanii was Areas surveyed in which the presence of Cercospora rodmanii was corlfirmed. .". o Areas previously surveyed in which the presence of Cercospora rodmanii has been confirmed. -G!OtOC'f -, Fig. 2 j 1-. I -....._ -"'---'''j '--'-

PAGE 40

-36-prior known locations bring fungus is now known to occur. to 12 sites-in which the See Figure 2 for present distribution of C. rodmanii in Florida. In attempts to evaluate the potential o'f C. rodmaniifor waterhyacinth control in Louisiana Corps of Engineers has transferred diseased plants from the Lake September of 1978 we accompanied Mr. E. Addor of the Corps on a survey of several of these sites. Plants were col1ec and returned to Gainesville for examination.-Cercospora rodmanii was present on plants from .4 of .9' locations -visi The fungus was present on plants. from a site North of Hayes LA; sites I and lIon Pe-c-an Island, LA; and site I in B.ayou Manchac. It was not present at either Sorrento II, Hayes West, Centerville C, Bayou Louise or Manchac IV sites. Results of these surveys indicate that C.rodinanii is becoming widespread in both Florida and Louisiana. There are indications based on observations that in several of these areas it is beginning to exert a degree of Rating 'scaTe for C. rodmanii damage. __ -._. _. c_ __ --!!Ii, -[the --prb b ---.-is that of evaluation of disease damage on a quantitative basis. Based on his years of experience with the disease, Dr. Conway was able to devise a rating scale system for C. rodmanii damage based on individual leaf damage. This -system is shown in Figure 3. The system worked exceptio 'n

PAGE 41

H til t:; './1 2. .. rillllIWUiiIO. _Mlllm till ,..,..... .... ....... "1 : I ,,--.. .. lit I.' rn-.wug,t =.Ilt It III __ ,r:-, .. Figure 3' .. RATING SCALE SYSTl!"'.M FOR DA1iAGE TO LEAVES OF WATERHYACINTH BY CERCOSPORA RODMA1HI. NUMERICAL RATD1G SYMPTO! o I' on leaf, I 2 3, 11 Leas than 50% Less than 25% of No spots on leaf of petiole. no petiolar spotting. Less than 2'5"% of leaf surface with spots J no.:' coalescence orpetiolar of leaf surface leaf surface 5 Less of leaf surface with spots, coalescence, 10% tipdieback, __ spo.tting. with spots, some spots, coaloscence, coalescence, some tip-diebnck anc:l .. Bpottin, .-,'., ... .,. ... ,. f.'"', ..... I ,. II .: 6 7 6 88 than 75% spots,' Ore a ter than 75% spots, D'eadleaf Blade, scence,(JO%)tip-coalescence,(6Q%) tip-Petiole green,but k,; 'increasing dieback, coalescing heavily spotted. !ol
PAGE 42

-3R-we Hi the Fish Prairie.:) tudies where inoculum concentration and spreaJ were variable being evaluated. It is, thelefore, recommenued that this system be used in all future work with C. rodmanii. It is readily adaptable to rating entire plant damage by simply rating all the leaves on a given plant and dividing the total rating by number of leaves per plant. Pathogenicity of PhycomYcetes to HydriIla. Phycomycetes are fungi frequently referred to as water molds. As the name implies they are adapted to sur-vival in an aquatic environment. Several species are para-sitic on higher plants which they attack under conditions of high moisture. Many of these pathogens have a wide host range and will attack several plant species. Most of these latter are soil inhabiting. We tested the susceptibility of hydrilla to 25 such pathogens belonging to three genera. Out of these, 3 would consistently attack hydrilla under conditions of this test (sprigs of hydrilla in test tubes of distilled water). Results are shown in Table 2. However, larger scale tests in gallon jugs and 5 gallon aquaria (inconsistent) were also obtained with unidentified species of Pythium and Phytophthora isolated from declining hydrilla in Orange Lake, FL. Despite this inconsistency, the search for pathogens of hydrilla in the Phycomycete group should be continued because of the adaption of this group of fungi to an aquatic mode of existense.

PAGE 43

I I I I I I I g i i -39-Table 2. Reaction of Hydrilla following inoculation with various Phycomycetes. Phycomycete Original host Aphanamyces cochliodes Beta vulgaris A. euteiches Pisum sp. Phytophthora cinnamoni Persea americana P. citrophthora P. cryptogea P. dreschleri P. erythroseEtica P. :ealmivora P. :earasitica P. parasitica P. stellata Citrus sp. Aster sp. Citrus sp. Solanum tuberosum Ficus sp. Lycopersicon esculentum Nicotiana tabacum unknown Pythium acanthi cum unknown P. aphanidermatum Chrysanthemum sp. P. carolinianum unknown P. deboryanum unknown P. graminicolum unknown P herbicoides ..... unknown P. irregulare unknown P. irregulare Caladium P. mrriotylum unknown P. Earoecandrum Zea mays P. Eolytylum unknown Hydrilla reactiona ++ +++ -(?) +

PAGE 44

P. splendens unknown P. ultimum unknown P. vexans unknown a+, ++, +++ = slight, moderate, and high degree of susceptibility indicated, -= not susceptible, -(?) = indefinite.

PAGE 45

1 I -41-Other fungi isolated from Hydril1a. During the summer of 1977, Dr. Olexa isolated over 175 cultures of fungi from declining hydril1a primarily from Remuda Canal in Southwest Florida and Orange Lake and-Rodman Reservoir in Notth Central Florida. Only one of these, tentatively identified as a microconidial Fusarium sp. was consistently pathogenic on hydrilla. However, pathogenicity level was considered too low to be of value in a biological control program. Therefore, the search for hydrilla patho-gens with biocontro1 potential must be continued. Effect of C. rodmanii on fish. One of the maj or concerns in the use of plant pathogens for biological control of aquatic weeds is that they will harm fish. To determine if C. rodmanii was detrimental, it was tested against the fish Gambusia affinis in a standard 96 hr. bioassy. Ground up mycelium and spores of C .rodnianii was placed in the fish containers at rates ranging from 0.4 gm/liter to 6.34 gl liter. The lowest rate corresponded to an inoculum level of 48 gm/M2 which was the inoculation -. .Ta te use di n.L ake Con cor d i san ,_ ..... "-"-"0-'-..cc. _,cce .. "-_< .. ,::. ":'C"'.' .cC :.:-::'C=='cc ,_ .. :;' --....... ::cc"'"c::. ... ... .,.:.'.c .. ::=.:-= .. .. .:.:..-.: .. : ..... -.: .. ,; "::::..:::'::":== Prairie. The highest rate is equivalent to a surface area rate of 800 g/M2 which is 4 times higher than the highest rate we have ever used to inoculated waterhyacinth with C. rodmanii. None of the fish in any of the treatments were adversely affected. In fact at the highest rate, the fish ate the fungus which was subsequently isolated from their

PAGE 46

-42-feces. Therefore based un this limited test, we feel that C. rodmanii poses no threat to fish but other species need to be tested.

PAGE 47

L ,.' -43-SURVEY OF HYDRILLA UNDERGOING ANNUAL DECLINE FOR PATHOGENIC BACTERIA Daryl E. McKinney PT 621 RESEARCH PROJECT

PAGE 48

! -44 -IntroL ,.ction Hydrilla verticillata (L. f.) Royle is a submersed aquatic macrophyte. It is a monocot and belongs to the Hydrocharitaceae family. The plant is thought to have been introduced to Florida from South America around 1940 (4). Since its introduction, it has spread to many major freshwater lakes and and is threatening all those not currently infested. It grows in dense mats that may quickly destroy the public usefulness of any body of water so infested. Florida may have been the first site of introduction, but the plant has now become a problem in many southeastern and southwestern states. In these areas and in northern Florida it grows similar to an annual plant. In early spring, as the water temperature increases and days become longer, new shoots arise from stem fragments, tubers, and turions that have survived the winter in the hydrosoil. Hydrilla soon outgrows native aquatic plants since it utilizes light more efficiently (6). It grows towards the surface of the water from depths as great as 40 to 50 feet. The hydrilla may then form dense, entangled mats depth (3). Throughout the summer, hydrilla stores starch in its stems, stolons, and underground rhizomes. By late summer the mat is at a maximum density. An increase in epiphytic growth is commonly observed at this time (2). As the summer passes into fall, hydrilla begins to undergo what

PAGE 49

I I i i -45-is known as annual decline. At this time of the year the weather is the warmest, with the growing season and light intensity near maximum. Plants undergoing annual decline exhibit chlorotic leaves and stems that may become trans-parent. Leaf abscission is common and stems fragment eas ily. These symptoms are ahiays associated with the sur-face growth of the hydrilla. There are several possible explanations for annual decline. It is possible that hydrilla loses an excessive amount of photosynthate, as dissolved organic matter (DOM) through its leaves and epiphytes then use the DOM. This would correlate with the observed growth increase of epip-hytes (1). These epiphytes (bacteria and algae) may then interfere with C02 diffusion or photosynthesis by the hydrilla (5). When photosynthetic processes are reduc.ed below equilibrium with the respiration processes the plant would begin to die. The epiphytes may produce toxic metabolites that damage hydrilla tissue. Another theory is that plant pathogens are involved in annual decline (2). This research project was initiated to study the po ?:si b iFt ,,tB,,:t .. _anl1 uGll.de c 1 i:Il 9fh Y drtl1a;J.$ -: __ '. __ "':":_-:::::-. __ ;--;" __ __ .. .. ;:,; __ ,-.;..;: .. -:"::.".;:.'_
PAGE 50

-46-bacterium would have to be reisolated from the test plants. Materials and Methods Isolation -Bacterial isolations were made from samples of hydrilla expressing symptoms of annual decline. The samples were collected on October 14, 1977, near the middle of Orange Lake. Isolations were made at the site of tion and also in the lab. At the site isolations were made by cutting necrotic hydrilla stem and leaf t'issue into approximately !.til pieces and surface sterilizing 20 of these pieces. Sterilization was accomplished by rinsing the hydri1la pieces with a 10% clorox solution in a petri plate for one minute followed by two rinses of sterile deionized water. The surface sterilized pieces were then plated on hydri1la infusion agar (10 g crushed hydril1a and 15 g Bacto agar in one liter of deionized water), nutrient agar, and potato dextrose agar. Plates were then incubated at 25 C for three days. Lab isolations were made from hydrilla spigs maintained in sterile deionized water. The sprigs were shaken vigorously in three sterile water rinses to remove most of the .. -1-::.2-. rod in small tubes containing 2 ml of sterile saline solu-tion. Loopfu1s of the resulting suspensions were streaked on hydril1a infusion agar, NA and PDA. Plates were incu-bated at 25C in the dark for three days. Seven apparently different bacteria were then selected

PAGE 51

-47-from the site and lab isolations based on colony morphology and color. Stock cultures of these seven bacteria were maintained on NA slant tubes under paraffin oil in the refrigerator. Inoculum preparation -Two liter flasks containing 500 ml of nutrient broth were ino,culated from stock cultures and Shake cultures at approximately 60 strokes per minute for 24 hours. Isolates six and seven grew extremely slow at these conditions. They were examined microscopically and found to be myceloid, apparently actinomycetes. They were not tested for pathogenicity due,to their slow growth and the fact that few actinomycetes have been found to be plant pathogens. The remaining five bacterial isolates were prepared for inoculum by first sedimenting them from the nutrient broth by centrifugation (10,000 K for 10 minutes). The supernatant was decanted and the pellet was resuspended in SU rol of sterile saline (0.85%) solution. Each isolate was then adjusted to 0.25 transmittance with a colorimeter. This equalled approximately a 10 8 cells/ml concentration. =-_-:0,,".'::' -Rodman Reservoir on October l8J 1977. Sprigs of hydrilla were washed thoroughly with running tapwater before being rinsed twice with sterile deionized water. Two inoculum systems were used. The first consisted of incubating an 80 to 100 mm long growing hydrilla shoot

PAGE 52

-48-in a 30 X 150 mm glass tube in a bacterial suspension. The suspension was prepared by adding 4 ml of a 108 cells/ml bacterial concentration to 36 ml of sterile water. This resulted in a 107 cells/ml concentration around each hydrilla sprig. Three replications were made of each treatment. A 4 ml saline solution was added to each of three control tubes. All the tubes were mixed with a Vortex mixer. The second inoculation method was similar to the first except tubes were vacuum infiltrated twomintues at 25 mm Hg vacuum after inoculation. All tubes were incubated at approximately 22 C on the lab windowsill for three weeks after inoculation. Disease assessment after three weeks was made by visually comparing inoculated tubes with the control tubes. Hydrilla sprigs were rated as either healthy (H), chlorotic (C) or necrotic (N), (see Table 1). Besides visually assessing the inoculated hydrilla, reisolations were made from all inoculated and control hydrilla sprigs. A central piece of each stem containing and_three,-le<;t1Les>w.l:!-__ __ sux,f,-ace' sterilized and rinsed : --: -.-.. -twice with sterile water before being crushed with a glass rod in a 2 ml saline solution. Results The seven colony types are listed in Table 1. The majority (4/7) of the colonies were gray or white. This

PAGE 53

-49-agrees with Berg's (2) finding of three white colony types commonly isolated from hydrilla undergoing annual decline. Colony types 2 and 3 may be the same bacterium, with 3 being a rough mutant. All bacterial types grew similarly on NA as they did on hydrilla infusion agar. This indicates a lack of specific growth requirements. Results from inoculated hydrilla sprigs show that the bacteria tested were not pathogenic to hydrilla CTable Z). The chlorosis observed was probably due to a nutrient deficiency and not a pathogenic response. Reisolations (Table 3) from the inoculated and control hydrilla resulted in a random pattern of bacteria reisolated. Many of the reisolations contained bacteria that had not been inocualted on that particular hydrilla sprig. These bacteria were compared to stock tubes and related visually on the basis of their simularities in gross morphology. Discussion Bacterial diseases are associated with enormous concentrations (10 9 cells/ml) of bacteria in diseased tissue. In concentrations of this that they -_" -c are not ions if more than one reisolation is made. There was no ponderance of anyone bacterial type found in any of the reisolations and none of the colony-types tested produced symptoms of annual decline in test inocualtions. Berg (2) previously isolated three white bacterial

PAGE 54

-50-TABLE 1. Description of the seven isolated bacteria grown on NA plates. Isolate 1 2 3 4 5 6 7 White, gummy, some slime production Gray, some slime, smooth colony Gray, some slime, rough colony Yellow, some slime (nonfluorescent on KMB) Yellow, copious slime, myce10id White, very slow growth, myceloid Pink, some slime, gummy, slow growth, myce10id

PAGE 55

, -51-, i r TABLE 2. Visual comparison of hydrilla sprigs three weeds after inoculation. Inocu1ati(m Isolate Inoculated Control method Tube 1 2 3 4 5 (saline only) Method 1 1 H* H H H H H 2 H H H H H H 3 H H C* H C .H Method 2 1 H C H C H H 2 H H H H H C 3 H H H H H H *H= Healthy; C= Chlorotic; N= Necrotic

PAGE 56

I N LI"l TABLt 3. Reisolations' days on NA). TREATMENT Inoculation Tube Control 1 2 3 4 Method 1 1 (5) 2 (2) (3) (4) 3 Method 2 1 2 (7) (6) 3 "'l"'.p-.... ... (4) (5) (1) (4)(5) (1) (2) (7) (2) (5) (2) (2) (5) (3) (4) (2) (3) (4) (4) (4) (7) (7) ( ) :i,;t\j contain bacterial type reisolated, compared with stock 'ilil' ;j'f.! ,,:'i ,;-) /.;':' iIi.;: if+ '!'::f 1t;:: 1 : i ;; ( I" d 'i.' 5 (5) (5) (7) (2) (5)

PAGE 57

I L -53-types from hydri1la expressing annual decline and inoculated unknown concentrations of these on healthy hydrilla. He found symptoms similar to annual decline only to occur when he mixed the three different bacteria and again inoculated unknown concentrations on hydrilla. He reasoned that ann.ual decline was due in part to toxins (or toxin) produced by the three bacteria. It is more probable that he used excessive numbers of bacteria in his inoculations and that normally non-toxic metabolites produced by the bacteria became concentrated to such an extent that they were toxie. His tests with the toxins found them to be non-specific on other aquatic plants, this supports the idea that they are super-concentrated metabolites and not toxins per see It is probable that the bacteria found in this survey were epiphytic and not associated directly with the symptoms of annual decline of hydrilla. This view is supported by the results of reiso1ations (Table 3) of inoculated and noninoculated hydrilla which indicate the presence of these bacteria on healthy hydri1la. I, __ _...:._ c--. _'::"";,,0i, 1. ALLEN, H. L. 1971. Primary chemoorgail-otrophy and nutritional interactions of epiphytic algae and bacteria on macrophytes in the littoral of a lake. Ecol. Monogr. 41:97-127. 2. BERG, R. H. 1977. Annual decline of the aquatic macrophyte Hydrilla verticillata (L. f.) Royle. Ph.D.

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-54-Dissertation, Univ. of FL. 4. HALLER, W. T. 1976. Hydrilla, a new and rapidly spreading aquatic weed problem. Fla. Ag. Expt. Station Circular S-245. 5. SAND-JENSEN, K. 1977. Effect of epiphytes on eelgrass photosynthesis. Aquat. Bot. 3:55-63. 6. VAN, T. K., W. T. HALLER and G. BOWES. 1976. Comparison of the photosynthetic characteristics of three submersed aquatic plants. Plant Physio1. 58:761-768. 7. WAITE, T. D. and R. MITCHELL. 1976 .. Some benevolent and antagonistic relationships betweenUlva lactuca and its microflors. Aquat. Bot. 2:13-22. I I

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I i I -55-COMPARISON OF THREE NOZZLE SYSTEMS FOR SPRAYING CERCOSPORA RODMANII by Deborah F. Reese

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.As a senior reseal"ch proj ect I undertook the assignment of determining if nozzle systems have any effect on the efficacy of biological control organisms. Under the .direction of Dr. Kenneth E. Conway and with the technical assistance of Richard Cullen, a field test was carried out on plots in Thrasher Pond on Fish Prairie, .south Micanopy, FL. The objectives of this experiment were to see if nozzel types have any affect on the size of inoculum particles and pathogenicity of Cercospora rodmanii, Conway, a biological control organism currently being experimented with for waterhyacinth control. Materials and Methods Nozzle The following nozzle types selected for testing: Delevan RD-lO, #45 core (raindrop); Delevan WR-25 (mister): and Delevan hollow cone, #10 disc (hollow cone). Each nozzle was used in conjunction with a Spray Systems gun jet #12GH, adapted with a Delevan gun #3160 and two 14 inch extensions for ease in covering the plots from a boat. A portable spray power rotary pump with a modified was calibrated to determine the amount of flow/second. The raindrop nozzle delivered 300 ml/sec., the mister 200 ml/ sec. Inocu:lunc,. amount used and method of application: Isolate

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-57-WH 9 of rodmanii, was grown on potato-dextrose broth with o.s \ yeast extract (PDBY) for approximately two and onehalf weeks. A concentration of 48 b/M2 wet weight mycelium was used. The mycelium was diluted with water to give the proper spray volume. Each plot received the same amount of inoculum, 6000 mI. To insure an even inoculation, the plots were sprayed. in the following series; 3000 ml of inoculum was applied to waterhyacinths in plot #33, 34, and 35. Then the remaining 3000 ml of the total 6000 ml was applied in series to plots #35, 34, and 33. Plots Frames (made from PVC pipe) 9 in2 were floated on the pond. Each frame contained approximately the same number, age, and degree of previous infection (this variable was unavoidable as field test with C. rodmariii for rate and effect is an ongoing experiment at this pond). The frames were numbered #33, 34, and 35. The inoculum was applied to waterhyacinths in frame #33 using the raindrop nozzle. Waterhyacinthsin frame #34 were inoculated using the mister nozzle. And waterhyacinths in frame #35 were inoculated .... damage before inoculation and at two and seven weeks after inoculation. Results Immediately after spraying, ten leaves per plot were

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-58-" removed. Each leaf was for average size of inoculum particles and average number of particles/unit area 2) b" 1 '-" ,"j,) cm uSlng a lnocu ar mlcroscope. Dimensions of ten particles per leaf were recorded and the area of an elispe formula was used to calculate the area of each particle. The unit area was chosen at random on each leaf. The follow-ing is a breakdown of data on particle size and number of particles/unit area: The leaves of ten waterhyacinth plants from each of the three plots were rat'ed us ing a system of numbers 0.9. Zero being no infection and nine being death and/or submersion of the leaf. For the purpose of this ',-,' --. ..-<.' .. we limited the data collected to inoculum out for this test. Therefore, only the leaves with damage .of 0-6 were counted. The following is a breakdown of damage caused by inoculum sprayed using the three nozzle types:

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t i i t I I 1 I \ \ Raindrop: (Plot #33) Total Damage Average damage/plant Average damage/leaf Mister: (Plot #34) Total Damage Average damage/plant Average damage/leaf Hollow cone: (Plot #35) Total Damage Average damage/plant Average damage/leaf -59-March 31 106 10.6 __ 2.0 102 10.2 1.8 107 10.7 1.9 April 26 170 17.0 2.2 165 16.5 2.3 146 14.6 2.2 May 31 158 15.8 2.2 162 16.2 2.3 122 12.2 2.0 Following inoculation plants in plot '33 had the greatest amount of total damage and average damage/plant. Plants in plot #34 had intermediate amount of total damage and average damage/plant. Plants in '35 had the least total damage and average damage/plant. But plants in plot '34 had the greatest amount of averagedamagelleaf, plot #33 intermediate and plot # 35 the least. The-average damage/ leaf was studied because each plant did not have the same amount of leaves. Visually Apri I Ulost tI1-plot -#35 ahd intermediate spotting and plants in plot #34 had the least spotting. On May 31, the plots were rated again to see if nozzle type had any affeet on the long-term pathogenicity of the inoculum. The plants in plot #34 had greatest total damage, average damage/plant, and average damage/leaf. The

PAGE 64

-60-' in plot #33 had intermediate total damage, average damage/plant and average damage/leaf. The plants in plot #35 had the least amount of total damage, average damage! plant and average damage/leaf. Visually it was hard to tell which plots had been sprayed with inoculum from different nozzles. and Recommendations Using the premise that the larger particle size of inoculum and the more inoculum particles/leaf is best, I concluded that the mister nozzle would be the best nozzle for spraying C. rodmanii. The nozzle delivered the largest particle and the most particles/leaf, although infection got a slower start. During the course of this test, inoculum sprayed by the mister nozzle produced the most damage on the plants. I believe the of data concerning total damage had too many variables. Not knowing exactly which leaves were inoculated and which leaves were already infected rodmanii satistically affected the data results. We should have tagged the oldest living leaf and I i i I j taggiing the youngest and oldest leaf before inoculation. Then we will rate those leaves between at the beginning and end of the test to insure that only those leaves inoculated directly with C. rodmanii are rated for statistical analysis. f

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} --,. -, I I j I -61-AND CONCLUSIONS During the past two and one-half years, considerable additional progresi has been made in reaching our goal of the utilization .of plant pathogens in biological control programs for aquatic weeds. The pathogen Cercospora rodmanii has been shown to be effective against waterhyacinth in tests in both Louisiana and Florida. Methods of culturing and dissemination of this fungus for biocontrol purposes have been developed and considerable basic information concerning the host parasite relationship has been elucidated. This fungus shows so much promise that the University has decided to apply for a patent for its use in biocontrol programs and Abbott Laboratories has entered into an agreement with the University to develop it into a marketable product form for possible worldwide distribution. Two exotic pathogens also show biocontrol potential in our tests. The rust fungus, Uredo eichhorniae, from Argentina appears to have potential in biocontrol programs for waterhyacinths.. A Dut<;h fungus, Fusarium roseum.. ..... .. _". with both. of these fungi has been slowed because of the necessity of conducting research on them in quarantine. In addition to the above studies, several other investigations have been conducted with other pathogens and potential pathogens on various weed host. As a result, we

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! :, -62-feel our program has advanced faster than anticipated and is nearing our goal, at least in some areas. Based on this work, we can conclude that our original proposition is correct -plant pathogens are viable candidates as biocontrol agents for aquatic weeds.

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-63-PROJECT PUBLICATIONS 1. CHARUDATTAN, R. 1973. Pathogenicity of fungi and bacteria from India to hydri11a and waterhyacinth. Hyacinth Contr. J. 11:44-48. 2. CHARUDATTAN, R. and C. Y. LIN. 1973. Penicillium, Aspergillus, and Trichoderma isolates toxic to .: hydrilla and other aquatic plants. Hyacinth Contra J. 12:70-73. 3. CHARUDATTAN, R. 1973. Evaluation of foreign pathogens as biocontrols of hydri11a and waterhyacinth in the U.S.A. Second International Congress of Plant Pathology, Proc. (Abstr. 0390). 4. CHARUDATTAN, R. 1974. Evaluation of foreign pathogens as biocontro1s of hydri11a and waterhyacinth in the U.S.A. WSSA Newsletter. 2:11 (Repring of No.3). 5. CHARUDATTAN, R., T. E. FREEMAN, K. E. CONWAY, and F. W. lETTLER. 1974. Studies on the use of plant pathogens in biological control of aquatic weeds in Florida. Proc. EWRC 4th International Symposium on Aquatic Weeds, Vienna, 144-151. 6. ,R. 'or. __ Penicillium, Aspergillus, and Tric'ho'derma toxic to aquatic plants. Proc. EWRC 4th International Symposium of Aquatic Weeds, Vienna. 142-143. (Abstr.). 7. CHARUDATTAN, R. 1975. Use of plant pathogens to control aquatic weeds. In Impact of the use of microorganisms on the aquatic environment. Ecological

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-64-Res. Series, U.S. bnvironmental Protection Agency, Corvallis, OR. 259 p. 8. CHARUDATTAN, R. 1975. Weed control with plant pathogens. Agrichemical Age. Jan.-Feb. 1975. 9. CHARUDATTAN, R. and K. E. CONWAY. 1975. Comparison of Uredo eichhorniae, the waterhyacinth rust with Uromyces pontederiae. Mycologia. 67:653-657. 10. CHARUDATTAN, R. and K. E. CONWAY. 1975. Mycolep-todiscus terrestris leaf-spot on waterhyacinth. Plant Dis. Reptr. 66:77-80. 11. CHARUDATTAN, R., K. E. CONWAY and T. E. FREEMAN. 1975. A blight of waterhyacinth, Eichhorinia crassipes caused by Bipolaris stenospi1a (He1minthosporium stenospi1um). Proc. Phytopathology Soc. 2:65 (Abstr.). 12. CHARUDATTAN, R., K. E. CONWAY and T. E. FREEMAN. 1976. A blight of waterhyacinth, Eichhorniae crassipes, caused by Bipo1aris stenospila (Helmirithosporium stenospi1um). Proc. Am. Phytopatto1. Soc. 2:65 (Abstr.) 13. CHARUDATTAN, R., D. E. McKINNEY, H. A. CORDO and A. GUIDO -_. -----Uredo eich;horniae, 1976 b iacon tro 1 age-nt .. national Sym. on BioI. Contr. Weeds. 210-213. 14. CHARUDATTAN. R. and D. E. McKINNEY. 1978. A Dutch isolate of Fusarium roseum 'Culmorum' may control Hydrilla verticillata in Florida. Proc. EWRs Sym. on Aquntic Weeds. 5:219-224. t

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.L- -''''''-''.''--'::--65-15. CHARUDATTAN, R. B. D. PERKINS, and R. C. LITTRELL. 1978. Effects of fungi and bacteria on the decline of arthropod-damaged waterhyacinth (Eichhorniae crassipes) in Florida. Weed Science 26:101-107. 16. CONWAY, K. E., T. E. FREEMAN and R. CHARUDATTAN. 1974. The fungal flora of waterhyacinths in Florida, Part I. Water Resources Research Center, Univ. of Florida Publication No. 30, Gainesville, FL. 17. CONWAY, K. E. and J. W. KIMBROUGH. 1975. A new Doratomyces from waterhyacinth. Mycotaxon. 2:127L 18. CONWAY, K. E. 1975. Procedures used to test endemic plant pathogens for biological control of waterhyacinth. Proe. Phytopathology Soc. 2:31 (Abstr.). 19. CONWAY, K. E. 1976. Cercospora rodmanii, a new pathogen of waterhyacinth with biological control potential. Canad. J. Bot 54:1079-1083. 20. K. E. 1976. Evaluation of Cercospora rodmanii as a biological control of waterhyacinth. pathology 66:914-917. 21. CONWAY, K. E. and T. E. FREEMAN. 1976. The potential .. .. waterhyacinths. Proc. IV International Sym. on BioI. Contr. of Weeds. 207-209. 22. CONWAY, K. E. and T. E. FREEMAN. 1977. Host speci-ficity of Cercospora rodmanii a potential biological control of waterhyacinth. Plant Dis. Reptr. 266.

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-66-I 23. CONWAY, K. E., T. E. FREEMAN and R. CHARUDATTAN. 1978. Development of Cercospora rodmanii as a biological control for Eichhorniae crassipes. Proc. EWRS Sym. on Aquatic Weeds. 5:225-230. 24. FREEMAN,. T. E. and F. W. ZETTLER. 1971. Rhizoctonia blight of waterhyacinth. Phytopathology 61:892 (Abstr.) 25. FREEMAN, T. E. and F. W. ZETTLER. 1972. A disease of waterhyacinth with biological control potential. Abs'tr. of 1972 meeting of Weed Sci. Soc. of America 61. 26. FREEMAN, T. E. 1973. Survival of sclerotia of Rhizoctonia solani in lake water. Plant Dis. Reptr. 57:601-602. 27. FREEMAN, T. E., F. W. ZETTLER, and R. CHARUDATTAN. 1973. Utilization of phytopathogens as biocontrols for aquatic weeds. l11 International Sym. on BioI. Contr. of Aquatic Weeds. Montpellier, France. 28. FREEMAN, T. E., R. C HARUDAT TAN and F. W. ZETTLER. 1973. Biological control of water weeds with plant pathogens. Univ. of Florida Water Resources Research ". --C 29. FREEMAN, T. E. and R. CHARUDATTAN. 1974. Occurrence of Cercospora piaropi on waterhyacinth in Florida. Plant Disease Reptri 58:277-278. 30. FREEMAN, T. E., F. W. ZETTLER and R. CHARUDATTAN.

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-67-1974. Phytopathogens as biocontro1s for aquatic weeds. PANS. 20:181-184. 31. FREEMAN, T. E., F. W. ZETTLER and R. CHARUDATTAN. 1974. Utilization of phytopathogens as biocontrols for aquatic weeds. Proc. Conf. on Intergrated Systems of Aquatic Plant Control. U.S. Army Engineer 97-102. 32. FREEMAN, T. E. 1975. Rhizoctoniosls of aquatic plants McGraw-Hill Encyclopedia of Science "and Tec'hriology Yearbook. 33. FREEMAN, T. R. CHARUDATTAN and K. E. CONWAY. 1975. Use of plant pathogens for bioregulation of aquatic macrophytes. 34. FREEMAN, T. E., R. CHARUDATTAN and K. E. CONWAY. 1976. of the use of plant path6geris in biological control of weeds. Proc. IV In,ternational Sym. on BioI. of Weeds. 201-206. 35. ;FREEMAN, T. E., R. CHARUDATTAN, K. E. CONWAY" F. W. ZETTLER AND R. D. MARTYN. 1Q76. Biological control of water weeds with plant path6gens. Fla. Water .". -. Res our c e s' Res ent er 36. FREEMAN, T. E. 1977. Bio1ogica1 control of aquatic weeds with plant pathogens. Aquatic Bot. 3:175-184. 37. FREEMAN, T. E. 1978. Biological control of aquatic weeds with plant pathogens. In E. O. Ganstad "Aquatic plant control in river basin management".

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-68-CRC publishers. West Palm Beach. 38. Gallstad, E. 0., T. E. FREEM..AN, F. W,. ZETTLER, R. E. RINTZ, R. CHARUDATTAN, K. E. CONWAY and H.E. HILL. 1974. Aquatic weed control with plant pathogens. U.S. Army Corps of Engineers. Waterways Experiment Station. Vicksburg, MS 62 p. 39. GOEDEN, R. E., L. A. ANDRES, T. E. FREEMAN, P. HARRIS, R. L. PIENKOWSKI, and C. R. WALKER. 1974. Present status of projects on the biocontrol of weeds with insects and,plant pathogens in the United States and Canada. Weed Science. 22:490-495. 40. HAYSLIP, H. F. 1972. Evaluation of Plant PathOgens as biocontrols of Eurasian watermilfoil (Myriophyllum spicatum L.) M. S. Thesis, Univ. of Florida, Gaines:ville. '41. HAYSLIP, H. F. ',aridP', W. ZETTLER. 1973. Past and current research on diseases of Eurasian watermilfoil (Myriophyllum spicatum L.) HyacinthContr. J. 11: 38-40. 42. HILL, H. R. 1972. Survey and evaluation of plant pathogens of al1igatorweed (Alternanthera phi1oxeroides (Mart.) Griseb.). M.S. Thesis, Univ. of Florida, ,.,' .c .. 43. HILL, H. R. and R. E. RINTZ. 1972. Observations' of declining water lettuce populations in Lake Izabel, Guatemala. Proc. Southern Weed Sci. Soc. 25:374-3BO. 44. HILL, H. R. and F. W. ZETTLER. 1973. A virus-like stunting disease of a11igatorweed from Florida. T I

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-69-Phytopathology. 63:443 (Abstr.). 45. HILL, H. R., F. W. ZETTLER and T. E. FREEMAN. 1972. Plant pathogens with potential for biological control of aquatic weeds. Proc. Southern Weed Sci. Soc. 25:388 (Abstr.). 46. JOYNER, B. G. 1972. Characterization of a Rhizoctonia sp. pathogenic to aquatic plants. M.S. Thesis, Univ. of Florida, Gainesville. 47. JOYNER, B. G. and T. E. FREEMAN. 1973. Pathogenicity of Rhizoctonia solani to aquatic plants. Phytopatho1. 63:681-685. 48. MARTYN, R. D. 1977. Disease resistance mechanisms in waterhyacinths and their significance in biocontro1 programs with phytopathogens. Ph.D. Dissertation, Univ. of Florida 204 p. 49. MARTYN, R. D. and T. E. FREEMAN. 1978. Evaluation of Ac'remoniumzonatum as a potential biocontro1 agent of waterhyacinth. Plant Dis. Reptr. 62:604. SO. McKINNEY, D. E. 1978. Germination and storage of an infection by uredeospores of Uromyces pontederiae Sl. RIDINGS, W. H. and blight of amazon 806 (Abstr.). 52. RIDINGS, W. H. and blight of amazon F. W. ZETTLER. sword plant. F. W. ZETTLER. sword plant. 1972. Ap.hanomyces Phytopathology. 62: 1973. Aphanomyces Phytopatho1. 62:289-295.

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::r1WWi'lMiW m -70-53. RINTZ, R. E. 1973. Zonal leafspot of Hyacinth Contra J. 11:41-44. 54. RINTZ, R. E. 1973. Location, identification and characterization of pathogens of the waterhyacinth. Ph.D. Dissertation, Univ. of Florida,Gainesville. 55. RINTZ, R. E. and T. E. FREEMAN. 1972. Fusarium roseum pathogenic to waterhyacinth in Florida. Phytopathology. 62:806 (Abstr.). 56. ZETTLER, F. W. and T. E. FREEMAN. 1972. Plant pathogens as biocontrols of aquatic weeds. Annu. Rev. Phyto-pathology. 10:455-470. 57. ZETTLER, F. W. and T. E. FREEMAN. 1973. Potential for the use of plant pathogens as biocontrol agents of weeds. Proceedings 2nd International Congress of Plant Pathology. St. Paul, Minn.