Citation
Integrated Control of Dodder (Cuscuta pentagona Engelm.) Using Glyphosate, Ammonium Sulfate, and the Biological Control Agent Alternaria destruens Simmons, sp. nov.

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Title:
Integrated Control of Dodder (Cuscuta pentagona Engelm.) Using Glyphosate, Ammonium Sulfate, and the Biological Control Agent Alternaria destruens Simmons, sp. nov.
Creator:
COOK, JENNIFER COLLEEN
Copyright Date:
2008

Subjects

Subjects / Keywords:
Alternaria ( jstor )
Diseases ( jstor )
Experimentation ( jstor )
Flood damage ( jstor )
Herbicides ( jstor )
Mycology ( jstor )
Quaternary ammonium compounds ( jstor )
Species ( jstor )
Sulfates ( jstor )
Wind velocity ( jstor )
City of Gainesville ( local )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Jennifer Colleen Cook. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2006
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649810195 ( OCLC )

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Full Text












INTEGRATED CONTROL OF DODDER (Cuscutapentagona Engelm.) USING
GLYPHOSATE, AMMONIUM SULFATE, AND THE BIOLOGICAL CONTROL
AGENT Alternaria destruens Simmons, sp. nov.















By

JENNIFER COLLEEN COOK


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































To my husband, Jeffrey Cook, for his love, understanding, and support. I could not have
done this without you.















ACKNOWLEDGMENTS

I would like to sincerely thank Dr. Raghavan Charudattan for his guidance,

patience, and support. In addition, I would like to express my deepest gratitude to Dr.

Erin Rosskopf, without whose support I would never have made it on my own through

this journey. I can't thank her enough. I would also like to acknowledge the members of

my committee, Drs. Greg MacDonald, William Stall, and Thomas Zimmerman for their

support and guidance. I would like to thank Dr. Tom Bewick for his counsel and

unending information on dodder; Jim DeValerio for his friendship and always being there

when I had a problem or needed an answer, and for his experimental and statistical

knowledge; Eldon Philman and Herman Brown for all of their assistance at the

greenhouse, including building some crazy stuff for me; Philip Ruck for providing me

citrus plants whenever I needed them; Camilla Yandoc for all her time in helping me with

my field study; Sylvan and John Cascino for providing SmolderTM to accomplish this

work; Dr. Portier for his statistical consulting services; Alana Den Breeyen, Abby

Guerra, and Linley Smith for all their time and help in getting me through the molecular

part of this research; Everyone in Charu's lab, past and present, who has assisted me with

this project; USDA-CSREES-Special Grants Program for the award of a TSTAR grant to

the University of the Virgin Islands and the University of Florida that supported this

research; and USDA-ARS-USHRL for a cooperative research agreement that partially

supported this research. A special thanks to Whitney Elmore for her friendship, input,

and shared misery of writing a dissertation. I might just miss those midnight emails! I









would also like to thank my closest friends, Angela Horton, Lesa Werkmeister, Lynn

Howard, Jill Bejarano, and Kim Rosiek for listening to me when I needed to cry, scream,

or whine. Finally, I would like to thank my mom Catherine who has always been there

no matter what, through good and bad times, and to push me to finish this degree when I

needed the motivation. Mom, you're my inspiration and I love you for it.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ......... .................................................................................... iii

LIST OF TABLES ............. ..... ......................... .......... ............. vii

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

ABSTRACT .............. ..................... .......... .............. xii

CHAPTER

1 L ITE R A TU R E R E V IE W ............................................................. ..........................1

T a x o n o m y ............................................................................................................
B iology and H ost R ange .......................................................................... ............... 2
Cuscuta pentagon Engelm ......... ........................... ... ................... ............... 6
C uscuta spp. in the U S.................... ................................ .. ......... ............ .....
Cuscuta spp. W orldwide....................... ........................... ............... 8
E conom ic Loss, D ispersal, and C control ............................................ .....................9
H e rb ic id e s ............................................................................................................. 1 0
G ly p h o sa te ...................... .. ............. .. .....................................................1 3
A m m onium Sulfate ......... ...... .................................................................. ............ 14
Alternaria destruens Simmons, sp. nov .............. .......... ........ .. ........... 15
B biological C control .......... ...................................................... ...................... ..... 17
Research Objective ........... .. ................. ....... ... ..... ..... .......... .... 18

2 EVALUATION OF THE HOST RANGE OF Alternaria destruens
(SM OLDERTM) ........... ........................ ....... ... .......... ... .. 20

M materials and M methods ....................................................................... ..................23
Results and Discussion .................................... ...... .. ...... ............. 25

3 FIELD EFFICACY OF Alternaria destruens (SMOLDERTM) AS A
BIOH ERBICID E OF Cuscuta......................................................_........................ 31

M materials and M methods ....................................................................... ..................32
Results and Discussion .................................... ...... .. ...... ............. 39









4 MOLECULAR PHYLOGENIC CHARACTERIZATION TO DIFFERENTIATE
Alternaria destruens Simmons, sp. nov. FROM VARIOUS Alternaria SPECIES
BASED ON INTERNAL TRANSCRIBED SPACER REGION ...........................50

M materials and M methods ....................................................................... ..................52
Results and Discussion .................................... ...... .. ...... ............. 56

5 DISPERSAL OF Alternaria destruens: A WIND TUNNEL STUDY......................65

M materials an d M eth od s .................................................................... .....................67
Results and Discussion .................................... ...... .. ...... ............. 69

6 EFFECTS OF Alternaria destruens SMOLDERST), GLYPHOSATE (ROUNDUP
PRO), AND AMMONIUM SULFATE INDIVIDUALLY AND INTEGRATED
FOR CONTROL OF Cuscutapentagona .................................................. 75

M materials and M methods ....................................................................... ..................80
Results and Discussion .................................... ...... .. ...... ............. 82

APPENDIX

A FEDERAL NOXIOUS WEED LIST (AS OF JANUARY 6, 2006).....................102

B W IN D SP E E D D A T A .................................................................... ..................... 103

C ENVIRONMENTAL DATA FOR GREENHOUSE TRIALS OF Alternaria
destruens, AMMONIUM SULFATE AT 0.125% W/V IN WATER, AND
GLYPHOSATE AT 0.02 kg (ae)/L OF ROUNDUP PRO ON Cuscuta
p en tag o n a ........................................................................ 10 7

D DATA FROM PRELIMINARY GREENHOUSE TRIAL ..................................... 111

L IST O F R E F E R E N C E S ...................................................................... ..................... 119

BIOGRAPHICAL SKETCH ............................................................. ............... 130
















LIST OF TABLES


Table p

2-1. Reaction of test plants to Alternaria destruens applied as SMOLDERm
formulated with PCC588 (United Agri Products, Greeley, CO)............................29

3-1. Means of area under the disease progress curve (AUDPC) for treatments on
Cuscutapentagona from Ft. Pierce field trial I..................... ................................. 45

3-2. Means of area under the disease progress curve (AUDPC) for treatments on
Cuscutapentagona from Ft. Pierce field trial II ............................................46

4-1. Sources of unknown isolates and treatments applied to plots from which these
isolates w ere recovered ...................... .. ...................................... ........... 60

4-2. Sources of Alternaria spp. used for DNA sequencing in this study..........................61

4-3. Fungal Isolates and the GenBank accession numbers for sequences used in
phylogenic analy ses.......... ..... ...................................................... ... .. .... .... .. 63

6-1. Regression equations and R2 values from greenhouse trials I and III (combined)
for the effects of disease or damage severity from treatments on Cuscuta
pentagon over time (days after treatment, DAT) .........................................89

6-2. Area under the disease or damage progress curve (AUDPC) means for all
treatments on Cuscutapentagona from greenhouse trials I and III (combined)......91

6-3. Area under the disease or damage progress curve (AUDPC) means for all
treatments on Cuscutapentagona from greenhouse trial II ...............................93

B-1. Ft. Pierce trial I average wind speed data........................ .................. .......... 103

B-2. Ft. Pierce trial II average wind speed data............... .......................................... 105

B-3. Gainesville field study average wind speed data..................................................106

D-1. Area under the disease or damage progress curve (AUDPC) means for all
preliminary glyphosate treatments on Cuscutapentagona in greenhouse trials I
an d II (com b in ed) ................................................................ ........ ......... 112

D-2. Area under the disease or damage progress curve (AUDPC) means for all
preliminary glyphosate treatments on Citrus spp. in greenhouse trials I and II.....114









D-3. Area under the disease or damage progress curve (AUDPC) means for all
preliminary ammonium sulfate treatments on Cuscutapentagona in greenhouse
trials I and II (combined)...................................... ........ ... ............... 116

D-4. Area under the disease or damage progress curve (AUDPC) means for all
ammonium sulfate treatments on Citrus spp. in greenhouse trials I and II
(com bined) ................................................................ ... ...........118
















LIST OF FIGURES


Figure p

1-1. C uscuta p entagona on citrus ............................................................ .....................3

1-2. Cuscutapentagona flowers and buds (A and B) ....................................................4

1-3. Cuscutapentagona haustorium (arrow) penetrating citrus ......................................5

1-4. A lternaria destruens spores .......................... ........... ...................... .............. 17

2-1. Capsicum chinense 'Habanero' pepper plants................................ ............... 27

2-2. Capsicum chinense 'Scotch Bonnet' pepper plants........................................28

2-3. Lactuca sativa 'G reen Ice' lettuce plants ..................... ..... .............. .................28

3-1. Header Canal Alternative Cropping Systems Research Farm in Ft. Pierce, FL........36

3-2. Simulated field study at the University of Florida in Gainesville .............................37

3-3. Experimental design of simulated field study containing 55 pots of Citrus spp.
parasitized with Cuscutapentagona ............................................. ............... 38

3-4. Distances used in simulated field study.................................................................. 38

3-5. Citrus spp. plants parasitized by Cuscutapentagona, the latter displaying
symptoms of treatments with Alternaria destruens + oil and an untreated control .39

3-6. Ft. Pierce fi eld trial I data ......... ................. ................................. ............... 45

3-7. Ft. Pierce field trial II data.................................... ......... ................................ 46

3-8. Disease severity over time (days after inoculation; DAI) on Cuscutapentagona
treated with Alternaria destruens + oil (PCC 588) in Circle I (0.91 m) and
untreated sentinel C. pentagon in Circle III (3.15 m) ........... ....... ............. 47

3-9. Disease severity over time (days after inoculation; DAI) on Cuscutapentagona
treated with Alternaria destruens + oil (PCC 588) in Circle I (0.91 m) and
untreated sentinel C. pentagon in Circle II (2.13 m)........................................... 48









3-10. Disease severity over time (days after inoculation; DAI) on untreated sentinel
Cuscutapentagona in Circles II (2.13 m) and III (3.15 m) .................................49

4-1. Rooted neighbor-joining dendrogram based on 507 bp from ITS 1, 5.8S, and
IT S 2 sequ en ces................................................. ................ 59

4-2. Rooted neighbor-joining dendrogram of ITS1, 5.8S, and ITS2 sequences ..............62

5-1. Results of the first experiment utilizing the wind tunnel to examine the effects of
wind tunnel length (m) from inoculum release on spore dispersal ofAlternaria
destruens ..................................... .................. ............... ......... 72

5-2. Results of the first experiment utilizing the wind tunnel to examine the effects of
wind speed (m s-) from inoculum release on spore dispersal ofAlternaria
destruens ..................................... .................. ............... ......... 73

5-3. Results of the second experiment utilizing the wind tunnel to examine the effects
of wind speed (m s-) and distance (m) from inoculum release on spore dispersal
of A lternaria destruens .................. ............................. .... .. .. .. ........ .... 74

6-1. Greenhouse trials I and III (combined) to determine the effects of disease or
damage severity from treatments on Cuscutapentagona over time (days after
treatm ent; D A T ) .................................................... ................ 8 8

6-2. Effects of treatments on disease or damage severity on Cuscutapentagona over
time (days after treatment; DAT) from greenhouse trials I and III (combined).......90

6-3. Effects of treatments on disease or damage severity on Cuscutapentagona over
time (days after treatment; DAT) from greenhouse trial II.................................92

6-4. Greenhouse trial II to determine the effect of time (DAT) on disease or damage
severity across all treatm ents......................................................... ............... 94

6-5. Stunting of regrowth of Cuscutapentagona treated with glyphosate at 0.02 kg
(ae)/L of Roundup Pro ............. .................. ................. .................................. 95

6-6. Effect of ammonium sulfate concentrations (0.50%, 0.25%, 0.125%, and 0%
(control) w/v in water) on the growth ofAlternaria destruens colony (mm day-1) .96

6-7. Inhibition of growth ofAlternaria destruens colony (mm day-) over time (DAT)
by ammonium sulfate at 0.50%, 0.25%, 0.125% and 0% ((CON) w/v in water) ....97

6-8. Effect of glyphosate concentrations (0.09, 0.04, 0.02 and 0 (control) kg (ae)/L of
Roundup Pro) on the growth ofAlternaria destruens colony (mm day-1) ............98

6-9. Inhibition of growth (mm day-) ofAlternaria destruens over time (DAT) by
glyphosate at (0.09, 0.04, 0.02 and 0 (CON)) kg (ae)/L of Roundup Pro) ...........99









6-10. Effect of oil (PCC 588) (7.5% v/v in water) on the growth of Alternaria
destruens compared to untreated control (no oil)........................ .................. 100

6-11. Effect of oil (PCC 588) at 7.5% v/v in water and control (CON) on the growth
(mm day-) of Alternaria destruens over time (DAT)................................ 101

C-1. Maximum and minimum temperatures (C) during greenhouse trial I.................107

C-2. Maximum and minimum temperatures (C) during greenhouse trial II ...............108

C-3. Maximum and minimum temperatures (C) during greenhouse trial III................ 109

C-4. Maximum relative humidity during all greenhouse trials................. .......... 110

D-1. Damage severity on Cuscutapentagona over time from six glyphosate
concentrations (0.09, 0.18, 0.36, 0.71, 1.42, and 0 (control) kg (ae)/L of
R oundup P ro) ...................................... ............................. ................ 111

D-2. Damage severity on Citrus spp. from six glyphosate concentrations
concentrations (0.09, 0.18, 0.36, 0.71, 1.42, and 0 (control) kg (ae)/L of
Roundup Pro), averaged over all days ............. ............... ....... ........... 113

D-3. Damage severity on Cuscutapentagona from seven ammonium sulfate
concentrations (0% (control), 1%, 2%, 3%, 4%, 5%, and 10% w/v in water) over
tim e (D A T ) ............................................................................................. ..... 115

D-4. Damage severity on Citrus spp. from seven ammonium sulfate concentrations
(0% (control), 1%, 2%, 3%, 4%, 5%, and 10% w/v in water) averaged over all
d a y s .......................................................................... 1 1 7










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

INTEGRATED CONTROL OF DODDER (Cuscutapentagona Engelm.) USING
GLYPHOSATE, AMMONIUM SULFATE, AND THE BIOLOGICAL CONTROL
AGENT Alternaria destruens Simmons, sp. nov.

By

Jennifer Colleen Cook

August 2006

Chair: Raghavan Charudattan
Major Department: Plant Pathology

Cuscutapentagona is an obligate parasite that is the most widespread and

aggressive Cuscuta species in the world. It causes a serious weed problem in many

economic and ornamental crops. Its minutely sized, easily dispersed, and highly viable

seed makes it difficult to control. Alternaria destruens has been developed as a

registered bioherbicide for Cuscuta species. A host-range study using 30 plants of

economic importance belonging to 8 families (Asteraceae, Convolvulaceae, Fabaceae,

Malvaceae, Meliaceae, Poaceae, Rutaceae, and Solanaceae) indicated that A. destruens

is pathogenic only to Cuscuta spp. In greenhouse trials of efficacy, seven treatments

were applied: an untreated control; oil (PCC 588); A. destruens (SmolderT WP); a

mixture ofA. destruens + oil; ammonium sulfate at 0.125% w/v in water; glyphosate at

0.02 kg (ae)/L of Roundup Pro; and a mixture of A. destruens + oil, glyphosate at 0.02

kg (ae)/L of Roundup Pro, and ammonium sulfate at 0.125%. By 35 days after

treatment, all Cuscuta plants treated with the last treatment were dead but not the host

plant, citrus. Efficacy ofA. destruens was further assessed in field trials. Four treatments

consisting of an untreated control, oil, A. destruens + oil, and A. destruens were tested,









but the levels of control did not differ among the treatments. To determine if this lack of

difference was due to pathogen's dispersal, Alternaria isolates from randomly chosen

Cuscuta tissue samples were collected from field and greenhouse trials and studied using

nuclear rDNA internal transcribed spacer (ITS) region, including ITS1, ITS2, and the

5.8S rDNA. Neighbor-joining analysis revealed that all 47 unknown isolates and the

reference A. destruens isolate were 100% identical. The results indicated a high degree

of conservation of the ITS region in A. destruens which made it impossible to conclude

that the isolates from field and greenhouse samples were the same as the reference isolate

used in these trials. Overall, this study has confirmed the effectiveness of an integrated

system consisting ofA. destruens, glyphosate, and ammonium sulfate to control Cuscuta

spp.














CHAPTER 1
LITERATURE REVIEW

Taxonomy

The name Cuscuta originates from the Arabic word "kushkut," which loosely

translates as "a tangled wisp of hair" (Austin 1980). When Cuscuta species (spp.) begin

to cover a plant, a mass of tangled yellow to orange threads with various diameters and

no leaves are produced. Cuscuta spp. are also known by various common names, such as

dodder, love vine, tangle gut, devil's gut, and strangleweed. The most common name,

dodder, possibly originates from the Old German word "dotter" which means yolk

(Dawson et al. 1994).

The description of the genus Cuscuta L. was first published in 1700 by

Tournefort. In 1932, Yuncker produced a worldwide monograph that divided the genus

into three subgenera based on the morphology of stigmas and styles. The first subgenus,

Monogyna (Englm.) Yunck., is the most distinctive and favors trees and shrubs as hosts

(Dawson et al. 1994). This subgenus has one style that is either partially or totally joined

with elongated to capitate stigma lobes (Guerra and Garcia 2004). The second subgenus,

Grammica (Lour.) Yunck., contains two styles, capitate stigmas, and is the most

diversified group with the majority of the species (Guerra and Garcia 2004). The last

subgenus is Cuscuta, which is characterized by elongated stigmas and two styles (Guerra

and Garcia 2004). This subgenus is currently under taxonomic revision (Garcia 2004).

There is some controversy regarding the family in which to place the genus

Cuscuta: either Convolvulaceae, or an entirely separate family Cuscutaceae. Based on









recent molecular studies, Cuscuta should remain in Convolvulaceae (Garcia 2004).

There are approximately 150 to 170 Cuscuta spp. worldwide; with the majority occurring

in North and South America (Ashton and Santana 1976; Dawson et al. 1994).

Biology and Host Range

Cuscuta species are obligate parasites that predominate disturbed habitats, which

preadapts them to becoming a serious weed problem (Figure 1-1). Cuscuta spp. are

found all over the world and parasitize mainly dicotyledonous and a few

monocotyledonous plants (Gaertner 1950; Dawson et al. 1994). Some species of Cuscuta

are very host specific, while others have a broad host range. Economically important

plants affected include alfalfa (Medicago sativa L.) (Dawson et al. 1984), rabbiteye

blueberry (Vaccinium ashei) (Dawson et al. 1984; Monaco and Mainland 1981), carrot

(Daucus carota L.) (CAB International 2005), citrus (Citrus spp.) (Ashton and Santana

1976), cranberry (Vaccinium macrocarpon Ait.) (Bewick et al. 1985), tomato

(Lycopersicon esculentum L.) (Ashton and Santana 1976), potato (Solanum tuberosum

L.) (Selleck et al. 1979), sugar beet (Beta vulgaris L.) (CAB International 2005), various

ornamental, native, and many weed species (Dawson et al. 1984) (Figure 1-2). Due to

the destructiveness and damaging effects of this parasitic plant, the Animal Plant Health

and Inspection Service (APHIS) of the United States Department of Agriculture (USDA)

has listed Cuscuta spp., other than the following species (Appendix A), as federal

noxious weeds (Tasker 2006). All movement throughout the United States or interstate

of these species is prohibited without a permit.






























Figure 1-1. Cuscutapentagona on citrus

The inability of Cuscuta spp. to parasitize many hosts may be due to various

interactions. The tomato defense system induces genes within the early stages of

haustorial development of Cuscuta reflexa Roxb. to stop parasitization (Werner et al.

2001). Cuscuta spp. growth on different hosts varies considerably (Nemli 1987).

Cuscuta spp. stems may twine around the host plant, but may not fully develop.

Therefore, the stems weaken and die. A host's anatomical structure may delay the

growth of the parasite, thereby, preventing the haustoria and hyphae from reaching the

vascular bundles of the host (Dawson et al. 1994).

Flowers produce seed capsules with 2 to 3 seeds, with each plant producing

several thousand seeds (Ashton and Santana 1976) (Figure 1-2). Seed coats are very hard

and newly matured seeds are impermeable to water (Dawson 1965; Gaertner 1950;

Hutchinson and Ashton 1980). Scarification must take place in order for seeds to

germinate, which in nature probably occurs through weathering, soil microbial action, or









other natural disturbances. Seeds can remain dormant and viable in the soil for up to 60

years depending on environmental conditions and the species (Gaertner 1950; Hutchinson

and Ashton 1980).


Figure 1-2. Cuscutapentagona flowers and buds (A and B)

Seedlings contain minute amounts of chlorophyll (both a and 0) and are capable

of photosynthesis at a low rate, but Cuscuta spp. are still dependent on the host to









complete its life cycle (Ashton and Santana 1976; Dawson et al. 1994; Zimmermann

1962). In addition, virtually 100% of the carbon Cuscuta spp. use is acquired from the

host's phloem (Jeschke et al. 1994).

Germination of seedlings occurs at temperature ranges between 15C and 38C

with optimum temperatures in the range of 30C to 33C (Hutchinson and Ashton 1980).

Once seedlings have emerged, elongation and circumnutation occurs. For seedling hooks

to open and twining to occur, seedlings must have sunlight or incandescent light (Dawson

et al. 1994). Once they find a vertical host, living or nonliving, they attach themselves

counterclockwise and are no longer connected to the soil. If the object the seedling

attaches to is a not a host or nonliving, the seedling will die after several days (Dawson et

al. 1994). Within 2 to 4 days of attaching to a suitable host, Cuscuta spp. produce a

haustorium (Dawson 1987) (Figure 1-3).



















Figure 1-3. Cuscutapentagona haustorium (arrow) penetrating citrus

The haustorium stops growing within the host tissue after 1 to 2 days and starts to

form searching hyphae that expands intra- and intercellularly along the middle lamellae









of the host cell wall (Dawson et al. 1994). Searching hyphae may increase in length up to

800 rm before connecting with the host plant's xylem and phloem (Dawson et al. 1994;

Vaughn 2003). In addition, the extensive growth may assist in fastening Cuscuta spp.

securely to the host (Vaughn 2003). Survival of Cuscuta spp. occurs through extracting

large amounts of translocated solutes from the phloem of the host. Assimilates and

minerals, such as, sucrose, phosphorus, and potassium are transported in the phloem from

source to sink (Ziegler 1975).

Once Cuscuta spp. has parasitized a host, it may grow as much as 8 cm per day,

with a single plant covering more than 3 m in diameter in one growing season (Dawson et

al. 1984). Cuscuta spp. forms a dense, thick mat over the host, which can eventually kill

the host. This is not only due to Cuscuta spp. transporting nutrients from the host, but

also from blocking out the sun and decreasing the amount of photosynthesis that can take

place in the host.

Cuscuta pentagon Engelm.

Cuscutapentagona, also known as bush-clover dodder, field dodder, five-angled

dodder, and lespedeza dodder, is the most widespread and aggressive Cuscuta species in

the world (Holm et al. 1997; Lanini and Kogan 2005; Parker and Wilson 1986). In

addition, this species is reported to be in 76 countries, provinces, and regions and in 46

United States (U.S.) (CAB International 2005; USDA, NRCS 2006). The broad range of

this species occurs from Argentina and Chile through the U.S. to Canada and from the

Cape of Good Hope almost to the Arctic in the Old World (Holm et al. 1997).

Cuscutapentagona is a serious weed problem of alfalfa in Australia, Hungary,

South Africa, and the U.S.; cabbage (Brassica oleracea L.), peppers (Capsicum spp. L.),

and potatoes in Hungary and the U.S.; carrots and onions (Allium cepa L.) in









Switzerland; legumes in Hungary and Pakistan; lentils (Lens culinaris Medik.) in Syria;

and tomatoes, sugar beets, and other vegetable crops in the U.S. (Dawson et al. 1994;

Holm et al. 1997). It is a significant weed problem of alfalfa in Saudi Arabia; carrots in

Italy; and dryland and irrigated crops, including vegetables, in Australia (Holm et al.

1997).

In addition to the crops listed previously, there are many others that C. pentagon

parasitizes. These being forage legumes in Australia, Chile, India, Italy, and Jordan;

beans (Phaseolus spp.), beets, peas (Pisum spp.), and various other vegetables in Canada,

India, Israel, Japan, the Netherlands, Pakistan, and Switzerland; pastures in South Africa

and Uganda; safflower (Carthamus tinctorius L.) in Italy; and sugar beets in Chile and

Turkey (Holm et al. 1997).

Cuscuta spp. in the U. S.

Currently, there are 49 species of Cuscuta found in the U.S., Puerto Rico, and the

U.S. Virgin Islands (USDA, NRCS 2006). The most problematic species, other than C.

pentagon, are C. approximate Bab. (smallseed alfalfa dodder or alfalfa dodder), C.

epilinum Weihe (flax dodder), C. gronovii Willd. ex J.A. Schultes (swamp dodder), and

C. indecora Choisy (large-seed dodder or bigseed alfalfa dodder).

Cuscuta approximate has many varieties with a wide host range; it is found in 12

states, and was introduced into the U.S. (Parker and Riches 1993; USDA, NRCS 2006).

Cuscuta epilinum Weihe has a very restricted host range, hence its common name of flax

dodder, was introduced into the U .S. and is found in 9 states (Parker and Riches 1993;

USDA, NRCS 2006). Cuscuta gronovii is generally found in wet places and along water

courses, is extremely invasive on cranberry, and is found in 44 states (Bewick et al. 1988;

USDA, NRCS 2006). Cuscuta indecora mainly attacks plants in the Leguminosae, is









native to the U.S., and is found in 35 states, Puerto Rico, and the U.S. Virgin Islands

(Parker and Riches 1993; USDA, NRCS 2006).

Cuscuta spp. Worldwide

There are numerous other Cuscuta spp. found throughout the world that cause

serious problems on forage legumes, herbaceous plants, shrubs, trees, and various crops.

Cuscuta chinensis Lam. (Chinese dodder) and C. obtusiflora Kunth (Peruvian dodder) are

mainly found in East Asia and Europe and are a major problem in soybeans in China (Li

1987; Parker and Riches 1993). Cuscuta epithymum (L.) L. (clover dodder) is found in

60 countries, provinces, and regions and in 26 U. S. states (CAB International 2005;

USDA, NRCS 2006). This species is a serious weed problem in alfalfa, cabbage, carrot,

peppers, potatoes, purple clover (Trifolium pratense L.), and tomatoes (CAB

International 2005; Holm et al. 1997; Parker and Riches 1993). Furthermore, C.

epithymum can be found parasitizing flax, orchards, ornamentals, and pastures (Holm et

al. 1997). Cuscuta europaea L. (greater dodder) is found in 58 countries, provinces, and

regions and in 2 U. S. states (CAB International 2005; USDA, NRCS 2006). Hosts of

this species include broad bean (Viciafaba L.), common hop (Humulus lupulus L.),

onion, raspberry (Rubus idaeus L.), and sugar beets (CAB International 2005). Cuscuta

japonica Choisy (Japanese dodder) is an East Asian species that parasitizes woody and

herbaceous hosts (Parker and Riches 1993). Cuscuta monogyna Vahl. (eastern dodder) is

found mainly in the Mediterranean and Middle East, but extends from France through

southern Europe, to Afghanistan (Parker and Riches 1993). This species is commonly

found on citrus, olive (Olea europaea L.) and other fruit and vegetable crops. Cuscuta

reflexa Roxb. (giant dodder) is an Asian species that ranges from India and the

Himalayas, extending through Pakistan to Afghanistan, south to Sri Lanka, China, and









into Indonesia (Parker and Riches 1993). This species mainly occurs on perennial trees

and shrubs such as Arabica coffee (Coffea arabica L.), citron (Citrus medical Linn.),

litchi (Litchi chinensis Sonn.), and peach (Prunuspersica (L.) Batsch.) (Parker and

Riches 1993).

Economic Loss, Dispersal, and Control

Extensive economic losses can occur from Cuscuta spp. infestations. Crops

impacted in Florida include blackeyed peas (Vigna unguiculata (L.) Walp.), blueberry,

eggplant (Solanum melongena L.), key lime (Citrus aurantiifolia (Christm.) Swingle),

sweet orange (Citrus x aurantium L.), and tomato (Alfieri et al. 1994). Yield losses as

high as 80% to 100% have been reported in cranberry fields in Massachusetts and

Wisconsin due to Cuscuta spp. (Devlin and Deubert 1980). Approximately 30,000 acres

of tomato crops in California are parasitized by Cuscuta spp. which can cause reduction

in crop yield by over 75% if not controlled (Lanini 2005). In the U.S. Virgin Islands, C.

americana L. (American dodder) infests many woody plants species, including citrus and

many ornamental species. Timber crops infested with this species have increased costs

and reduced income and yield. Furthermore, ornamental species parasitized by C.

americana are either killed or seriously reduced in value.

Dispersal of Cuscuta spp. is either by seeds or vegetative propagation. Seeds are

small, but heavy and not adapted for wind dispersal (Dawson et al. 1984); they are

primarily dispersed by humans (Dawson et al. 1994). Seeds are also easily dispersed

through contaminated crop seed (Dawson et al. 1984). Cuscuta spp. is also easily

transferable to other plants by vegetative propagation. Placement of actively growing

Cuscuta spp. stems on host plants usually results in the formation of haustoria and thus, a

new Cuscuta plant. In the U.S. Virgin Islands birds have been observed using Cuscuta









spp. stem tissue, which can include seeds, in nest building (T. Zimmerman, UVI-AES-

Biotechnology & Agroforestry, Kingshill, St. Croix, VI, personal communication). This

is believed to aid in transport of Cuscuta spp. between trees. Cuscuta spp. can also be

used as a living bridge to transport pathogens, such as plant viruses and mycoplasma-like

organisms from one plant to another (Agrios 1997).

Control of Cuscuta spp. is very difficult, with the best method being exclusion,

i.e., not to introduce it into a farm or field. Mechanical removal by seed machines is an

option, but these devices are often slow and they do not always remove all of the Cuscuta

spp. seed and sometimes remove the crop seed. Tillage can be useful because the

seedlings are easily dislodged from the soil or die when buried, but if they are not buried

completely, they can resurface. Physically removing Cuscuta spp. by hand is time-

consuming and although Cuscuta spp. generally grow as annuals, they can survive in the

tissue of perennial host plants under some conditions (Dawson et al. 1984).

Crop rotations with cereal grains or forage grasses for two or more years can

significantly decrease Cuscuta spp. seed banks (Dawson et al. 1984). Seedlings emerge

and die because they rarely will parasitize plants in the family Gramineae. Another

cultural method that can be used to control Cuscuta spp. is shade. Formation of a canopy

from heavy crop cover can suppress seedling emergence (Dawson 1966).

Herbicides

Soil-applied herbicides can be very effective in controlling Cuscuta spp. primarily

because control occurs prior to attachment to host. The first soil-applied herbicide used

was chlorpropham (1-methylethyl (3-chlorophenyl) carbamate), which was a plant

growth regulator that terminated seedling growth prior to emergence. Unfortunately, this

herbicide is no longer available for use in the U.S.









Other preemergent herbicides available for Cuscuta spp. control are dichlobenil

(2,6-dichlorobenzonitrile), pendimethalin (N-(1-ethylpropyl)-3,4-dimethyl-2,6-

dinitrobenzenamine), prodiamine (2,4-dinitro-N3,N3-dipropyl-6-(trifluoromethyl)-1,3-

benzenediamine), pronamide (3,5-dichloro-N(1,1-dimethyl-2-propynyl) benzamide), and

trifluralin (a,a,a-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) (Nice 2005; Ristau 1996).

Prodiamine and pendimethalin control Cuscuta spp. more efficiently than trifluralin

(Dawson 1987; Dawson et al. 1994).

Preemergent herbicides may not provide season-long control and repeated

treatments may be necessary. Therefore, postemergent applications of imazamox (2-[4,5-

dihydro-4-methyl-4-(1-methylethyl)-5-oxo-l1H-imidazol-2-yl]-5-(methoxymethyl)-3-

pyridinecarboxylic acid) or imazethapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-

oxo- 1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid) may also be used but since

these herbicides are used after attachment, success may be limited (Nice 2005; Ristau

1996).

Foliar applications of diquat (6,7-dihydrodipyrido[1,2-a:2', '-c]pyrazinediium

dibromide) or paraquat (1, '-dimethyl-4,4'-bipyridinium dichloride) provide partial

selectivity in alfalfa; Cuscuta spp. and the host plant foliage will be destroyed, but the

host plant may regrow from crowns after treatment (Dawson 1987; Dawson et al. 1994).

Other nonselective post-attachment herbicides that will provide complete kill to the host

plant and Cuscuta spp. are 2,4-D ((2,4-dichlorophenoxy)acetic acid) or glyphosate (N-

(phosphonomethyl)glycine) (Dawson et al. 1994).

Preemergent herbicides labeled for citrus in Florida are diuron (N'-(3,4-

dichlorophenyl)-N,N-dimethylurea), norflurazon (4-chloro-5-(methylamino)-2-(3-









(trifluoromethyl)phenyl)-3(2H)-pyridazinone), oryzalin (4-(dipropylamino)-3,5-

dinitrobenzenesulfonamide), and thiazopyr (methyl 2-(difluoromethyl)-5-(4,5-dihydro-2-

thiazolyl)-4-(2-methylpropyl)-6- (trifluoromethyl)-3-pyridinecarboxylate) (Futch 2001).

Postemergence herbicides are glyphosate, paraquat, and sethoxydim (2-[1-

(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen- 1-one) (Futch

2001).

Preplant incorporated herbicides labeled for peppers in Florida are bensulide

(O,O-bis(1-methylethyl) S-[2-[(phenylsulfonyl)amino] ethyl]phosphorodithioate),

napropamid (N,N-diethyl-2-(1-naphthalenyloxy)propanamide), and trifluralin (Stall and

Gilreath 2005a). Preemergent herbicides applied are bensulide, clomozone (2-[(2-

chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone), which can be applied to all

peppers except banana peppers (Capsicum annuum L.), and paraquat (Stall and Gilreath

2005a). Halosulfuron (3-chloro-5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]

amino]sulfonyl]-1-methyl-1H-pyrazole-4-carboxylic acid) can be applied in the row

middle and sethoxydim can be applied postemergent (Stall and Gilreath 2005a).

Preplant incorporated herbicides labeled for tomatoes in Florida are napropamid

and trifluralin, while preemergent herbicides used are paraquat or pelargonic acid

(nonanoic acid) (Stall and Gilreath 2005b). Herbicides used postemergent are clethodim

((E,E)-(+)-2-[1-[[(3-chloro-2-propenyl)oxy]imino]propyl]-5-[2-(ethylthio)propyl]-3-

hydroxy-2-cyclohexen- -one), halosulfuron, metribuzin (4-amino-6-(1,1-dimethylethyl)-

3-(methylthio)-1,2,4-triazin-5(4H)-one), rimsulfuron (N-[[(4,6-dimethoxy-2-

pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide), and sethoxydim









(Stall and Gilreath 2005b). Halosulfuron and rimsulfuron are also applied to row middles

(Stall and Gilreath 2005b).

Glyphosate

Glyphosate, a phloem-mobile herbicide, moves in the symplast via source to sink

(McAllister and Haderlie 1985). The primary mode of action of glyphosate is to inhibit

5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; E.C. 2.5.1.19), which is the key

enzyme in the shikimate pathway. Inhibition of EPSPS prevents the production of

chorismate which is required for biosynthesis of the aromatic amino acids phenylalanine,

tyrosine, and tryptophan (Amrhein et al. 1980; Geiger and Bestman 1990; Hollander and

Amrhein 1980).

Dawson and Saghir sprayed glyphosate over the top of parasitized alfalfa, which

resulted in injury and death to the Cuscuta spp. but not to the alfalfa (Dawson and Saghir

1983). The highest rate of glyphosate (0.30 kg per ha) provided sufficient delay for the

parasitized alfalfa to recover prior to regrowth of Cuscuta spp. (Dawson and Saghir

1983). Even though all exposed shoots of Cuscuta spp. were killed by the glyphosate,

some haustoria were still embedded within the alfalfa stems and regenerated. All alfalfa

foliage in the control plots were destroyed by Cuscuta spp. (Dawson and Saghir 1983).

Fer found that 14C-labeled glyphosate was more concentrated in the apical part of

the Cuscuta spp. shoots, especially in the buds, than in the haustorial coil (Fer 1984).

Furthermore, at 4 days after treatment (DAT), Cuscuta spp. had absorbed 75% of the

glyphosate from the hosts' phloem. Fer concluded from these studies that phloem-mobile

herbicides should be able to selectively control Cuscuta spp. (Fer 1984).

Bewick et al. conducted studies with 14C-labeled glyphosate on carrot parasitized

with Cuscuta gronovii (Bewick et al. 1991). They determined that at 1 DAT Cuscuta









tissue had absorbed as much 14C-labeled glyphosate as any sink in the carrot. At 45

DAT, Cuscuta tissue contained most of the 14C-labeled glyphosate in regard to all other

physiological sinks, except the petiole of the treated carrot leaf (Bewick et al. 1991).

Ammonium Sulfate

Ammonium sulfate, (NH4)2SO4, is formed by reacting ammonia with sulfuric acid

to form a stable ammonium salt. This compound is soluble in water and insoluble in

liquid ammonia or alcohol. The toxicity of the ammonium ion enhances this compound's

capacity for weed control (Brian 1976). The plant cell rapidly absorbs the ammonia and

the cell sap, which is normally acidic, may become alkaline due to the effect of the

ammonia (Harvey 1911). Rapid cell death ensues from increased alkalinity and from the

toxicity of ammonia (Brian 1976). Excess concentrations of ammonium ions or ammonia

are toxic to plants, although the exact biochemical causes of toxicity are not understood

(Given 1979; Smith and Vandenn Born 1992, Young et al. 2003).

Several theories have been proposed as to how ammonium sulfate increases

herbicide absorption. Studies conducted with tomato fruit, which has a well-developed

cuticle, confirmed large increases in uptake when sethoxydim was applied with

ammonium sulfate (Wanamarta et al. 1993). The cuticle is a barrier to sethoxydim

penetration, but with the addition of ammonium sulfate the capability of the cuticle to

block herbicide absorption was reduced. Ammonium salts ofbentazon (3-(1-

methylethyl)-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide) formed from the

addition of ammonium sulfate were more readily absorbed and demonstrated increased

transcuticular movement compared with calcium, magnesium, or sodium salts of

bentazon (Wanamarta et al. 1993). Ammonium sulfate is related to increased permeation

of the herbicide through the plasma membrane (Young et al. 2003). Several researchers









have hypothesized that the acidification of the spray solution by the addition of

ammonium sulfate allows a greater proportion of weak-acid herbicides to remain in their

nondissociated form, which allows the herbicide to diffuse across the plasma membrane

more easily (Gronwald et al. 1993; Smith and Vanden Born 1992). When the herbicide

enters the cell, the higher pH of the cytoplasm causes dissociation of weak-acid

herbicides, thus efficiently trapping the herbicide inside the cell (Young et al. 2003).

Ammonium sulfate is also used as a surfactant to increase activity in many

herbicides, including glyphosate (Nalewaja and Matysiak 1993; O'Sullivan et al. 1981;

Turner and Loader 1975). Hard water contains polyvalent cations that antagonize the

activity of glyphosate (Nalewaja and Matysiak 1991, 1993). Thelen et al. (1995)

determined that the glyphosate molecule reacts with Ca+ and other cations to form a

less-readily absorbed glyphosate-Ca salt that decreases herbicidal activity. When

ammonium sulfate is added into the tank mix, the ammonium ion binds with the

glyphosate molecule, preventing the formation of glyphosate-Ca salt, which in turn

increases herbicidal absorption (Gronwald et al. 1993; Pratt et al. 2003). In addition,

ammonium sulfate alters spray droplet morphology by delaying or preventing the

crystallization of glyphosate on the leaf surface, thereby allowing more time for

glyphosate to penetrate the cuticle (MacIsaac et al. 1991).

Alternaria destruens Simmons, sp. nov.

The genus Alternaria Nees was first described in 1816 with the type species A.

tenuis Nees, which was later redescribed as A. alternate (Fr.:Fr.) Keissl. (Simmons

1967). Taxonomically, Alternaria falls under mitosporic fungi anamorphicc fungi) and

has no known sexual stage (Noyd 2000). The majority ofAlternaria spp. are

saprophytic, but some species are pathogenic and produce a range of plant diseases









throughout the world. Depending on the pathogen, symptoms typically appear as blights

and leaf spots, but damping-off of seedlings, and fruit, stem or tuber rots can also occur

(Agrios 1997).

Simmons (1992) has described over 100 species of Alternaria worldwide.

However, the number of described species fluctuates due to other taxonomists merging

several existing species ofAlternaria into one species or by taking one species and

dividing it into several different species (Rotem 1994). These differences are most likely

due to errors of taxonomic placement due to variability of morphological characteristics

under a variety of environmental conditions (Thomma 2003).

The most important conidial characteristic in identifying a species is spore

conidiumm) dimension, including that of the beak (Rotem 1994). Spore length varies

significantly between species, while spore width is similar. However, spore length,

including the beak, can vary within the same species causing an overlap. This is due to a

number of factors, but most commonly by the type of substrate. Therefore, most

taxonomists give measured dimensions in ranges rather than as means and provide the

substrate on which the fungus was grown (Rotem 1994).

In 1984, Bewick et al. isolated an Alternaria spp. from a diseased C. gronovii

plant growing in an uncultivated marsh in Wisconsin (Bewick et al. 1986). In subsequent

field studies, this pathogen was shown to provide 92% control of C. gronovii (Bewick et

al. 1987). No disease was observed on, nor was the pathogen recovered from any of the

host plants of dodder. In 1990, this pathogen was patented as a bioherbicide for

suppression of Cuscuta spp. (Bewick et al. 2000). In 1998, Simmons identified this

isolate as belonging to Alternaria destruens. Simmons described A. destruens as a small-








spored (25-40 (- 46) x 10-13 gm), brevicatenate (each conidiophore initially produces a
single unbranched chain of 4-8 conidia, which may increase to 10-12 conidia with age)
species in which interspersed secondary condiophores range from extremely short to
exceedingly long, (3-) 20-80 mrn, in a single chain (Simmons 1998) (Figure 1-4)


10
10 IJM


Figure 1-4. Alternaria destruens spores
Biological Control
The use of herbicides to control and manage weed populations may not be
conducive to all situations. Therefore, other means of control are necessary, such as
biological control. Biological control is grouped into three strategies: the classical
approach, the augmentative approach, and the inundative approach (Rosskopf et al.
1999). The classical approach or inoculative method involves applying small doses of


57 pm


10 pm
10 Pm


10 pm


@,a ""A









inoculum over a large weed population to produce a spreading epidemic (Charudattan

1988). This approach is beneficial when exotic plants become problematic due to the

absence of natural enemies and herbicide usage is not cost effective (Rosskopf et al.

1999). In the augmentative approach, a small amount of inoculum is released to initiate

an epidemic (Charudattan 1988). In the inundative or bioherbicide approach, inoculum is

applied in massive doses to the weed population to stimulate epidemic conditions (Harley

and Forno 1992; Scheepens et al. 2001). Bioherbicides are based on fungi, bacteria, or

viruses that are industrially developed and registered through the U.S. Environmental

Protection Agency to manage or reduce weed populations. The mycoherbicide (a fungus

used as a herbicide) approach can be successful when substantial quantities of inoculum

are used to help compensate for natural restrictions to disease development (Bewick et al.

1987; Charudattan 1988). Through manipulation, some fungi that normally cause

sporadic or endemic disease levels on their hosts can be developed into effective

bioherbicides. (Charudattan 1988; Rosskopf et al. 1999).

Research Objective

Current management methods are insufficient for controlling dodder. Therefore,

the main objectives of this research were (1) to determine the host range of A. destruens,

a bioherbicide agent, in greenhouse trials using plants of importance to Florida, the U.S.

Virgin Islands, and the Caribbean in general; (2) to evaluate the effect ofA. destruens on

C. pentagon in the field; (3) to determine whether A. destruens would spread from a

point source to surrounding uninfected C. pentagon plants; (4) to use internal

transcribed spacing regions to differentiate unknown Alternaria isolates from our

reference isolate of A. destruens; (5) to determine the capability of A. destruens spores to

be dispersed through a simulated wind tunnel; (6) to evaluate the effects of various






19


concentrations of glyphosate (Roundup Pro) and ammonium sulfate on the growth of A.

destruens on Citrus spp.; and (7) develop an integrated control method for C. pentagon

using glyphosate, ammonium sulfate, and the biological control agent A. destruens.














CHAPTER 2
EVALUATION OF THE HOST RANGE OF ALTERNARIA DESTRUENS
SMOLDERSTM)

Simmons (1992) has described over 100 species of the genus Alternaria Nees

worldwide. While the majority of Alternaria species are saprophytic, many are

pathogenic and can cause a multitude of plant diseases over a wide host range. Some

species of Alternaria have a restricted host range, such as, A. solani Sorauer which causes

early blight only on potato (Solanum tuberosum L.) and tomato (Lycopersicon

esculentum Mill.) and Alternaria leaf spot on Jerusalem cherry (Solanum pseudocapsicum

L.) (APSnet 2006). While other species of Alternaria have a wide host range such as A.

alternate that causes Alternaria blight on chickpea (Cicer arietinum L.), lentil (Lens

culinaris Medik.), pea (Pisum sativum L.), and pigeonpea (Cajanus cajan (L.) Millsp.);

Alternaria fruit spot on papaya (Caricapapaya L.); Alternaria leaf spot on African daisy

(Gerberajamesonii H. Bolus ex J. D. Hook), almond (Prunus dulcis (Mill.) Webb),

asparagus (Asparagus officinalis L.), beet (Beta vulgaris L.), garden dalia (Dahlia spp.),

mango (Mangifera indica L.), and rose (Rosa spp.); Alternaria rot on apple (Malus x

domestic Borkh.) and grape (Vitis spp.); Alternaria spot and fruit rot on apricot (Prunus

armeniaca L.); Alternaria spot and veinal necrosis on peanut (Arachis hypogaea L.); and

seed mold on red clover (Trifolium pratense L.) (APSnet 2006).

Alternaria spp. have also been developed as biological control agents or

bioherbicides, which are based on fungi, bacteria, or viruses that are industrially

developed and registered through the United States Environmental Protection Agency to









manage or reduce weed populations. The mycoherbicide (fungi used as herbicides)

approach can be successful when substantial quantities of inoculum are used to help

compensate for natural restrictions to disease development (Bewick et al. 1987). By

using certain adjuvants, such as humectants, uv-protectants, and other materials that can

alter the microclimate on leaf surfaces some fungi that normally cause sporadic or

endemic disease levels on their hosts can be used efficiently as weed-control agents

(Charudattan 1988; Rosskopf et al. 1999).

Alternaria eichhorniae Nag Raj and Ponnappa is a pathogen of waterhyacinth

(Eichhornia crassipes (Mart.) Solms), which is one of the world's worst aquatic weeds

(Holm et al. 1977). This weed creates serious problems throughout much of the tropical

and subtropical regions of the world where it affects water flow, water quality,

navigation, and human health and wellbeing. Shabana et al. reported that A. eichhorniae

is a safe and effective bioherbicide for control of waterhyacinth (Shabana et al. 2001;

Shabana et al. 1997). Another potential mycoherbicide that is being evaluated for

waterhyacinth is A. alternate (Fr.:Fr.) Keissl. (Babu et al. 2004). This pathogen was

isolated from diseased waterhyacinth; but in India, where this weed also causes severe

problems (Babu et al. 2003). Another bioherbicide, A. cassiae Jurair and Khan, was

isolated from diseased sicklepod (Cassia obtusifolia L.) and was found to have a

restricted host range of coffee senna (Cassia occidentalis L.), showy crotalaria

(Crotalaria spectabilis Roth) and sicklepod (Walker 1982). This bioherbicide effectively

reduced sicklepod plants 95-100% in field trials (Walker and Riley 1982).

In 1984, Bewick et al. isolated an Alternaria spp. from a diseased Cuscuta

gronovii Willd. ex J.A. Schultes (swamp dodder) plant growing in an uncultivated marsh









in Wisconsin (Bewick et al. 1986). Subsequent field studies with this pathogen resulted

in 92% control of C. gronovii (Bewick et al. 1987). No disease was observed or

recovered on any of the host plants that the Cuscuta had parasitized. Thus, the potential

ofAlternaria destruens as a biological control agent for C. gronovii was established.

In 1990, the Alternaria sp. was patented as a bioherbicide for suppression of

Cuscuta spp. (Bewick et al. 2000). In 1998, Simmons identified this isolate as belonging

to Alternaria destruens. Simmons described A. destruens as a small-spored (25-40 (- 46)

x 10-13 rm), brevicatenate (each conidiophore initially produces a single unbranched

chain of 4-8 conidia, which may increase to 10-12 conidia with age) species in which

interspersed secondary condiophores range from extremely short to exceedingly long,

(3-) 20-80 rim, in a single chain (Simmons 1998).

Alternaria destruens has been shown to have a restricted host range, but more

information is necessary, especially in regard to crop plants grown in Florida and the U.S.

Virgin Islands where it is proposed to be used to control Cuscuta spp. Host range or

host-specificity testing is conducted to determine if a plant pathogen, which is being

considered for a biological control agent, can develop under optimal conditions for

disease development on a group of plants closely related to the target weed species

(Harley and Forno 1992; Rosskopf et al. 1999). This group of plants should also include

closely related crop plants, native, and weed plant species (Rosskopf et al. 1999).

Alternaria destruens has been field-tested in crops such as alfalfa, carrot, celery,

cranberry, potato, and spearmint (Bewick et al. 1987), but further testing is needed to

establish the effectiveness of this biocontrol agent as a control for Cuscuta in diverse

crops of other geographical areas. Therefore, the objective of this study was to determine









the host range of A. destruens in greenhouse trials using plants of importance to Florida,

the U.S. Virgin Islands, and the Caribbean in general.

Materials and Methods

Test Plants

A total of 30 plants of economic and commercial importance comprising 19

species and 8 families (Asteraceae, Convolvulaceae, Fabaceae, Malvaceae, Meliaceae,

Poaceae, Rutaceae, and Solanaceae) were chosen from Florida and the U. S. Virgin

Islands. All studies were conducted in greenhouses at the University of Florida in

Gainesville from the spring of 2002 through the fall of 2002.

All plants except Citrus spp. ('Smooth Flat Seville' 7 months old), Hibiscus

rose-sinensis ('Brilliant Red' Hibiscus approximately 2 to 4 years old), Ipomoea

batatas ('Puerto Rican Bunch' Sweet Potato seedlings), and Swietenia mahagoni

(Mahogany 3 years old) were either seeded directly or seeded in plastic trays (53 cm x

27 cm x 6 cm) and then transplanted into 9.5 cm x 9.5 cm plastic pots. Each pot

contained one or two plants depending on the size and growth habit of the species. All

seeds were grown in a commercial potting medium (Metro-Mix 300; Sun Gro

Horticulture Canada Ltd., Seba Beach, AB Canada) and fertilized with Rainbow

Premium Plant Nutrients (Royster Clark, Inc., Mulberry, FL). Plants were then placed in

a greenhouse (35/29 + 5C day/night temperature) for 4 weeks and rated weekly. Light

intensity was measured in the greenhouse with a quantum/radiometer/photometer (LI-

COR Model LI-185B, Lincoln, NE) and ranged from 13,500-15,000 lux.

Inoculation and Treatments

Three treatments consisting of an untreated control, oil (PCC 588; United Agri

Products, Greeley, CO), and A. destruens + oil were applied to each plant species. Each









study was conducted in a randomized complete block design with 4 replications and was

repeated once.

A container of A. destruens spores (SmolderTM WP) was supplied by Sylvan

Bioproducts, Inc., Kittanning, PA. Spores (1.28 g) were stirred with 55.5 ml of water for

15 min. Oil (PCC 588), at 7.5%, was added to the spore mixture and mixed for another

15 min. Spore counts were determined to be 1.8 x 105 spores per ml in the spray mixture.

When using oil alone, the same mixing procedure was followed with oil added to 55.5 ml

of water. All treatments were used within 1 h of mixing.

All plants were inoculated with a hand-held sprayer at 100 ml per m2 until the

excess liquid ran off the foliage. After inoculation, plants were placed at 260C for 16 h in

a dew chamber (100% relative humidity) and then moved to the greenhouse. Plants were

rated weekly for 4 weeks.

Disease Rating

Symptoms were assessed on a scale of 1 to 10, with 1 being no symptoms and 10

being a dead plant. Plants that were rated at 1 to 2 were considered immune (none or few

leaf spots observed; slight stunting), plants rated at 3 to 6 resistant (hypersensitive

response (HR); 25% of the leaf covered in spots; stunting), and plants rated at 7 to 10

susceptible (severe stunting; blighted; plant death). Plants exhibiting HR developed

small necrotic spots or lesions on the leaves. These spots initially would be small, black,

circular spots that slowly developed into larger irregular lesions. The host reactions were

determined by comparing the A. destruens + oil treatment reaction with oil alone and the

untreated control.









Results and Discussion

Fourteen plants in seven families (Convolvulaceae, Fabaceae, Malvaceae,

Meliaceae, Poaceae, Rutaceae, and Solanaceae) were found to be immune to A.

destruens + oil (Table 2-1). These species were Capsicum annuum ('Hungarian Hot

Wax' Pepper), Capsicum frutescens ('Tabasco' Pepper), Citrus sp. ('Smooth Flat

Seville'), Crotalariajuncea ('Tropic Sun' Sunn hemp), Hibiscus rose-sinensis ('Brilliant

Red' Hibiscus), Ipomoea batatas ('Puerto Rican Bunch' Sweet Potato), I. nil ('Scarlet

O'Hara' Momingglory), L tricolor ('Heavenly Blue' Morningglory), x imperialism

('Rose Silk' Morningglory), Leucaena collinsii (Chalip), L. diversifolia (Wild Tamarind),

L. retusa (Littleleaf Leadtree), Swietenia mahagoni (Mahogany), and Zea mays ('Florida

Stay Sweet' Corn).

Sixteen plants in five families (Asteraceae, Convolvulaceae, Fabaceae, Poaceae,

and Solanaceae) were found to be resistant (Table 2-1). These species and cultivars were

Lactuca sativa ('Bibb' Lettuce), L. sativa ('Black Seeded' Lettuce), L. sativa ('Iceberg'

Lettuce) L. sativa ('Green Ice' Lettuce), I. nil ('Minibar Rose' Morningglory), L.

shannonii, Sorghum bicolor var. A3TX398/TX398, Capsicum annuum ('California

Belle' Pepper), C. annuum ('Capistrano' Pepper), C. annuum ('Jalapeno M' Pepper),

Capsicum chinense ('Habanero Red' Pepper), C. chinense ('Scotch Bonnet' Pepper),

Lycopersicon esculentum ('Agriset 761' Tomato), L. esculentum ('Beefsteak' Tomato),

L. esculentum ('Florida 47' Tomato), and L. esculentum ('Florida 91' Tomato). No

plants were found to be susceptible.

Symptoms on pepper cultivars varied. Slight stunting from oil and A. destruens +

oil was observed on 'California Belle' in addition to an HR from A. destruens + oil.

Plants that developed an HR would develop small necrotic spots or lesions on the leaves.









Stunting and phytotoxicity from oil and stunting and initial HR from A. destruens + oil

were observed on 'Capistrano' and 'Jalapeno M'. 'Habanero' and 'Scotch Bonnet' each

exhibited stunting and initial phytotoxicity from oil and A. destruens + oil, which also

exhibited an initial HR (Figures 2-1 and 2-2). Only an initial HR from A. destruens + oil

was observed on 'Tabasco' and no symptoms were observed on the 'Hungarian Hot

Wax' peppers.

All tomato cultivars reacted with similar symptoms. An HR from A. destruens +

oil was observed in all cultivars, while only 'Florida 47' exhibited slight stunting from oil

and A. destruens + oil. All lettuce cultivars also exhibited an HR to A. destruens + oil in

the first study. However, when this study was repeated, only 'Iceberg' and 'Green Ice'

exhibited an initial HR to A. destruens + oil (Figure 2-3).

Momingglory symptoms also varied between cultivars. 'Minibar Rose' exhibited

a slight HR from A. destruens + oil. 'Heavenly Blue' and 'Rose Silk' exhibited stunting

from A. destruens + oil, while no symptoms were observed in 'Scarlet O'Hara'.

Foliar streaking from the oil was observed on the sweet corn, but disappeared as

the plants grew. In addition, slight stunting was caused by oil and A. destruens + oil.

Initial stunting was also observed on the sunn hemp from oil and A. destruens + oil.

Sorghum plants initially exhibited stunting and phytotoxicity from oil and an HR from A.

destruens + oil.

All Leucaena species exhibited initial phytotoxicity symptoms from oil and A.

destruens + oil. Only L. shannonii exhibited an HR to A. destruens + oil. Stunting was

also observed in all four species inoculated with oil and A. destruens + oil, although, L.

retusa only exhibited stunting from oil.









Oil, either alone or with A. destruens, caused foliar streaking, phytotoxicity, or

stunting (Table 2-1), on the majority of the plant cultivars, while A. destruens + oil

caused mostly HR. New growth on plants initially exhibiting an HR, stunting, or

phytotoxicity from either oil or A. destruens + oil subsequently grew healthy and without

symptoms. This level of phytotoxicity followed by normal growth would be acceptable

for most growers of fruits and vegetables, but not for those crops where the leaves are the

produce, i.e. lettuce. Therefore, A. destruens would need to be mixed with another

surfactant that produces less phytotoxic results, especially on the first set of leaves.

These results, in addition to studies conducted by Bewick et al. (1987), reveal that

A. destruens is not likely to pose a risk to nontarget plants. In addition, this demonstrates

that A. destruens has a restricted host range and is safe for use on crop plants in Florida,

the U.S. Virgin Islands, and the Caribbean in general.











Control Oil A. destruens + Oil


Figure 2-1. Capsicum chinense 'Habanero' pepper plants. Control plant is normal with
no visible damage, oil plant exhibited phytotoxicity, and Alternaria destruens
+ oil plant exhibited slight phytotoxicity and HR.


























A. destruens + Oil


Figure 2-2. Capsicum chinense 'Scotch Bonnet' pepper plants. Control plant is normal
with no visible damage, oil plant is slightly stunted, and Alternaria destruens
+ oil plant exhibits phytotoxicity and HR.


Figure 2-3. Lactuca sativa 'Green Ice' lettuce plants. Control and oil plants are normal
with no visible damage and Alternaria destruens + oil plant exhibited only an
HR.













Table 2-1. Reaction of test plants to Alternaria destruens applied as SMOLDERTM formulated with PCC588 (United
Agri Products, Greeley, CO).


Latin Name


Cultivar/Variety


Common Name


Lactuca sativa L.
L. sativa L.
L. sativa L.
L. sativa L.


Bibb
Black Seeded
Iceberg
Green Ice


Convolvulaceae


Ipomoea batatas L. (Lam)
I. nil (L.) Roth
I. nil (L.) Roth
I. tricolor L.
I. x imperialis (L.) Roth


Puerto Rican Bunch
Minibar Rose
Scarlet O'Hara
Heavenly Blue
Rose Silk


Sweet Potato
Momingglory
Momingglory
Momingglory
Mominaglorv


Crotalaria juncea L.
Leucaena collinsii Britton & Rose
L. diversifolia (Schltdl.) Benth.
L. retusa Benth.
L. shannonii J. D. Smith

Hibiscus rose-sinensis L.

Swietenia mahagoni (L.) Jacq.


Zea mays L.
Sorghum bicolor
Citrus sp.


Tropic Sun


Brilliant Red


Florida Stay
var. A3TX398/TX398
Smooth Flat Seville


Sunn Hemp
Chalip
Wild Tamarind
Littleleaf Leadtree


Hibiscus

Mahogany

Sweet Corn
Sorghum
Citrus


Family


Asteraceae


Overall
Host
Reaction"


Host
Reaction
to Oilb


Lettuce
Lettuce
Lettuce
Lettuce


Fabaceae


Malvaceae

Meliaceae

Poaceae

Rutaceae


ST
ST, PH
ST, PH
ST, PH
ST, PH


FS, ST
ST, PH
NR













Solanaceae Capsicum annuum L. California Belle Pepper R ST
C. annuum L. Capistrano Pepper R ST, PH
C. annuum L. Hungarian Hotwax Pepper I NR
C. annuum L. Jalapeno M Pepper R ST, PH
Capsicum chinense Jacq. Habanera Red Pepper R ST, PH
C. chinense Jacq. Scotch Bonnet Pepper R ST, PH
Capsicum frutescens L. Tabasco Pepper I PH
Lycopersicon esculentum L. Agriset 761 Tomato R NR
L. esculentum L. Beefsteak Tomato R NR
L. esculentum L. Florida 47 Tomato R ST
L. esculentum L. Florida 91 Tomato R NR
a I = Immune (plants that were rated at 0 to 2 (none or few leaf spots observed; slight stunting)); R= Resistant (plants
rated at 3 to 6 (hypersensitive response (HR); fourth of leaf covered in spots; stunting)); S = Susceptible (plants rated at
7 to 10 (severe stunting; blighted; plant death)).
b FS = Foliar streaking; NR = no reaction; PH = phytotoxicity; ST = stunting














CHAPTER 3
FIELD EFFICACY OF ALTERNARIA DESTRUENS (SMOLDERTM) AS A
BIOHERBICIDE OF CUSCUTA

The genus Alternaria Nees contains plant pathogens that have been recorded in

almost every country in the world (Rotem 1994). Some of these pathogens produce

diseases that can be quite devastating. One such pathogen, A. alternate (Fr.:Fr.) Keissl.,

causes Alternaria brown spot of tangerine (Citrus reticulata), tangerine hybrids, and

tangelos (C. reticulata x C. paradise) (Peever et al. 2004; Timmer et al. 2003; Timmer et

al. 2000). This pathogen was first reported on Emperor Mandarin in Australia in 1903

(Cobb 1903). Currently, Alternaria brown spot is found in Argentina, Brazil (Peres et al.

2003), Israel (Solel 1991), Peru (Marin et al. 2006), South Africa (Schutte et al. 1992),

Spain (Vicent 2000), Turkey (Canihos et al. 1997) and the U.S. (Whiteside 1976).

The Alternaria spp. disease cycle contains no teleomorph (Timmer 1999) and

conidia are produced primarily on mature or senescent leaves during high humidity or

when leaves are slightly wet (Timmer et al. 2003). Conidial release is triggered by

sudden changes in relative humidity or rainfall and the optimum temperature for infection

is 27C (Canihos et al. 1999; Timmer et al. 1998). Infection takes place on young fruit,

leaves, and twigs with symptoms appearing in 24 h or less (Timmer et al. 2003). Infected

fruit will form brown to black lesions which vary in size, infected twigs will die back,

and infected young shoots will form brown lesions 1 to 10 mm in diameter (Timmer et al.

2003). The fruit will either abscise or contain blemishes, which will greatly reduce yield

and marketability (Timmer et al. 2003).









An Alternaria species that has the potential to cause devastation on just one weed

was isolated from diseased Cuscuta gronovii Willd. ex J.A. Schultes plants growing in an

uncultivated marsh in Wisconsin (Bewick et al. 1986) and was identified as Alternaria

destruens Simmons, sp. nov. (Simmons 1998). This fungus disease or damage progress

was found to be highly pathogenic and in field studies it yielded 92% control of C.

gronovii (Bewick et al. 1987). In 1990, this pathogen was patented as a bioherbicide for

suppression of Cuscuta spp. (Bewick et al. 2000).

Cuscuta species are obligate parasites that predominate disturbed habitats, which

preadapts them to becoming a serious weed problem. There are approximately 150 to

170 Cuscuta species worldwide; with the majority occurring in North and South America

(Ashton and Santana 1976; Dawson et al. 1994). Cuscuta spp. parasitize mainly

dicotyledonous and a few monocotyledonous plants (Gaertner 1950; Dawson et al. 1994).

Reports of anecdotal evidence of rapid spread of A. destruens in the field have led

researchers to speculate about the dispersal capability of this pathogen (T. A. Bewick,

USDA-CSREES-NPS, Washington, DC, personal communication). Therefore, the

objectives of this study were (1) to evaluate the effect of A. destruens on C. pentagon in

the field and (2) to determine whether A. destruens would spread from a point source to

surrounding uninfected C. pentagon plants.

Materials and Methods

Ft. Pierce Field Study

Field trials were conducted at the Header Canal Alternative Cropping Systems

Research Farm in Ft. Pierce, FL from December 2003 through April 2004. 'Tropic Sun'

Sunn hemp (Crotalariajuncea L.) seeds were sown in early November 2003 directly into

the field. Seeds were obtained from Peaceful Valley Farm Supply, Grass Valley, CA. At









the time of inoculation, both the Sunn hemp and C. pentagon, the latter naturally present

in this field, were at the flowering stage. This species of Cuscuta flowers within days

after attachment to a host and had parasitized the Sunn hemp throughout the field.

The experiment was conducted as a randomized complete block design with eight

replications and was repeated once. A total of 32 plots (1 m2) were randomly distributed

throughout the 0.39-acre (Figure 3-1). Four treatments consisting of an untreated control,

oil (PCC 588; United Agri Products, Greeley, CO), A. destruens + oil, and A. destruens

were applied with 8 replications per treatment. A. destruens spores (Smolderm WP) were

supplied by Sylvan Bioproducts, Inc., Kittanning, PA. Spores (19.26 g) were stirred with

832.45 ml of water for 15 min. Spore counts were determined to be 1.8 x 107 spores per

ml in the spray mixture. Oil (PCC 588), at 7.5%, was added to the spore mixture and

mixed for another 15 min. When using oil or A. destruens alone, the same mixing

procedure was followed as previously stated plus 832.45 ml of water. The plots were

inoculated with a hand-held sprayer at 100 ml per m2 and the treatments were applied

within 1 h of mixing the spores. The field trial was repeated in an adjacent field and was

initiated 3 weeks later.

Gainesville Field Study

A simulated field study was conducted outdoors at the University of Florida in

Gainesville from July 2005 through August 2005 (Figure 3-2). Five-month old Citrus

spp. ('Smooth Flat Seville'; Phillip Rucks Citrus Nursery, Frostproof, FL) in 1-gallon

pots were parasitized with C. pentagon. At the time of treatment, the Cuscuta was

approximately 1-month old and flowering.









Two treatments consisting of an untreated control and A. destruens + oil (PCC

588; United Agri Products, Greeley, CO) were applied. Spores (1.28 g) were stirred with

55.5 ml of water for 15 min before oil (PCC 588) at 7.5% was added, and the spore

mixture was mixed for another 15 min. The 10 treated plants were positioned in a circle

then inoculated, and then the remaining plants were placed into two outer circles (Figure

3-2). This was done to decrease chances of initial contamination and spread of A.

destruens to the control plants. Plants were placed in saucers, which were watered

regularly.

The experimental design consisted of three concentric circles with 55 pots (Figure

3-3). The distances from the center pot were as follows: the first circle was 0.91 m; the

second circle was 2.13 m; and the third circle was 3.35 m (Figure 3-4). Each pot was

spaced 0.63 m in the first circle, 0.79 m in the second circle, and 0.76 m in the third

circle. The first circle and center pot, which contained 10 plants, were the treated area,

while the second and third circles had the untreated sentinel plants. This study was

conducted once.

Disease Rating

The Ft. Pierce trials were observed and rated weekly, while the Gainesville study

was rated every 5 days for disease symptoms. Disease symptoms as well as phytotoxic

damage from the oil were expressed as necrotic spots or blight on dodder. Hence, there

was no need to use different rating scales or systems and both the disease and damage

were assessed on a scale of 1 to 10, with 1 being no symptoms and 10 being a dead plant.

Plants that were rated at 1 to 2 were considered immune (none or few leaf spots observed;

slight stunting), plants rated at 3 to 6 resistant (hypersensitive response (HR); 25% of the









leaf covered in spots; stunting), and plants rated at 7 to 10 susceptible (severe stunting;

blighted; plant death). Plants exhibiting HR developed small necrotic spots or lesions on

the leaves (Figure 3-5).

All data were subjected to analysis of variance (ANOVA) and means were

separated using Fisher's protected least significant difference (LSD) at P= 0.05 in SAS

(SAS 1999). In addition, all data were analyzed using Area Under the Disease or

Damage Progress Curve (AUDPC), which is used to quantify the progress of disease or

damage over time by integrating all factors of an epidemic, such as, environment, host,

and pathogen effects (Campbell and Madden 1991). AUDPC values for each trial were

calculated using the formula I [(y, + y(,1+))/2]*(t(,+l +1- t,), where i = 1, 2, 3, ...n-1,

where y, is the disease or damage severity at the ith evaluation, and t, is time at the ith

evaluation (Shaner and Finney 1977). AUDPC values were subjected to analysis of

variance (ANOVA) and means separated by Tukey's Studentized Range (HSD) test at

P=0.05 in SAS (SAS 1999).


































Figure 3-1. Header Canal Alternative Cropping Systems Research Farm in Ft. Pierce, FL. Each of the 32 m2 plots were indicated
with 2 sticks with red ribbon. Plots were changed for Trial II.


611-



































4-






Figure 3-2. Simulated field study at the University of Florida in Gainesville. Fifty-five pots of Citrus spp. parasitized with Cuscuta
pentagon were placed in three concentric circles.





























Figure 3-3. Experimental design of simulated field study containing 55 pots of Citrus
spp. parasitized with Cuscutapentagona. The black pots in the middle were
treated with A. destruens + oil (PCC 588) and the white pots were untreated
controls.


Figure 3-4. Distances used in simulated field study. White pots were 0.91 m from the
center pot and spaced 0.63 m apart. Grey pots were 2.13 m from the center
pot and spaced 0.79 m apart. Black pots were 3.35 m from the center pot and
spaced 0.76 m apart.




































Figure 3-5. Citrus spp. plants parasitized by Cuscutapentagona, the latter displaying
symptoms of treatments with Alternaria destruens + oil and an untreated
control. 0 = no symptoms; 1 = 1-10% (tip necrosis; stems starting to wilt and
become necrotic; 2 = 11-35% (slightly more stem necrosis; flowers starting to
senesce), 3 = 36-65% (over half of the stems are dead or dying; clusters of
flowers senescing); 4 = 66-90% (the majority of the stems and flowers are
dead or dying; some healthy flowers and stems may still be present), and 5 =
91-100% (plant death).

Results and Discussion

Ft. Pierce Field Study

Symptoms of necrosis were observed on C. pentagon one week after inoculation

(WAI) in both trials. Due to significant differences between trials (P=0.0019), data were

analyzed separately. Within each trial, the effects of treatment for trial I (P=0.0001) and


trial II (P=0.0124), and time (P=0.0001) were significant.









Trial I had significant treatment differences during the third (P=0.0217) and

fourth (P=0.0061) WAI where A. destruens (Fungus) and A. destruens + oil (Fungus +

oil) and oil and the untreated control were not significantly different from each other.

Ratings of disease severity on C. pentagon from A. destruens and A. destruens + oil

were 3.8 and 4.2, respectively, while the phototoxic damage rating or natural senescence,

respectively, from the oil and the untreated control were 2.1. By the fifth WAI, the

untreated control was not significantly different from the fungal treatments. At 6 WAI

(P=0.0767), there were no differences between any treatments and disease severity

ratings ranged from 3.9 to 4.7 (Figure 3-6).

Trial II had no significant differences among treatments except for A. destruens at

6 WAI (P=0.056) where the disease severity of C. pentagon reached 4.4 (Figure 3-7).

At 7 WAI, the level of disease incidence had reached an average of 4.1 in all treatments.

Regrowth of C. pentagon at 8 WAI was apparent in eight plots, which was almost half

as much as in trial I, where regrowth was evident in 15 plots.

AUDPC values for trial I indicated that there were no significant differences

between A. destruens + oil (186.38) and A. destruens (179.38) (Table 3-1). Nor were

there significant differences between A. destruens and the untreated control (143.06)

(Table 3-1). AUDPC values for trial II had no significant differences among the

treatments (Table 3-2).

Gainesville Field Study

Symptoms of disease on C. pentagon were observed 5 days after inoculation

(DAI) on all treated plants. Likewise, by 5 DAI untreated controls also started to exhibit

signs of disease. When comparing all three circles together, DAI and distance were both









significant (P=0.0001). Therefore, the circles were compared with one another to

determine if there were any differences among them. DAI was significant (P=0.0001) in

circles I (0.91 m) and III (3.15 m) (Figure 3-8); while circles I (0.91 m) and II (2.13 m)

had a DAI and distance interaction (P=0.0214) (Figure 3-9). In circles II (2.13 m) and III

(3.15 m), both DAI and distance were both significant (P=0.0001) (Figure 3-10).

There were no significant differences between 5, 10, and 15 DAI in any circle

comparison, nor was there a difference between 25 and 30 DAI between circles I and II.

Disease progressed linearly over time (circles I and III combined) and by 30 DAI the

disease severity rating was a 4.3 (Figure 3-8). Disease severity increased proportionally

over time in circles I and II, with circle I having the higher disease severity, but at 30

DAI circle II surpassed circle I, which were the treated plants (Figure 3-9). Pots placed

in the outermost circle, which were 3.35 m from the center, had higher disease ratings

than those closer at 2.13 m (Figure 3-10).

All Cuscuta plants in the Gainesville study were not in contact, at any time, with

any of the surrounding pots, which decreased the probability of the Cuscuta spreading the

A. destruens by contact. This was not possible at the Ft. Pierce field location due to the

nature of the study, but untreated plots were randomly placed from treated plots. In

previous greenhouse studies (Chapter 6) the Cuscuta and pots were separated as in the

Gainesville study, but all of the untreated controls still died after an initial lag period

when they were healthy.

One explanation for this occurrence is that there could be volatile signaling

between Cuscuta plants. If this theory holds true, then as the inoculated Cuscuta plants

in the greenhouse began to senesce, they would release senescence-inducing volatiles into









the air, which would be transmitted to the Cuscuta on the untreated plants. This in effect

would let the untreated Cuscuta recognize that senescence signal and they would start to

die.

A second explanation is that the A. destruens sporulates abundantly and as a

result, spreading rapidly around the greenhouse to the untreated controls. This theory is

more plausible in light of data presented herein. It is evident that disease ratings in the

untreated controls, in both the Ft. Pierce and the Gainesville studies, can be attributed to

the movement ofA. destruens spores from treated to untreated areas.

The main dispersal method for distribution ofAlternaria spores is through

airborne movement and to a lesser extent through splash dispersal (Rotem 1994). The

large size of Alternaria spores enables them to float in the air and therefore disperse from

their site of production (Gregory 1973). Alternaria conidia are firmly attached to

conidiophores and have to be detached by relatively strong winds (Rotem 1994). Studies

conducted on A. dauci estimated that wind velocities above 2-3 m s-' (approximately 7-11

km/h) were required to release large numbers of conidia (Strandberg 1977). In addition,

wind speeds do not have to be steady and brief gusts of otherwise moderate winds can

facilitate spore release (Aylor 1990).

Daily average wind speeds during the first Ft. Pierce trial ranged from 1.34 m s-1

to 4.02 m s-1 (Table B-1) (FAWN 2006), which would be sufficient to have spread A.

destruens spores to untreated areas in the field. In addition, there were three days where

recorded wind speeds ranged from 4.47 m s-1 to 5.36 m s-1. Daily average wind speeds

for the second trial at Ft. Pierce were even higher than the first trial and ranged from 1.79

m s-1 to 4.47 m s-1 (Table B-2) (FAWN 2006).









These results suggest the probability that A. destruens was already infecting C.

pentagon in the field prior to implementation of the second trial because there were no

significant differences between treatments and this species ofAlternaria has not been

found in the state of Florida (Farr et al. 2006). However, no symptoms were present at

the time of inoculation during the second trial and isolations were not made at that time to

confirm infection. Although, it is known that members of the genus Alternaria

frequently cause quiescent infections in which the fungus enters the tissue where it

remains dormant until environmental conditions favor infection (Thomma 2003).

Therefore, this could possibly indicate that the fungus produces secondary inoculum.

The Gainesville study daily average wind speeds ranged only from 0.89 m s-1 to

1.79 m s-1 (Table B-3) and no reported wind gusts (FAWN 2006). During this period,

there were two storms with heavy rain and one thunderstorm with winds reported at

25.72 m s-1 (NCDC 2006). The rain and winds from these storms would be adequate to

disperse A. destruens spores to untreated plants and infect the Cuscuta. This could also

explain the higher disease severity on the pots located furthest away from the treatment

site. Unfortunately, the design of the Gainesville study was not suitable to gather wind

direction and only one trial was completed.

In conclusion, no differences were observed between A. destruens or A. destruens

+ oil in either trial. Therefore, oil may not be needed when applying A. destruens. In

addition, it appears as though A. destruens spores are capable of rapid dispersal

throughout the field to untreated control areas, although more research is needed is this

area. This capacity may enhance this bioherbicide's efficacy and potential for use, but

may also present some limitations. Studies conducted using a simulated wind tunnel






44


further support my conclusion on the spore dispersal of A. destruens (Chapter 5). Finally,

these results indicate that Alternaria destruens can be used as a stand alone treatment, but

would be more effective if used in conjunction as an integrated control method for

Cuscuta spp.










-- Untreated Oil Fungus + Oil Fungus


0 1 2 3 4 5 6 7 8
WAI


Figure 3-6. Ft. Pierce field trial I data. Disease severity from Alternaria destruens, with
and without oil (PCC 588), oil only, and an untreated control on Cuscuta
pentagon over weeks after inoculation (WAI).


Table 3-1. Means of area under the disease progress curve (AUDPC) for treatments on
Cuscutapentagona from Ft. Pierce field trial I.

Treatment Mean AUDPC"
A. destruens + Oil 186.38 a

A. destruens 179.38 ab

Untreated Control 143.06 bc

Oil 128.63 c

a Means with the same letter are not significantly different according
to Tukey's Studentized Range (HSD) test at P=0.05.










-- Untreated Oil Fungus + Oil -- Fungus


0 1 2 3 4 5 6 7 8
WAI


Figure 3-7. Ft. Pierce field trial II data. Disease severity from Alternaria destruens, with
and without oil (PCC 588), oil only, and an untreated control on Cuscuta
pentagon over weeks after inoculation (WAI).


Table 3-2. Means of area under the disease progress curve (AUDPC) for treatments on
Cuscutapentagona from Ft. Pierce field trial II.


Treatment


Mean AUDPCa


A. destruens 159.69 a

Oil 133.44 a

A. destruens + Oil 128.63 a

Untreated Control 128.19 a

a Means with the same letter are not significantly different according
to Tukey's Studentized Range (HSD) test at P=0.05.







47



5



4





ci
1










y = 0.694x 0.7143 R2 0.
0
0 5 10 15 20 25 30

DAI


Figure 3-8. Disease severity over time (days after inoculation; DAI) on Cuscuta
pentagon treated with Alternaria destruens + oil (PCC 588) in Circle I
(0.91 m) and untreated sentinel C. pentagon in Circle III (3.15 m).







48


- 0.91 m -A- 2.13 m


or


N


0 5 10 15 20 25 30
DAI


Figure 3-9. Disease severity over time (days after inoculation; DAI) on Cuscuta
pentagon treated with Alternaria destruens + oil (PCC 588) in Circle I
(0.91 m) and untreated sentinel C. pentagon in Circle II (2.13 m).






49


-A- 2.13 m -- 3.15 m


0 5 10 15 20 25 30
DAI


Figure 3-10. Disease severity over time (days after inoculation; DAI) on untreated
sentinel Cuscutapentagona in Circles II (2.13 m) and III (3.15 m).














CHAPTER 4
MOLECULAR PHYLOGENIC CHARACTERIZATION TO DIFFERENTIATE
ALTERNARIA DESTRUENS SIMMONS, SP. NOV. FROM VARIOUS ALTERNARIA
SPECIES BASED ON INTERNAL TRANSCRIBED SPACER REGION

The genus Alternaria Nees was first described in 1816 with the type species A.

tenuis Nees, which was later redescribed as A. alternate (Fr.:Fr.) Keissl. (Simmons

1967). The majority ofAlternaria spp. are saprophytic, but some species are pathogenic

and produce a range of plant diseases throughout the world on numerous economically

important plants. Alternaria leaf blight of carrot (Daucus carota) is caused by A. dauci

(Kiuhn) Groves & Skolko. This pathogen creates small irregular shaped lesions, which

are dark brown to black in color, on the margins and tips of carrot leaflets (Gugino et al.

2004). The causal agent of Alternaria late blight of pistachio (Pistacia vera L.) is A.

alternate that infects both foliage and fruit. On foliage, large necrotic lesions, that are

black in the center and surrounded by a chlorotic halo, eventually occupy the entire leaf

(Pryor and Michailides 2002). Infection on the fruit is characterized by small necrotic

spots on the hull of immature nuts that are surrounded by a red halo (Pryor and

Michailides 2002). In citrus, Alternaria brown spot, caused by A. alternate, is a serious

disease of tangerines (Citrus reticulata), tangerine hybrids, and tangelos (C. reticulata x

C. paradise) (Peever et al. 2004; Timmer et al. 2000). This pathogen attacks young

leaves, fruit, and twigs, causing small black necrotic spots (Timmer et al. 2000). Black

rot of citrus, caused by A. alternate, occurs postharvest and may appear in the field prior

to harvest. This disease develops most commonly on navel oranges (C. sinenesis (L.)

Osbeck) in the field, and on lemons (C. limon (L.) Burm.) and tangerines in storage









(Brown and McCornack 1972; Peever et al. 2005). Infection occurs through natural

openings or wounds in the stylar "blossom" end of the fruit, which is where brown to

blackish discoloration develops in diseased fruits (Peever et al. 2005).

In 1984, Bewick et al. isolated an Alternaria spp. from a diseased Cuscuta

gronovii plant growing in an uncultivated marsh in Wisconsin (Bewick et al. 1986). In

1998, Simmons described this isolate as belonging to Alternaria destruens Simmons, sp.

nov. Simmons described A. destruens as a small-spored (25-40 (- 46) x 10-13 pm),

brevicatenate (each conidiophore initially produces a single unbranched chain of 4-8

conidia, which may increase to 10-12 conidia with age) species in which interspersed

secondary condiophores range from extremely short to exceedingly long, (3-) 20-80 rim,

in a single chain (Simmons 1998) (Figure 1-4).

Simmons (1992) has described over 100 species of Alternaria worldwide.

However, the number of species described fluctuates due to other taxonomists merging

several existing species of Alternaria into one species or by taking one species and

dividing it into several different species (Rotem 1994). These differences are most likely

due to the variability of morphological characteristics under a variety of environmental

conditions (Thomma 2003).

Conidium morphology and patterns of conidium catenulation are the only means

available to morphologically differentiate between Alternaria species and to identify

Alternaria isolates (Simmons 1992). The most important conidial characteristic in

identifying a species is spore conidiumm) dimension, including that of the beak (Rotem

1994). Spore length varies significantly between species, while spore width is similar.

However, spore length, including the beak, can vary within the same species causing an









overlap. This is due to a number of factors, but most commonly by the type of substrate.

Therefore, most taxonomists give measured dimensions in ranges rather than as means

(Rotem 1994).

Analysis of ribosomal DNA (rDNA) sequences has become a frequent tool that

has been used to establish molecular phylogenetic relationships within many groups of

fungi (White et al. 1990). A review of the literature has revealed that characterization

and differentiation of small-spored Alternaria species uses a wide variety of molecular

techniques, such as, random amplification of polymorphic DNA (RAPD) fragment

patterns analysis (Roberts et al. 2000); restriction fragment length polymorphism (RFLP)

analysis (Sharma and Tewari 1998); and sequencing of rDNA internal transcribed spacer

(ITS) regions, which are frequently used for analysis of fungal taxa at or below the

species level (Chou and Wu 2002; Pryor and Gilbertson 2000).

While spore morphology is a valid tool for species description, a more reliable

method is needed for field diagnostic purposes. Based on past and current molecular

studies of small-spored Alternaria, the objective of this study was to use internal

transcribed spacing regions to differentiate known and unknown Alternaria isolates.

Materials and Methods

Sampling Material

Cuscutapentagona stems and flowers were collected from three previous studies

conducted at the University of Florida in Gainesville, FL. The first two studies were

conducted with plants grown in a greenhouse in the spring of 2005, while the third study

was conducted with field-grown plants in the summer of 2005. The Cuscuta collected

from the greenhouse studies were inoculated with seven treatments consisting of: an

untreated control; oil (PCC 588, United Agri Products, Greeley, CO) at 7.5%; ammonium









sulfate at 0.125% w/v in water; glyphosate (N-(phosphonomethyl)glycine) at 0.02 kg

(ae)/L of Roundup Pro; A. destruens (SmolderT WP, obtained from Sylvan Bioproducts,

Inc., Kittanning, PA); A. destruens + oil; and a mixture of A. destruens + oil, glyphosate,

and ammonium sulfate (the latter two at the preceding concentrations). The Cuscuta

collected from the field study were either treated with A. destruens + oil or an untreated

control.

Fungal Cultures

For morphological identification of the species and to obtain single-spore

cultures, the fungi were grown to produce spores in culture. To stimulate sporulation,

plant tissue from 47 randomly chosen samples (Table 4-1) was placed onto plates

containing water agar (15 g of agar [Fisher Chemical, Fairlawn, NJ] per liter of H20).

The plates were stored at 220C. Approximately 1 to 2 days later, Alternaria spores were

visible, which were then transferred to new water agar plates. This process was

continued until pure cultures were obtained. Single germinating colonies were then

transferred onto plates containing potato dextrose agar (PDA) (39 g of PDA [Difco,

Detroit, MI] per liter of H20). All plates were stored in the dark at 220C for

approximately 10 days.

In addition to the unknown 47 isolates, 4 known Alternaria spp., Alternaria

cassiae Jurair and Khan, A. eichhorniae Nag Raj and Ponnappa, A. macrospora

Zimmermann, and A. tenuis Nees (Table 4-2), were used for comparison. These isolates

were prepared in the same manner as previously stated. GenBank isolates used for

comparison are listed in Table 4-3.









DNA Extraction

Mycelium on the surface of the PDA plates was scraped and ground to a powder

with liquid nitrogen. DNA was then extracted from the mycelial powder using the

DNeasy Plant Mini Kit (QIAGEN, Valencia, CA).

PCR Amplification

The nuclear rDNA internal transcribed spacer (ITS) region, including ITS1 and

ITS2 and the 5.8S rDNA, was amplified with primer pairs ITS4 and ITS5 by polymerase

chain reaction (PCR) (White et al. 1990). The forward primer ITS5 was 5'-GGA AGT

AAA AGT CGT AAC AAG G-3' and the reverse primer was ITS4 5'-TCC TCC GCT

TAT TGA TAT GC-3' (Integrated DNA Technologies, Inc., Coralville, IN). Reactions

were carried out in volumes of 50 pl containing 33.7 pl of double distilled H20; 5 pl of

10x buffer (Promega, Madison, WI); 5 pl of 50 mM MgC12 (Promega, Madison, WI); 1 [l

of an equal mixture of 10 mM dNTPs (Promega, Madison, WI);1 Il of 10 |M of each

primer; 0.25 [l of 5 units per pl Taq DNA polymerase (Promega, Madison, WI); and 3 pl

template DNA.

A PTC-200 Peltier Thermal cycler (GMI, Inc., Ramsey, MN) was used for

amplification. Cycling conditions used an initial denaturation step of 3 min at 940C, with

30 cycles of 94C for 30 s, 50C for 30 s, and 72C for 45 s. The last cycle included an

incubation step at 720C for 10 min and then storage at 40C.

Bands were resolved by electrophoresis in a 1% agarose gel in IX TAE buffer

(0.004 Tris-acetate, 0.001 M EDTA, pH 8.0) stained with 1% ethidium bromide (100V

for 50 min). Ten pl of PCR product, 2 pl of loading buffer (Bio-Rad, Hercules, CA), and









8 .il of a 100 base-pair ladder (Fisher Scientific International, Inc., Hampton, NH) were

loaded into the gel, run and visualized under ultraviolet light.

PCR products were purified by using a QIAquick PCR purification Kit

(QIAGEN, Valencia, CA). DNA samples were analyzed by electrophoresis, as

previously stated, except that 10 .il of a X DNA/Hind III ladder (InvitrogenTM, Carlsbad,

CA) was used. The purified products were sent to the DNA Sequencing Core Laboratory

at the University of Florida and sequenced with a Perkin Elmer, Applied Biosystems

model 373A or 377 automated DNA sequencer.

Phylogenetic Analysis

DNA forward and reverse sequences were combined, trimmed to 507 bp, and any

incongruences were edited in Sequencher (version 4.5 Gene Codes Corporation, Ann

Arbor, MI). Alignment of the sequence files were conducted using ClustalX v. 1.83.1

(Thompson et al. 1997) and manual adjustments of sequence alignments were performed

using the data editor program of MacClade Phylogenetic Software (version 4.08; Sinauer

Associates, Inc., Sunderland, MA). Phylogenetic analyses were performed with

programs contained in PAUP Phylogenetic Software (version 4.0 8, Sinauer Associates,

Inc., Sunderland, MA). Alternaria cassiae, A. eichhorniae, A. macrospora, and A. tenuis

were designated as outgroups. Phylogenetic trees were constructed using distance

methods because heuristic searches for the parsimony analysis yielded too many trees.

Distance-based trees were generated with neighbor-joining using the Jukes-Cantor one-

parameter algorithm. The significance of the branches in the neighbor-joining trees was

tested with 10,000 bootstrap replicates to assess the statistical support of each clade.









Results and Discussion

Neighbor-joining analysis revealed that all 47 unknown isolates, which were

recovered from the greenhouse- and field-grown Cuscuta plants, and the reference A.

destruens isolate, were 100% identical (Figure 4-1). The first clade contained the defined

outgroups, A. cassiae and A. macrospora, which were supported with a bootstrap value of

100%. The second and third clades contained A. tenuis and A. eichhorniae with a

bootstrap value of 99% and 86%, respectively.

To determine if the sequence obtained was unique, a Basic Local Alignment

Search Tool (BLAST) search was performed using the previously obtained A. destruens

ITS sequence. This query revealed 100 other sequences that were similar, with 82 of

them being from Alternaria. These 82 Alternaria sequences were analyzed in PAUP

with the original A. destruens and 47 unknown Alternaria isolates. All 82 Alternaria

sequences from the BLAST search were 100% identical to the Alternaria in this study

(data not shown).

A GenBank search was then conducted for other Alternaria ITS sequences, to

determine if the A. destruens ITS sequence, that resulted from this study, was unique.

This search produced a listing of numerous, highly divergent Alternaria spp., of which 61

were randomly chosen and analyzed in PAUP with the original A. destruens ITS

sequence (Table 4-1). This analysis grouped the original A. destruens with eight different

Alternaria spp., which had the same ITS sequence, all in the same clade. This could be

due to misidentification in GENBANK because ITS sequences are typically species

specific. In addition, another A. destruens ITS sequence was found in the GENBANK

search, which grouped with this clade (Figure 4-2). This isolate of A. destruens also

contained the nuclear rDNA region, ITS1 and ITS2, and the 5.8S rDNA.









The other Alternaria species grouped into various clades, many of which

contained the same species, but some of which contained different species. This could

once again be due to either misidentification based on morphology or the different

species are clustering together based upon Alternaria species-groups identified by

Simmons (1992).

The results of this study confirmed that the unknown isolates grouped with A.

destruens. However, ITS is highly conserved within A. destruens, therefore it was not

possible to confirm that the isolates collected were the same as the isolate inoculated in

the greenhouse and field trials. This is in agreement with Serdani et al. (2002) who found

that a high degree of interspecies similarity exists within Alternaria in both ITS 1 and

ITS 2. Serdani et al. (2002) also indicated that ITS genes are not appropriate for

differentiating between closely related Alternaria species. In addition, Peever et al.

(2004) and Pryor and Michailides (2002) failed to differentiate small-spored Alternaria

species due to a lack of variation in nuclear ribosomal ITS sequences.

Currently, A. destruens has been reported to occur naturally only in Wisconsin

(Bewick et al. 1986). There are no reports of natural infestations ofA. destruens in the

state of Florida. In addition, A. destruens has never been recorded as a pathogen on C.

pentagon in Florida or in any other part of the world. Therefore, it is highly probable

that the isolates collected are the same as the one used in my research studies.

In conclusion, the data support concurrence with Simmons (E. G. Simmons,

Research Associate at Wabash College, Crawfordsville, Indiana, personal

communication) that ITS sequences are very conservative and extremely unreliable in

revealing any true differences or relationships between Alternaria species. Further






58


studies need to be conducted to amplify and sequence a more highly variable region of

the genome such as introns or microsatellites, which would identify more polymorphisms

between isolates within a species. In addition, isolation and sequencing of a naturally

occurring A. destruens from Florida would lend further credence to differentiation of

isolates.












Altemana cassie
A macrospora
A tennis
86 A eichomiae
11-403
1-106
1-505
SFS-2
SFS-10
11-501
SFS-37
SFS-7
1-304
SFS-30
1-105
1-507
SFS-8
11-306
11-104
1-502
1-202
1-402
1-101
1-102
SFS-45
SFS-54
SFS-47
11-107
SFS-52
11-303
11-301
1-205
11-406
11-103
11-203
SFS-3
SFS-9
SFS-39
11-407
1-406
11-205
1-206
SFS-32
SFS-5
1-403
11-302
SFS-6
A. destrens
11-505
11-305
SFS-4
SFS-1
0.005 substitutions/site


Figure 4-1. Rooted neighbor-joining dendrogram based on 507 bp from ITS 1, 5.8S, and
ITS2 sequences. Alternaria cassiae, A. eichhorniae, A. macrospora, and A.
tenuis were designated as outgroups. Bootstrap values generated from 10,000
replications are indicated. A bar indicates genetic distances.










Table 4-1. Sources of unknown isolates and treatments applied to
plots from which these isolates were recovered.a


Isolateb
1-101
1-102
1-105
1-106
1-202
1-205
1-206
1-304
1-402
1-403
1-406
1-502
1-505
I-507
11-103
II-104
11-107
11-203
11-205
11-301
11-302
11-303
11-305
11-306


Treatments
U
A.d.O
GLY
AS
A.d.O
GLY
MIX
A.d.
U
AS
0


Isolate
11-403
11-406
11-407
11-501
11-505
SFS-1
SFS-2
SFS-3
SFS-4
SFS-5
SFS-6
SFS-7
SFS-8
SFS-9
SFS-10
SFS-30
SFS-32
SFS-37
SFS-39
SFS-45
SFS-47
SFS-52
SFS-54


MIX
A.d.O
A.d.
AS
O
A.d.
MIX
O
U
A.d.O
AS
MIX
GLY


Treatment
A.d.
AS
GLY
A.d.O
U
A.d.O
A.d.O
A.d.O
A.d.O
A.d.O
A.d.O
A.d.O
A.d.O
A.d.O
A.d.O
U
U
U
U
U
U
U
U


a All isolates were used for DNA sequencing in this study.
b Isolates beginning with SFS are from the field study, while
all other isolates are from the greenhouse studies.
c Treatment designations are as follows: A.d. = Alternaria
destruens, A.d.O = A. destruens + oil, AS = ammonium
sulfate, GLY = glyphosate, MIX = A. destruens + oil,
glyphosate, and ammonium sulfate, O = oil, U = untreated
control.







61





Table 4-2. Sources ofAlternaria spp. used for DNA sequencing in this study.

Isolate Original Source
Alternaria cassiae" Soil sample; Marianna and Quincy, FL
A. destruens SmolderT WP; Sylvan Bioproducts, Inc., Kittanning, PA
A. eichhorniae" Dr. Yasser Shabana; Mansoura, Egypt
A. macrosporaa Dr. C. Doug Boyette; USDA-ARS, Stoneville, MS
A. tenuisa Late Dr. George E. Templeton; University of Arkansas,
Fayetteville, AR
a Stored in the culture collection of Dr. R. Charudattan, Plant Pathology
Department,University of Florida, Gainesville.













135
082 A. ziniae
896
689 A. macrospora
134
136 A. tagetica
420
469 A. macrospora

070 A- so/ani
683 A- a/i
705 I A ajr
576 A. solani
342 A. macrospora
130
468 A.dauci
467
040
639
038
637 A- panax
036
035
984
463
253 A. brassicae
086
714
074
073
014 A- radicina
133
704

0 .7 i1 nfectoria
060
goo
948 A. triticina
095
474
703 A. japonica
63g
343 |
713 A- he/ianthi
682 A. palaridui
568A. altemata
944A. gaixea
945A. mal
702A. palandmi
699 A. altenata
A. destrueas
712 A. teuissima
585 A. mali
587 A. longipe
582 A. wall
457 A. gaisen
581 I
571 A. loagipes
940 A. tenissima
0098 I
575 A. mali
681 A. destrueas
338 A. longipes


Figure 4-2. Rooted neighbor-joining dendrogram ofITS1, 5.8S, and ITS2 sequences.
Bootstrap values generated from 10,000 replications are indicated. Isolates
are specified by the last three numbers of the GenBank accession number
followed by the genus and species. Alternaria destruens without a number is
the isolate used in this study.










Table 4-3. Fungal Isolates and the GenBank accession numbers for sequences used in
phylogenic analyses.


Species
Alternaria alli isolate Alt34
A. alli isolate Alt35
A. alternate
A. alternate isolate EGS34-016
A. brassicae
A. brassicae AB 11
A. brassicae gz01-6-6
A. brassicae strain AB57
A. brassicae strain BMP 21-61-02
A. dauci isolate CHHADA
A. dauci strain BMP 21-31-09
A. dauci strain BMP 21-31-16
A. destruens isolate Altl
A. gaisen strain AGM
A. gaisen strain EGS90-0512
A. gaisen strain LCL
A. helianthi
A. helianthi strain B
A. infectoria isolate 4A
A. infectoria isolate 4B
A. japonica
A. japonica strain ATCC 13618
A. japonica strain ATCC 13618
A. longipes
A. longipes strain ALM
A. longipes strain ALS
A. macrospora
A. macrospora strain B
A. macrospora strain DGG Ams 1
A. mali strain AMM
A. mali strain EGS 38-029


GenBank Accession Number
DQ323705.1
DQ323683.1
DQ059568.1
DQ323699.1
AY154714.1
U05253.1
AF392984.1
AY372686.1
AF229463.1
AF267130.1
AF229467.1
AF229468.1
DQ323681.1
AF314581.1
AY762944.1
AF314574.1
AY154713.1
DQ156343.1
Y17066.1
Y17067.1
AY 154703.1
AY376639.1
AF229474.1
DQ156338.1
AF314571.1
AF314587.1
AY154689.1
DQ156342.1
AF229469.1
AF314582.1
AY762945.1










A. mali strain PGC
A. mali strain PGX
A. palandui isolate Alt 10
A. palandui isolate Alt9
A. panax isolate AP 2035
A. panax isolate AP 2036
A. panax isolate BC 2085
A. panax isolate BC 2086
A. panax isolate JAT 2120
A. panax isolate JAT 2125
A. radicina
A. radicina isolate AR018
A. radicina isolate AR07
A. radicina isolate CHHAR1
A. radicina strain ATCC6503
A. solani isolate CBS 111.44
A. solani isolate ICMP 6519-79
A. solani strain AS3
A. tg eficctL
A. thgec-ati isolate CHHAT1
A. tugeticu isolate CHHAT2
A. tenuissima isolate EGS34-015
A. tenuissima isolate Po56
A. tenuissima strain IA287
A. triticina
A. triticina
A. triticina strain EGS
A. zinniae

A. zinniae isolate CHHAZ1

A. zinniae strain AZ33


AF314575.1
AF314585.1
DQ323682.1
DQ323702.1
AY898635.1
AY898636.1
AY898637.1
AY898638.1
AY898639.1
AY898640.1
AY154704.1
DQ394074.1
DQ394073.1
AF267133.1
AF307014.1
Y17070.1
Y17069.1
AF314576.1
DQ100420.1
AF267134.1
AF267136.1
DQ323698.1
AY513940.1
AY154712.1
DQ117960.1
AY 154695.1
AY762948.1
AY154696.1

AF267135.1

AY372682.1














CHAPTER 5
DISPERSAL OF ALTERNARIA DESTRUENS: A WIND TUNNEL STUDY

Disease in a field does not occur uniformly in space due to either environmental

and/or dispersal gradients of inoculum (R. Berger, University of Florida, Gainesville, FL,

personal communication). To investigate these dispersal gradients, complicating

environmental factors must be controlled. In a study evaluating competing models of

inoculum and disease spread, Cowger et al. were able to attribute the velocity of wheat

stripe rust across space and time based on dissemination from specific disease foci

(Cowger et al. 2005). In the case of dispersal gradients, differential dissemination of

inoculum away from the source may be influenced by mortality of the pathogen's

propagules, movement by insects, or even simple diffusion (R. Berger, University of

Florida, Gainesville, FL, personal communication).

Wadia et al. (1998) determined that Passalorapersonata (Berk. and M. A. Curtis)

S. A. Khan and M. Kamal conidia were more effectively removed from sporulating

lesions and distributed over longer distances than by steady winds at the same wind

speeds. Inoculum dispersal is commonly neglected in the simulation of disease, which

according to Waggoner is necessary for prediction and forecasting models to accurately

explain an epidemic (Waggoner 1983). In the case of a foliar disease caused by a fungal

pathogen, an accurate prediction model based on a wind tunnel study would be of direct

use in identifying and evaluating disease development and spread as well as disease

management strategies.









In 1984, Bewick et al. isolated an Alternaria spp. from a diseased Cuscuta

gronovii Willd. ex J.A. Schultes (swamp dodder) plant growing in an uncultivated marsh

in Wisconsin (Bewick et al. 1986). In 1998, E. G. Simmons identified the isolate as

belonging to Alternaria destruens Simmons, sp. nov. (Simmons 1998). Simmons

described A. destruens as small-spored with conidial size ranging from 25-40 (- 46) x 10-

13 rim, unbranched, and brevicatenate with 4-8 conidia. Secondary conidiophores range

from very short to remarkably long, (3-) 20-80 rim, and in a single chain (Simmons

1998).

The main dispersal method for distribution ofAlternaria spp. spores is through

airborne movement and to a lesser extent through splash dispersal (Rotem 1994). The

rate at which Alternaria spp. spores fall from the air and how these spores are removed

from the air by impaction and deposition processes are determined by the spore shape and

size (McCartney and Fitt 1985). Alternaria spp. spores are relatively large in size, which

enables them to float in the air and therefore disperse from their site of production

(Gregory 1973). Alternaria spp. conidia are firmly attached to conidiophores and have to

be detached by relatively strong winds (Rotem 1994). Studies conducted on A. dauci

(Kiuhn) Groves & Skolko estimated that wind velocities above 2-3 m s-1 (approximately

7-11 km/h) were required to release large numbers of conidia (Strandberg 1977). Even

low to moderate wind speeds can effectively facilitate spore release just as easily as brief

or sustained gusts (Aylor 1990).

To investigate the dispersal ofA. destruens spores within a field situation, a wind

tunnel study is an effective means to correlate inoculum dispersal to disease severity over

increasing wind speeds. Hence, an accurate prediction model of spread of disease caused









by A. destruens based on a wind tunnel study would be of direct use to identify and

evaluate bioherbicide management strategies to control Cuscuta spp.

In previous field (Chapter 3) and greenhouse studies (Chapter 6) investigating A.

destruens, untreated control plants have been consistently diseased although attempts

were made to protect these plants during the application of the biological control agent.

It is hypothesized that this infection is a result of wind-carried conidia originating from

the inoculated plants. Therefore, the objective of this study was to erect a wind tunnel to

determine the capability of A. destruens spores to be dispersed at certain wind speeds and

determine how far those spores would travel. This simulated wind dispersal of A.

destruens spores may shed light on the observed rapid field spread of this pathogen and

help identify strategies for best deployment of this bioherbicide in the field.

Materials and Methods

Wind Tunnel

Studies were conducted outdoors, in an open-unshaded area, during the summer

of 2005 at the University of Florida at Gainesville. A wind tunnel was constructed with 5

PVC pipes (10.16 cm x 3 m) that had a PVC pipe increaser-reducer (7.62 cm x 10.16 cm)

attached to the beginning of the first PVC pipe for the insertion of the Vac 'N' Mulch

Blower/Vac (model # BV2000, Black and Decker). The blower was calibrated with a

rheostat (3PN1010V, Statco Inc., Dayton, OH) by holding each speed for 10 sec. Each

speed had 10 replications and then the average speed was taken. A total of 18 speeds

were calibrated and 6 representative speeds (2.1, 4.1, 7.4, 9.2, 11.6, and 15.6 m s-1) were

chosen for the study. Wind speed was measured with an anemometer (Kestrel 1000,

Nielsen-Kellerman, Boothwyn, PA) within +/- 3% (+0.1/-0.3 m s-1) wind velocity

accuracy.









Microscope slides covered in petroleum jelly (Vaseline, Unilever, Trumbull, CT)

were hung with duct tape at the top of each PVC pipe before the pipes were connected.

A petri plate (60 mm x 15 mm) was taped at the edge of the first PVC pipe with a piece

of 5.5-cm filter paper (Whatman) taped on top of the plate. Alternaria destruens spores

(SmolderTM WP) were supplied by Sylvan Bioproducts, Inc., Kittanning, PA. Using a

hemocytometer, spore counts were determined to be 1.8 x 107 spores per g. Alternaria

destruens spores (0.1 g) were deposited on top of the filter paper prior to each wind speed

run. Prior to each run, the blower was set to the correct wind speed to attain a steady air

flow, and then inserted into the PVC pipe increaser-reducer for 10 sec. The slides were

then taken out of the tunnel, covered with a piece of clear scotch tape, and put into

separate petri plates. An area of 5.08 cm x 2.54 cm of spores was counted on all slides.

After each run, the filter paper was changed and air was blown through the PVC piping

for 10 sec to clear any remaining spores. Each speed was replicated 3 times.

When attempting to repeat this experiment, the location was changed to the inside

of a large greenhouse. This was to minimize possible wind gusts that may have happened

in the first trial. In addition, the method of spore collection changed. This was due to the

number of spores collected and problems encountered with spore counting in the first

trial. The scotch tape and petroleum jelly made it difficult to accurately collect and count

all of the spores present. Therefore, in this experiment, glycerin (Fisher Scientific, Fair

Lawn, NJ) was used to coat the slides and they were laid diagonally in petri plates that

were taped at the end of each PVC pipe.

Because different agents were used to trap the spores, petroleum jelly in the first

repetition and glyercine in the second, these repetitions were treated as separate









experiments. All data were subjected to analysis of variance (ANOVA) and the means

were separated using Fisher's protected least significant difference (LSD) at P= 0.05 in

SAS (SAS 1999).

Results and Discussion

In the first experiment, the effects of length (P=0.0059) and speed (P=0.0001)

were both significant, and in the second experiment, there was a length by speed

interaction (P=0.0001).

In the first trial, the spore number decreased linearly as wind tunnel length

increased (Figure 5.1). There were no significant differences between the first two tunnel

lengths of 3.0 m and 6.1 m, nor were there differences between the last three lengths of

9.1 m, 12.2 m, and 15.2 m. The two lowest speeds of 2.1 m s-1 and 4.1 m s-1 were not

significantly different from each other (Figure 5.2). Likewise, the four highest speeds

(7.4, 9.2, 11.6, and 15.6 m s-) were also not significantly different from each other

(Figure 5.2).

In the second experiment, the number of spores trapped decreased with tunnel

length, but increased at higher wind speeds (Figure 5-3). The last two tunnel lengths

(12.2 m and 15.2 m) had the lowest number of spores trapped (138 and 68), while the

second and third tunnel lengths (6.1 m and 9.1 m) had the most spores trapped (239 and

207) at the highest speed of 15.6 m s-1 (Figure 5-3).

Dispersal of spores in dry weather is either by release under the force of gravity or

removal from the host mainly during fast, turbulent wind (Aylor 1990). Many spore

types require a certain amount of direct mechanical force to be applied to the spore for it

to be detached (Aylor 1975). In addition, a threshold wind speed must be surpassed,

which is unique for each pathogen. Spores of each species are usually removed over a









range of wind speeds with the total amount of spores detached from a condiophore

increasing rapidly as wind velocity increases (Aylor 1990). Although, once a certain

percentage of spores are released, it is difficult to remove additional spores at any

realistic speed (Waggoner 1973).

The results of this study are in accordance with other simulated wind tunnel

studies (Chen et al. 2003; Wadia et al. 1998). At higher wind speeds, the numbers of

spores that are dispersed from a source reach a peak and then decrease with increasing

distance. The highest amounts of A. destruens spores were collected at the distances of

6.1 m and 9.1 m with a wind speed of 15.6 m s1. The average number of spores

collected between these two distances was 223. Once the distance reached 15.2 m at that

same speed, the number of spores decreased to 68.

At these wind speeds and distances, dispersal of A. destruens would be more than

enough to reach untreated controls in the field and greenhouse. In addition, this study did

not include wind gusts which have been shown to be more effective in dispersing spores

further from a source than steady winds (Wadia et al. 1998).

In conclusion, this pathogen's capability of spreading rapidly and over great

distances can be an advantage when applying A. destruens in the field. Making use of

wind patterns flowing parallel to the field where applications of this bioherbicide are to

be made may aid in field coverage and could reduce additional treatments ofA.

destruens. Applications could also be made in certain areas of the field, i.e. divide the

field into quadrants and then use wind patterns to decide which quadrants to treat with A.

destruens that would best flow to untreated areas in the field.






71


Although the results of these wind tunnel studies confirm that large numbers of A.

destruens spores are capable of spreading far from a source point, the methodology of

this study could be vastly improved by using potted plants inoculated with A. destruens.

This would give a better representation of how the spores disperse when airborne from a

diseased plant. Overall, these experiments provide some insight into the dispersal

patterns of A. destruens when airborne.












40





a 1
E v30




















Figure 5-1. Results of the first experiment utilizing the wind tunnel to examine the
o 20


c<
a 10





3 6.1 9.1 12.2 15.2
TUNNEL LENGTH (m)




Figure 5-1. Results of the first experiment utilizing the wind tunnel to examine the
effects of wind tunnel length (m) from inoculum release on spore dispersal of
Alternaria destruens.










120


m 100


m- 80
-* t 3.0
0 -6.1
S60- -- 9.1

Zc 15.2
4 40 -


20 -


0
0 2.1 4.1 7.4 9.2 11.6 15.6

WIND SPEED (m s-)



Figure 5-2. Results of the first experiment utilizing the wind tunnel to examine the
effects of wind speed (m s1) from inoculum release on spore dispersal of
Alternaria destruens.










240

W 200

Sn 160 + 3.0
-- 6.1
s 120 \9.1
|c i \ 12.2
c-- 15.2
1 80 -

40 -

0
0 2.1 4.1 7.4 9.2 11.6 15.6
WIND SPEED (m s )


Figure 5-3. Results of the second experiment utilizing the wind tunnel to examine the
effects of wind speed (m s-) and distance (m) from inoculum release on spore
dispersal of Alternaria destruens.














CHAPTER 6
EFFECTS OF ALTERNARIA DESTRUENS SMOLDERSTM), GLYPHOSATE
(ROUNDUP PRO), AND AMMONIUM SULFATE INDIVIDUALLY AND
INTEGRATED FOR CONTROL OF CUSCUTA PENTAGON

Cuscuta species are obligate parasites that predominate disturbed habitats, which

preadapts them to becoming a serious weed problem. There are approximately 150 to

170 Cuscuta spp. worldwide; with the majority occurring in North and South America

(Ashton and Santana 1976; Dawson et al. 1994). Cuscuta spp. parasitize mainly

dicotyledonous and a few monocotyledonous plants with some species of Cuscuta being

very host specific, while others have a broad host range. (Gaertner 1950; Dawson et al.

1994).

Control of Cuscuta spp. is very difficult, with the best method being exclusion,

i.e., not to introduce it into a farm or field. Mechanical removal by seed cleaning

machines is an option, but these devices are often slow and they do not always remove all

of the Cuscuta spp. seed and sometimes remove the crop seed. Tillage can be useful

because the seedlings are easily dislodged from the soil or die when buried, but if they

are not buried completely, they can resurface. Physically removing Cuscuta spp. by hand

is time-consuming and although Cuscuta spp. generally behave as annuals, they can

survive in the tissue of perennial host plants under some conditions (Dawson et al. 1984).

Use of preemergent herbicides may not provide season-long control and repeated

treatments may be necessary.

Foliar applications of diquat (6,7-dihydrodipyrido[1,2-a:2', '-c]pyrazinediium

dibromide) or paraquat (1, '-dimethyl-4,4'-bipyridinium dichloride) provide partial









selectivity in alfalfa, Cuscuta spp. and the host plant foliage will be destroyed, but the

host plant may regrow from crowns after treatment (Dawson 1987; Dawson et al. 1994).

Other nonselective post-attachment herbicides that will provide complete kill to the host

plant as well as Cuscuta spp. are 2,4-D ((2,4-dichlorophenoxy)acetic acid) or glyphosate

(N-(phosphonomethyl)glycine) (Dawson et al. 1994).

Glyphosate, a phloem-mobile herbicide, moves in the symplast via source to sink

(McAllister and Haderlie 1985). The primary mode of action of glyphosate is to inhibit

5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; E.C. 2.5.1.19), which is the key

enzyme in the shikimate pathway. Inhibition of EPSPS prevents the production of

chorismate which is required for biosynthesis of the aromatic amino acids phenylalanine,

tyrosine, and tryptophan (Amrhein et al. 1980; Geiger and Bestman 1990; Hollander and

Amrhein 1980).

Dawson and Saghir sprayed glyphosate over the top of parasitized alfalfa, which

resulted in injury and death to the Cuscuta spp. but not to the alfalfa (Dawson and Saghir

1983). The highest rate of glyphosate (0.30 kg per ha) provided sufficient delay for the

parasitized alfalfa to recover prior to regrowth of Cuscuta spp. (Dawson and Saghir

1983). Even though all exposed shoots of Cuscuta spp. were killed by the glyphosate,

some haustoria were still embedded within the alfalfa stems and regenerated. All alfalfa

foliage in the control plots were destroyed by Cuscuta spp. (Dawson and Saghir 1983).

Fer found that 14C-labeled glyphosate was more concentrated in the apical part of

the Cuscuta spp. shoots, especially in the buds, than in the haustorial coil (Fer 1984).

Furthermore, at 4 days after treatment (DAT), Cuscuta spp. had absorbed 75% of the









glyphosate from the hosts' phloem. Fer concluded from these studies that phloem-mobile

herbicides should be able to selectively control Cuscuta spp. (Fer 1984).

Bewick et al. conducted studies with 14C-labeled glyphosate on carrot parasitized

with Cuscuta gronovii (Bewick et al. 1991). They determined that at 1 DAT Cuscuta

tissue had absorbed as much 14C-labeled glyphosate as any sink in the carrot. At 45

DAT, Cuscuta tissue contained most of 14C-labeled glyphosate in regard to all other

physiological sinks, except the petiole of the treated carrot leaf (Bewick et al. 1991).

Ammonium sulfate, (NH4)2804, is used as a surfactant to increase phytotoxicity

in many herbicides, including glyphosate (Nalewaja and Matysiak 1993; O'Sullivan et al.

1981; Turner and Loader 1975). Hard water contains polyvalent cations that interfere

with the activity of glyphosate (Nalewaja and Matysiak 1991, 1993). Thelen et al. (1995)

determined that the glyphosate molecule reacts with Ca" and other cations to form a

less-readily absorbed glyphosate-Ca salt that decreases herbicidal activity. When

ammonium sulfate is added into the tank mix, the ammonium ion binds with the

glyphosate molecule, preventing the formation of glyphosate-Ca salt, which in turn

increases herbicide's absorption (Gronwald et al. 1993; Pratt et al. 2003).

Several theories have been proposed as to how ammonium sulfate increases

herbicide absorption. Studies conducted with tomato fruit, which has a well-developed

cuticle, confirmed large increases in uptake when sethoxydim (2-[1-(ethoxyimino)butyl]-

5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen- -one) was applied with ammonium

sulfate (Wanamarta et al. 1993). The cuticle is a barrier to sethoxydim penetration, but

with the addition of ammonium sulfate the capability of the cuticle to block herbicide

absorption was reduced. Ammonium salts of bentazon (3-(1-methylethyl)-(1H)-2,1,3-









benzothiadiazin-4(3H)-one 2,2-dioxide) formed from the addition of ammonium sulfate

were more readily absorbed and demonstrated increased transcuticular movement

compared with calcium, magnesium, or sodium salts of bentazon (Wanamarta et al.

1993). Ammonium sulfate is related to increased permeation of the herbicide through the

plasma membrane (Young et al. 2003). Several researchers have hypothesized that the

acidification of the spray solution by the addition of ammonium sulfate allows a greater

proportion of weak-acid herbicides to remain in their nondissociated form, which allows

the herbicide to diffuse across the plasma membrane more easily (Gronwald et al. 1993;

Smith and Vanden Born 1992). When the herbicide enters the cell, the higher pH of the

cytoplasm causes dissociation of weak-acid herbicides, thus efficiently trapping the

herbicide inside the cell (Young et al. 2003).

Ammonium sulfate is formed by reacting ammonia with sulfuric acid to form a

stable ammonium salt. It is soluble in water and insoluble in liquid ammonia or alcohol.

The toxicity of the ammonium ion enhances this compound's capacity for weed control

(Brian 1976). The plant cell rapidly absorbs the ammonia and the cell sap, which is

normally acidic, may become alkaline due to the effect of ammonia (Harvey 1911).

Rapid cell death ensues from increased alkalinity and from the toxicity of ammonia

(Brian 1976). Excess concentrations of ammonium ions or ammonia are toxic to plants,

although the exact biochemical causes of toxicity are still being studied (Given 1979;

Smith and Vandenn Born 1992, Young et al. 2003).

Another option to control dodder is the use of a bioherbicide. Bioherbicides are

biological control agents based on fungi, bacteria, or viruses that are industrially

developed and registered through the United States Environmental Protection Agency as









a biopesticides to manage or reduce weed populations. The mycoherbicide (fungi used as

herbicides) approach can be successful when substantial quantities of inoculum are used

to help compensate for natural restrictions to disease development (Bewick et al. 1987;

Charudattan 1988). Through manipulation, some fungi that normally cause sporadic or

endemic disease levels on their hosts can be developed into effective bioherbicides

(Charudattan 1988; Rosskopf et al. 1999).

In 1984, Bewick et al. isolated an Alternaria spp. from a diseased C. gronovii

Willd. ex J.A. Schultes plant growing in an uncultivated marsh in Wisconsin (Bewick et

al. 1986). The majority ofAlternaria spp. are saprophytic, but some species are

pathogenic and produce a range of plant diseases throughout the world. Depending on

the pathogen, symptoms typically appear as blights and leaf spots, but damping-off of

seedlings, as well as fruit, stem or tuber rots can also occur (Agrios 1997). The

Alternaria isolate recovered by Bewick was highly virulent and field studies with this

pathogen resulted in 92% control of C. gronovii (Bewick et al. 1987). No disease was

observed on, nor was the pathogen recovered from any of the host plants of dodder. In

1990, this pathogen was patented as a bioherbicide for suppression of Cuscuta spp.

(Bewick et al. 2000), and in 1998, Simmons identified the species as Alternaria destruens

Simmons, sp. nov. (Simmons 1998).

Currently, there are no integrated control methods for Cuscuta spp. let alone a

single economical control method. Therefore, the objectives of this study were to 1)

evaluate the effects of various concentrations of glyphosate (Roundup Pro) and

ammonium sulfate on the growth ofA. destruens on Citrus spp. and 2) evaluate the









effects ofA. destruens, glyphosate, and ammonium sulfate individually and in

combination, the latter as an integrated control method, on C. pentagon.

Materials and Methods

Greenhouse Test Plants

Ten-month-old Citrus spp. ('Smooth Flat Seville'; Phillip Rucks Citrus Nursery,

Frostproof, FL) in 1-gallon pots were parasitized with C. pentagon. At the time of

treatment, the Cuscuta spp. was approximately 4-weeks old.

Inoculation and Treatments

All studies were conducted in the greenhouses at the University of Florida in

Gainesville from the spring of 2005 through the summer of 2005. Maximum relative

humidity for all greenhouse trials (Figure C-4) and maximum and minimum temperatures

for greenhouse trial I (Figure C-l), trial II (Figure C-2), and trial III (Figure C-3) were

measured using a HOBO Pro RH/Temp Data Logger (H08-032-08) (Onset Computer

Corporation, Bourne, MA). Seven treatments were applied consisting of: an untreated

control; oil (PCC 588, United Agri Products, Greeley, CO) at 7.5%; ammonium sulfate at

0.125% w/v in water; glyphosate (N-(phosphonomethyl)glycine) at 0.02 kg (ae)/L of

Roundup Pro; A. destruens (SmolderTM WP, obtained from Sylvan Bioproducts, Inc.,

Kittanning, PA); A. destruens + oil; and a mixture of A. destruens + oil, glyphosate, and

ammonium sulfate (the latter two at the preceding concentrations). Glyphosate and

ammonium sulfate concentrations were determined from previous preliminary

greenhouse studies on amounts that would successfully kill C. pentagon, but not Citrus

spp. (Appendix D). Fungal suspensions were prepared as described for the greenhouse

trial in Chapter 2. Using a hemocytometer, spore counts were standardized to 1.8 x 107









spores per ml. Each study was conducted in a randomized complete block design with 5

replications and was performed twice.

Plants were inoculated with a hand-held sprayer at 100 ml per m2 and all

treatments were used within 1 h of mixing. All data were analyzed using Area Under the

Disease or Damage Progress Curve (AUDPC), which is used to quantify disease or

damage progress over time by integrating all factors of an epidemic, such as,

environment, host, and pathogen effects (Campbell and Madden 1991). AUDPC values

for each trial were calculated using the formula I [(y, + y(, +))/2]*(t(+l)+l- t,), where i =

1, 2, 3, ...n-1, where y, is the disease or damage severity at the ith evaluation, and t, is time

at the ith evaluation (Shaner and Finney 1977). All data and AUDPC values were

subjected to analysis of variance (ANOVA) and means separated by Tukey's Studentized

Range (HSD) test at P=0.05 in SAS (SAS 1999).

Disease or Phytotoxic Damage Rating

Plants were observed and rated weekly. Disease symptoms as well as the

phytotoxic damage from glyphosate or ammonium sulfate were expressed as necrotic

spots or blight on dodder. Phytotoxic damage from ammonium sulfate or glyphosate on

Citrus spp. plants in the preliminary studies were expressed as leaf and stem senescence.

Hence there was no need to use different rating scales or systems and both the disease

and damage were assessed on a severity scale of 0 to 5, with 0 = no symptoms; 1 = 1-

10% (tip necrosis; stems starting to wilt and become necrotic; 2 = 11-35% (slightly more

stem necrosis; flowers starting to senesce), 3 = 36-65% (over half of the stems are dead

or dying; clusters of flowers senescing); 4 = 66-90% (the majority of the stems and









flowers are dead or dying; some healthy flowers and stems may still present), and 5 = 91-

100% (plant death) (Chapter 3, Figure 3-5).

Growth Inhibition Study

The treatments consisted of three glyphosate concentrations (0.09, 0.04, and 0.02

kg (ae)/L of Roundup Pro), three ammonium sulfate concentrations (0.50%, 0.25%, and

0.125% w/v in water), one oil (PCC 588) concentration (7.5% v/v in water), and an

untreated control. Each treatment was mixed into 200 ml of potato dextrose agar (PDA)

(39 g of PDA [Difco, Detroit, MI] per liter of H20), which was then poured in 20 ml

aliquots into petri plates. All treatments were replicated 10 times.

Single-spore isolations of A. destruens were made as described in Chapter 4.

Twelve-day-old agar plugs (6 mm) ofA. destruens were placed in the center of each plate

and stored at 22C. Plates were measured every other day over a 10-day period. All data

were subjected to analysis of variance (ANOVA) and the means were separated using

Fisher's protected least significant difference (LSD) at P= 0.05 in SAS (SAS 1999).

Results and Discussion

Greenhouse Studies

Due to lower treatment effects in the second trial, this study was repeated a third

time. The lower effects were most likely due to environmental conditions at the time,

such as a greater number of cloudy days which would have slowed the growth of Cuscuta

spp.

Due to no significant differences in variance between trials I and III (P=5.030),

the data were combined, although, there was a treatment by DAT interaction (P=0.0210).

The second trial was significantly different from trials I and III (P=0.0001) and was

analyzed separately. Trial II also had significant treatment (P=0.0001) and DAT









(P=0.0001) effects. In all trials, disease or damage severity progressed linearly as DAT

increased, except for the mixture and the A. destruens + oil treatments in trial I and III

(combined), which were better fit with a quadratic curve (Figures 6.1 and 6.4).

Regression equations and R2 values are listed in Table 6-1.

In preliminary greenhouse trials, glyphosate and ammonium sulfate

concentrations that caused the highest dodder mortality levels were determined to be

glyphosate at 1.42, 0.71, and 0.36 kg (ae)/L of Roundup Pro and ammonium sulfate at

0.10% w/v in water (Appendix D). In addition, the effects of damage severity from

glyphosate and ammonium sulfate concentrations increased over time (DAT) (Figures D-

1 and D-3). There were no significant differences between the two lowest concentrations

of glyphosate where the AUDPC values were 110.81 and 105.69, respectively (Table D-

1), therefore for further testing the lowest concentration of 0.09 kg (ae)/L of Roundup

Pro was used in addition to two lesser concentrations of 0.04 and 0.02 kg (ae)/L of

Roundup Pro. The effects of glyphosate on Citrus spp. were significantly different in

both trials (P=0.0001) (Figure D-2). AUDPC values in trial II, as compared to trial I,

were higher which could be due to possible environmental effects at the time of

application which may have increased the phytotoxic damage on the Citrus spp. plants

(Table D-2). The only AUDPC value in trial I that was significantly different from the

other AUDPC values was the untreated control. In trial II, there were no significant

differences between AUDPC values (Table D-2). In the ammonium sulfate study, the

highest concentration was not significantly different from the untreated control; therefore,

the concentrations were increased to 0.125%, 0.25%, and 0.5% (Table D-3). The effects

of ammonium sulfate on Citrus spp. were not significantly different and therefore both









trials were combined (Figure D-4). AUDPC values indicated no differences between

concentrations (Table D-4).

In the greenhouse trials I and III (combined), the AUDPC value for the mixture

treatment (88.90) had the highest disease or damage severity rating out of all treatments

(Table 6.2). In trial II, the AUDPC values were all the same except for the oil treatment

(28.9) (Figure 6.3 and Table 6.3). In addition, severe stunting after treatment with

glyphosate was observed on all regrowths of Cuscuta spp. (Figure 6.5).

Growth Inhibition

Results from the growth-inhibition study indicated that ammonium sulfate at any

concentration did not inhibit A. destruens growth (Figures 6.6 and 6.7), while glyphosate

at the highest concentration (0.09 kg (ae)/L) had the greatest inhibition (Figures 6.8 and

6.9). Oil and the untreated control were not significantly different (Figures 6.10 and

6.11).

The mixture treatment provided the best control of Cuscuta spp. in trials I and III

(combined) with an AUDPC value of 88.90. By 35 DAT, all Cuscuta spp. plants treated

with the mixture treatment were dead. Citrus plants were not damaged by any treatment,

even though glyphosate, despite the low rate used, injured and at times completely killed

the Cuscuta spp. parasitizing the plants. Cuscuta spp. treated with glyphosate alone

regrew with vegetative tissue that was severely distorted and stunted. This has been

observed in other studies where glyphosate alone was used to control Cuscuta spp.

(Dawson and Saghir 1983).

Phytotoxic damage from ammonium sulfate, glyphosate, and oil (PCC 588) and

disease severity from A. destruens could not be differentiated when rating Cuscuta spp.









plants. All injury symptoms appeared the same on Cuscuta spp. Although, each

treatment separately in trials I and III (combined) were not significantly different, but

when mixed together were significantly different from all other treatments.

Some formulations of glyphosate have exhibited fungicidal properties either by

inhibiting, stimulating, or producing a synergistic interaction with various pathogenic and

saprophytic fungal species (Anderson and Kolmer 2005; Chakravarty and Chatarpaul

1990; Wardle and Parkinson 1992). Studies have found that glyphosate produces an

inhibitory effect on Drechslera teres (Sacc.) Shoemaker (Toubia-Rahme et al. 1995),

Phytophthora spp. (Kassaby and Hepworth 1987; Utkhede 1982), Puccinia graminis

Pers.:Pers. f sp. tritici Eriks. & E. Henn. (Anderson and Kolmer 2005), Puccinia triticina

Eriks. (Anderson and Kolmer 2005), Pyrenophora tritici-repentis (Died.) Drechs.

(Fernandez et al. 1998), Rhynchosporium secalis (Oudem) J. J. Davis (Turkington et al.

2001), and Septoria nodorum Berk. (Harris and Grossbard 1979); while producing a

stimulatory effect on Pyrenophora teres Drechs (Turkington et al. 2001), Pythium

coloratum Vaartaja and Pythium ultimum Trow (Descalzo et al. 1998).

Other species, such as, Fusarium solani (Mart.) Sacc. (Njiti et al. 2003; Sanogo et

al. 2000; Sanogo et al. 2001) and Rhizoctonia solani Kuhn (Black et al. 1996;

Harikrishnan and Yang 2002; Smiley et al. 1992) have had effects from glyphosate

ranging from inhibitory, to stimulatory, or to no effects. As to why there are differences

among species or even within species from glyphosate, the answer lie in differences in

herbicidal formulations (i.e. the active ingredient, inert ingredients, or an interaction

between the two) rather than the glyphosate itself (Morj an et al. 2002).









The growth-inhibition studies done with A. destruens and glyphosate did

demonstrate that at higher concentrations glyphosate did impede the growth of the

pathogen, therefore, the lowest concentration 0.02 kg (ae)/L of Roundup Pro was chosen

for this study. The results from the AUDPC values indicate that glyphosate did not

appear to have any inhibiting affect on A. destruens, but rather stimulate the pathogen.

However, studies to determine if there were any differences in spore production were not

done.

Ammonium sulfate was added to the mixture treatment to increase the absorption

rate of glyphosate and enhance foliar penetration. AUDPC values of ammonium sulfate

and glyphosate alone indicate that there were no significant differences between the two

treatments at the concentrations used in this study. Although, when ammonium sulfate,

like glyphosate, was added to the mixture treatment, there was a significant difference in

the AUDPC value (Figure 6-2 and Table 6-2).

Oil (PCC 588) was added with A. destruens, to reduce the surface tension, which

improves coverage over the plants surface, keeps the surface moist longer, and aids in

attachment of the pathogen to the plant. Although, in trials I and III (combined), there

were no significant differences between the AUDPC values of A. destruens + oil (56.50)

and A. destruens without oil (49.95). The same is true for trial II where the AUDPC

values for A. destruens + oil (33.70) and A. destruens (38.10) are not significantly

different. Therefore, based on this study oil (PCC 588) may not be needed when

applying A. destruens.

This study provides evidence that by adding glyphosate and ammonium sulfate to

A. destruens a synergistic effect was produced and better control of Cuscuta spp. was






87


obtained than by using A. destruens alone or either of the other treatments alone. In light

of the current control methods, this combination of a bioherbicide, herbicide, and

nonionic surfactant appears to be the best integrated control method for Cuscuta spp.




Full Text

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INTEGRATED CONTROL OF DODDER ( Cuscuta pentagona Engelm.) USING GLYPHOSATE, AMMONIUM SULFATE, AND THE BIOLOGICAL CONTROL AGENT Alternaria destruens Simmons, sp. nov. By JENNIFER COLLEEN COOK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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To my husband, Jeffrey Cook, for his love, understanding, and support. I could not have done this without you.

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ACKNOWLEDGMENTS I would like to sincerely thank Dr. Raghavan Charudattan for his guidance, patience, and support. In addition, I would like to express my deepest gratitude to Dr. Erin Rosskopf, without whose support I would never have made it on my own through this journey. I cant thank her enough. I would also like to acknowledge the members of my committee, Drs. Greg MacDonald, William Stall, and Thomas Zimmerman for their support and guidance. I would like to thank Dr. Tom Bewick for his counsel and unending information on dodder; Jim DeValerio for his friendship and always being there when I had a problem or needed an answer, and for his experimental and statistical knowledge; Eldon Philman and Herman Brown for all of their assistance at the greenhouse, including building some crazy stuff for me; Philip Ruck for providing me citrus plants whenever I needed them; Camilla Yandoc for all her time in helping me with my field study; Sylvan and John Cascino for providing Smolder to accomplish this work; Dr. Portier for his statistical consulting services; Alana Den Breeyen, Abby Guerra, and Linley Smith for all their time and help in getting me through the molecular part of this research; Everyone in Charus lab, past and present, who has assisted me with this project; USDA-CSREES-Special Grants Program for the award of a TSTAR grant to the University of the Virgin Islands and the University of Florida that supported this research; and USDA-ARS-USHRL for a cooperative research agreement that partially supported this research. A special thanks to Whitney Elmore for her friendship, input, and shared misery of writing a dissertation. I might just miss those midnight emails! I iii

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would also like to thank my closest friends, Angela Horton, Lesa Werkmeister, Lynn Howard, Jill Bejarano, and Kim Rosiek for listening to me when I needed to cry, scream, or whine. Finally, I would like to thank my mom Catherine who has always been there no matter what, through good and bad times, and to push me to finish this degree when I needed the motivation. Mom, youre my inspiration and I love you for it. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES ............................................................................................................vii LIST OF FIGURES ...........................................................................................................ix ABSTRACT ......................................................................................................................xii CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Taxonomy.....................................................................................................................1 Biology and Host Range...............................................................................................2 Cuscuta pentagona Engelm..........................................................................................6 Cuscuta spp. in the U. S................................................................................................7 Cuscuta spp. Worldwide...............................................................................................8 Economic Loss, Dispersal, and Control.......................................................................9 Herbicides...................................................................................................................10 Glyphosate..................................................................................................................13 Ammonium Sulfate.....................................................................................................14 Alternaria destruens Simmons, sp. nov......................................................................15 Biological Control......................................................................................................17 Research Objective.....................................................................................................18 2 EVALUATION OF THE HOST RANGE OF Alternaria destruens (SMOLDER )............................................................................................................20 Materials and Methods...............................................................................................23 Results and Discussion...............................................................................................25 3 FIELD EFFICACY OF Alternaria destruens (SMOLDER ) AS A BIOHERBICIDE OF Cuscuta....................................................................................31 Materials and Methods...............................................................................................32 Results and Discussion...............................................................................................39 v

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4 MOLECULAR PHYLOGENIC CHARACTERIZATION TO DIFFERENTIATE Alternaria destruens Simmons, sp. nov. FROM VARIOUS Alternaria SPECIES BASED ON INTERNAL TRANSCRIBED SPACER REGION..............................50 Materials and Methods...............................................................................................52 Results and Discussion...............................................................................................56 5 DISPERSAL OF Alternaria destruens: A WIND TUNNEL STUDY......................65 Materials and Methods...............................................................................................67 Results and Discussion...............................................................................................69 6 EFFECTS OF Alternaria destruens (SMOLDER ), GLYPHOSATE (ROUNDUP PRO ), AND AMMONIUM SULFATE INDIVIDUALLY AND INTEGRATED FOR CONTROL OF Cuscuta pentagona...................................................................75 Materials and Methods...............................................................................................80 Results and Discussion...............................................................................................82 APPENDIX A FEDERAL NOXIOUS WEED LIST (AS OF JANUARY 6, 2006)........................102 B WIND SPEED DATA...............................................................................................103 C ENVIRONMENTAL DATA FOR GREENHOUSE TRIALS OF Alternaria destruens, AMMONIUM SULFATE AT 0.125% W/V IN WATER, AND GLYPHOSATE AT 0.02 kg (ae)/L OF ROUNDUP PRO ON Cuscuta pentagona.................................................................................................................107 D DATA FROM PRELIMINARY GREENHOUSE TRIAL......................................111 LIST OF REFERENCES.................................................................................................119 BIOGRAPHICAL SKETCH...........................................................................................130 vi

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LIST OF TABLES Table page 2-1. Reaction of test plants to Alternaria destruens applied as SMOLDER formulated with PCC588 (United Agri Products, Greeley, CO)..............................29 3-1. Means of area under the disease progress curve (AUDPC) for treatments on Cuscuta pentagona from Ft. Pierce field trial I........................................................45 3-2. Means of area under the disease progress curve (AUDPC) for treatments on Cuscuta pentagona from Ft. Pierce field trial II......................................................46 4-1. Sources of unknown isolates and treatments applied to plots from which these isolates were recovered............................................................................................60 4-2. Sources of Alternaria spp. used for DNA sequencing in this study..........................61 4-3. Fungal Isolates and the GenBank accession numbers for sequences used in phylogenic analyses..................................................................................................63 6-1. Regression equations and R 2 values from greenhouse trials I and III (combined) for the effects of disease or damage severity from treatments on Cuscuta pentagona over time (days after treatment, DAT)...................................................89 6-2. Area under the disease or damage progress curve (AUDPC) means for all treatments on Cuscuta pentagona from greenhouse trials I and III (combined)......91 6-3. Area under the disease or damage progress curve (AUDPC) means for all treatments on Cuscuta pentagona from greenhouse trial II.....................................93 B-1. Ft. Pierce trial I average wind speed data...............................................................103 B-2. Ft. Pierce trial II average wind speed data..............................................................105 B-3. Gainesville field study average wind speed data....................................................106 D-1. Area under the disease or damage progress curve (AUDPC) means for all preliminary glyphosate treatments on Cuscuta pentagona in greenhouse trials I and II (combined)...................................................................................................112 D-2. Area under the disease or damage progress curve (AUDPC) means for all preliminary glyphosate treatments on Citrus spp. in greenhouse trials I and II.....114 vii

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D-3. Area under the disease or damage progress curve (AUDPC) means for all preliminary ammonium sulfate treatments on Cuscuta pentagona in greenhouse trials I and II (combined)........................................................................................116 D-4. Area under the disease or damage progress curve (AUDPC) means for all ammonium sulfate treatments on Citrus spp. in greenhouse trials I and II (combined).............................................................................................................118 viii

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LIST OF FIGURES Figure page 1-1. Cuscuta pentagona on citrus.......................................................................................3 1-2. Cuscuta pentagona flowers and buds (A and B).........................................................4 1-3. Cuscuta pentagona haustorium (arrow) penetrating citrus.........................................5 1-4. Alternaria destruens spores.......................................................................................17 2-1. Capsicum chinense Habanero pepper plants...........................................................27 2-2. Capsicum chinense Scotch Bonnet pepper plants...................................................28 2-3. Lactuca sativa Green Ice lettuce plants..................................................................28 3-1. Header Canal Alternative Cropping Systems Research Farm in Ft. Pierce, FL........36 3-2. Simulated field study at the University of Florida in Gainesville.............................37 3-3. Experimental design of simulated field study containing 55 pots of Citrus spp. parasitized with Cuscuta pentagona........................................................................38 3-4. Distances used in simulated field study.....................................................................38 3-5. Citrus spp. plants parasitized by Cuscuta pentagona, the latter displaying symptoms of treatments with Alternaria destruens + oil and an untreated control.39 3-6. Ft. Pierce field trial I data..........................................................................................45 3-7. Ft. Pierce field trial II data.........................................................................................46 3-8. Disease severity over time (days after inoculation; DAI) on Cuscuta pentagona treated with Alternaria destruens + oil (PCC 588) in Circle I (0.91 m) and untreated sentinel C. pentagona in Circle III (3.15 m)............................................47 3-9. Disease severity over time (days after inoculation; DAI) on Cuscuta pentagona treated with Alternaria destruens + oil (PCC 588) in Circle I (0.91 m) and untreated sentinel C. pentagona in Circle II (2.13 m)..............................................48 ix

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3-10. Disease severity over time (days after inoculation; DAI) on untreated sentinel Cuscuta pentagona in Circles II (2.13 m) and III (3.15 m).....................................49 4-1. Rooted neighbor-joining dendrogram based on 507 bp from ITS 1, 5.8S, and ITS2 sequences.........................................................................................................59 4-2. Rooted neighbor-joining dendrogram of ITS1, 5.8S, and ITS2 sequences...............62 5-1. Results of the first experiment utilizing the wind tunnel to examine the effects of wind tunnel length (m) from inoculum release on spore dispersal of Alternaria destruens...................................................................................................................72 5-2. Results of the first experiment utilizing the wind tunnel to examine the effects of wind speed (m s -1 ) from inoculum release on spore dispersal of Alternaria destruens...................................................................................................................73 5-3. Results of the second experiment utilizing the wind tunnel to examine the effects of wind speed (m s -1 ) and distance (m) from inoculum release on spore dispersal of Alternaria destruens.............................................................................................74 6-1. Greenhouse trials I and III (combined) to determine the effects of disease or damage severity from treatments on Cuscuta pentagona over time (days after treatment; DAT).......................................................................................................88 6-2. Effects of treatments on disease or damage severity on Cuscuta pentagona over time (days after treatment; DAT) from greenhouse trials I and III (combined).......90 6-3. Effects of treatments on disease or damage severity on Cuscuta pentagona over time (days after treatment; DAT) from greenhouse trial II......................................92 6-4. Greenhouse trial II to determine the effect of time (DAT) on disease or damage severity across all treatments....................................................................................94 6-5. Stunting of regrowth of Cuscuta pentagona treated with glyphosate at 0.02 kg (ae)/L of Roundup Pro ............................................................................................95 6-6. Effect of ammonium sulfate concentrations (0.50%, 0.25%, 0.125%, and 0% (control) w/v in water) on the growth of Alternaria destruens colony (mm day -1 ).96 6-7. Inhibition of growth of Alternaria destruens colony (mm day -1 ) over time (DAT) by ammonium sulfate at 0.50%, 0.25%, 0.125% and 0% ((CON) w/v in water)....97 6-8. Effect of glyphosate concentrations (0.09, 0.04, 0.02 and 0 (control) kg (ae)/L of Roundup Pro ) on the growth of Alternaria destruens colony (mm day -1 ).............98 6-9. Inhibition of growth (mm day -1 ) of Alternaria destruens over time (DAT) by glyphosate at (0.09, 0.04, 0.02 and 0 (CON)) kg (ae)/L of Roundup Pro )............99 x

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6-10. Effect of oil (PCC 588) (7.5% v/v in water) on the growth of Alternaria destruens compared to untreated control (no oil)...................................................100 6-11. Effect of oil (PCC 588) at 7.5% v/v in water and control (CON) on the growth (mm day -1 ) of Alternaria destruens over time (DAT)............................................101 C-1. Maximum and minimum temperatures ( o C) during greenhouse trial I...................107 C-2. Maximum and minimum temperatures ( o C) during greenhouse trial II.................108 C-3. Maximum and minimum temperatures ( o C) during greenhouse trial III................109 C-4. Maximum relative humidity during all greenhouse trials.......................................110 D-1. Damage severity on Cuscuta pentagona over time from six glyphosate concentrations (0.09, 0.18, 0.36, 0.71, 1.42, and 0 (control) kg (ae)/L of Roundup Pro ).......................................................................................................111 D-2. Damage severity on Citrus spp. from six glyphosate concentrations concentrations (0.09, 0.18, 0.36, 0.71, 1.42, and 0 (control) kg (ae)/L of Roundup Pro ), averaged over all days.................................................................113 D-3. Damage severity on Cuscuta pentagona from seven ammonium sulfate concentrations (0% (control), 1%, 2%, 3%, 4%, 5%, and 10% w/v in water) over time (DAT).............................................................................................................115 D-4. Damage severity on Citrus spp. from seven ammonium sulfate concentrations (0% (control), 1%, 2%, 3%, 4%, 5%, and 10% w/v in water) averaged over all days.........................................................................................................................117 xi

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INTEGRATED CONTROL OF DODDER (Cuscuta pentagona Engelm.) USING GLYPHOSATE, AMMONIUM SULFATE, AND THE BIOLOGICAL CONTROL AGENT Alternaria destruens Simmons, sp. nov. By Jennifer Colleen Cook August 2006 Chair: Raghavan Charudattan Major Department: Plant Pathology Cuscuta pentagona is an obligate parasite that is the most widespread and aggressive Cuscuta species in the world. It causes a serious weed problem in many economic and ornamental crops. Its minutely sized, easily dispersed, and highly viable seed makes it difficult to control. Alternaria destruens has been developed as a registered bioherbicide for Cuscuta species. A host-range study using 30 plants of economic importance belonging to 8 families (Asteraceae, Convolvulaceae, Fabaceae, Malvaceae, Meliaceae, Poaceae, Rutaceae, and Solanaceae) indicated that A. destruens is pathogenic only to Cuscuta spp. In greenhouse trials of efficacy, seven treatments were applied: an untreated control; oil (PCC 588); A. destruens (Smolder WP); a mixture of A. destruens + oil; ammonium sulfate at 0.125% w/v in water; glyphosate at 0.02 kg (ae)/L of Roundup Pro ; and a mixture of A. destruens + oil, glyphosate at 0.02 kg (ae)/L of Roundup Pro and ammonium sulfate at 0.125%. By 35 days after treatment, all Cuscuta plants treated with the last treatment were dead but not the host plant, citrus. Efficacy of A. destruens was further assessed in field trials. Four treatments consisting of an untreated control, oil, A. destruens + oil, and A. destruens were tested, xii

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but the levels of control did not differ among the treatments. To determine if this lack of difference was due to pathogens dispersal, Alternaria isolates from randomly chosen Cuscuta tissue samples were collected from field and greenhouse trials and studied using nuclear rDNA internal transcribed spacer (ITS) region, including ITS1, ITS2, and the 5.8S rDNA. Neighbor-joining analysis revealed that all 47 unknown isolates and the reference A. destruens isolate were 100% identical. The results indicated a high degree of conservation of the ITS region in A. destruens which made it impossible to conclude that the isolates from field and greenhouse samples were the same as the reference isolate used in these trials. Overall, this study has confirmed the effectiveness of an integrated system consisting of A. destruens, glyphosate, and ammonium sulfate to control Cuscuta spp. xiii

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CHAPTER 1 LITERATURE REVIEW Taxonomy The name Cuscuta originates from the Arabic word kushkut, which loosely translates as a tangled wisp of hair (Austin 1980). When Cuscuta species (spp.) begin to cover a plant, a mass of tangled yellow to orange threads with various diameters and no leaves are produced. Cuscuta spp. are also known by various common names, such as dodder, love vine, tangle gut, devils gut, and strangleweed. The most common name, dodder, possibly originates from the Old German word dotter which means yolk (Dawson et al. 1994). The description of the genus Cuscuta L. was first published in 1700 by Tournefort. In 1932, Yuncker produced a worldwide monograph that divided the genus into three subgenera based on the morphology of stigmas and styles. The first subgenus, Monogyna (Englm.) Yunck., is the most distinctive and favors trees and shrubs as hosts (Dawson et al. 1994). This subgenus has one style that is either partially or totally joined with elongated to capitate stigma lobes (Guerra and Garcia 2004). The second subgenus, Grammica (Lour.) Yunck., contains two styles, capitate stigmas, and is the most diversified group with the majority of the species (Guerra and Garcia 2004). The last subgenus is Cuscuta, which is characterized by elongated stigmas and two styles (Guerra and Garcia 2004). This subgenus is currently under taxonomic revision (Garcia 2004). There is some controversy regarding the family in which to place the genus Cuscuta: either Convolvulaceae, or an entirely separate family Cuscutaceae. Based on 1

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2 recent molecular studies, Cuscuta should remain in Convolvulaceae (Garcia 2004). There are approximately 150 to 170 Cuscuta spp. worldwide; with the majority occurring in North and South America (Ashton and Santana 1976; Dawson et al. 1994). Biology and Host Range Cuscuta species are obligate parasites that predominate disturbed habitats, which preadapts them to becoming a serious weed problem (Figure 1-1). Cuscuta spp. are found all over the world and parasitize mainly dicotyledonous and a few monocotyledonous plants (Gaertner 1950; Dawson et al. 1994). Some species of Cuscuta are very host specific, while others have a broad host range. Economically important plants affected include alfalfa (Medicago sativa L.) (Dawson et al. 1984), rabbiteye blueberry (Vaccinium ashei) (Dawson et al. 1984; Monaco and Mainland 1981), carrot (Daucus carota L.) (CAB International 2005), citrus (Citrus spp.) (Ashton and Santana 1976), cranberry (Vaccinium macrocarpon Ait.) (Bewick et al. 1985), tomato (Lycopersicon esculentum L.) (Ashton and Santana 1976), potato (Solanum tuberosum L.) (Selleck et al. 1979), sugar beet (Beta vulgaris L.) (CAB International 2005), various ornamental, native, and many weed species (Dawson et al. 1984) (Figure 1-2). Due to the destructiveness and damaging effects of this parasitic plant, the Animal Plant Health and Inspection Service (APHIS) of the United States Department of Agriculture (USDA) has listed Cuscuta spp., other than the following species (Appendix A), as federal noxious weeds (Tasker 2006). All movement throughout the United States or interstate of these species is prohibited without a permit.

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3 Figure 1-1. Cuscuta pentagona on citrus The inability of Cuscuta spp. to parasitize many hosts may be due to various interactions. The tomato defense system induces genes within the early stages of haustorial development of Cuscuta reflexa Roxb. to stop parasitization (Werner et al. 2001). Cuscuta spp. growth on different hosts varies considerably (Nemli 1987). Cuscuta spp. stems may twine around the host plant, but may not fully develop. Therefore, the stems weaken and die. A hosts anatomical structure may delay the growth of the parasite, thereby, preventing the haustoria and hyphae from reaching the vascular bundles of the host (Dawson et al. 1994). Flowers produce seed capsules with 2 to 3 seeds, with each plant producing several thousand seeds (Ashton and Santana 1976) (Figure 1-2). Seed coats are very hard and newly matured seeds are impermeable to water (Dawson 1965; Gaertner 1950; Hutchinson and Ashton 1980). Scarification must take place in order for seeds to germinate, which in nature probably occurs through weathering, soil microbial action, or

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4 other natural disturbances. Seeds can remain dormant and viable in the soil for up to 60 years depending on environmental conditions and the species (Gaertner 1950; Hutchinson and Ashton 1980). Figure 1-2. Cuscuta pentagona flowers and buds (A and B) Seedlings contain minute amounts of chlorophyll (both and ) and are capable of photosynthesis at a low rate, but Cuscuta spp. are still dependent on the host to

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5 complete its life cycle (Ashton and Santana 1976; Dawson et al. 1994; Zimmermann 1962). In addition, virtually 100% of the carbon Cuscuta spp. use is acquired from the hosts phloem (Jeschke et al. 1994). Germination of seedlings occurs at temperature ranges between 15 o C and 38 o C with optimum temperatures in the range of 30 o C to 33 o C (Hutchinson and Ashton 1980). Once seedlings have emerged, elongation and circumnutation occurs. For seedling hooks to open and twining to occur, seedlings must have sunlight or incandescent light (Dawson et al. 1994). Once they find a vertical host, living or nonliving, they attach themselves counterclockwise and are no longer connected to the soil. If the object the seedling attaches to is a not a host or nonliving, the seedling will die after several days (Dawson et al. 1994). Within 2 to 4 days of attaching to a suitable host, Cuscuta spp. produce a haustorium (Dawson 1987) (Figure 1-3). Figure 1-3. Cuscuta pentagona haustorium (arrow) penetrating citrus The haustorium stops growing within the host tissue after 1 to 2 days and starts to form searching hyphae that expands intraand intercellularly along the middle lamellae

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6 of the host cell wall (Dawson et al. 1994). Searching hyphae may increase in length up to 800 m before connecting with the host plants xylem and phloem (Dawson et al. 1994; Vaughn 2003). In addition, the extensive growth may assist in fastening Cuscuta spp. securely to the host (Vaughn 2003). Survival of Cuscuta spp. occurs through extracting large amounts of translocated solutes from the phloem of the host. Assimilates and minerals, such as, sucrose, phosphorus, and potassium are transported in the phloem from source to sink (Ziegler 1975). Once Cuscuta spp. has parasitized a host, it may grow as much as 8 cm per day, with a single plant covering more than 3 m in diameter in one growing season (Dawson et al. 1984). Cuscuta spp. forms a dense, thick mat over the host, which can eventually kill the host. This is not only due to Cuscuta spp. transporting nutrients from the host, but also from blocking out the sun and decreasing the amount of photosynthesis that can take place in the host. Cuscuta pentagona Engelm. Cuscuta pentagona, also known as bush-clover dodder, field dodder, five-angled dodder, and lespedeza dodder, is the most widespread and aggressive Cuscuta species in the world (Holm et al. 1997; Lanini and Kogan 2005; Parker and Wilson 1986). In addition, this species is reported to be in 76 countries, provinces, and regions and in 46 United States (U.S.) (CAB International 2005; USDA, NRCS 2006). The broad range of this species occurs from Argentina and Chile through the U.S. to Canada and from the Cape of Good Hope almost to the Arctic in the Old World (Holm et al. 1997). Cuscuta pentagona is a serious weed problem of alfalfa in Australia, Hungary, South Africa, and the U.S.; cabbage (Brassica oleracea L.), peppers ( Capsicum spp. L.) and potatoes in Hungary and the U.S.; carrots and onions (Allium cepa L.) in

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7 Switzerland; legumes in Hungary and Pakistan; lentils (Lens culinaris Medik.) in Syria; and tomatoes, sugar beets, and other vegetable crops in the U.S. (Dawson et al. 1994; Holm et al. 1997). It is a significant weed problem of alfalfa in Saudi Arabia; carrots in Italy; and dryland and irrigated crops, including vegetables, in Australia (Holm et al. 1997). In addition to the crops listed previously, there are many others that C. pentagona parasitizes. These being forage legumes in Australia, Chile, India, Italy, and Jordan; beans (Phaseolus spp.), beets, peas (Pisum spp.), and various other vegetables in Canada, India, Israel, Japan, the Netherlands, Pakistan, and Switzerland; pastures in South Africa and Uganda; safflower ( Carthamus tinctorius L.) in Italy; and sugar beets in Chile and Turkey (Holm et al. 1997). Cuscuta spp. in the U. S. Currently, there are 49 species of Cuscuta found in the U.S., Puerto Rico, and the U.S. Virgin Islands (USDA, NRCS 2006). The most problematic species, other than C. pentagona, are C. approximata Bab. (smallseed alfalfa dodder or alfalfa dodder), C. epilinum Weihe (flax dodder), C. gronovii Willd. ex J.A. Schultes (swamp dodder) and C. indecora Choisy (large-seed dodder or bigseed alfalfa dodder). Cuscuta approximata has many varieties with a wide host range; it is found in 12 states, and was introduced into the U.S. (Parker and Riches 1993; USDA, NRCS 2006). Cuscuta epilinum Weihe has a very restricted host range, hence its common name of flax dodder, was introduced into the U .S. and is found in 9 states ( Parker and Riches 1993; USDA, NRCS 2006). Cuscuta gronovii is generally found in wet places and along water courses, is extremely invasive on cranberry, and is found in 44 states (Bewick et al. 1988; USDA, NRCS 2006). Cuscuta indecora mainly attacks plants in the Leguminosae, is

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8 native to the U.S., and is found in 35 states, Puerto Rico, and the U.S. Virgin Islands (Parker and Riches 1993; USDA, NRCS 2006). Cuscuta spp. Worldwide There are numerous other Cuscuta spp. found throughout the world that cause serious problems on forage legumes, herbaceous plants, shrubs, trees, and various crops. Cuscuta chinensis Lam. (Chinese dodder) and C. obtusiflora Kunth (Peruvian dodder) are mainly found in East Asia and Europe and are a major problem in soybeans in China (Li 1987; Parker and Riches 1993). Cuscuta epithymum (L.) L. (clover dodder) is found in 60 countries, provinces, and regions and in 26 U. S. states (CAB International 2005; USDA, NRCS 2006). This species is a serious weed problem in alfalfa, cabbage, carrot, peppers, potatoes, purple clover ( Trifolium pratense L.), and tomatoes ( CAB International 2005; Holm et al. 1997; Parker and Riches 1993). Furthermore, C. epithymum can be found parasitizing flax, orchards, ornamentals, and pastures (Holm et al. 1997). Cuscuta europaea L. (greater dodder) is found in 58 countries, provinces, and regions and in 2 U. S. states (CAB International 2005; USDA, NRCS 2006). Hosts of this species include broad bean (Vicia faba L.), common hop ( Humulus lupulus L.), onion, raspberry ( Rubus idaeus L.), and sugar beets (CAB International 2005). Cuscuta japonica Choisy (Japanese dodder) is an East Asian species that parasitizes woody and herbaceous hosts (Parker and Riches 1993). Cuscuta monogyna Vahl. (eastern dodder) is found mainly in the Mediterranean and Middle East, but extends from France through southern Europe, to Afghanistan (Parker and Riches 1993). This species is commonly found on citrus, olive ( Olea europaea L. ) and other fruit and vegetable crops. Cuscuta reflexa Roxb. (giant dodder) is an Asian species that ranges from India and the Himalayas, extending through Pakistan to Afghanistan, south to Sri Lanka, China, and

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9 into Indonesia (Parker and Riches 1993). This species mainly occurs on perennial trees and shrubs such as Arabica coffee (Coffea arabica L.), citron (Citrus medica Linn.), litchi (Litchi chinensis Sonn.), and peach (Prunus persica (L.) Batsch.) (Parker and Riches 1993). Economic Loss, Dispersal, and Control Extensive economic losses can occur from Cuscuta spp. infestations. Crops impacted in Florida include blackeyed peas (Vigna unguiculata (L.) Walp.), blueberry, eggplant (Solanum melongena L.), key lime ( Citrus aurantiifolia (Christm.) Swingle ), sweet orange (Citrus x aurantium L.), and tomato (Alfieri et al. 1994). Yield losses as high as 80% to 100% have been reported in cranberry fields in Massachusetts and Wisconsin due to Cuscuta spp. (Devlin and Deubert 1980). Approximately 30,000 acres of tomato crops in California are parasitized by Cuscuta spp. which can cause reduction in crop yield by over 75% if not controlled (Lanini 2005). In the U.S. Virgin Islands, C. americana L. (American dodder) infests many woody plants species, including citrus and many ornamental species. Timber crops infested with this species have increased costs and reduced income and yield. Furthermore, ornamental species parasitized by C. americana are either killed or seriously reduced in value. Dispersal of Cuscuta spp. is either by seeds or vegetative propagation. Seeds are small, but heavy and not adapted for wind dispersal (Dawson et al. 1984); they are primarily dispersed by humans (Dawson et al. 1994). Seeds are also easily dispersed through contaminated crop seed (Dawson et al. 1984). Cuscuta spp. is also easily transferable to other plants by vegetative propagation. Placement of actively growing Cuscuta spp. stems on host plants usually results in the formation of haustoria and thus, a new Cuscuta plant. In the U.S. Virgin Islands birds have been observed using Cuscuta

PAGE 23

10 spp. stem tissue, which can include seeds, in nest building (T. Zimmerman, UVI-AES-Biotechnology & Agroforestry, Kingshill, St. Croix, VI, personal communication). This is believed to aid in transport of Cuscuta spp. between trees. Cuscuta spp. can also be used as a living bridge to transport pathogens, such as plant viruses and mycoplasma-like organisms from one plant to another (Agrios 1997). Control of Cuscuta spp. is very difficult, with the best method being exclusion, i.e., not to introduce it into a farm or field. Mechanical removal by seed machines is an option, but these devices are often slow and they do not always remove all of the Cuscuta spp. seed and sometimes remove the crop seed. Tillage can be useful because the seedlings are easily dislodged from the soil or die when buried, but if they are not buried completely, they can resurface. Physically removing Cuscuta spp. by hand is time-consuming and although Cuscuta spp. generally grow as annuals, they can survive in the tissue of perennial host plants under some conditions (Dawson et al. 1984). Crop rotations with cereal grains or forage grasses for two or more years can significantly decrease Cuscuta spp. seed banks (Dawson et al. 1984). Seedlings emerge and die because they rarely will parasitize plants in the family Gramineae. Another cultural method that can be used to control Cuscuta spp. is shade. Formation of a canopy from heavy crop cover can suppress seedling emergence (Dawson 1966). Herbicides Soil-applied herbicides can be very effective in controlling Cuscuta spp. primarily because control occurs prior to attachment to host. The first soil-applied herbicide used was chlorpropham (1-methylethyl (3-chlorophenyl) carbamate), which was a plant growth regulator that terminated seedling growth prior to emergence. Unfortunately, this herbicide is no longer available for use in the U.S.

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11 Other preemergent herbicides available for Cuscuta spp. control are dichlobenil (2,6-dichlorobenzonitrile), pendimethalin (N-(1-ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzenamine), prodiamine (2,4-dinitro-N 3 ,N 3 -dipropyl-6-(trifluoromethyl)-1,3-benzenediamine), pronamide (3,5-dichloro-N(1,1-dimethyl-2-propynyl) benzamide), and trifluralin (a,a,a-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) (Nice 2005; Ristau 1996). Prodiamine and pendimethalin control Cuscuta spp. more efficiently than trifluralin (Dawson 1987; Dawson et al. 1994). Preemergent herbicides may not provide season-long control and repeated treatments may be necessary. Therefore, postemergent applications of imazamox (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-(methoxymethyl)-3-pyridinecarboxylic acid) or imazethapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid) may also be used but since these herbicides are used after attachment, success may be limited (Nice 2005; Ristau 1996). Foliar applications of diquat (6,7-dihydrodipyrido[1,2-a:2,1-c]pyrazinediium dibromide) or paraquat (1,1-dimethyl-4,4-bipyridinium dichloride) provide partial selectivity in alfalfa; Cuscuta spp. and the host plant foliage will be destroyed, but the host plant may regrow from crowns after treatment (Dawson 1987; Dawson et al. 1994). Other nonselective post-attachment herbicides that will provide complete kill to the host plant and Cuscuta spp. are 2,4-D ((2,4-dichlorophenoxy)acetic acid) or glyphosate (N-(phosphonomethyl)glycine) (Dawson et al. 1994). Preemergent herbicides labeled for citrus in Florida are diuron ( N '-(3,4-dichlorophenyl)N N -dimethylurea), norflurazon (4-chloro-5-(methylamino)-2-(3

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12 (trifluoromethyl)phenyl)-3(2 H )-pyridazinone), oryzalin (4-(dipropylamino)-3,5-dinitrobenzenesulfonamide), and thiazopyr (methyl 2-(difluoromethyl)-5-(4,5-dihydro-2-thiazolyl)-4-(2-methylpropyl)-6(trifluoromethyl)-3-pyridinecarboxylate) (Futch 2001). Postemergence herbicides are glyphosate, paraquat, and sethoxydim (2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one) (Futch 2001). Preplant incorporated herbicides labeled for peppers in Florida are bensulide ( O O -bis(1-methylethyl) S -[2-[(phenylsulfonyl)amino]ethyl]phosphorodithioate), napropamid ( N N -diethyl-2-(1-naphthalenyloxy)propanamide), and trifluralin (Stall and Gilreath 2005a). Preemergent herbicides applied are b ensulide, clomozone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone), which can be applied to all peppers except banana peppers ( Capsicum annuum L.), and paraquat (Stall and Gilreath 2005a). Halosulfuron (3-chloro-5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl] amino]sulfonyl]-1-methyl-1 H -pyrazole-4-carboxylic acid) can be applied in the row middle and sethoxydim can be applied postemergent (Stall and Gilreath 2005a). Preplant incorporated herbicides labeled for tomatoes in Florida are napropamid and trifluralin, while preemergent herbicides used are paraquat or pelargonic acid (nonanoic acid) (Stall and Gilreath 2005b). Herbicides used postemergent are clethodim (( E E )-()-2-[1-[[(3-chloro-2-propenyl)oxy]imino]propyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one), halosulfuron, metribuzin (4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4 H )-one), rimsulfuron ( N -[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide), and sethoxydim

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13 (Stall and Gilreath 2005b). Halosulfuron and rimsulfuron are also applied to row middles (Stall and Gilreath 2005b). Glyphosate Glyphosate, a phloem-mobile herbicide, moves in the symplast via source to sink (McAllister and Haderlie 1985). The primary mode of action of glyphosate is to inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; E.C. 2.5.1.19), which is the key enzyme in the shikimate pathway. Inhibition of EPSPS prevents the production of chorismate which is required for biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan (Amrhein et al. 1980; Geiger and Bestman 1990; Hollnder and Amrhein 1980). Dawson and Saghir sprayed glyphosate over the top of parasitized alfalfa, which resulted in injury and death to the Cuscuta spp. but not to the alfalfa (Dawson and Saghir 1983). The highest rate of glyphosate (0.30 kg per ha) provided sufficient delay for the parasitized alfalfa to recover prior to regrowth of Cuscuta spp. (Dawson and Saghir 1983). Even though all exposed shoots of Cuscuta spp. were killed by the glyphosate, some haustoria were still embedded within the alfalfa stems and regenerated. All alfalfa foliage in the control plots were destroyed by Cuscuta spp. (Dawson and Saghir 1983). Fer found that 14 C-labeled glyphosate was more concentrated in the apical part of the Cuscuta spp. shoots, especially in the buds, than in the haustorial coil (Fer 1984). Furthermore, at 4 days after treatment (DAT), Cuscuta spp. had absorbed 75% of the glyphosate from the hosts phloem. Fer concluded from these studies that phloem-mobile herbicides should be able to selectively control Cuscuta spp. (Fer 1984). Bewick et al. conducted studies with 14 C-labeled glyphosate on carrot parasitized with Cuscuta gronovii (Bewick et al. 1991). They determined that at 1 DAT Cuscuta

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14 tissue had absorbed as much 14 C-labeled glyphosate as any sink in the carrot. At 45 DAT, Cuscuta tissue contained most of the 14 C-labeled glyphosate in regard to all other physiological sinks, except the petiole of the treated carrot leaf (Bewick et al. 1991). Ammonium Sulfate Ammonium sulfate, (NH 4 ) 2 SO 4 is formed by reacting ammonia with sulfuric acid to form a stable ammonium salt. This compound is soluble in water and insoluble in liquid ammonia or alcohol. The toxicity of the ammonium ion enhances this compounds capacity for weed control (Brian 1976). The plant cell rapidly absorbs the ammonia and the cell sap, which is normally acidic, may become alkaline due to the effect of the ammonia (Harvey 1911). Rapid cell death ensues from increased alkalinity and from the toxicity of ammonia (Brian 1976). Excess concentrations of ammonium ions or ammonia are toxic to plants, although the exact biochemical causes of toxicity are not understood (Given 1979; Smith and Vandenn Born 1992, Young et al. 2003). Several theories have been proposed as to how ammonium sulfate increases herbicide absorption. Studies conducted with tomato fruit, which has a well-developed cuticle, confirmed large increases in uptake when sethoxydim was applied with ammonium sulfate (Wanamarta et al. 1993). The cuticle is a barrier to sethoxydim penetration, but with the addition of ammonium sulfate the capability of the cuticle to block herbicide absorption was reduced. Ammonium salts of bentazon (3-(1-methylethyl)-(1 H )-2,1,3-benzothiadiazin-4(3 H )-one 2,2-dioxide) formed from the addition of ammonium sulfate were more readily absorbed and demonstrated increased transcuticular movement compared with calcium, magnesium, or sodium salts of bentazon (Wanamarta et al. 1993). Ammonium sulfate is related to increased permeation of the herbicide through the plasma membrane (Young et al. 2003). Several researchers

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15 have hypothesized that the acidification of the spray solution by the addition of ammonium sulfate allows a greater proportion of weak-acid herbicides to remain in their nondissociated form, which allows the herbicide to diffuse across the plasma membrane more easily (Gronwald et al. 1993; Smith and Vanden Born 1992). When the herbicide enters the cell, the higher pH of the cytoplasm causes dissociation of weak-acid herbicides, thus efficiently trapping the herbicide inside the cell (Young et al. 2003). Ammonium sulfate is also used as a surfactant to increase activity in many herbicides, including glyphosate (Nalewaja and Matysiak 1993; OSullivan et al. 1981; Turner and Loader 1975). Hard water contains polyvalent cations that antagonize the activity of glyphosate (Nalewaja and Matysiak 1991, 1993). Thelen et al. (1995) determined that the glyphosate molecule reacts with Ca ++ and other cations to form a less-readily absorbed glyphosate-Ca salt that decreases herbicidal activity. When ammonium sulfate is added into the tank mix, the ammonium ion binds with the glyphosate molecule, preventing the formation of glyphosate-Ca salt, which in turn increases herbicidal absorption (Gronwald et al. 1993; Pratt et al. 2003). In addition, ammonium sulfate alters spray droplet morphology by delaying or preventing the crystallization of glyphosate on the leaf surface, thereby allowing more time for glyphosate to penetrate the cuticle (MacIsaac et al. 1991). Alternaria destruens Simmons, sp. nov. The genus Alternaria Nees was first described in 1816 with the type species A. tenuis Nees, which was later redescribed as A. alternata (Fr.:Fr.) Keissl. (Simmons 1967). Taxonomically, Alternaria falls under mitosporic fungi (anamorphic fungi) and has no known sexual stage (Noyd 2000). The majority of Alternaria spp. are saprophytic, but some species are pathogenic and produce a range of plant diseases

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16 throughout the world. Depending on the pathogen, symptoms typically appear as blights and leaf spots, but damping-off of seedlings, and fruit, stem or tuber rots can also occur (Agrios 1997). Simmons (1992) has described over 100 species of Alternaria worldwide. However, the number of described species fluctuates due to other taxonomists merging several existing species of Alternaria into one species or by taking one species and dividing it into several different species (Rotem 1994). These differences are most likely due to errors of taxonomic placement due to variability of morphological characteristics under a variety of environmental conditions (Thomma 2003). The most important conidial characteristic in identifying a species is spore (conidium) dimension, including that of the beak (Rotem 1994). Spore length varies significantly between species, while spore width is similar. However, spore length, including the beak, can vary within the same species causing an overlap. This is due to a number of factors, but most commonly by the type of substrate. Therefore, most taxonomists give measured dimensions in ranges rather than as means and provide the substrate on which the fungus was grown (Rotem 1994). In 1984, Bewick et al. isolated an Alternaria spp. from a diseased C. gronovii plant growing in an uncultivated marsh in Wisconsin (Bewick et al. 1986). In subsequent field studies, this pathogen was shown to provide 92% control of C. gronovii (Bewick et al. 1987). No disease was observed on, nor was the pathogen recovered from any of the host plants of dodder. In 1990, this pathogen was patented as a bioherbicide for suppression of Cuscuta spp. (Bewick et al. 2000). In 1998, Simmons identified this isolate as belonging to Alternaria destruens. Simmons described A. destruens as a small

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17 spored (25-40 (46) x 10-13 m), brevicatenate (each conidiophore initially produces a single unbranched chain of 4-8 conidia, which may increase to 10-12 conidia with age) species in which interspersed secondary condiophores range from extremely short to exceedingly long, (3-) 20-80 m, in a single chain (Simmons 1998) (Figure 1-4) Figure 1-4. Alternaria destruens spores Biological Control The use of herbicides to control and manage weed populations may not be conducive to all situations. Therefore, other means of control are necessary, such as biological control. Biological control is grouped into three strategies: the classical approach, the augmentative approach, and the inundative approach (Rosskopf et al. 1999). The classical approach or inoculative method involves applying small doses of

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18 inoculum over a large weed population to produce a spreading epidemic (Charudattan 1988). This approach is beneficial when exotic plants become problematic due to the absence of natural enemies and herbicide usage is not cost effective (Rosskopf et al. 1999). In the augmentative approach, a small amount of inoculum is released to initiate an epidemic (Charudattan 1988). In the inundative or bioherbicide approach, inoculum is applied in massive doses to the weed population to stimulate epidemic conditions (Harley and Forno 1992; Scheepens et al. 2001). Bioherbicides are based on fungi, bacteria, or viruses that are industrially developed and registered through the U.S. Environmental Protection Agency to manage or reduce weed populations. The mycoherbicide (a fungus used as a herbicide) approach can be successful when substantial quantities of inoculum are used to help compensate for natural restrictions to disease development (Bewick et al. 1987; Charudattan 1988). Through manipulation, some fungi that normally cause sporadic or endemic disease levels on their hosts can be developed into effective bioherbicides. (Charudattan 1988; Rosskopf et al. 1999). Research Objective Current management methods are insufficient for controlling dodder. Therefore, the main objectives of this research were (1) to determine the host range of A. destruens, a bioherbicide agent, in greenhouse trials using plants of importance to Florida, the U.S. Virgin Islands, and the Caribbean in general; (2) to evaluate the effect of A. destruens on C. pentagona in the field; (3) to determine whether A. destruens would spread from a point source to surrounding uninfected C. pentagona plants; (4) to use internal transcribed spacing regions to differentiate unknown Alternaria isolates from our reference isolate of A. destruens; (5) to determine the capability of A. destruens spores to be dispersed through a simulated wind tunnel; (6) to evaluate the effects of various

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19 concentrations of glyphosate (Roundup Pro ) and ammonium sulfate on the growth of A. destruens on Citrus spp.; and (7) develop an integrated control method for C. pentagona using glyphosate, ammonium sulfate, and the biological control agent A. destruens.

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CHAPTER 2 EVALUATION OF THE HOST RANGE OF ALTERNARIA DESTRUENS (SMOLDER ) Simmons (1992) has described over 100 species of the genus Alternaria Nees worldwide. While the majority of Alternaria species are saprophytic, many are pathogenic and can cause a multitude of plant diseases over a wide host range. Some species of Alternaria have a restricted host range, such as, A. solani Sorauer which causes early blight only on potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.) and Alternaria leaf spot on Jerusalem cherry (Solanum pseudocapsicum L.) (APSnet 2006). While other species of Alternaria have a wide host range such as A. alternata that causes Alternaria blight on chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.), pea (Pisum sativum L.), and pigeonpea (Cajanus cajan (L.) Millsp.); Alternaria fruit spot on papaya (Carica papaya L.); Alternaria leaf spot on African daisy (Gerbera jamesonii H. Bolus ex J. D. Hook), almond (Prunus dulcis (Mill.) Webb), asparagus (Asparagus officinalis L.), beet (Beta vulgaris L.), garden dalia (Dahlia spp.), mango (Mangifera indica L.), and rose (Rosa spp.); Alternaria rot on apple (Malus domestica Borkh.) and grape (Vitis spp.); Alternaria spot and fruit rot on apricot (Prunus armeniaca L.); Alternaria spot and veinal necrosis on peanut (Arachis hypogaea L.); and seed mold on red clover (Trifolium pratense L.) (APSnet 2006). Alternaria spp. have also been developed as biological control agents or bioherbicides, which are based on fungi, bacteria, or viruses that are industrially developed and registered through the United States Environmental Protection Agency to 20

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21 manage or reduce weed populations. The mycoherbicide (fungi used as herbicides) approach can be successful when substantial quantities of inoculum are used to help compensate for natural restrictions to disease development (Bewick et al. 1987). By using certain adjuvants, such as humectants, uv-protectants, and other materials that can alter the microclimate on leaf surfaces some fungi that normally cause sporadic or endemic disease levels on their hosts can be used efficiently as weed-control agents (Charudattan 1988; Rosskopf et al. 1999). Alternaria eichhorniae Nag Raj and Ponnappa is a pathogen of waterhyacinth (Eichhornia crassipes (Mart.) Solms), which is one of the worlds worst aquatic weeds (Holm et al. 1977). This weed creates serious problems throughout much of the tropical and subtropical regions of the world where it affects water flow, water quality, navigation, and human health and wellbeing. Shabana et al. reported that A. eichhorniae is a safe and effective bioherbicide for control of waterhyacinth (Shabana et al. 2001; Shabana et al. 1997). Another potential mycoherbicide that is being evaluated for waterhyacinth is A. alternata (Fr.:Fr.) Keissl. (Babu et al. 2004). This pathogen was isolated from diseased waterhyacinth; but in India, where this weed also causes severe problems (Babu et al. 2003). Another bioherbicide, A. cassiae Jurair and Khan, was isolated from diseased sicklepod (Cassia obtusifolia L.) and was found to have a restricted host range of coffee senna (Cassia occidentalis L.), showy crotalaria (Crotalaria spectabilis Roth) and sicklepod (Walker 1982). This bioherbicide effectively reduced sicklepod plants 95-100% in field trials (Walker and Riley 1982). In 1984, Bewick et al. isolated an Alternaria spp. from a diseased Cuscuta gronovii Willd. ex J.A. Schultes (swamp dodder) plant growing in an uncultivated marsh

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22 in Wisconsin (Bewick et al. 1986). Subsequent field studies with this pathogen resulted in 92% control of C. gronovii (Bewick et al. 1987). No disease was observed or recovered on any of the host plants that the Cuscuta had parasitized. Thus, the potential of Alternaria destruens as a biological control agent for C. gronovii was established. In 1990, the Alternaria sp. was patented as a bioherbicide for suppression of Cuscuta spp. (Bewick et al. 2000). In 1998, Simmons identified this isolate as belonging to Alternaria destruens. Simmons described A. destruens as a small-spored (25-40 (46) x 10-13 m), brevicatenate (each conidiophore initially produces a single unbranched chain of 4-8 conidia, which may increase to 10-12 conidia with age) species in which interspersed secondary condiophores range from extremely short to exceedingly long, (3-) 20-80 m, in a single chain (Simmons 1998). Alternaria destruens has been shown to have a restricted host range, but more information is necessary, especially in regard to crop plants grown in Florida and the U.S. Virgin Islands where it is proposed to be used to control Cuscuta spp. Host range or host-specificity testing is conducted to determine if a plant pathogen, which is being considered for a biological control agent, can develop under optimal conditions for disease development on a group of plants closely related to the target weed species (Harley and Forno 1992; Rosskopf et al. 1999). This group of plants should also include closely related crop plants, native, and weed plant species (Rosskopf et al. 1999). Alternaria destruens has been field-tested in crops such as alfalfa, carrot, celery, cranberry, potato, and spearmint (Bewick et al. 1987), but further testing is needed to establish the effectiveness of this biocontrol agent as a control for Cuscuta in diverse crops of other geographical areas. Therefore, the objective of this study was to determine

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23 the host range of A. destruens in greenhouse trials using plants of importance to Florida, the U.S. Virgin Islands, and the Caribbean in general. Materials and Methods Test Plants A total of 30 plants of economic and commercial importance comprising 19 species and 8 families (Asteraceae, Convolvulaceae, Fabaceae, Malvaceae, Meliaceae, Poaceae, Rutaceae, and Solanaceae) were chosen from Florida and the U. S. Virgin Islands. All studies were conducted in greenhouses at the University of Florida in Gainesville from the spring of 2002 through the fall of 2002. All plants except Citrus spp. (Smooth Flat Seville 7 months old), Hibiscus rose-sinensis (Brilliant Red Hibiscus approximately 2 to 4 years old), Ipomoea batatas (Puerto Rican Bunch Sweet Potato seedlings), and Swietenia mahagoni (Mahogany 3 years old) were either seeded directly or seeded in plastic trays (53 cm x 27 cm x 6 cm) and then transplanted into 9.5 cm x 9.5 cm plastic pots. Each pot contained one or two plants depending on the size and growth habit of the species. All seeds were grown in a commercial potting medium (Metro-Mix 300; Sun Gro Horticulture Canada Ltd., Seba Beach, AB Canada) and fertilized with Rainbow Premium Plant Nutrients (Royster Clark, Inc., Mulberry, FL). Plants were then placed in a greenhouse (35/29 + 5 o C day/night temperature) for 4 weeks and rated weekly. Light intensity was measured in the greenhouse with a quantum/radiometer/photometer (LI-COR Model LI-185B, Lincoln, NE) and ranged from 13,500,000 lux. Inoculation and Treatments Three treatments consisting of an untreated control, oil (PCC 588; United Agri Products, Greeley, CO), and A. destruens + oil were applied to each plant species. Each

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24 study was conducted in a randomized complete block design with 4 replications and was repeated once. A container of A. destruens spores (Smolder WP) was supplied by Sylvan Bioproducts, Inc., Kittanning, PA. Spores (1.28 g) were stirred with 55.5 ml of water for 15 min. Oil (PCC 588), at 7.5%, was added to the spore mixture and mixed for another 15 min. Spore counts were determined to be 1.8 x 10 5 spores per ml in the spray mixture. When using oil alone, the same mixing procedure was followed with oil added to 55.5 ml of water. All treatments were used within 1 h of mixing. All plants were inoculated with a hand-held sprayer at 100 ml per m 2 until the excess liquid ran off the foliage. After inoculation, plants were placed at 26C for 16 h in a dew chamber (100% relative humidity) and then moved to the greenhouse. Plants were rated weekly for 4 weeks. Disease Rating Symptoms were assessed on a scale of 1 to 10, with 1 being no symptoms and 10 being a dead plant. Plants that were rated at 1 to 2 were considered immune (none or few leaf spots observed; slight stunting), plants rated at 3 to 6 resistant (hypersensitive response (HR); 25% of the leaf covered in spots; stunting), and plants rated at 7 to 10 susceptible (severe stunting; blighted; plant death). Plants exhibiting HR developed small necrotic spots or lesions on the leaves. These spots initially would be small, black, circular spots that slowly developed into larger irregular lesions. The host reactions were determined by comparing the A. destruens + oil treatment reaction with oil alone and the untreated control.

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25 Results and Discussion Fourteen plants in seven families (Convolvulaceae, Fabaceae, Malvaceae, Meliaceae, Poaceae, Rutaceae, and Solanaceae) were found to be immune to A. destruens + oil (Table 2-1). These species were Capsicum annuum (Hungarian Hot Wax Pepper), Capsicum frutescens (Tabasco Pepper), Citrus sp. (Smooth Flat Seville), Crotalaria juncea (Tropic Sun Sunn hemp), Hibiscus rose-sinensis (Brilliant Red Hibiscus), Ipomoea batatas (Puerto Rican Bunch Sweet Potato), I. nil (Scarlet OHara Morningglory), I. tricolor (Heavenly Blue Morningglory), I. x imperialis (Rose Silk Morningglory), Leucaena collinsii (Chalip), L. diversifolia (Wild Tamarind), L. retusa (Littleleaf Leadtree), Swietenia mahagoni (Mahogany), and Zea mays (Florida Stay Sweet Corn). Sixteen plants in five families (Asteraceae, Convolvulaceae, Fabaceae, Poaceae, and Solanaceae) were found to be resistant (Table 2-1). These species and cultivars were Lactuca sativa (Bibb Lettuce), L. sativa (Black Seeded Lettuce), L. sativa (Iceberg Lettuce) L. sativa (Green Ice Lettuce), I. nil (Minibar Rose Morningglory), L. shannonii, Sorghum bicolor var. A3TX398/TX398, Capsicum annuum (California Belle Pepper), C. annuum (Capistrano Pepper), C. annuum (Jalapeno M Pepper), Capsicum chinense (Habanero Red Pepper), C. chinense (Scotch Bonnet Pepper), Lycopersicon esculentum (Agriset 761 Tomato), L. esculentum (Beefsteak Tomato), L. esculentum (Florida 47 Tomato), and L. esculentum (Florida 91 Tomato). No plants were found to be susceptible. Symptoms on pepper cultivars varied. Slight stunting from oil and A. destruens + oil was observed on California Belle in addition to an HR from A. destruens + oil. Plants that developed an HR would develop small necrotic spots or lesions on the leaves.

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26 Stunting and phytotoxicity from oil and stunting and initial HR from A. destruens + oil were observed on Capistrano and Jalapeno M. Habanero and Scotch Bonnet each exhibited stunting and initial phytotoxicity from oil and A. destruens + oil, which also exhibited an initial HR (Figures 2-1 and 2-2). Only an initial HR from A. destruens + oil was observed on Tabasco and no symptoms were observed on the Hungarian Hot Wax peppers. All tomato cultivars reacted with similar symptoms. An HR from A. destruens + oil was observed in all cultivars, while only Florida 47 exhibited slight stunting from oil and A. destruens + oil. All lettuce cultivars also exhibited an HR to A. destruens + oil in the first study. However, when this study was repeated, only Iceberg and Green Ice exhibited an initial HR to A. destruens + oil (Figure 2-3). Morningglory symptoms also varied between cultivars. Minibar Rose exhibited a slight HR from A. destruens + oil. Heavenly Blue and Rose Silk exhibited stunting from A. destruens + oil, while no symptoms were observed in Scarlet OHara. Foliar streaking from the oil was observed on the sweet corn, but disappeared as the plants grew. In addition, slight stunting was caused by oil and A. destruens + oil. Initial stunting was also observed on the sunn hemp from oil and A. destruens + oil. Sorghum plants initially exhibited stunting and phytotoxicity from oil and an HR from A. destruens + oil. All Leucaena species exhibited initial phytotoxicity symptoms from oil and A. destruens + oil. Only L. shannonii exhibited an HR to A. destruens + oil. Stunting was also observed in all four species inoculated with oil and A. destruens + oil, although, L. retusa only exhibited stunting from oil.

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27 Oil, either alone or with A. destruens, caused foliar streaking, phytotoxicity, or stunting (Table 2-1), on the majority of the plant cultivars, while A. destruens + oil caused mostly HR. New growth on plants initially exhibiting an HR, stunting, or phytotoxicity from either oil or A. destruens + oil subsequently grew healthy and without symptoms. This level of phytotoxicity followed by normal growth would be acceptable for most growers of fruits and vegetables, but not for those crops where the leaves are the produce, i.e. lettuce. Therefore, A. destruens would need to be mixed with another surfactant that produces less phytotoxic results, especially on the first set of leaves. These results, in addition to studies conducted by Bewick et al. (1987), reveal that A. destruens is not likely to pose a risk to nontarget plants. In addition, this demonstrates that A. destruens has a restricted host range and is safe for use on crop plants in Florida, the U.S. Virgin Islands, and the Caribbean in general. Figure 2-1. Capsicum chinense Habanero pepper plants. Control plant is normal with no visible damage, oil plant exhibited phytotoxicity, and Alternaria destruens + oil plant exhibited slight phytotoxicity and HR.

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28 Figure 2-3. Lactuca sativa Green Ice lettuce plants. Control and oil plants are normal with no visible damage and Alternaria destruens + oil plant exhibited only an HR. Figure 2-2. Capsicum chinense Scotch Bonnet pepper plants. Control plant is normal with no visible damage, oil plant is slightly stunted, and Alternaria destruens + oil plant exhibits phytotoxicity and HR.

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Table 2-1. Reaction of test plants to Alternaria destruens applied as SMOLDER formulated with PCC588 (United Agri Products, Greeley, CO). 29 Family Latin Name Cultivar/Variety Common Name Overall Host Reaction a Host Reaction to Oil b Asteraceae Lactuca sativa L. Bibb Lettuce R PH L. sativa L. Black Seeded Lettuce R PH L. sativa L. Iceberg Lettuce R PH L. sativa L. Green Ice Lettuce R PH Convolvulaceae Ipomoea batatas L. (Lam) Puerto Rican Bunch Sweet Potato I NR I. nil (L.) Roth Minibar Rose Morningglory R NR I. nil (L.) Roth Scarlet OHara Morningglory I NR I. tricolor L. HeavenlyBlue Morningglory I ST I. x imperialis (L.) Roth Rose Silk Morningglory I ST Fabaceae Crotalaria juncea L. Tropic Sun Sunn Hemp I ST Leucaena collinsii Britton & Rose Chalip I ST, PH L. diversifolia (Schltdl.) Benth. Wild Tamarind I ST, PH L. retusa Benth. Littleleaf Leadtree I ST, PH L. shannonii J. D. Smith R ST, PH Malvaceae Hibiscus rose-sinensis L. Brilliant Red Hibiscus I NR Meliaceae Swietenia mahagoni (L.) Jacq. Mahogany I NR Poaceae Zea mays L. Florida Stay Sweet Corn I FS, ST Sorghum bicolor var. A3TX398/TX398 Sorghum R ST, PH Rutaceae Citrus sp. SmoothFlatSeville Citrus I NR

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30 Solanaceae Capsicum annuum L. California Belle Pepper R ST C. annuum L. Capistrano Pepper R ST, PH C. annuum L. Hungarian Hotwax Pepper I NR C. annuum L. Jalapeno M Pepper R ST, PH Capsicum chinense Jacq. Habanera Red Pepper R ST, PH C. chinense Jacq. Scotch Bonnet Pepper R ST, PH Capsicum frutescens L. Tabasco Pepper I PH Lycopersicon esculentum L. Agriset 761 Tomato R NR L. esculentum L. Beefsteak Tomato R NR L. esculentum L. Florida 47 Tomato R ST L. esculentum L. Florida 91 Tomato R NR a I = Immune (plants that were rated at 0 to 2 (none or few leaf spots observed; slight stunting)); R = Resistant (plants rated at 3 to 6 (hypersensitive response (HR); fourth of leaf covered in spots; stunting)); S = Susceptible (plants rated at 7 to 10 (severe stunting; blighted; plant death)). b FS = Foliar streaking; NR = no reaction; PH = phytotoxicity; ST = stunting

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CHAPTER 3 FIELD EFFICACY OF ALTERNARIA DESTRUENS (SMOLDER ) AS A BIOHERBICIDE OF CUSCUTA The genus Alternaria Nees contains plant pathogens that have been recorded in almost every country in the world (Rotem 1994). Some of these pathogens produce diseases that can be quite devastating. One such pathogen, A. alternata (Fr.:Fr.) Keissl., causes Alternaria brown spot of tangerine (Citrus reticulata), tangerine hybrids, and tangelos (C. reticulata x C. paradisi) (Peever et al. 2004; Timmer et al. 2003; Timmer et al. 2000). This pathogen was first reported on Emperor Mandarin in Australia in 1903 (Cobb 1903). Currently, Alternaria brown spot is found in Argentina, Brazil (Peres et al. 2003), Israel (Solel 1991), Peru (Marin et al. 2006), South Africa (Schutte et al. 1992), Spain (Vicent 2000), Turkey (Canihos et al. 1997) and the U.S. (Whiteside 1976). The Alternaria spp. disease cycle contains no teleomorph (Timmer 1999) and conidia are produced primarily on mature or senescent leaves during high humidity or when leaves are slightly wet (Timmer et al. 2003). Conidial release is triggered by sudden changes in relative humidity or rainfall and the optimum temperature for infection is 27 o C (Canihos et al. 1999; Timmer et al. 1998). Infection takes place on young fruit, leaves, and twigs with symptoms appearing in 24 h or less (Timmer et al. 2003). Infected fruit will form brown to black lesions which vary in size, infected twigs will die back, and infected young shoots will form brown lesions 1 to 10 mm in diameter (Timmer et al. 2003). The fruit will either abscise or contain blemishes, which will greatly reduce yield and marketability (Timmer et al. 2003). 31

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32 An Alternaria species that has the potential to cause devastation on just one weed was isolated from diseased Cuscuta gronovii Willd. ex J.A. Schultes plants growing in an uncultivated marsh in Wisconsin (Bewick et al. 1986) and was identified as Alternaria destruens Simmons, sp. nov. (Simmons 1998). This fungus disease or damage progress was found to be highly pathogenic and in field studies it yielded 92% control of C. gronovii (Bewick et al. 1987). In 1990, this pathogen was patented as a bioherbicide for suppression of Cuscuta spp. (Bewick et al. 2000). Cuscuta species are obligate parasites that predominate disturbed habitats, which preadapts them to becoming a serious weed problem. There are approximately 150 to 170 Cuscuta species worldwide; with the majority occurring in North and South America (Ashton and Santana 1976; Dawson et al. 1994). Cuscuta spp. parasitize mainly dicotyledonous and a few monocotyledonous plants (Gaertner 1950; Dawson et al. 1994). Reports of anecdotal evidence of rapid spread of A. destruens in the field have led researchers to speculate about the dispersal capability of this pathogen (T. A. Bewick, USDA-CSREES-NPS, Washington, DC, personal communication). Therefore, the objectives of this study were (1) to evaluate the effect of A. destruens on C. pentagona in the field and (2) to determine whether A. destruens would spread from a point source to surrounding uninfected C. pentagona plants. Materials and Methods Ft. Pierce Field Study Field trials were conducted at the Header Canal Alternative Cropping Systems Research Farm in Ft. Pierce, FL from December 2003 through April 2004. Tropic Sun Sunn hemp (Crotalaria juncea L.) seeds were sown in early November 2003 directly into the field. Seeds were obtained from Peaceful Valley Farm Supply, Grass Valley, CA. At

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33 the time of inoculation, both the Sunn hemp and C. pentagona, the latter naturally present in this field, were at the flowering stage. This species of Cuscuta flowers within days after attachment to a host and had parasitized the Sunn hemp throughout the field. The experiment was conducted as a randomized complete block design with eight replications and was repeated once. A total of 32 plots (1 m 2 ) were randomly distributed throughout the 0.39-acre (Figure 3-1). Four treatments consisting of an untreated control, oil (PCC 588; United Agri Products, Greeley, CO), A. destruens + oil, and A. destruens were applied with 8 replications per treatment. A. destruens spores (Smolder WP) were supplied by Sylvan Bioproducts, Inc., Kittanning, PA. Spores (19.26 g) were stirred with 832.45 ml of water for 15 min. Spore counts were determined to be 1.8 x 10 7 spores per ml in the spray mixture. Oil (PCC 588), at 7.5%, was added to the spore mixture and mixed for another 15 min. When using oil or A. destruens alone, the same mixing procedure was followed as previously stated plus 832.45 ml of water. The plots were inoculated with a hand-held sprayer at 100 ml per m 2 and the treatments were applied within 1 h of mixing the spores. The field trial was repeated in an adjacent field and was initiated 3 weeks later. Gainesville Field Study A simulated field study was conducted outdoors at the University of Florida in Gainesville from July 2005 through August 2005 (Figure 3-2). Five-month old Citrus spp. (Smooth Flat Seville; Phillip Rucks Citrus Nursery, Frostproof, FL) in 1-gallon pots were parasitized with C. pentagona. At the time of treatment, the Cuscuta was approximately 1-month old and flowering.

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34 Two treatments consisting of an untreated control and A. destruens + oil (PCC 588; United Agri Products, Greeley, CO) were applied. Spores (1.28 g) were stirred with 55.5 ml of water for 15 min before oil (PCC 588) at 7.5% was added, and the spore mixture was mixed for another 15 min. The 10 treated plants were positioned in a circle then inoculated, and then the remaining plants were placed into two outer circles (Figure 3-2). This was done to decrease chances of initial contamination and spread of A. destruens to the control plants. Plants were placed in saucers, which were watered regularly. The experimental design consisted of three concentric circles with 55 pots (Figure 3-3). The distances from the center pot were as follows: the first circle was 0.91 m; the second circle was 2.13 m; and the third circle was 3.35 m (Figure 3-4). Each pot was spaced 0.63 m in the first circle, 0.79 m in the second circle, and 0.76 m in the third circle. The first circle and center pot, which contained 10 plants, were the treated area, while the second and third circles had the untreated sentinel plants. This study was conducted once. Disease Rating The Ft. Pierce trials were observed and rated weekly, while the Gainesville study was rated every 5 days for disease symptoms. Disease symptoms as well as phytotoxic damage from the oil were expressed as necrotic spots or blight on dodder. Hence, there was no need to use different rating scales or systems and both the disease and damage were assessed on a scale of 1 to 10, with 1 being no symptoms and 10 being a dead plant. Plants that were rated at 1 to 2 were considered immune (none or few leaf spots observed; slight stunting), plants rated at 3 to 6 resistant (hypersensitive response (HR); 25% of the

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35 leaf covered in spots; stunti ng), and plants rated at 7 to 10 susceptible (severe stunting; blighted; plant death). Plants exhibiting HR developed small necrotic spots or lesions on the leaves (Figure 3-5). All data were subjected to analysis of variance (ANOVA) and means were separated using Fishers prot ected least significant differe nce (LSD) at P= 0.05 in SAS (SAS 1999). In addition, all data were analyzed using Area Under the Disease or Damage Progress Curve (AUDPC), which is used to quantify the progress of disease or damage over time by integrating all factors of an epidemic, such as, environment, host, and pathogen effects (Campbell and Madden 199 1). AUDPC values for each trial were calculated using the formula [(y i + y ( i+1) )/2]*(t ( i+1) +1t i ), where i = 1, 2, 3, n-1, where y i is the disease or damage severity at the i th evaluation, and t i is time at the i th evaluation (Shaner and Finney 1977). AUDPC va lues were subjected to analysis of variance (ANOVA) and means separated by Tu key's Studentized Range (HSD) test at P=0.05 in SAS (SAS 1999).

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36 Figure 3-1. Header Canal Alternative Cropping Systems Research Farm in Ft. Pierce, FL. Each of the 32 m 2 plots were indicated with 2 sticks with red ribbon. Plots were changed for Trial II.

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37 Figure 3-2. Simulated field study at the University of Florida in Gainesville. Fifty-five pots of Citrus spp. parasitized with Cuscuta pentagona were placed in three concentric circles.

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38 Figure 3-3. Experimental design of simulated field study containing 55 pots of Citrus spp. parasitized with Cuscuta pentagona. The black pots in the middle were treated with A. destruens + oil (PCC 588) and the white pots were untreated controls. Figure 3-4. Distances used in simulated field study. White pots were 0.91 m from the center pot and spaced 0.63 m apart. Grey pots were 2.13 m from the center pot and spaced 0.79 m apart. Black pots were 3.35 m from the center pot and spaced 0.76 m apart.

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39 Figure 3-5. Citrus spp. plants parasitized by Cuscuta pentagona, the latter displaying symptoms of treatments with Alternaria destruens + oil and an untreated control. 0 = no symptoms; 1 = 1-10% (tip necrosis; stems starting to wilt and become necrotic; 2 = 11-35% (slightly more stem necrosis; flowers starting to senesce), 3 = 36-65% (over half of the stems are dead or dying; clusters of flowers senescing); 4 = 66-90% (the majority of the stems and flowers are dead or dying; some healthy flowers and stems may still be present), and 5 = 91-100% (plant death). Results and Discussion Ft. Pierce Field Study Symptoms of necrosis were observed on C. pentagona one week after inoculation (WAI) in both trials. Due to significant differences between trials (P=0.0019), data were analyzed separately. Within each trial, the effects of treatment for trial I (P=0.0001) and trial II (P=0.0124), and time (P=0.0001) were significant.

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40 Trial I had significant treatment differences during the third (P=0.0217) and fourth (P=0.0061) WAI where A. destruens (Fungus) and A. destruens + oil (Fungus + oil) and oil and the untreated control were not significantly different from each other. Ratings of disease severity on C. pentagona from A. destruens and A. destruens + oil were 3.8 and 4.2, respectively, while the phototoxic damage rating or natural senescence, respectively, from the oil and the untreated control were 2.1. By the fifth WAI, the untreated control was not significantly different from the fungal treatments. At 6 WAI (P=0.0767), there were no differences between any treatments and disease severity ratings ranged from 3.9 to 4.7 (Figure 3-6). Trial II had no significant differences among treatments except for A. destruens at 6 WAI (P=0.056) where the disease severity of C. pentagona reached 4.4 (Figure 3-7). At 7 WAI, the level of disease incidence had reached an average of 4.1 in all treatments. Regrowth of C. pentagona at 8 WAI was apparent in eight plots, which was almost half as much as in trial I, where regrowth was evident in 15 plots. AUDPC values for trial I indicated that there were no significant differences between A. destruens + oil (186.38) and A. destruens (179.38) (Table 3-1). Nor were there significant differences between A. destruens and the untreated control (143.06) (Table 3-1). AUDPC values for trial II had no significant differences among the treatments (Table 3-2). Gainesville Field Study Symptoms of disease on C. pentagona were observed 5 days after inoculation (DAI) on all treated plants. Likewise, by 5 DAI untreated controls also started to exhibit signs of disease. When comparing all three circles together, DAI and distance were both

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41 significant (P=0.0001). Therefore, the circles were compared with one another to determine if there were any differences among them. DAI was significant (P=0.0001) in circles I (0.91 m) and III (3.15 m) (Figure 3-8); while circles I (0.91 m) and II (2.13 m) had a DAI and distance interaction (P=0.0214) (Figure 3-9). In circles II (2.13 m) and III (3.15 m), both DAI and distance were both significant (P=0.0001) (Figure 3-10). There were no significant differences between 5, 10, and 15 DAI in any circle comparison, nor was there a difference between 25 and 30 DAI between circles I and II. Disease progressed linearly over time (circles I and III combined) and by 30 DAI the disease severity rating was a 4.3 (Figure 3-8). Disease severity increased proportionally over time in circles I and II, with circle I having the higher disease severity, but at 30 DAI circle II surpassed circle I, which were the treated plants (Figure 3-9). Pots placed in the outermost circle, which were 3.35 m from the center, had higher disease ratings than those closer at 2.13 m (Figure 3-10). All Cuscuta plants in the Gainesville study were not in contact, at any time, with any of the surrounding pots, which decreased the probability of the Cuscuta spreading the A. destruens by contact. This was not possible at the Ft. Pierce field location due to the nature of the study, but untreated plots were randomly placed from treated plots. In previous greenhouse studies (Chapter 6) the Cuscuta and pots were separated as in the Gainesville study, but all of the untreated controls still died after an initial lag period when they were healthy. One explanation for this occurrence is that there could be volatile signaling between Cuscuta plants. If this theory holds true, then as the inoculated Cuscuta plants in the greenhouse began to senesce, they would release senescence-inducing volatiles into

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42 the air, which would be transmitted to the Cuscuta on the untreated plants. This in effect would let the untreated Cuscuta recognize that senescence signal and they would start to die. A second explanation is that the A. destruens sporulates abundantly and as a result, spreading rapidly around the greenhouse to the untreated controls. This theory is more plausible in light of data presented herein. It is evident that disease ratings in the untreated controls, in both the Ft. Pierce and the Gainesville studies, can be attributed to the movement of A. destruens spores from treated to untreated areas. The main dispersal method for distribution of Alternaria spores is through airborne movement and to a lesser extent through splash dispersal (Rotem 1994). The large size of Alternaria spores enables them to float in the air and therefore disperse from their site of production (Gregory 1973). Alternaria conidia are firmly attached to conidiophores and have to be detached by relatively strong winds (Rotem 1994). Studies conducted on A. dauci estimated that wind velocities above 2-3 m s -1 (approximately 7-11 km/h) were required to release large numbers of conidia (Strandberg 1977). In addition, wind speeds do not have to be steady and brief gusts of otherwise moderate winds can facilitate spore release (Aylor 1990). Daily average wind speeds during the first Ft. Pierce trial ranged from 1.34 m s -1 to 4.02 m s -1 (Table B-1) (FAWN 2006), which would be sufficient to have spread A. destruens spores to untreated areas in the field. In addition, there were three days where recorded wind speeds ranged from 4.47 m s -1 to 5.36 m s -1 Daily average wind speeds for the second trial at Ft. Pierce were even higher than the first trial and ranged from 1.79 m s -1 to 4.47 m s -1 (Table B-2) (FAWN 2006).

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43 These results suggest the probability that A. destruens was already infecting C. pentagona in the field prior to implementation of the second trial because there were no significant differences between treatments and this species of Alternaria has not been found in the state of Florida (Farr et al. 2006). However, no symptoms were present at the time of inoculation during the second trial and isolations were not made at that time to confirm infection. Although, it is known that members of the genus Alternaria frequently cause quiescent infections in which the fungus enters the tissue where it remains dormant until environmental conditions favor infection (Thomma 2003). Therefore, this could possibly indicate that the fungus produces secondary inoculum. The Gainesville study daily average wind speeds ranged only from 0.89 m s -1 to 1.79 m s -1 (Table B-3) and no reported wind gusts (FAWN 2006). During this period, there were two storms with heavy rain and one thunderstorm with winds reported at 25.72 m s -1 (NCDC 2006). The rain and winds from these storms would be adequate to disperse A. destruens spores to untreated plants and infect the Cuscuta. This could also explain the higher disease severity on the pots located furthest away from the treatment site. Unfortunately, the design of the Gainesville study was not suitable to gather wind direction and only one trial was completed. In conclusion, no differences were observed between A. destruens or A. destruens + oil in either trial. Therefore, oil may not be needed when applying A. destruens. In addition, it appears as though A. destruens spores are capable of rapid dispersal throughout the field to untreated control areas, although more research is needed is this area. This capacity may enhance this bioherbicides efficacy and potential for use, but may also present some limitations. Studies conducted using a simulated wind tunnel

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44 further support my conclusion on the spore dispersal of A. destruens (Chapter 5). Finally, these results indicate that Alternaria destruens can be used as a stand alone treatment, but would be more effective if used in conjunction as an integrated control method for Cuscuta spp.

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45 0123456012345678WAIDISEASE OR DAMAGE SEVERITY Untreated Oil Fungus + Oil Fungus Figure 3-6. Ft. Pierce field trial I data. Disease severity from Alternaria destruens, with and without oil (PCC 588), oil only, and an untreated control on Cuscuta pentagona over weeks after inoculation (WAI). Table 3-1. Means of area under the disease progress curve (AUDPC) for treatments on Cuscuta pentagona from Ft. Pierce field trial I. Treatment Mean AUDPC a A. destruens + Oil 186.38 a A. destruens 179.38 ab Untreated Control 143.06 bc Oil 128.63 c a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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46 0123456012345678WAIDISEASE OR DAMAGE SEVERITY Untreated Oil Fungus + Oil Fungus Figure 3-7. Ft. Pierce field trial II data. Disease severity from Alternaria destruens, with and without oil (PCC 588), oil only, and an untreated control on Cuscuta pentagona over weeks after inoculation (WAI). Table 3-2. Means of area under the disease progress curve (AUDPC) for treatments on Cuscuta pentagona from Ft. Pierce field trial II. Treatment Mean AUDPC a A. destruens 159.69 a Oil 133.44 a A. destruens + Oil 128.63 a Untreated Control 128.19 a a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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47 y = 0.694x 0.7143 R2 = 0.986012345051015202530DAIDISEASE SEVERITY Figure 3-8. Disease severity over time (days after inoculation; DAI) on Cuscuta pentagona treated with Alternaria destruens + oil (PCC 588) in Circle I (0.91 m) and untreated sentinel C. pentagona in Circle III (3.15 m).

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48 012345051015202530DAIDISEASE SEVERITY 0.91 m 2.13 m Figure 3-9. Disease severity over time (days after inoculation; DAI) on Cuscuta pentagona treated with Alternaria destruens + oil (PCC 588) in Circle I (0.91 m) and untreated sentinel C. pentagona in Circle II (2.13 m).

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49 012345051015202530DAIDISEASE SEVERITY 2.13 m 3.15 m Figure 3-10. Disease severity over time (days after inoculation; DAI) on untreated sentinel Cuscuta pentagona in Circles II (2.13 m) and III (3.15 m).

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CHAPTER 4 MOLECULAR PHYLOGENIC CHARACTERIZATION TO DIFFERENTIATE ALTERNARIA DESTRUENS SIMMONS, SP. NOV. FROM VARIOUS ALTERNARIA SPECIES BASED ON INTERNAL TRANSCRIBED SPACER REGION The genus Alternaria Nees was first described in 1816 with the type species A. tenuis Nees, which was later redescribed as A. alternata (Fr.:Fr.) Keissl. (Simmons 1967). The majority of Alternaria spp. are saprophytic, but some species are pathogenic and produce a range of plant diseases throughout the world on numerous economically important plants. Alternaria leaf blight of carrot (Daucus carota) is caused by A. dauci (Khn) Groves & Skolko. This pathogen creates small irregular shaped lesions, which are dark brown to black in color, on the margins and tips of carrot leaflets (Gugino et al. 2004). The causal agent of Alternaria late blight of pistachio (Pistacia vera L.) is A. alternata that infects both foliage and fruit. On foliage, large necrotic lesions, that are black in the center and surrounded by a chlorotic halo, eventually occupy the entire leaf (Pryor and Michailides 2002). Infection on the fruit is characterized by small necrotic spots on the hull of immature nuts that are surrounded by a red halo (Pryor and Michailides 2002). In citrus, Alternaria brown spot, caused by A. alternata, is a serious disease of tangerines (Citrus reticulata), tangerine hybrids, and tangelos (C. reticulata x C. paradisi) (Peever et al. 2004; Timmer et al. 2000). This pathogen attacks young leaves, fruit, and twigs, causing small black necrotic spots (Timmer et al. 2000). Black rot of citrus, caused by A. alternata, occurs postharvest and may appear in the field prior to harvest. This disease develops most commonly on navel oranges (C. sinenesis (L.) Osbeck) in the field, and on lemons (C. limon (L.) Burm.) and tangerines in storage 50

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51 (Brown and McCornack 1972; Peever et al. 2005). Infection occurs through natural openings or wounds in the stylar "blossom" end of the fruit, which is where brown to blackish discoloration develops in diseased fruits (Peever et al. 2005). In 1984, Bewick et al. isolated an Alternaria spp. from a diseased Cuscuta gronovii plant growing in an uncultivated marsh in Wisconsin (Bewick et al. 1986). In 1998, Simmons described this isolate as belonging to Alternaria destruens Simmons, sp. nov. Simmons described A. destruens as a small-spored (25-40 (46) x 10-13 m), brevicatenate (each conidiophore initially produces a single unbranched chain of 4-8 conidia, which may increase to 10-12 conidia with age) species in which interspersed secondary condiophores range from extremely short to exceedingly long, (3-) 20-80 m, in a single chain (Simmons 1998) (Figure 1-4). Simmons (1992) has described over 100 species of Alternaria worldwide. However, the number of species described fluctuates due to other taxonomists merging several existing species of Alternaria into one species or by taking one species and dividing it into several different species (Rotem 1994). These differences are most likely due to the variability of morphological characteristics under a variety of environmental conditions (Thomma 2003). Conidium morphology and patterns of conidium catenulation are the only means available to morphologically differentiate between Alternaria species and to identify Alternaria isolates (Simmons 1992). The most important conidial characteristic in identifying a species is spore (conidium) dimension, including that of the beak (Rotem 1994). Spore length varies significantly between species, while spore width is similar. However, spore length, including the beak, can vary within the same species causing an

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52 overlap. This is due to a number of factors, but most commonly by the type of substrate. Therefore, most taxonomists give measured dimensions in ranges rather than as means (Rotem 1994). Analysis of ribosomal DNA (rDNA) sequences has become a frequent tool that has been used to establish molecular phylogenetic relationships within many groups of fungi (White et al. 1990). A review of the literature has revealed that characterization and differentiation of small-spored Alternaria species uses a wide variety of molecular techniques, such as, random amplification of polymorphic DNA (RAPD) fragment patterns analysis (Roberts et al. 2000); restriction fragment length polymorphism (RFLP) analysis (Sharma and Tewari 1998); and sequencing of rDNA internal transcribed spacer (ITS) regions, which are frequently used for analysis of fungal taxa at or below the species level (Chou and Wu 2002; Pryor and Gilbertson 2000). While spore morphology is a valid tool for species description, a more reliable method is needed for field diagnostic purposes. Based on past and current molecular studies of small-spored Alternaria, the objective of this study was to use internal transcribed spacing regions to differentiate known and unknown Alternaria isolates. Materials and Methods Sampling Material Cuscuta pentagona stems and flowers were collected from three previous studies conducted at the University of Florida in Gainesville, FL. The first two studies were conducted with plants grown in a greenhouse in the spring of 2005, while the third study was conducted with field-grown plants in the summer of 2005. The Cuscuta collected from the greenhouse studies were inoculated with seven treatments consisting of: an untreated control; oil (PCC 588, United Agri Products, Greeley, CO) at 7.5%; ammonium

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53 sulfate at 0.125% w/v in water; glyphosate ( N -(phosphonomethyl)glycine) at 0.02 kg (ae)/L of Roundup Pro ; A. destruens (Smolder WP, obtained from Sylvan Bioproducts, Inc., Kittanning, PA); A. destruens + oil; and a mixture of A. destruens + oil, glyphosate, and ammonium sulfate (the latter two at the preceding concentrations). The Cuscuta collected from the field study were either treated with A. destruens + oil or an untreated control. Fungal Cultures For morphological identification of the species and to obtain single-spore cultures, the fungi were grown to produce spores in culture. To stimulate sporulation, plant tissue from 47 randomly chosen samples (Table 4-1) was placed onto plates containing water agar (15 g of agar [Fisher Chemical, Fairlawn, NJ] per liter of H 2 O). The plates were stored at 22 o C. Approximately 1 to 2 days later, Alternaria spores were visible, which were then transferred to new water agar plates. This process was continued until pure cultures were obtained. Single germinating colonies were then transferred onto plates containing potato dextrose agar (PDA) (39 g of PDA [Difco, Detroit, MI] per liter of H 2 O). All plates were stored in the dark at 22 o C for approximately 10 days. In addition to the unknown 47 isolates, 4 known Alternaria spp., Alternaria cassiae Jurair and Khan, A. eichhorniae Nag Raj and Ponnappa, A. macrospora Zimmermann, and A. tenuis Nees (Table 4-2), were used for comparison. These isolates were prepared in the same manner as previously stated. GenBank isolates used for comparison are listed in Table 4-3.

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54 DNA Extraction Mycelium on the surface of the PDA plates was scraped and ground to a powder with liquid nitrogen. DNA was then extracted from the mycelial powder using the DNeasy Plant Mini Kit (QIAGEN, Valencia, CA). PCR Amplification The nuclear rDNA internal transcribed spacer (ITS) region, including ITS1 and ITS2 and the 5.8S rDNA, was amplified with primer pairs ITS4 and ITS5 by polymerase chain reaction (PCR) (White et al. 1990). The forward primer ITS5 was 5-GGA AGT AAA AGT CGT AAC AAG G-3 and the reverse primer was ITS4 5-TCC TCC GCT TAT TGA TAT GC-3 (Integrated DNA Technologies, Inc., Coralville, IN). Reactions were carried out in volumes of 50 l containing 33.7 l of double distilled H 2 O; 5 l of 10x buffer (Promega, Madison, WI); 5 l of 50 mM MgCl 2 (Promega, Madison, WI);1 l of an equal mixture of 10 mM dNTPs (Promega, Madison, WI);1 l of 10 M of each primer; 0.25 l of 5 units per l Taq DNA polymerase (Promega, Madison, WI); and 3 l template DNA. A PTC-200 Peltier Thermal cycler (GMI, Inc., Ramsey, MN) was used for amplification. Cycling conditions used an initial denaturation step of 3 min at 94 o C, with 30 cycles of 94 o C for 30 s, 50 o C for 30 s, and 72 o C for 45 s. The last cycle included an incubation step at 72 o C for 10 min and then storage at 4 o C. Bands were resolved by electrophoresis in a 1% agarose gel in 1X TAE buffer (0.004 Tris-acetate, 0.001 M EDTA, pH 8.0) stained with 1% ethidium bromide (100V for 50 min). Ten l of PCR product, 2 l of loading buffer (Bio-Rad, Hercules, CA), and

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55 8 l of a 100 base-pair ladder (Fisher Scientific International, Inc., Hampton, NH) were loaded into the gel, run and visualized under ultraviolet light. PCR products were purified by using a QIAquick PCR purification Kit (QIAGEN, Valencia, CA). DNA samples were analyzed by electrophoresis, as previously stated, except that 10 l of a DNA/Hind III ladder (Invitrogen, Carlsbad, CA) was used. The purified products were sent to the DNA Sequencing Core Laboratory at the University of Florida and sequenced with a Perkin Elmer, Applied Biosystems model 373A or 377 automated DNA sequencer. Phylogenetic Analysis DNA forward and reverse sequences were combined, trimmed to 507 bp, and any incongruences were edited in Sequencher (version 4.5 Gene Codes Corporation, Ann Arbor, MI). Alignment of the sequence files were conducted using ClustalX v. 1.83.1 (Thompson et al. 1997) and manual adjustments of sequence alignments were performed using the data editor program of MacClade Phylogenetic Software (version 4.08; Sinauer Associates, Inc., Sunderland, MA). Phylogenetic analyses were performed with programs contained in PAUP Phylogenetic Software (version 4.0 Sinauer Associates, Inc., Sunderland, MA). Alternaria cassiae, A. eichhorniae, A. macrospora, and A. tenuis were designated as outgroups. Phylogenetic trees were constructed using distance methods because heuristic searches for the parsimony analysis yielded too many trees. Distance-based trees were generated with neighbor-joining using the Jukes-Cantor one-parameter algorithm. The significance of the branches in the neighbor-joining trees was tested with 10,000 bootstrap replicates to assess the statistical support of each clade.

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56 Results and Discussion Neighbor-joining analysis revealed that all 47 unknown isolates, which were recovered from the greenhouseand field-grown Cuscuta plants, and the reference A. destruens isolate, were 100% identical (Figure 4-1). The first clade contained the defined outgroups, A. cassiae and A. macrospora, which were supported with a bootstrap value of 100%. The second and third clades contained A. tenuis and A. eichhorniae with a bootstrap value of 99% and 86%, respectively. To determine if the sequence obtained was unique, a Basic Local Alignment Search Tool (BLAST) search was performed using the previously obtained A. destruens ITS sequence. This query revealed 100 other sequences that were similar, with 82 of them being from Alternaria. These 82 Alternaria sequences were analyzed in PAUP with the original A. destruens and 47 unknown Alternaria isolates. All 82 Alternaria sequences from the BLAST search were 100% identical to the Alternaria in this study (data not shown). A GenBank search was then conducted for other Alternaria ITS sequences, to determine if the A. destruens ITS sequence, that resulted from this study, was unique. This search produced a listing of numerous, highly divergent Alternaria spp., of which 61 were randomly chosen and analyzed in PAUP with the original A. destruens ITS sequence (Table 4-1). This analysis grouped the original A. destruens with eight different Alternaria spp., which had the same ITS sequence, all in the same clade. This could be due to misidentification in GENBANK because ITS sequences are typically species specific. In addition, another A. destruens ITS sequence was found in the GENBANK search, which grouped with this clade (Figure 4-2). This isolate of A. destruens also contained the nuclear rDNA region, ITS1 and ITS2, and the 5.8S rDNA.

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57 The other Alternaria species grouped into various clades, many of which contained the same species, but some of which contained different species. This could once again be due to either misidentification based on morphology or the different species are clustering together based upon Alternaria species-groups identified by Simmons (1992). The results of this study confirmed that the unknown isolates grouped with A. destruens. However, ITS is highly conserved within A. destruens, therefore it was not possible to confirm that the isolates collected were the same as the isolate inoculated in the greenhouse and field trials. This is in agreement with Serdani et al. (2002) who found that a high degree of interspecies similarity exists within Alternaria in both ITS 1 and ITS 2. Serdani et al. (2002) also indicated that ITS genes are not appropriate for differentiating between closely related Alternaria species. In addition, Peever et al. (2004) and Pryor and Michailides (2002) failed to differentiate small-spored Alternaria species due to a lack of variation in nuclear ribosomal ITS sequences. Currently, A. destruens has been reported to occur naturally only in Wisconsin (Bewick et al. 1986). There are no reports of natural infestations of A. destruens in the state of Florida. In addition, A. destruens has never been recorded as a pathogen on C. pentagona in Florida or in any other part of the world. Therefore, it is highly probable that the isolates collected are the same as the one used in my research studies. In conclusion, the data support concurrence with Simmons (E. G. Simmons, Research Associate at Wabash College, Crawfordsville, Indiana, personal communication) that ITS sequences are very conservative and extremely unreliable in revealing any true differences or relationships between Alternaria species. Further

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58 studies need to be conducted to amplify and sequence a more highly variable region of the genome such as introns or microsatellites, which would identify more polymorphisms between isolates within a species. In addition, isolation and sequencing of a naturally occurring A. destruens from Florida would lend further credence to differentiation of isolates.

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59 Figure 4-1. Rooted neighbor-joining dendrogram based on 507 bp from ITS 1, 5.8S, and ITS2 sequences. Alternaria cassiae, A. eichhorniae, A. macrospora, and A. tenuis were designated as outgroups. Bootstrap values generated from 10,000 replications are indicated. A bar indicates genetic distances.

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60 Table 4-1. Sources of unknown isolates and treatments applied to plots from which these isolates were recovered. a Isolate b Treatment c Isolate Treatment I-101 U II-403 A.d. I-102 A.d.O II-406 AS I-105 GLY II-407 GLY I-106 AS II-501 A.d.O I-202 A.d.O II-505 U I-205 GLY SFS-1 A.d.O I-206 MIX SFS-2 A.d.O I-304 A.d. SFS-3 A.d.O I-402 U SFS-4 A.d.O I-403 AS SFS-5 A.d.O I-406 O SFS-6 A.d.O I-502 MIX SFS-7 A.d.O I-505 A.d.O SFS-8 A.d.O I-507 A.d. SFS-9 A.d.O II-103 AS SFS-10 A.d.O II-104 O SFS-30 U II-107 A.d. SFS-32 U II-203 MIX SFS-37 U II-205 O SFS-39 U II-301 U SFS-45 U II-302 A.d.O SFS-47 U II-303 AS SFS-52 U II-305 MIX SFS-54 U II-306 GLY a All isolates were used for DNA sequencing in this study. b Isolates beginning with SFS are from the field study, while all other isolates are from the greenhouse studies. c Treatment designations are as follows: A.d. = Alternaria destruens, A.d.O = A. destruens + oil, AS = ammonium sulfate, GLY = glyphosate, MIX = A. destruens + oil, glyphosate, and ammonium sulfate, O = oil, U = untreated control.

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61 Table 4-2. Sources of Alternaria spp. used for DNA sequencing in this study. Isolate Original Source Alternaria cassiae a Soil sample; Marianna and Quincy, FL A. destruens Smolder WP; Sylvan Bioproducts, Inc., Kittanning, PA A. eichhorniae a Dr. Yasser Shabana; Mansoura, Egypt A. macrospora a Dr. C. Doug Boyette; USDA-ARS, Stoneville, MS A. tenuis a Late Dr. George E. Templeton; University of Arkansas, Fayetteville, AR a Stored in the culture collection of Dr. R. Charudattan, Plant Pathology Department,University of Florida, Gainesville.

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62 Figure 4-2. Rooted neighbor-joining dendrogram of ITS1, 5.8S, and ITS2 sequences. Bootstrap values generated from 10,000 replications are indicated. Isolates are specified by the last three numbers of the GenBank accession number followed by the genus and species. Alternaria destruens without a number is the isolate used in this study.

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63 Table 4-3. Fungal Isolates and the GenBank accession numbers for sequences used in phylogenic analyses. Species GenBank Accession Number Alternaria alli isolate Alt34 DQ323705.1 A. alli isolate Alt35 DQ323683.1 A. alternata DQ059568.1 A. alternata isolate EGS34-016 DQ323699.1 A. brassicae AY154714.1 A. brassicae AB11 U05253.1 A. brassicae gz01-6-6 AF392984.1 A. brassicae strain AB57 AY372686.1 A. brassicae strain BMP 21-61-02 AF229463.1 A. dauci isolate CHHADA AF267130.1 A. dauci strain BMP 21-31-09 AF229467.1 A. dauci strain BMP 21-31-16 AF229468.1 A. destruens isolate Alt1 DQ323681.1 A. gaisen strain AGM AF314581.1 A. gaisen strain EGS90-0512 AY762944.1 A. gaisen strain LCL AF314574.1 A. helianthi AY154713.1 A. helianthi strain B DQ156343.1 A. infectoria isolate 4A Y17066.1 A. infectoria isolate 4B Y17067.1 A. japonica AY154703.1 A. japonica strain ATCC 13618 AY376639.1 A. japonica strain ATCC 13618 AF229474.1 A. longipes DQ156338.1 A. longipes strain ALM AF314571.1 A. longipes strain ALS AF314587.1 A. macrospora AY154689.1 A. macrospora strain B DQ156342.1 A. macrospora strain DGG Ams1 AF229469.1 A. mali strain AMM AF314582.1 A. mali strain EGS 38-029 AY762945.1

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64 A. mali strain PGC AF314575.1 A. mali strain PGX AF314585.1 A. palandui isolate Alt10 DQ323682.1 A. palandui isolate Alt9 DQ323702.1 A. panax isolate AP 2035 AY898635.1 A. panax isolate AP 2036 AY898636.1 A. panax isolate BC 2085 AY898637.1 A. panax isolate BC 2086 AY898638.1 A. panax isolate JAT 2120 AY898639.1 A. panax isolate JAT 2125 AY898640.1 A. radicina AY154704.1 A. radicina isolate AR018 DQ394074.1 A. radicina isolate AR07 DQ394073.1 A. radicina isolate CHHAR1 AF267133.1 A. radicina strain ATCC6503 AF307014.1 A. solani isolate CBS 111.44 Y17070.1 A. solani isolate ICMP 6519-79 Y17069.1 A. solani strain AS3 AF314576.1 A. tagetica DQ100420.1 A. tagetica isolate CHHAT1 AF267134.1 A. tagetica isolate CHHAT2 AF267136.1 A. tenuissima isolate EGS34-015 DQ323698.1 A. tenuissima isolate Po56 AY513940.1 A. tenuissima strain IA287 AY154712.1 A. triticina DQ117960.1 A. triticina AY154695.1 A. triticina strain EGS AY762948.1 A. zinniae AY154696.1 A. zinniae isolate CHHAZ1 AF267135.1 A. zinniae strain AZ33 AY372682.1

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CHAPTER 5 DISPERSAL OF ALTERNARIA DESTRUENS: A WIND TUNNEL STUDY Disease in a field does not occur uniformly in space due to either environmental and/or dispersal gradients of inoculum (R. Berger, University of Florida, Gainesville, FL, personal communication). To investigate these dispersal gradients, complicating environmental factors must be controlled. In a study evaluating competing models of inoculum and disease spread, Cowger et al. were able to attribute the velocity of wheat stripe rust across space and time based on dissemination from specific disease foci (Cowger et al. 2005). In the case of dispersal gradients, differential dissemination of inoculum away from the source may be influenced by mortality of the pathogens propagules, movement by insects, or even simple diffusion (R. Berger, University of Florida, Gainesville, FL, personal communication). Wadia et al. (1998) determined that Passalora personata (Berk. and M. A. Curtis) S. A. Khan and M. Kamal conidia were more effectively removed from sporulating lesions and distributed over longer distances than by steady winds at the same wind speeds. Inoculum dispersal is commonly neglected in the simulation of disease, which according to Waggoner is necessary for prediction and forecasting models to accurately explain an epidemic (Waggoner 1983). In the case of a foliar disease caused by a fungal pathogen, an accurate prediction model based on a wind tunnel study would be of direct use in identifying and evaluating disease development and spread as well as disease management strategies. 65

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66 In 1984, Bewick et al. isolated an Alternaria spp. from a diseased Cuscuta gronovii Willd. ex J.A. Schultes (swamp dodder) plant growing in an uncultivated marsh in Wisconsin (Bewick et al. 1986). In 1998, E. G. Simmons identified the isolate as belonging to Alternaria destruens Simmons, sp. nov. (Simmons 1998). Simmons described A. destruens as small-spored with conidial size ranging from 25-40 (46) x 10-13 m, unbranched, and brevicatenate with 4-8 conidia. Secondary conidiophores range from very short to remarkably long, (3-) 20-80 m, and in a single chain (Simmons 1998). The main dispersal method for distribution of Alternaria spp. spores is through airborne movement and to a lesser extent through splash dispersal (Rotem 1994). The rate at which Alternaria spp. spores fall from the air and how these spores are removed from the air by impaction and deposition processes are determined by the spore shape and size (McCartney and Fitt 1985). Alternaria spp. spores are relatively large in size, which enables them to float in the air and therefore disperse from their site of production (Gregory 1973). Alternaria spp. conidia are firmly attached to conidiophores and have to be detached by relatively strong winds (Rotem 1994). Studies conducted on A. dauci (Khn) Groves & Skolko estimated that wind velocities above 2-3 m s -1 (approximately 7-11 km/h) were required to release large numbers of conidia (Strandberg 1977). Even low to moderate wind speeds can effectively facilitate spore release just as easily as brief or sustained gusts (Aylor 1990). To investigate the dispersal of A. destruens spores within a field situation, a wind tunnel study is an effective means to correlate inoculum dispersal to disease severity over increasing wind speeds. Hence, an accurate prediction model of spread of disease caused

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67 by A. destruens based on a wind tunnel study would be of direct use to identify and evaluate bioherbicide management strategies to control Cuscuta spp. In previous field (Chapter 3) and greenhouse studies (Chapter 6) investigating A. destruens, untreated control plants have been consistently diseased although attempts were made to protect these plants during the application of the biological control agent. It is hypothesized that this infection is a result of wind-carried conidia originating from the inoculated plants. Therefore, the objective of this study was to erect a wind tunnel to determine the capability of A. destruens spores to be dispersed at certain wind speeds and determine how far those spores would travel. This simulated wind dispersal of A. destruens spores may shed light on the observed rapid field spread of this pathogen and help identify strategies for best deployment of this bioherbicide in the field. Materials and Methods Wind Tunnel Studies were conducted outdoors, in an open-unshaded area, during the summer of 2005 at the University of Florida at Gainesville. A wind tunnel was constructed with 5 PVC pipes (10.16 cm x 3 m) that had a PVC pipe increaser-reducer (7.62 cm x 10.16 cm) attached to the beginning of the first PVC pipe for the insertion of the Vac N Mulch Blower/Vac (model # BV2000, Black and Decker). The blower was calibrated with a rheostat (3PN1010V, Statco Inc., Dayton, OH) by holding each speed for 10 sec. Each speed had 10 replications and then the average speed was taken. A total of 18 speeds were calibrated and 6 representative speeds (2.1, 4.1, 7.4, 9.2, 11.6, and 15.6 m s -1 ) were chosen for the study. Wind speed was measured with an anemometer (Kestrel 1000, Nielsen-Kellerman, Boothwyn, PA) within +/3% (+0.1/-0.3 m s -1 ) wind velocity accuracy.

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68 Microscope slides covered in petroleum jelly (Vaseline Unilever, Trumbull, CT) were hung with duct tape at the top of each PVC pipe before the pipes were connected. A petri plate (60 mm x 15 mm) was taped at the edge of the first PVC pipe with a piece of 5.5-cm filter paper (Whatman) taped on top of the plate. Alternaria destruens spores (Smolder WP) were supplied by Sylvan Bioproducts, Inc., Kittanning, PA. Using a hemocytometer, spore counts were determined to be 1.8 x 10 7 spores per g. Alternaria destruens spores (0.1 g) were deposited on top of the filter paper prior to each wind speed run. Prior to each run, the blower was set to the correct wind speed to attain a steady air flow, and then inserted into the PVC pipe increaser-reducer for 10 sec. The slides were then taken out of the tunnel, covered with a piece of clear scotch tape, and put into separate petri plates. An area of 5.08 cm x 2.54 cm of spores was counted on all slides. After each run, the filter paper was changed and air was blown through the PVC piping for 10 sec to clear any remaining spores. Each speed was replicated 3 times. When attempting to repeat this experiment, the location was changed to the inside of a large greenhouse. This was to minimize possible wind gusts that may have happened in the first trial. In addition, the method of spore collection changed. This was due to the number of spores collected and problems encountered with spore counting in the first trial. The scotch tape and petroleum jelly made it difficult to accurately collect and count all of the spores present. Therefore, in this experiment, glycerin (Fisher Scientific, Fair Lawn, NJ) was used to coat the slides and they were laid diagonally in petri plates that were taped at the end of each PVC pipe. Because different agents were used to trap the spores, petroleum jelly in the first repetition and glyercine in the second, these repetitions were treated as separate

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69 experiments. All data were subjected to analysis of variance (ANOVA) and the means were separated using Fishers protected least significant difference (LSD) at P= 0.05 in SAS (SAS 1999). Results and Discussion In the first experiment, the effects of length (P=0.0059) and speed (P=0.0001) were both significant, and in the second experiment, there was a length by speed interaction (P=0.0001). In the first trial, the spore number decreased linearly as wind tunnel length increased (Figure 5.1). There were no significant differences between the first two tunnel lengths of 3.0 m and 6.1 m, nor were there differences between the last three lengths of 9.1 m, 12.2 m, and 15.2 m. The two lowest speeds of 2.1 m s -1 and 4.1 m s -1 were not significantly different from each other (Figure 5.2). Likewise, the four highest speeds (7.4, 9.2, 11.6, and 15.6 m s -1 ) were also not significantly different from each other (Figure 5.2). In the second experiment, the number of spores trapped decreased with tunnel length, but increased at higher wind speeds (Figure 5-3). The last two tunnel lengths (12.2 m and 15.2 m) had the lowest number of spores trapped (138 and 68), while the second and third tunnel lengths (6.1 m and 9.1 m) had the most spores trapped (239 and 207) at the highest speed of 15.6 m s -1 (Figure 5-3). Dispersal of spores in dry weather is either by release under the force of gravity or removal from the host mainly during fast, turbulent wind (Aylor 1990). Many spore types require a certain amount of direct mechanical force to be applied to the spore for it to be detached (Aylor 1975). In addition, a threshold wind speed must be surpassed, which is unique for each pathogen. Spores of each species are usually removed over a

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70 range of wind speeds with the total amount of spores detached from a condiophore increasing rapidly as wind velocity increases (Aylor 1990). Although, once a certain percentage of spores are released, it is difficult to remove additional spores at any realistic speed (Waggoner 1973). The results of this study are in accordance with other simulated wind tunnel studies (Chen et al. 2003; Wadia et al. 1998). At higher wind speeds, the numbers of spores that are dispersed from a source reach a peak and then decrease with increasing distance. The highest amounts of A. destruens spores were collected at the distances of 6.1 m and 9.1 m with a wind speed of 15.6 m s -1 The average number of spores collected between these two distances was 223. Once the distance reached 15.2 m at that same speed, the number of spores decreased to 68. At these wind speeds and distances, dispersal of A. destruens would be more than enough to reach untreated controls in the field and greenhouse. In addition, this study did not include wind gusts which have been shown to be more effective in dispersing spores further from a source than steady winds (Wadia et al. 1998). In conclusion, this pathogens capability of spreading rapidly and over great distances can be an advantage when applying A. destruens in the field. Making use of wind patterns flowing parallel to the field where applications of this bioherbicide are to be made may aid in field coverage and could reduce additional treatments of A. destruens. Applications could also be made in certain areas of the field, i.e. divide the field into quadrants and then use wind patterns to decide which quadrants to treat with A. destruens that would best flow to untreated areas in the field.

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71 Although the results of these wind tunnel studies confirm that large numbers of A. destruens spores are capable of spreading far from a source point, the methodology of this study could be vastly improved by using potted plants inoculated with A. destruens. This would give a better representation of how the spores disperse when airborne from a diseased plant. Overall, these experiments provide some insight into the dispersal patterns of A. destruens when airborne.

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72 y = -7.0669x + 50.023 R2 = 0.9201020304036.19.112.215.2TUNNEL LENGTH (m)MEAN NUMBER OF SPORES PER 5.08 cm x 2.54 cm Figure 5-1. Results of the first experiment utilizing the wind tunnel to examine the effects of wind tunnel length (m) from inoculum release on spore dispersal of Alternaria destruens.

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73 02040608010012002.14.17.49.211.615.6WIND SPEED (m s-1)MEAN NUMBER OF SPORES PER 5.08 cm x 2.54 cm 3.0 6.1 9.1 12.2 15.2 Figure 5-2. Results of the first experiment utilizing the wind tunnel to examine the effects of wind speed (m s -1 ) from inoculum release on spore dispersal of Alternaria destruens.

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74 0408012016020024002.14.17.49.211.615.6WIND SPEED (m s-1)MEAN NUMBER OF SPORES PER 5.08 cm x 2.54 cm 3.0 6.1 9.1 12.2 15.2 Figure 5-3. Results of the second experiment utilizing the wind tunnel to examine the effects of wind speed (m s -1 ) and distance (m) from inoculum release on spore dispersal of Alternaria destruens.

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CHAPTER 6 EFFECTS OF ALTERNARIA DESTRUENS (SMOLDER ), GLYPHOSATE (ROUNDUP PRO ), AND AMMONIUM SULFATE INDIVIDUALLY AND INTEGRATED FOR CONTROL OF CUSCUTA PENTAGONA Cuscuta species are obligate parasites that predominate disturbed habitats, which preadapts them to becoming a serious weed problem. There are approximately 150 to 170 Cuscuta spp. worldwide; with the majority occurring in North and South America (Ashton and Santana 1976; Dawson et al. 1994). Cuscuta spp. parasitize mainly dicotyledonous and a few monocotyledonous plants with some species of Cuscuta being very host specific, while others have a broad host range. (Gaertner 1950; Dawson et al. 1994). Control of Cuscuta spp. is very difficult, with the best method being exclusion, i.e., not to introduce it into a farm or field. Mechanical removal by seed cleaning machines is an option, but these devices are often slow and they do not always remove all of the Cuscuta spp. seed and sometimes remove the crop seed. Tillage can be useful because the seedlings are easily dislodged from the soil or die when buried, but if they are not buried completely, they can resurface. Physically removing Cuscuta spp. by hand is time-consuming and although Cuscuta spp. generally behave as annuals, they can survive in the tissue of perennial host plants under some conditions (Dawson et al. 1984). Use of preemergent herbicides may not provide season-long control and repeated treatments may be necessary. Foliar applications of diquat (6,7-dihydrodipyrido[1,2-a:2,1-c]pyrazinediium dibromide) or paraquat (1,1-dimethyl-4,4-bipyridinium dichloride) provide partial 75

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76 selectivity in alfalfa, Cuscuta spp. and the host plant foliage will be destroyed, but the host plant may regrow from crowns after treatment (Dawson 1987; Dawson et al. 1994). Other nonselective post-attachment herbicides that will provide complete kill to the host plant as well as Cuscuta spp. are 2,4-D ((2,4-dichlorophenoxy)acetic acid) or glyphosate (N-(phosphonomethyl)glycine) (Dawson et al. 1994). Glyphosate, a phloem-mobile herbicide, moves in the symplast via source to sink (McAllister and Haderlie 1985). The primary mode of action of glyphosate is to inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; E.C. 2.5.1.19), which is the key enzyme in the shikimate pathway. Inhibition of EPSPS prevents the production of chorismate which is required for biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan (Amrhein et al. 1980; Geiger and Bestman 1990; Hollnder and Amrhein 1980). Dawson and Saghir sprayed glyphosate over the top of parasitized alfalfa, which resulted in injury and death to the Cuscuta spp. but not to the alfalfa (Dawson and Saghir 1983). The highest rate of glyphosate (0.30 kg per ha) provided sufficient delay for the parasitized alfalfa to recover prior to regrowth of Cuscuta spp. (Dawson and Saghir 1983). Even though all exposed shoots of Cuscuta spp. were killed by the glyphosate, some haustoria were still embedded within the alfalfa stems and regenerated. All alfalfa foliage in the control plots were destroyed by Cuscuta spp. (Dawson and Saghir 1983). Fer found that 14 C-labeled glyphosate was more concentrated in the apical part of the Cuscuta spp. shoots, especially in the buds, than in the haustorial coil (Fer 1984). Furthermore, at 4 days after treatment (DAT), Cuscuta spp. had absorbed 75% of the

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77 glyphosate from the hosts phloem. Fer concluded from these studies that phloem-mobile herbicides should be able to selectively control Cuscuta spp. (Fer 1984). Bewick et al. conducted studies with 14 C-labeled glyphosate on carrot parasitized with Cuscuta gronovii (Bewick et al. 1991). They determined that at 1 DAT Cuscuta tissue had absorbed as much 14 C-labeled glyphosate as any sink in the carrot. At 45 DAT, Cuscuta tissue contained most of 14 C-labeled glyphosate in regard to all other physiological sinks, except the petiole of the treated carrot leaf (Bewick et al. 1991). Ammonium sulfate, (NH 4 ) 2 SO 4 is used as a surfactant to increase phytotoxicity in many herbicides, including glyphosate (Nalewaja and Matysiak 1993; OSullivan et al. 1981; Turner and Loader 1975). Hard water contains polyvalent cations that interfere with the activity of glyphosate (Nalewaja and Matysiak 1991, 1993). Thelen et al. (1995) determined that the glyphosate molecule reacts with Ca ++ and other cations to form a less-readily absorbed glyphosate-Ca salt that decreases herbicidal activity. When ammonium sulfate is added into the tank mix, the ammonium ion binds with the glyphosate molecule, preventing the formation of glyphosate-Ca salt, which in turn increases herbicides absorption (Gronwald et al. 1993; Pratt et al. 2003). Several theories have been proposed as to how ammonium sulfate increases herbicide absorption. Studies conducted with tomato fruit, which has a well-developed cuticle, confirmed large increases in uptake when sethoxydim (2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one) was applied with ammonium sulfate (Wanamarta et al. 1993). The cuticle is a barrier to sethoxydim penetration, but with the addition of ammonium sulfate the capability of the cuticle to block herbicide absorption was reduced. Ammonium salts of bentazon (3-(1-methylethyl)-(1 H )-2,1,3

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78 benzothiadiazin-4(3 H )-one 2,2-dioxide) formed from the addition of ammonium sulfate were more readily absorbed and demonstrated increased transcuticular movement compared with calcium, magnesium, or sodium salts of bentazon (Wanamarta et al. 1993). Ammonium sulfate is related to increased permeation of the herbicide through the plasma membrane (Young et al. 2003). Several researchers have hypothesized that the acidification of the spray solution by the addition of ammonium sulfate allows a greater proportion of weak-acid herbicides to remain in their nondissociated form, which allows the herbicide to diffuse across the plasma membrane more easily (Gronwald et al. 1993; Smith and Vanden Born 1992). When the herbicide enters the cell, the higher pH of the cytoplasm causes dissociation of weak-acid herbicides, thus efficiently trapping the herbicide inside the cell (Young et al. 2003). Ammonium sulfate is formed by reacting ammonia with sulfuric acid to form a stable ammonium salt. It is soluble in water and insoluble in liquid ammonia or alcohol. The toxicity of the ammonium ion enhances this compounds capacity for weed control (Brian 1976). The plant cell rapidly absorbs the ammonia and the cell sap, which is normally acidic, may become alkaline due to the effect of ammonia (Harvey 1911). Rapid cell death ensues from increased alkalinity and from the toxicity of ammonia (Brian 1976). Excess concentrations of ammonium ions or ammonia are toxic to plants, although the exact biochemical causes of toxicity are still being studied (Given 1979; Smith and Vandenn Born 1992, Young et al. 2003). Another option to control dodder is the use of a bioherbicide. Bioherbicides are biological control agents based on fungi, bacteria, or viruses that are industrially developed and registered through the United States Environmental Protection Agency as

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79 a biopesticides to manage or reduce weed populations. The mycoherbicide (fungi used as herbicides) approach can be successful when substantial quantities of inoculum are used to help compensate for natural restrictions to disease development (Bewick et al. 1987; Charudattan 1988). Through manipulation, some fungi that normally cause sporadic or endemic disease levels on their hosts can be developed into effective bioherbicides (Charudattan 1988; Rosskopf et al. 1999). In 1984, Bewick et al. isolated an Alternaria spp. from a diseased C. gronovii Willd. ex J.A. Schultes plant growing in an uncultivated marsh in Wisconsin (Bewick et al. 1986). The majority of Alternaria spp. are saprophytic, but some species are pathogenic and produce a range of plant diseases throughout the world. Depending on the pathogen, symptoms typically appear as blights and leaf spots, but damping-off of seedlings, as well as fruit, stem or tuber rots can also occur (Agrios 1997). The Alternaria isolate recovered by Bewick was highly virulent and field studies with this pathogen resulted in 92% control of C. gronovii (Bewick et al. 1987). No disease was observed on, nor was the pathogen recovered from any of the host plants of dodder. In 1990, this pathogen was patented as a bioherbicide for suppression of Cuscuta spp. (Bewick et al. 2000), and in 1998, Simmons identified the species as Alternaria destruens Simmons, sp. nov. (Simmons 1998). Currently, there are no integrated control methods for Cuscuta spp. let alone a single economical control method. Therefore, the objectives of this study were to 1) evaluate the effects of various concentrations of glyphosate (Roundup Pro ) and ammonium sulfate on the growth of A. destruens on Citrus spp. and 2) evaluate the

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80 effects of A. destruens, glyphosate, and ammonium sulfate individually and in combination, the latter as an integrated control method, on C. pentagona. Materials and Methods Greenhouse Test Plants Ten-month-old Citrus spp. (Smooth Flat Seville; Phillip Rucks Citrus Nursery, Frostproof, FL) in 1-gallon pots were parasitized with C. pentagona. At the time of treatment, the Cuscuta spp. was approximately 4-weeks old. Inoculation and Treatments All studies were conducted in the greenhouses at the University of Florida in Gainesville from the spring of 2005 through the summer of 2005. Maximum relative humidity for all greenhouse trials (Figure C-4) and maximum and minimum temperatures for greenhouse trial I (Figure C-1), trial II (Figure C-2), and trial III (Figure C-3) were measured using a HOBO Pro RH/Temp Data Logger (H08-032-08) (Onset Computer Corporation, Bourne, MA). Seven treatments were applied consisting of: an untreated control; oil (PCC 588, United Agri Products, Greeley, CO) at 7.5%; ammonium sulfate at 0.125% w/v in water; glyphosate ( N -(phosphonomethyl)glycine) at 0.02 kg (ae)/L of Roundup Pro ; A. destruens (Smolder WP, obtained from Sylvan Bioproducts, Inc., Kittanning, PA); A. destruens + oil; and a mixture of A. destruens + oil, glyphosate, and ammonium sulfate (the latter two at the preceding concentrations). Glyphosate and ammonium sulfate concentrations were determined from previous preliminary greenhouse studies on amounts that would successfully kill C. pentagona, but not Citrus spp. (Appendix D). Fungal suspensions were prepared as described for the greenhouse trial in Chapter 2. Using a hemocytometer, spore counts were standardized to 1.8 x 10 7

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81 spores per ml. Each study was conducted in a randomized complete block design with 5 replications and was performed twice. Plants were inoculated with a hand-held sprayer at 100 ml per m 2 and all treatments were used within 1 h of mixing. All data were analyzed using Area Under the Disease or Damage Progress Curve (AUDPC), which is used to quantify disease or damage progress over time by integrating all factors of an epidemic, such as, environment, host, and pathogen effects (Campbell and Madden 1991). AUDPC values for each trial were calculated using the formula [(y i + y (i+1) )/2]*(t (i+1) +1t i ), where i = 1, 2, 3, n-1, where y i is the disease or damage severity at the i th evaluation, and t i is time at the i th evaluation (Shaner and Finney 1977). All data and AUDPC values were subjected to analysis of variance (ANOVA) and means separated by Tukey's Studentized Range (HSD) test at P=0.05 in SAS (SAS 1999). Disease or Phytotoxic Damage Rating Plants were observed and rated weekly. Disease symptoms as well as the phytotoxic damage from glyphosate or ammonium sulfate were expressed as necrotic spots or blight on dodder. Phytotoxic damage from ammonium sulfate or glyphosate on Citrus spp. plants in the preliminary studies were expressed as leaf and stem senescence. Hence there was no need to use different rating scales or systems and both the disease and damage were assessed on a severity scale of 0 to 5, with 0 = no symptoms; 1 = 1-10% (tip necrosis; stems starting to wilt and become necrotic; 2 = 11-35% (slightly more stem necrosis; flowers starting to senesce), 3 = 36-65% (over half of the stems are dead or dying; clusters of flowers senescing); 4 = 66-90% (the majority of the stems and

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82 flowers are dead or dying; some healthy flowers and stems may still present), and 5 = 91-100% (plant death) (Chapter 3, Figure 3-5). Growth Inhibition Study The treatments consisted of three glyphosate concentrations (0.09, 0.04, and 0.02 kg (ae)/L of Roundup Pro ), three ammonium sulfate concentrations (0.50%, 0.25%, and 0.125% w/v in water), one oil (PCC 588) concentration (7.5% v/v in water), and an untreated control. Each treatment was mixed into 200 ml of potato dextrose agar (PDA) (39 g of PDA [Difco, Detroit, MI] per liter of H 2 O), which was then poured in 20 ml aliquots into petri plates. All treatments were replicated 10 times. Single-spore isolations of A. destruens were made as described in Chapter 4. Twelve-day-old agar plugs (6 mm) of A. destruens were placed in the center of each plate and stored at 22 o C. Plates were measured every other day over a 10-day period. All data were subjected to analysis of variance (ANOVA) and the means were separated using Fishers protected least significant difference (LSD) at P= 0.05 in SAS (SAS 1999). Results and Discussion Greenhouse Studies Due to lower treatment effects in the second trial, this study was repeated a third time. The lower effects were most likely due to environmental conditions at the time, such as a greater number of cloudy days which would have slowed the growth of Cuscuta spp. Due to no significant differences in variance between trials I and III (P=5.030), the data were combined, although, there was a treatment by DAT interaction (P=0.0210). The second trial was significantly different from trials I and III (P=0.0001) and was analyzed separately. Trial II also had significant treatment (P=0.0001) and DAT

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83 (P=0.0001) effects. In all trials, disease or damage severity progressed linearly as DAT increased, except for the mixture and the A. destruens + oil treatments in trial I and III (combined), which were better fit with a quadratic curve (Figures 6.1 and 6.4). Regression equations and R 2 values are listed in Table 6-1. In preliminary greenhouse trials, glyphosate and ammonium sulfate concentrations that caused the highest dodder mortality levels were determined to be glyphosate at 1.42, 0.71, and 0.36 kg (ae)/L of Roundup Pro and ammonium sulfate at 0.10% w/v in water (Appendix D). In addition, the effects of damage severity from glyphosate and ammonium sulfate concentrations increased over time (DAT) (Figures D-1 and D-3). There were no significant differences between the two lowest concentrations of glyphosate where the AUDPC values were 110.81 and 105.69, respectively (Table D-1), therefore for further testing the lowest concentration of 0.09 kg (ae)/L of Roundup Pro was used in addition to two lesser concentrations of 0.04 and 0.02 kg (ae)/L of Roundup Pro The effects of glyphosate on Citrus spp. were significantly different in both trials (P=0.0001) (Figure D-2). AUDPC values in trial II, as compared to trial I, were higher which could be due to possible environmental effects at the time of application which may have increased the phytotoxic damage on the Citrus spp. plants (Table D-2). The only AUDPC value in trial I that was significantly different from the other AUDPC values was the untreated control. In trial II, there were no significant differences between AUDPC values (Table D-2). In the ammonium sulfate study, the highest concentration was not significantly different from the untreated control; therefore, the concentrations were increased to 0.125%, 0.25%, and 0.5% (Table D-3). The effects of ammonium sulfate on Citrus spp. were not significantly different and therefore both

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84 trials were combined (Figure D-4). AUDPC values indicated no differences between concentrations (Table D-4). In the greenhouse trials I and III (combined), the AUDPC value for the mixture treatment (88.90) had the highest disease or damage severity rating out of all treatments (Table 6.2). In trial II, the AUDPC values were all the same except for the oil treatment (28.9) (Figure 6.3 and Table 6.3). In addition, severe stunting after treatment with glyphosate was observed on all regrowths of Cuscuta spp. (Figure 6.5). Growth Inhibition Results from the growth-inhibition study indicated that ammonium sulfate at any concentration did not inhibit A. destruens growth (Figures 6.6 and 6.7), while glyphosate at the highest concentration (0.09 kg (ae)/L) had the greatest inhibition (Figures 6.8 and 6.9). Oil and the untreated control were not significantly different (Figures 6.10 and 6.11). The mixture treatment provided the best control of Cuscuta spp. in trials I and III (combined) with an AUDPC value of 88.90. By 35 DAT, all Cuscuta spp. plants treated with the mixture treatment were dead. Citrus plants were not damaged by any treatment, even though glyphosate, despite the low rate used, injured and at times completely killed the Cuscuta spp. parasitizing the plants. Cuscuta spp. treated with glyphosate alone regrew with vegetative tissue that was severely distorted and stunted. This has been observed in other studies where glyphosate alone was used to control Cuscuta spp. (Dawson and Saghir 1983). Phytotoxic damage from ammonium sulfate, glyphosate, and oil (PCC 588) and disease severity from A. destruens could not be differentiated when rating Cuscuta spp.

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85 plants. All injury symptoms appeared the same on Cuscuta spp. Although, each treatment separately in trials I and III (combined) were not significantly different, but when mixed together were significantly different from all other treatments. Some formulations of glyphosate have exhibited fungicidal properties either by inhibiting, stimulating, or producing a synergistic interaction with various pathogenic and saprophytic fungal species (Anderson and Kolmer 2005; Chakravarty and Chatarpaul 1990; Wardle and Parkinson 1992). Studies have found that glyphosate produces an inhibitory effect on Drechslera teres (Sacc.) Shoemaker (Toubia-Rahme et al. 1995), Phytophthora spp. (Kassaby and Hepworth 1987; Utkhede 1982), Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn. (Anderson and Kolmer 2005), Puccinia triticina Eriks. (Anderson and Kolmer 2005), Pyrenophora tritici-repentis (Died.) Drechs. (Fernandez et al. 1998), Rhynchosporium secalis (Oudem) J. J. Davis (Turkington et al. 2001), and Septoria nodorum Berk. (Harris and Grossbard 1979); while producing a stimulatory effect on Pyrenophora teres Drechs (Turkington et al. 2001), Pythium coloratum Vaartaja and Pythium ultimum Trow (Descalzo et al. 1998). Other species, such as, Fusarium solani (Mart.) Sacc. (Njiti et al. 2003; Sanogo et al. 2000; Sanogo et al. 2001) and Rhizoctonia solani Khn (Black et al. 1996; Harikrishnan and Yang 2002; Smiley et al. 1992) have had effects from glyphosate ranging from inhibitory, to stimulatory, or to no effects. As to why there are differences among species or even within species from glyphosate, the answer lie in differences in herbicidal formulations (i.e. the active ingredient, inert ingredients, or an interaction between the two) rather than the glyphosate itself (Morjan et al. 2002).

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86 The growth-inhibition studies done with A. destruens and glyphosate did demonstrate that at higher concentrations glyphosate did impede the growth of the pathogen, therefore, the lowest concentration 0.02 kg (ae)/L of Roundup Pro was chosen for this study. The results from the AUDPC values indicate that glyphosate did not appear to have any inhibiting affect on A. destruens, but rather stimulate the pathogen. However, studies to determine if there were any differences in spore production were not done. Ammonium sulfate was added to the mixture treatment to increase the absorption rate of glyphosate and enhance foliar penetration. AUDPC values of ammonium sulfate and glyphosate alone indicate that there were no significant differences between the two treatments at the concentrations used in this study. Although, when ammonium sulfate, like glyphosate, was added to the mixture treatment, there was a significant difference in the AUDPC value (Figure 6-2 and Table 6-2). Oil (PCC 588) was added with A. destruens, to reduce the surface tension, which improves coverage over the plants surface, keeps the surface moist longer, and aids in attachment of the pathogen to the plant. Although, in trials I and III (combined), there were no significant differences between the AUDPC values of A. destruens + oil (56.50) and A. destruens without oil (49.95). The same is true for trial II where the AUDPC values for A. destruens + oil (33.70) and A. destruens (38.10) are not significantly different. Therefore, based on this study oil (PCC 588) may not be needed when applying A. destruens. This study provides evidence that by adding glyphosate and ammonium sulfate to A. destruens a synergistic effect was produced and better control of Cuscuta spp. was

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87 obtained than by using A. destruens alone or either of the other treatments alone. In light of the current control methods, this combination of a bioherbicide, herbicide, and nonionic surfactant appears to be the best integrated control method for Cuscuta spp.

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88 -1012345605101520253035DATDISEASE OR DAMAGE SEVERITY ALT ALTOIL AOGLAS AS125 GLY 0.02 kg (ae)/L OIL UNT Figure 6-1. Greenhouse trials I and III (combined) to determine the effects of disease or damage severity from Alternaria destruens, with (ALTOIL) or without (ALT) oil (PCC 588), ammonium sulfate at 0.125% (AS.125) w/v in water, glyphosate at 0.02 kg (ae)/L of Roundup Pro(GLY 0.02), a mixture of A. destruens + oil, glyphosate, and ammonium sulfate (the latter two at the above-mentioned concentrations) (AOGLAS), oil (PCC 588) only (OIL), and an untreated control (UNT) on Cuscuta pentagona over time (days after treatment; DAT).

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89 Table 6-1. Regression equations and R 2 values from greenhouse trials I and III (combined) for the effects of disease or damage severity from treatments on Cuscuta pentagona over time (days after treatment, DAT). Treatments Regression Equation R 2 A. destruens + Oil + Glyphosate at 0.02 kg (ae)/L of Roundup Pro Ammonium Sulfate 0.125% y = -0.0964x2 + 1.6107x 1.6893 R 2 = 0.99 Glyphosate at 0.02 kg (ae)/L of Roundup Pro y = 0.7155x 0.8821 R 2 = 0.98 Oil y = 0.6893x 0.8893 R 2 = 0.98 A. destruens + Oil y = -0.0506x2 + 0.9827x 1.1446 R 2 = 0.96 Ammonium Sulfate 0.125% y = 0.6548x 0.8464 R 2 = 0.97 A. destruens y = 0.6917x 1.1 R 2 = 0.95 Untreated Control y = 0.5583x 0.875 R 2 = 0.98

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90 0102030405060708090AOGLASGLY0.02OILALTOILAS.125ALTUNTTREATMENTSAUDPC a b b b b b b Figure 6-2. Effects of treatments on disease or damage severity on Cuscuta pentagona over time (days after treatment; DAT) from greenhouse trials I and III (combined). Area under the disease or damage progress curve (AUDPC) means within a column followed by the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05. AOGLAS = a mixture of Alternaria destruens + oil (PCC 588), glyphosate at 0.02 kg (ae)/L of Roundup Pro and ammonium sulfate at 0.125% w/v in water; GLY0.02 = glyphosate at 0.02 kg (ae)/L of Roundup Pro ; AS.125 = ammonium sulfate at 0.125% w/v in water; ALT = A. destruens; OIL = oil (PCC 588); ALTOIL = A. destruens + oil; UNT = untreated control.

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91 Table 6-2. Area under the disease or damage progress curve (AUDPC) means for all treatments on Cuscuta pentagona from greenhouse trials I and III (combined). Treatments AUDPC a A. destruens + Oil + Glyphosate at 0.02 kg (ae)/L of Roundup Pro Ammonium Sulfate 0.125% 88.90 a Glyphosate at 0.02 kg (ae)/L of Roundup Pro 61.15 b Oil 58.65 b A. destruens + Oil 56.50 b Ammonium Sulfate 0.125% 53.55 b A. destruens 49.95 b Untreated Control 42.25 b a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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92 0102030405060AOGLASAS.125UNTGLY 0.02ALTALTOILOILTREATMENTSAUDPC a ab ab ab ab ab b Figure 6-3. Effects of treatments on disease or damage severity on Cuscuta pentagona over time (days after treatment; DAT) from greenhouse trial II. Area under the disease or damage progress curve (AUDPC) means within a column followed by the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05. AOGLAS = a mixture of Alternaria destruens + oil (PCC 588), glyphosate at 0.02 kg (ae)/L of Roundup Pro and ammonium sulfate at 0.125% w/v in water; GLY 0.02 = glyphosate at 0.02 kg (ae)/L of Roundup Pro ; AS.125 = ammonium sulfate at 0.125% w/v in water; ALT = A. destruens; OIL = oil (PCC 588); ALTOIL = A. destruens + oil; UNT = untreated control.

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93 Table 6-3. Area under the disease or damage progress curve (AUDPC) means for all treatments on Cuscuta pentagona from greenhouse trial II. Treatments AUDPC a A. destruens + Oil + Glyphosate at 0.02 kg (ae)/L of Roundup Pro Ammonium Sulfate 0.125% 59.70 a Ammonium Sulfate 0.125% 51.60 ab Untreated Control 40.90 ab Glyphosate at 0.02 kg (ae)/L of of Roundup Pro 38.80 ab A. destruens 38.10 ab A. destruens + Oil 33.70 ab Oil 28.90 b a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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94 y = 0.1182x 0.4786 R2 = 0.97450123450102030DATDISEASE OR DAMAGE SEVERITY 40 Figure 6-4. Greenhouse trial II to determine the effect of time (DAT) on disease or damage severity across all treatments.

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95 Figure 6-5. Stunting of regrowth of Cuscuta pentagona treated with glyphosate at 0.02 kg (ae)/L of Roundup Pro

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96 Figure 6-6. Effect of ammonium sulfate concentrations (0.50%, 0.25%, 0.125%, and 0% (control) w/v in water) on the growth of Alternaria destruens colony (mm day -1 ).

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97 012345678902468DATCOLONY GROWTH (mm day-1) AS.50 AS.25 AS.125 CON y = 0.87x + 0.33 R 2 = 0.98 y = 0.86x + 0.59 R 2 = 0.96 y = 0.84x + 0.47 R 2 = 0.98 y = 0.81x + 0.39 R 2 = 0.99 10 Figure 6-7. Inhibition of growth of Alternaria destruens colony (mm day -1 ) over time (DAT) by ammonium sulfate at 0.50%, 0.25%, 0.125% and 0% ((CON) w/v in water).

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98 Figure 6-8. Effect of glyphosate concentrations (0.09, 0.04, 0.02 and 0 (control) kg (ae)/L of Roundup Pro ) on the growth of Alternaria destruens colony (mm day -1 ).

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99 012345678902468DATCOLONY GROWTH (mm day-1) GLY0.09 GLY0.04 GLY0.02 CON y = 0.20x + 0.20 R 2 = 0.95 y = 0.41x + 0.49 R 2 = 0.93 y = 0.62x + 0.37 R 2 = 0.98 y = 0.81x + 0.39 R 2 = 0.99 10 Figure 6-9. Inhibition of growth (mm day -1 ) of Alternaria destruens over time (DAT) by glyphosate at (0.09, 0.04, 0.02 and 0 (CON)) kg (ae)/L of Roundup Pro ).

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100 Figure 6-10. Effect of oil (PCC 588) (7.5% v/v in water) on the growth of Alternaria destruens compared to untreated control (no oil).

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101 y = 0.8528x + 0.3142 R2 = 0.99y = 0.8134x + 0.3948 R2 = 0.99012345678902468DATCOLONY GROWTH (mm day-1) OIL CON 10 Figure 6-11. Effect of oil (PCC 588) at 7.5% v/v in water and control (CON) on the growth (mm day -1 ) of Alternaria destruens over time (DAT). There were no significant differences between the oil treatment and the control.

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APPENDIX A FEDERAL NOXIOUS WEED LIST (AS OF JANUARY 6, 2006) Cuscuta spp. (dodders), other than following species: Cuscuta americana L. Cuscuta harperi Small Cuscuta applanata Engelm. Cuscuta howelliana Rubtzoff Cuscuta approximata Bab. Cuscuta indecora Choisy Cuscuta attenuata Waterfall Cuscuta jepsonii Yunck. Cuscuta boldinghii Urban Cuscuta leptantha Engelm. Cuscuta brachycalyx (Yuncker) Yunck. Cuscuta mitriformis Engelm. Cuscuta californica Hooker & Arnott Cuscuta nevadensis I. M. Johnston Cuscuta campestris Yunck. Cuscuta obtusiflora Kunth Cuscuta cassytoides Nees ex Engelm. Cuscuta occidentalis Millsp. ex Mill & Nuttall Cuscuta ceanothii Behr Cuscuta odontolepis Engelm. Cuscuta cephalanthii Engelm. Cuscuta pentagona Engelm. Cuscuta compacta Juss. ex Choisy Cuscuta planiflora Ten. Cuscuta corylii Engelm. Cuscuta plattensis A. Nels. Cuscuta cuspidata Engelm. Cuscuta polygonorum Engelm. Cuscuta decipiens Yunck. Cuscuta rostrata Shuttlew. ex Engelm. & Gray Cuscuta dentatasquamata Yunck. Cuscuta runyonii Yunck. Cuscuta denticulata Engelm. Cuscuta salina Engelm. Cuscuta epilinum Weihe Cuscuta sandwichiana Choisy Cuscuta epithymum (L.) L. Cuscuta squamata Engelm. Cuscuta erosa Yunck. Cuscuta suaveolens Ser. Cuscuta europaea L. Cuscuta suksdorfii Yunck. Cuscuta exalta Engelm. Cuscuta tuberculata Brandeg. Cuscuta fasciculata Yunck. Cuscuta umbellata Kunth Cuscuta glabrior (Engelm.) Yunck. Cuscuta umbrosa Beyr. ex Hook. Cuscuta globulosa Benth. Cuscuta veatchii Brandeg. Cuscuta glomerata Choisy Cuscuta warneri Yunck. Cuscuta gronovii Willd. ex J.A. Schultes 102

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APPENDIX B WIND SPEED DATA Table B-1. Ft. Pierce trial I average wind speed data. Collected by the Florida Automated Weather Network (FAWN). Date Wind Speed (m s -1 ) Date Wind Speed (m s -1 ) 3 Dec 2003 4.92 1 Jan 2004 1.34 4 Dec 2003 4.02 2 Jan 2004 1.34 5 Dec 2003 1.34 3 Jan 2004 2.24 6 Dec 2003 3.58 4 Jan 2004 1.79 7 Dec 2003 3.13 5 Jan 2004 1.34 8 Dec 2003 1.79 6 Jan 2004 2.68 9 Dec 2003 2.24 7 Jan 2004 5.36 10 Dec 2003 3.58 8 Jan 2004 1.79 11 Dec 2003 2.24 9 Jan 2004 1.34 12 Dec 2003 1.34 10 Jan 2004 4.02 13 Dec 2003 1.34 11 Jan 2004 4.47 14 Dec 2003 3.13 12 Jan 2004 1.79 15 Dec 2003 2.68 13 Jan 2004 1.34 16 Dec 2003 2.24 14 Jan 2004 1.34 17 Dec 2003 3.13 15 Jan 2004 2.24 18 Dec 2003 2.24 16 Jan 2004 1.79 19 Dec 2003 2.68 17 Jan 2004 2.24 20 Dec 2003 2.68 18 Jan 2004 2.24 21 Dec 2003 3.58 19 Jan 2004 1.79 22 Dec 2003 1.79 20 Jan 2004 3.13 23 Dec 2003 1.34 21 Jan 2004 1.79 24 Dec 2003 2.24 22 Jan 2004 1.79 25 Dec 2003 2.68 23 Jan 2004 2.24 26 Dec2003 2.68 24 Jan 2004 1.34 103

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104 27 Dec 2003 2.68 25 Jan 2004 1.79 28 Dec 2003 2.68 26 Jan 2004 3.13 29 Dec 2003 2.68 27 Jan 2004 3.58 30 Dec 2003 1.79 28 Jan 2004 3.13 31 Dec 2003 1.34 29 Jan 2004 2.24

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105 Table B-2. Ft. Pierce trial II average wind speed data. Collected by the Florida Automated Weather Network (FAWN). Date Wind Speed (m s -1 ) Date Wind Speed (m s -1 ) 23 Feb 2004 2.24 23 Mar 2004 5.81 24 Feb 2004 3.13 24 Mar 2004 5.81 25 Feb 2004 2.68 25 Mar 2004 5.36 26 Feb 2004 2.68 26 Mar 2004 6.26 27 Feb 2004 4.02 27 Mar 2004 4.02 28 Feb 2004 4.92 28 Mar 2004 1.79 29 Feb 2004 3.13 29 Mar 2004 3.13 1 Mar 2004 4.02 30 Mar 2004 2.24 2 Mar 2004 3.58 31 Mar 2004 2.68 3 Mar 2004 2.24 1 Apr 2004 2.68 4 Mar 2004 3.58 2 Apr 2004 2.68 5 Mar 2005 4.02 3 Apr 2004 2.68 6 Mar 2004 2.68 4 Apr 2004 2.68 7 Mar 2004 2.24 5 Apr 2004 2.24 8 Mar 2004 2.68 6 Apr 2004 2.24 9 Mar 2004 1.79 7 Apr 2004 2.68 10 Mar 2004 3.58 8 Apr 2004 3.58 11 Mar 2004 2.68 9 Apr 2004 2.24 12 Mar 2004 1.34 10 Apr 2004 2.24 13 Mar 2004 2.68 11 Apr 2004 3.13 14 Mar 2004 3.13 12 Apr 2004 3.13 15 Mar 2004 3.58 13 Apr 2004 4.47 16 Mar 2004 3.13 14 Apr 2004 3.58 17 Mar 2004 2.24 15 Apr 2004 2.68 18 Mar 2004 1.79 16 Apr 2004 2.68 19 Mar 2004 2.24 17 Apr 2004 3.58 20 Mar 2004 4.47 18 Apr 2004 4.47 21 Mar 2004 1.79 19 Apr 2004 3.58 22 Mar 2004 4.02

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106 Table B-3. Gainesville field study average wind speed data. Collected by the Florida Automated Weather Network (FAWN). Date Wind Speed (m s -1 ) Date Wind Speed (m s -1 ) 15 Jul 2005 1.34 1 Aug 2005 1.34 16 Jul 2005 1.34 2 Aug 2005 1.34 17 Jul 2005 1.34 3 Aug 2005 1.34 18 Jul 2005 1.34 4 Aug 2005 1.34 19 Jul 2005 1.79 5 Aug 2005 1.34 20 Jul 2005 1.34 6 Aug 2005 1.34 21 Jul 2005 1.34 7 Aug 2005 0.89 22 Jul 2005 1.34 8 Aug 2005 1.34 23 Jul 2005 2.24 9 Aug 2005 1.34 24 Jul 2005 1.79 10 Aug 2005 1.34 25 Jul 2005 1.79 11 Aug 2005 1.34 26 Jul 2005 0.89 12 Aug 2005 0.89 27 Jul 2005 0.89 13 Aug 2005 0.89 28 Jul 2005 0.89 14 Aug 2005 2.24 29 Jul 2005 1.34 15 Aug 2005 1.34 30 Jul 2005 1.79 16 Aug 2005 1.34 31 Jul 2005 1.34 17 Aug 2005 0.45

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APPENDIX C ENVIRONMENTAL DATA FOR GREENHOUSE TRIALS OF ALTERNARIA DESTRUENS, AMMONIUM SULFATE AT 0.125% W/V IN WATER, AND GLYPHOSATE AT 0.02 KG (AE)/L OF ROUNDUP PRO ON CUSCUTA PENTAGONA 051015202530354045501357911131517192123252729313335DATTEMPERATURE (C) Max. Temp. (C) Min. Temp. (C) Figure C-1. Maximum and minimum temperatures ( o C) during greenhouse trial I to determine the effects of disease or damage severity from Alternaria destruens, with (ALTOIL) or without oil (PCC 588) (ALT), ammonium sulfate at 0.125% (AS.125) w/v in water, glyphosate at 0.02 kg (ae)/L of Roundup Pro (GLY 0.02), a mixture of A. destruens + oil, glyphosate, and ammonium sulfate (the latter two at the above-mentioned concentrations) (AOGLAS), oil (PCC 588) only (OIL) and an untreated control (UNT) on Cuscuta pentagona over time (days after treatment; DAT). Data were recorded with a HOBO Pro RH/Temp Data Logger (H08-032-08) (Onset Computer Corporation, Bourne, MA). 107

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108 051015202530354045501357911131517192123252729313335DATTEMPERATURE (C) Max. Temp. (C) Min. Temp. (C) Figure C-2. Maximum and minimum temperatures ( o C) during greenhouse trial II to determine the effects of disease or damage severity from Alternaria destruens, with (ALTOIL) or without oil (PCC 588) (ALT), ammonium sulfate at 0.125% (AS.125) w/v in water, glyphosate at 0.02 kg (ae)/L of Roundup Pro (GLY 0.02), a mixture of A. destruens + oil, glyphosate, and ammonium sulfate (the latter two at the above-mentioned concentrations) (AOGLAS), oil (PCC 588) only (OIL) and an untreated control (UNT) on Cuscuta pentagona over time (days after treatment; DAT). Data were recorded with a HOBO Pro RH/Temp Data Logger (H08-032-08) (Onset Computer Corporation, Bourne, MA).

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109 051015202530354045501357911131517192123252729313335DATTEMPERATURE (C) Max. Temp. (C) Min. Temp. (C) Figure C-3. Maximum and minimum temperatures ( o C) during greenhouse trial III to determine the effects of disease or damage severity from Alternaria destruens, with (ALTOIL) or without oil (PCC 588) (ALT), ammonium sulfate at 0.125% (AS.125) w/v in water, glyphosate at 0.02 kg (ae)/L of Roundup Pro (GLY 0.02), a mixture of A. destruens + oil, glyphosate, and ammonium sulfate (the latter two at the above-mentioned concentrations) (AOGLAS), oil (PCC 588) only (OIL) and an untreated control (UNT) on Cuscuta pentagona over time (days after treatment; DAT). Data were recorded with a HOBO Pro RH/Temp Data Logger (H08-032-08) (Onset Computer Corporation, Bourne, MA).

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110 0204060801001357911131517192123252729313335DATMAXIMUM RELATIVE HUMIDITY STUDY I STUDY II STUDY III Figure C-4. Maximum relative humidity during all greenhouse trials to determine the effects of disease or damage severity from Alternaria destruens, with (ALTOIL) or without oil (PCC 588) (ALT), ammonium sulfate at 0.125% (AS.125) w/v in water, glyphosate at 0.02 kg (ae)/L of Roundup Pro (GLY 0.02), a mixture of A. destruens + oil, glyphosate, and ammonium sulfate (the latter two at the above-mentioned concentrations) (AOGLAS), oil (PCC 588) only (OIL) and an untreated control (UNT) on Cuscuta pentagona over time (days after treatment; DAT). Data were recorded with a HOBO Pro RH/Temp Data Logger (H08-032-08) (Onset Computer Corporation, Bourne, MA).

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APPENDIX D DATA FROM PRELIMINARY GREENHOUSE TRIAL 0123450716232936DATDAMAGE SEVERITY 0 kg (ae)/L 0.09 kg (ae)/L 0.18 kg (ae)/L 0.36 kg (ae)/L 0.71 kg (ae)/L 1.42 kg (ae)/L Figure D-1. Damage severity on Cuscuta pentagona over time from six glyphosate concentrations (0.09, 0.18, 0.36, 0.71, 1.42, and 0 (control) kg (ae)/L of Roundup Pro ). Preliminary greenhouse trials (I and II) were conducted from May through August 2004. No significant differences between trials were detected; therefore data were combined for both trials. 111

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112 Table D-1. Area under the disease or damage progress curve (AUDPC) means for all preliminary glyphosate treatments on Cuscuta pentagona in greenhouse trials I and II (combined). Treatments AUDPC a Glyphosate at 1.42 kg (ae)/L of Roundup Pro 134.31 a Glyphosate at 0.71 kg (ae)/L of Roundup Pro 131.75 a Glyphosate at 0.36 kg (ae)/L of Roundup Pro 125.37 ab Glyphosate at 0.18 kg (ae)/L of Roundup Pro 110.81 bc Glyphosate at 0.09 kg (ae)/L of Roundup Pro 105.69 c Glyphosate at 0.0 kg (ae)/L of Roundup Pro 62.25 d a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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113 010203040506070809000.090.180.360.711.42GLYPHOSATE CONCENTRATIONS (kg (ae)/L)AUDPC TRIAL I TRIAL II A A A A a ab ab A A ab ab b Figure D-2. Damage severity on Citrus spp. from six glyphosate concentrations concentrations (0.09, 0.18, 0.36, 0.71, 1.42, and 0 (control) kg (ae)/L of Roundup Pro ), averaged over all days. Preliminary greenhouse trials (I and II) were conducted from May through August 2004. Due to significant differences between trials (P=0.0001), data were analyzed separately. Area under the disease or damage progress curve (AUDPC) means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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114 Table D-2. Area under the disease or damage progress curve (AUDPC) means for all preliminary glyphosate treatments on Citrus spp. in greenhouse trials I and II. TREATMENTS AUDPC a TRIAL I TRIAL II Glyphosate at 1.42 kg (ae)/L of Roundup Pro 48.38 a 63.88 a Glyphosate at 0.71 kg (ae)/L of Roundup Pro 41.00 ab 78.63 a Glyphosate at 0.36 kg (ae)/L of Roundup Pro 35.88 ab 74.38 a Glyphosate at 0.18 kg (ae)/L of Roundup Pro 13.50 ab 35.63 a Glyphosate at 0.09 kg (ae)/L of Roundup Pro 15.50 ab 23.13 a Glyphosate at 0.0 kg (ae)/L of Roundup Pro 2.63 b 52.50 a a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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115 y = -0.2209x2 + 2.5422x 2.301 R2 = 0.9996y = -0.1917x2 + 2.4013x 2.4019 R2 = 0.99010123450714212835DATDAMAGE SEVERITY TRIAL I TRIAL II Figure D-3. Damage severity on Cuscuta pentagona from seven ammonium sulfate concentrations (0% (control), 1%, 2%, 3%, 4%, 5%, and 10% w/v in water) over time (DAT). Preliminary greenhouse trials (I and II) were conducted from July through August 2003. Due to significant differences between trials (P=0.0165), data were analyzed separately. No significant differences between treatments were detected in each trial; therefore data for treatments were combined within each trial.

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116 Table D-3. Area under the disease or damage progress curve (AUDPC) means for all preliminary ammonium sulfate treatments on Cuscuta pentagona in greenhouse trials I and II (combined). Treatments AUDPC a Ammonium Sulfate at 0% 103.25 a Ammonium Sulfate at 1% 116.37 a Ammonium Sulfate at 2% 109.81 a Ammonium Sulfate at 3% 111.12 a Ammonium Sulfate at 4% 112.00 a Ammonium Sulfate at 5% 113.75 a Ammonium Sulfate at 10% 104.12 a a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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117 051015200%1%2%3%4%5%10%AMMONIUM SULFATE CONCENTRATIONSAUDPC a a a a a a a Figure D-4. Damage severity on Citrus spp. from seven ammonium sulfate concentrations (0% (control), 1%, 2%, 3%, 4%, 5%, and 10% w/v in water) averaged over all days. Preliminary greenhouse trials (I and II) were conducted from July through August 2003. No significant differences between trials were detected; therefore data were combined for both trials. Area under the disease or damage progress curve (AUDPC) means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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118 Table D-4. Area under the disease or damage progress curve (AUDPC) means for all ammonium sulfate treatments on Citrus spp. in greenhouse trials I and II (combined). Treatments AUDPC a Ammonium Sulfate at 0% 7.87 a Ammonium Sulfate at 1% 13.12 a Ammonium Sulfate at 2% 13.12 a Ammonium Sulfate at 3% 13.12 a Ammonium Sulfate at 4% 15.75 a Ammonium Sulfate at 5% 16.18 a Ammonium Sulfate at 10% 12.25 a a Means with the same letter are not significantly different according to Tukey's Studentized Range (HSD) test at P=0.05.

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BIOGRAPHICAL SKETCH Jennifer Colleen Shaffer (Cook) was born September 4, 1968 in Pittsburgh, PA. Her parents, Catherine and Ed, raised her and her brother, Robert, in the small town of Midway, PA. In August of 1986 she began college at the Pennsylvania State University, majoring in biology. After 2 years, she decided to take a year off and join the Pennsylvania Air National Guard with her brother. She continued her education at Behrend College, a branch campus of Penn State, and earned her Bachelor of Science degree in biology in June 1991. While deciding what to do with her life, Jennifer worked full time at the national headquarters for the Air National Guard at Andrews Air Force Base, MD. She married Jeffrey Cook and they eventually moved to Raleigh, NC where she received her Master of Science degree in horticulture in 2001. In January 2002, she received a research assistantship to pursue her Doctor of Philosophy degree in plant pathology at the University of Florida. After graduation Jennifer would like to continue to work in the field of plant pathology and eventually retire from the military. 130