Physiological aspects of hydrilla Hydrilla verticillata (L.f.) Royle growth and reproduction

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Title:
Physiological aspects of hydrilla Hydrilla verticillata (L.f.) Royle growth and reproduction
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xii, 113 leaves : ill. ; 29 cm.
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MacDonald, Gregory E., 1963-
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Agronomy thesis Ph.D
Dissertations, Academic -- Agronomy -- UF
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Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 100-112).
Statement of Responsibility:
by Gregory E. MacDonald.
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Typescript.
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Vita.

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University of Florida
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PHYSIOLOGICAL ASPECTS OF
HYDRILLA [Hydrilla verticillata (L.f.) Royle]
GROWTH AND REPRODUCTION















By

GREGORY E. MacDONALD


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


1994





















In memory of George E. MacDonald; your love, support, guidance and

understanding are the basis for all I have attained.












ACKNOWLEDGEMENTS


For their support and guidance during my tenure at the University of Florida

I wish to express my sincere appreciation to my committee members: Dr. Thomas

Bewick, Dr. George Bowes, Dr. William Haller, and Dr. Michael Kane. I would like

to extend my most sincere appreciation to my major advisor, Dr. Donn Shilling, for

his patience, understanding and guidance throughout my tenure at Florida. I would

also like to acknowledge the advice and support of Drs. Daniel Colvin, Ken

Langeland, and Bert McCarty.

In addition, I wish to acknowledge Christine Kelly, Bob Querns, Ron Kern,

Jim Gaffney, Jigna Thakore, Sean and Cindy Ragland, Laura Rankin, Margaret

Glenn, Jan Miller, Jamey Carter, Sandra McDonald, Brian Smith, Ken Smith,

Carlene Chase, Eric Bish, and Ron Doong. Without their valuable time and

assistance this research would have been extremely difficult, if not impossible.

For the two most important women in my life, I wish to express my deepest

gratitude to my wife Mickey and my mother Marilyn MacDonald. No words can

adequately describe the value of their support. I would also like to thank my

brother, Tim MacDonald, for his support and friendship.

During my tenure at the University of Florida I have had the opportunity to

build lasting friendships. The experiences I have shared will always be cherished.













TABLE OF CONTENTS


ACKNOWLEDGEMENTS ..............................................................

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

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

ABSTRACT ..................................................................................................

CHAPTERS

I INTRODUCTION ............................................................

II EVALUATION OF AN ENZYME LINKED
IMMUNOASSAY PROCEDURE FOR THE
ANALYSIS OF ABSCISIC ACID
IN HYDRILLA ............................................................

Introduction ..............................................................
Materials and Methods ................................................
Elution Profiles and Column/ABA
Characterization ..................................................
ELISA Internal Standardization
for ABA in Hydrilla ......................................
Results and Discussion ................................................

III THE INTERACTIVE EFFECT OF PHOTOPERIOD
AND FLURIDONE ON THE GROWTH,
REPRODUCTION, AND BIOCHEMISTRY OF
HYDRILLA [Hydrilla verticillata
(L.f.) Royle] ..............................................................

Introduction ..............................................................
Materials and Methods ................................................
Chlorophyll and Carotenoid Analyses ............
Anthocyanin Analysis .....................................
Abscisic Acid Analysis .....................................

iv


PAGE

iii

vii

x

xi



1




20

20
23

23

25
26





39

39
43
45
45
46







Statistical Analysis ................................................. 46
Results and Discussion ................................................. 47


IV THE INFLUENCE OF FLURIDONE, PHOTOPERIOD,
AND PLANT GROWTH REGULATORS ON THE
GROWTH AND REPRODUCTION OF MONOECIOUS
AND DIOECIOUS HYDRILLA (Hydrilla verticillata
(L.f.) R oyle] ............................................................. 62
Introduction ........................................................... 62
M materials and M ethods ................................................. 65
Photoperiodic Response ..................................... 67
Effect of Fluridone ................................................. 67
Effect of Exogenous Abscisic Acid and
Gibberellic Acid ................................................ 68
Statistical Analysis ................................................. 68
Results and Discussion ................................................. 69

V SUMMARY AND CONCLUSIONS ........................ 89

APPENDICES

A ELISA PLATE COATING
PROCEDURE ................................................ 91

B ELISA SAMPLE QUANTIFICATION ............. 92

C ELISA STOCK SOLUTIONS AND
STANDARDS .................................................. 93

D ELISA BUFFERS AND SOLUTIONS ............. 94

E ELISA STANDARD CURVE AND SAMPLE
ABA DETERMINATION .................................... 95

F ABSCISIC ACID COLUMN EXTRACTION
PROCEDURE ................................................. 96

G THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF MATURE HYDRILLA
ROOT CROWNS ................................................ 97







H THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF MATURE HYDRILLA
ROOT CROWNS ................................................ 98

I THE EFFECT OF PHOTOPERIOD AND
FLURIDONE ON THE ABSCISIC ACID
CONTENT OF YOUNG HYDRILLA ............ 99

LITERATURE CITED ........................................................................ 100

BIOGRAPHICAL SKETCH ............................................................. 113












LIST OF TABLES

TABLE PAGE

2.1 The effect of elution volume and methanol
concentration on abscisic acid C18 column retention
under neutral pH conditions ..................................... 27

2.2 The effect of elution volume and methanol
concentration on abscisic acid C18 column retention
under low pH conditions ................................................. 28

2.3 The effect of plant tissue and column extraction on
the recovery of abscisic acid. Abscisic acid was added
before or after column clean-up .................................... 30

3.1 The effect of photoperiod and fluridone on the dry
weight of mature hydrilla plants .................................... 48

3.2 The effect of photoperiod and fluridone on the dry
weight of young hydrilla plants .................................... 49

3.3 The effect of photoperiod and fluridone on the
subterranean turion production of
m ature hydrilla plants ................................................. 51

3.4 The effect of photoperiod and fluridone on the
subterranean turion production of young
hydrilla ........................................................................... 52

3.5 The effect of photoperiod and fluridone on the
chlorophyll content of mature hydrilla plants ............. 55

3.6 The effect of photoperiod and fluridone on the
carotenoid content of mature hydrilla plants ............. 56

3.7 The effect of photoperiod and fluridone on the
anthocyanin content of mature hydrilla plants ............. 57








3.8 The effect of fluridone concentration on the
abscisic acid content of mature hydrilla apical stem
segments, average across photoperiodic regime ............. 60

4.1 The effect of photoperiod on the growth of 8 week
old dioecious and monoecious hydrilla ......................... 70

4.2 The effect of photoperiod on the axillary turion
production of 8 week old dioecious and monoecious
hydrilla ........................................................................... 71

4.3 The effect of photoperiod on the subterranean turion
production of 8 week old dioecious and monoecious
hydrilla ........................................................................... 72

4.4 The effect of fluridone on the growth of dioecious
and monoecious hydrilla. Plants were grown under
long-day conditions for 12 weeks ..................... 74

4.5 The effect of fluridone on the axillary turion
production of monoecious hydrilla. Plants were
grown under long-day conditions for 12 weeks ............. 76

4.6 Axillary turion production by 8 week old, long-day grown
dioecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications ............... 78

4.7 Axillary turion production by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications ........................ 79

4.8 Axillary turion weight per plant by 8 week old, long-day
grown dioecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications ........................ 80

4.9 Axillary turion weight per plant by 8 week old, long-day
grown monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications ....................... 81

4.10 Percent weight of axillary turions produced by 8 week old,
long-day grown dioecious hydrilla as influenced by
exogenous abscisic and gibberellic acid
applications ........................................................................... 83









4.11 Percent weight of axillary turions produced by 8 week
old, long-day grown monoecious hydrilla as influenced by
exogenous abscisic and gibberellic acid
applications ........................................................................... 84

4.12 Shoot biomass production by 8 week old, long-day grown
dioecious hydrilla as influenced by exogenous abscisic
and gibberellic acid applications ...................................... 85

4.13 Shoot biomass production by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous
abscisic and gibberellic acid applications ........................ 86













LIST OF FIGURES


FIGURE PAGE

2.1 Internal abscisic acid standardization for column
cleanup procedure with hydrilla tissue ........................ 32

2.2 Internal standardization of abscisic acid quantification
using ELISA with hydrilla stem tissue. Range of
values was 0.02 to 0.8 pmol ..................................... 34

2.3 Internal standardization of abscisic acid quantification
using ELISA with hydrilla stem tissue. Range of
values was 0.2 to 5.0 pmol ................................................ 35

2.4 Internal standardization of abscisic acid quantification
using ELISA with hydrilla turion tissue. Range of
values was 0.02 to 0.6 pmol ..................................... 36

2.5 Internal standardization of abscisic acid quantification
using ELISA with hydrilla turion tissue. Range of
values was 0.2 to 5.0 pmol ................................................ 37












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

PHYSIOLOGICAL ASPECTS OF HYDRILLA
[Hydrilla verticillata (L.f.) Royle]
GROWTH AND REPRODUCTION

By

Gregory E. MacDonald

August, 1994


Chairman: Dr. D.G. Shilling
Major Department: Agronomy

The goal of this research was to provide a better understanding of the

fundamental processes involved in hydrilla growth and reproduction. Abscisic acid

from hydrilla tissue was analyzed by enzyme-linked immunoassay. Although solid-

phase extraction provided good purification of abscisic acid present in hydrilla tissue,

no clean up of the extract was necessary prior to analysis. Crude, unpurified extracts

of hydrilla stem and turion tissue were analyzed by ELISA. Validation of abscisic

acid extraction and analysis procedure was verified by internal standardization.

Accurate quantification could be obtained from plant extracts containing 0.02 to 5.0

pmol of abscisic acid. Short days promoted subterranean turion formation but this

effect was reduced by long days and 5 and 10 ppb fluridone. Fluridone caused a

significant reduction in chlorophyll and carotenoid levels and the abscisic acid







content of mature plant shoot tips. The concentration of abscisic acid in young

plants was higher under short days. These studies provide further evidence that

fluridone can be used as a fall herbicide treatment to reduce turion formation in

hydrilla. The growth of both biotypes was reduced under short-day conditions.

Monoecious hydrilla formed axillary and subterranean turions regardless of

photoperiod, while dioecious hydrilla formed only subterranean turions only under

short-day conditions. Fluridone reduced the growth of both biotypes and reduced

turion production in monoecious hydrilla. Exogenously applied abscisic acid at 0.1,

1.0, and 10 tyM induced axillary turion formation by dioecious hydrilla under long day
conditions and abscisic acid at 1.0 and 10 /M induced axillary turion formation by

monoecious hydrilla. This process could be reversed by gibberellic acid in both

biotypes. However, subterranean turion formation was not observed under any

treatment for the dioecious biotype, but was observed in control monoecious hydrilla

plants. Monoecious hydrilla could be induced to flower by 50 1M gibberellic acid
applications. This study indicates abscisic acid alone does not control axillary turion

formation in monoecious and dioecious hydrilla, but rather a balance between

abscisic acid and gibberellic acid may be the key to regulation of this phenological

process.












CHAPTER I
INTRODUCTION

Hydrilla [Hydrilla verticillata (L.f.) Royle] is an exotic, submersed aquatic

vascular plant that causes major problems in the freshwater ecosystems of Florida

and the southeastern United States (Haller, 1976). Hydrilla interferes with

navigation, flood control and most recreational water activities, including fishing,

through a displacement of native vegetation (Langeland, 1990). Hydrilla was

discovered in 1959 in Florida, in a Miami canal. It was also reported in Crystal

River during the same period and was originally called Florida Elodea (Blackburn

et al., 1969).

Hydrilla is a monotypic genus in the family Hydrocharitaceae, taxonomically

an ancient monocot family, with the center of origin thought to be tropical Asia

(Cooke and Luond, 1982). However, hydrilla is cosmopolitan, and is reported to be

broadly distributed throughout Germany, Lithuania, England, Poland, the upper Nile

of Africa, southeast Asia, Australia, Madagascar, India, China, Japan and the

southern United States (Lazor, 1978). A review of hydrilla by Mitra (1955) provided

the first detailed description of the species with historical background information.

Linnaeus filius first described hydrilla as Serpicula verticillata and classified it in the

Haloragaceae family. Hydrilla was later placed into the Hydrocharitaceae family

under the name Hydrilla ovalifolia but it was later changed to Hydrilla verticillata.







2
Hydrilla has been characterized by many scientists, as indicated by several describing

authors including Presl., Royle, and Casp. Today, Hydrilla verticillata (Lf.) Royle or

Casp. is the scientific name used to denote hydrilla. Langeland et al. (1992) showed

hydrilla to be endopolyploid, with monoecious hydrilla being triploid and dioecious

hydrilla being diploid; but, evidence from root tip karyotypes suggest sexual

compatibility across hydrilla populations and biotypes.

Anatomically, hydrilla is considered to be a primitive plant with reduced

vasculature. The xylem tissue is vestigial and phloem members are greatly reduced

(Yeo et al., 1984). The leaves are comprised of two contiguous epidermal layers with

cuneate plastid inclusions, lacking in starch granules characteristic of most

monocots. The upper epidermis is much thicker than the lower layer (Pendland,

1979). Hydrilla leaves are borne in whorls of 4 or 5, with a distinctive spiny midrib

on the abaxial side of the leaf, distinguishing this species from elodea. Plants are

attached to the hydrosoil through fibrous roots and may resprout from root crowns

at the surface of the hydrosoil (Haller, 1976). Flowers are borne on stems near the

water surface (Cooke and Luond, 1982). Male and female flowers may be borne on

the same monoeciouss) or separate (dioecious) plants. The control of floral

production is not well known but Pieterse metal. (1984), reported that low nitrogen

and phosphorus concentrations stimulated flower production in monoecious hydrilla.

Hydrilla regrows in the spring, as water temperature begins to increase, from

root crowns and/or subterranean turions in the hydrosoil (Haller, 1976). Shoot

growth is regulated by light quality with green light (prominent at deeper depths)







3

promoting stem elongation and red light (prominent at the surface) stimulating

branching (Van et al., 1977). Hydrilla has also been shown to increase the level of

chlorophyll b at greater depths, enabling the plant to use longer wavelengths of light

for photosynthesis (Van et al., 1977).

The development of a thick, entangled growth at the water surface (mat)

occurs when the shoots reach the water surface. This forms a dense canopy and

reduces light penetration in the first 0.3 m by 95% (Haller and Sutton, 1975). The

rapid growth of hydrilla has been reported to be 4.2 g dry wt'm2"day' (DeBusk et

al. 1981). Hydrilla is extremely competitive, forming large, monotypic stands.

Studies on the photosynthetic characteristics revealed hydrilla to have a CO2

compensation point of 44 1LL'-1 with light saturation occurring at 600 to 700

1mol'm-2"s1 PPFD (Van et al., 1976). Hydrilla can adapt to low light levels with a

light compensation point of 10 to 12 Amol'm-2s-1 PPFD (Bowes et al., 1977).

Holaday and Bowes (1980) reported hydrilla could exist with low or normal CO2

compensation points. Those plants with low compensation points had high activity

of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) and pyruvate, Pi dikinase (E.C.

2.7.9.1) enzyme levels, suggesting that hydrilla is capable of both C3 and C4

photosynthetic metabolism and the change was due to low CO2 concentrations in the

water (Barko and Smart, 1981; Holaday et al., 1983). Salvucci and Bowes (1983)

implicated carbonic anhydrase (E.C. 4.2.1.1) in the reduction of photorespiration in

hydrilla during periods of low CO2 compensation points. Ascencio and Bowes (1983)

also reported a major increase in PEP carboxylase under these conditions.







4
Collectively, these characteristics allow hydrilla to utilize the free CO2 from the water

sooner after sunrise than other aquatic plants due its low light compensation point.

Furthermore, hydrilla can maintain net photosynthesis under low CO2 levels through

the activity of PEPcarboxylase. This increases hydrilla's competiveness, decreasing

competition from other species and allows hydrilla to maintain the luxuriant growth

of the mat under low CO2 and high 02 conditions. In addition, Kulshreshtha and

Gopal (1983) reported allelopathic properties of hydrilla on species of Ceratophyllum,

further increasing the competitive advantage of this species.

Several studies have evaluated the effect of mineral nutrition on hydrilla

growth. Sutton (1985) suggested the growth of hydrilla is controlled by nutrients in

the root zone, while Spencer (1986) showed hydrilla could grow equally well under

low or high organic matter sediment composition. Stewart (1984) showed phosphorus

in the rooting media was the limiting factor in growth for hydrilla. Basiouny and

Garrard (1984) demonstrated that calcium and phosphorus uptake in hydrilla was

passive while potassium, copper, iron and manganese uptake required metabolic

energy. Iron appeared to be particularly important for hydrilla growth with

tremendous iron uptake and an exceptionally high iron to manganese ratio required

for optimum growth (Basiouny et al., 1977b). Basiouny et al. (1977a) showed iron

was absorbed by roots and leaves of hydrilla and translocated throughout the plant.

Hydrilla is extremely difficult to manage and control because spread can occur

through a variety of mechanisms including fragmentation and specialized dormant

buds called turions (Sculthorpe, 1967). Langeland and Sutton (1980) reported







5
regrowth from hydrilla fragments with a single node and Sutton metal. (1980) reported

one shoot tip will produce as much biomass as 16 tips. Seed production has been

reported for the monoecious biotype but does not occur in dioecious hydrilla in the

United States.

Turions can be formed in the axials of leaves, or at the ends of positively

geotropic rhizomes which extend into the hydrosoil (Haller, 1976; Yeo et al., 1984).

Subterranean turions (tubers) can remain dormant for as long as 5 years (Van and

Stewart, 1990). Miller et al. (1976) reported that increased tuber size and density

were correlated to increased water depth. There appears to be an environmentally

enforced dormancy of tubers, preventing rapid depletion of the tuber bank (Van and

Stewart, 1990). Basiouny metal. (1978b) showed gibberellic acid, ethephon, thiourea

and storage at 5 C to increase turion sprouting, and hypothesized the existence of

two types of dormancy in hydrilla subterranean turions. Stewart (1969) found

gibberellic acid enhanced sprouting of subterranean turions while IAA and 2,4-D

enhanced axillary turion development. Subterranean turions are thought to maintain

hydrilla within a given area, possibly through periods of drought. Work by Basiouny

et al. (1978a) showed that tubers are better able to survive drought than axillary

turions with almost 17% germination rate for tubers dried for 64 hours.

Axillary turions are smaller than subterranean turions and are thought to

function in dispersal (Thullen, 1990). These structures are generally formed on

detached floating mats of hydrilla (Thullen, 1990; Miller et al., 1993). Furthermore,

axillary turions could last only 1 year in the hydrosoil (Van and Stewart, 1990),









presumably due to their smaller size.

Turion formation in dioecious hydrilla has been shown to be a photoperiodic

response with a critical photoperiod of 13 hours for subterranean turion formation

(Van et al., 1978a). Haller et al. (1976) reported subterranean turion production

occurred in north Florida between October and April. Klaine and Ward (1984)

demonstrated the turion formation response was phytochrome mediated, with red

light (650 nm) stimulation and far-red light (750 nm) repression of turion formation

in the dioecious biotype. Exogenous ABA applications induced turion formation

under non-inductive conditions. Van metal. (1978b) also showed exogenous ABA

promoted turion formation under 16 hour daylengths. Axillary turion formation

appears to respond similarly to subterranean turion formation with regard to

photoperiod. Miller metal. (1993) showed hydrilla axillary turion production was

similar to subterranean turion formation -- occurring in the fall and winter months

only.

Both the monoecious and dioecious hydrilla biotypes occur in the United

States. The dioecious female plant occurs throughout Florida, the southeast and

California, whereas monoecious hydrilla is found in Washington D.C., Virginia,

Maryland and North Carolina. Besides a difference in flowering, there seems to be

several distinct differences between the two biotypes in terms of vegetative growth

and turion production.

Monoecious hydrilla produces subterranean turions under both 10 and 16 hour

photoperiods, with a greater number of turions produced under short-day conditions.







7
The growth of monoecious hydrilla is generally prostrate, near the hydrosoil with

many horizontal stems and higher shoot densities than dioecious hydrilla. In

comparative studies, Steward and Van (1987) found that the monoecious biotype

tolerated cooler temperatures and was able to sprout at lower temperatures. Ames

et al, (1986) also reported greater growth of monoecious hydrilla under cooler

temperatures compared to dioecious.

Subterranean turion production has been shown to be greatest under short

days for monoecious hydrilla with more turions produced than the dioecious form,

but there was a decline in biomass accumulation with short days. Monoecious

hydrilla also appears to able to reproduce much more rapidly than dioecious, forming

new subterranean turions in 4 weeks as compared to 8 weeks for dioecious hydrilla

(Van, 1989). Spencer et al. (1987) reported subterranean turions to be nearly 2 to

5 times as large as axillary turions, with monoecious subterranean turions smaller

than those of dioecious hydrilla. In addition, Ames et al. (1987) showed percent

sprouting was higher in monoecious than dioecious hydrilla. These studies indicate

monoecious hydrilla possesses an annual growth habit, along with rapid and prolific

turion production, adapting this biotype to northern areas which have cooler

temperatures and short growing seasons.

Earlier work suggested monoecious hydrilla was photoperiodic in response to

turion formation. Spencer and Anderson (1987) showed that monoecious hydrilla did

not produce subterranean turions under daylengths greater than 14 hours and

production increased as daylength decreased. They also reported photo-interruption







8
of monoecious hydrilla inhibited turion formation, similar to long-day grown plants

(Anderson and Spencer, 1986). However, monoecious hydrilla appears only to be

delayed in the formation of turion under long-day conditions, producing turions after

8 weeks under long-day conditions (Van, 1989).

Management of hydrilla is difficult due to rapid growth rate and prolific turion

formation. Cultural management schemes, such as drawdowns to deplete tuber

populations have had limited success (Haller et al., 1976) and biocontrol agents, such

as grass carp, are unpredictable forms of control. Martyn (1985) used grass carp to

control 9000 acres of hydrilla in a 20,000 acre lake in Texas. All hydrilla was

removed after 2 years but over 270,000 fish were necessary. Insect biocontrol has

been studied, with several introductions but these have met with only limited success.

The herbicides diquat, endothall and copper sulfate, alone or in combination

provide good initial control but regrowth of the hydrilla quickly occurs. Fluridone

(1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(1H)-pyridinone) has been shown

to be very effective for hydrilla control, but contact time and concentration are

critical issues affecting the efficacy of a given fluridone treatment (Van and Stewart,

1985; Haller et al., 1990; Fox et al., 1991).

Fluridone affects plant tissue by decreasing pigment levels (Maas and Dunlap,

1989). This is due to a decrease in carotenoid levels followed by a subsequent

decrease in chlorophyll content due to excess photooxidative stress (Devlin et al.,

1978). Bartels and Watson (1978) demonstrated the mechanism of action of

fluridone to be carotenoid inhibition and this effect could be overcome by adding 5-







9
aminolevulinic acid (Fletcher t al., 1984). The actual site is an inhibition of the

conversion of phytoene to phytofluene in the terpenoid biosynthetic pathway (Mayer

tg 1989). Fluridone was reported to be a non-competitive enzymatic inhibitor,

with reversible binding. Fluridone causes similar effects on hydrilla tissue, decreasing

the chlorophyll and carotenoid content, but interestingly, increasing anthocyanin

content (Doong et ., 1993).

Fluridone was originally marketed for weed control in cotton (Gossypium

hirsutium L.). Translocation studies showed fluridone to be translocated

apoplastically following root absorption. Tolerant species, such as cotton, show

limited translocation and root uptake (Berard gLaL, 1978). Banks and Merkle (1978)

reported good control with fluridone of many annual weeds in cotton but also

reported some soil persistence.

Fluridone is an off-white crystalline solid, with a melting point of 151-154 C

and a water solubility of 12 ppm (McCowen et al., 1979). It is classified as a

pyridinone herbicide with a molecular weight of 329.3 (McCowen et al., 1979).

Mossler etal (1991) reported microbial breakdown for fluridone was very slow and

variable. Aqueous degradation rates from lake collected microbial consortia did not

correlate to previous fluridone use patterns in the aquatic environment. Fluridone

has a fairly high affinity to clay or organic matter (Mossler et al, 1993). Therefore,

slow-release formulations could only be effectively used in aquatic ecosystems with

a sandy bottom substrate or in situations where the fluridone pellet does not contact

the hydrosoil.







10
Ultraviolet light breakdown is the major degradation mechanism in the natural

environment. Photolytic degradation of fluridone is highest at 297 to 325 nm with

a half-life of 26 hours although some degradation can occur at 325 to 355 where the

photolytic half-life was 840 hours (Mossier et al., 1989).

Fluridone has also been shown to decrease the levels of ABA within plant

tissue (Jones and Davies, 1991). Abscisic acid is derived from carotenoid precursors

so the negative effect of fluridone on carotenoid levels also translates to lower ABA

content. Abscisic acid is thought to be involved in turion formation in hydrilla, and

fluridone could be used to regulate this process. Turion formation in hydrilla can be

inhibited by fluridone under short-day conditions at concentrations greater than 5

ppb (MacDonald et al., 1993), but the effect on ABA and subsequent inhibition of

turion formation was not determined.

Abscisic acid (ABA) is a plant hormone involved in a multitude of

physiological responses including stomatal opening (Cornish and Zeevaart, 1985), bud

and seed dormancy (Quatrano, 1987; Barros and Neill, 1986), freeze tolerance

(Johnson-Flanagan et al., 1991; Reaney and Gusta, 1987), and tuber and turion

formation (Smart and Trewavas, 1984). ABA is formed through the carotenoid

biosynthetic pathway by the conversion of 9'-cis-neoxanthin to xanthin (Xan).

Xanthin is subsequently converted to ABA aldehyde and finally to cis-ABA (Gamble

and Mullet, 1986; Zeevaart et al., 1989; Li and Walton, 1990; Parry and Horgan,

1991). Light can cause isomerization to trans-ABA but only cis-ABA is thought to

be the biologically active form. ABA biosynthesis is regulated by the production of









xanthin, not the conversion of xanthin to ABA (Parry and Horgan, 1991).

ABA has been implicated in the seed dormancy of many species (Hole et al.

1989; Singh and Browning, 1991; Le Page-Degivry and Garello, 1992), but the exact

mechanism not well understood. Morris et al. (1991) indicated that ABA induced

dormancy in wheat seeds at the gene level while Ozga and Dennis (1991) showed

changes in ABA in apple seeds were not related to dormancy. In a review, Quatrano

(1987) reported that ABA has been shown to modulate gene expression in cultured

embryos of certain crops. During seed development the increase in endogenous

ABA promotes maturation of the embryo and represses seedling growth. This is

accomplished by modulating gene sets, positively affecting the maturation set and

negatively affecting the germination set. As the seed begins to lose water, the

amount of ABA and the embryo sensitivity to ABA decreases and the dehydration

of the seed, not ABA, prevents germination.

Subbaiah and Powell (1992) reported that the chilling requirement of apple

seed is not related to ABA changes during stratification, and chilling, not ABA, was

required for germination. Also the decline in ABA was largely due to leaching of

the seed coat and nuclear membrane while ABA content of embryo remained

constant. They postulated that ABA leaching from the seed allows certain promotive

forces to develop, but these can only occur after the chilling requirement was met.

Earlier work also supports this contention. Schopfer et al. (1979) showed

ABA could reversibly arrest embryo development by inhibiting water uptake, not by

control of DNA, RNA or protein synthesis. Release from dormancy is not due to







12

lower ABA levels, but rather to a loss of sensitivity to ABA. Oishi and Bewley

(1990) found the decline in ABA will permit germination of maize kernels, but only

drying will allow the aleurone layer to become sensitive to gibberellic acid.

Gibberellic acid induces a-amylase activity, causing the remobilization of food
reserves for seedling growth.

Mutations are often used to study the role of ABA. Koornnef et al., 1989

used three ABA mutants of Arabidopsis thaliana, termed abi loci, and found all

mutations reduced dormancy. However, one mutation did not effect the water

relations of leaf tissue. They hypothesized that the mutations affect different tissue-

specific receptors. Research by McCarty (unpublished) indicated that there was an

ABA receptor molecule complex on a promoter sequence of RNA. When ABA

binds to the promoter, RNA transcription begins and a specific enzyme(s) or event

is initiated.

There have been several indications that ABA plays an important role in plant

growth, especially under water-stressed conditions. Saab et al. (1990) showed that

ABA maintains primary root growth and inhibits shoot growth of maize seedlings

under low water potentials. In later work, Saab metal. (1992) suggested a differential

response to endogenous ABA by root and mesocotyl growing zones of maize. At low

water potentials, ABA had the greatest effects near the root tip and this effect

decreased from the tip. Mesocotyl tissue is more responsive to ABA in those cells

away from the meristematic region. Porter (1981) suggested that endogenous levels

of ABA in different plant organs could be a factor in the directional control of







13
assimilate transport in plants. Hoffmann-Benning and Kende (1992) reported that

the growth of rice was regulated by a ratio of gibberellic acid and abscisic acid, as

a growth promoter and inhibitor, respectively.

Interesting work on the effects of ABA on aquatic macrophytes has also been

reported. Kane and Albert (1987) demonstrated that ABA, possibly as an indicator

of water-stress, caused changes in aerial leaf morphology and vasculature in Hippuris

vulgaris. ABA also caused aerial leaf development inMyriophyllum and Proserpinaca,

and suggested possible ABA/ethylene interactions (Kane and Albert, 1989). Goliber

and Feldman (1989) measured the ABA content of the aerial leaves of Hippuris

vulgaris and showed much higher levels. They suggested the effect of osmotic stress

is the cue in aerial leaf development.

There is considerable changes in the compartmentation of ABA during light

and dark periods due to pH changes, with a 2-fold increase of ABA in the apoplast

in the dark. ABA can irreversibly conjugate to glucose forming ABA-glucoside.

This form is inactive and is restricted to the vacuole, and may be a sequestration

mechanism by which ABA is removed from the active pool. ABA has also been

shown to be a major factor in the control of stomatal opening through regulation of

guard cell turgor pressure (Zeevaart and Creelman, 1988). Guard cells under stress

appear to release ABA, initiating closure. Water-stressed roots can also produce

ABA that is translocated to the leaves and effects stomatal closure (Zeevaart and

Creelman, 1988).







14

Hartung (1983) reported the site of action of ABA in guard cells to be on the

outer surface of the plasmalemma. Raschke (1987) reported ABA prevented

stomatal opening by blocking H+ extrusion and K+ influx, and initiated rapid closure .

by K+ release. Recent work suggested that ABA acts on a G-protein at the

membrane surface, activating phospholipase C, which catalyzes the hydrolysis of

phosphotidalinositol-pyrophosate to inositol-3-phosphate (IP3) and diglyceralhyde.

IP3 then causes intracellular Ca2+ release, blocking K+ channels and lowering

stomatal opening. There is also some evidence that internal ABA may have some

control over this process as well.

Abscisic acid is also thought to be involved in the process of turion and tuber

formation in hydrilla and other species. Tubers are among the most common form

of vegetative reproductive structures and are produced by several species including

potato (Solanum tuberosum) and jerusalem artichoke (Helianthus tuberosum).

Tubers are swollen underground stems possessing multiple nodes and greatly reduced

leaf characteristics (Cutter, 1978). In potato, the morphology of tuber formation has

been well documented. Tubers are formed from stem tissue, generally at the ends

of basal stolons (Peterson, et aL, 1985), and are initiated in the youngest elongating

internode (Peterson, et al, 1985). The pith cells in this area enlarge, followed by

rapid cell division of the perimedullary parenchyma and cortical cells. Cell division

occurs in early tuber development and final tuber size is achieved through cell

enlargement (Reeve L al., 1973).







15
Turions are similar to tubers but are morphologically less complex, derived

from leaf tissue, with little differentiation of the stem tissue. Turion are often found

in freshwater aquatic species, such as green milfoil (Myriophyllum verticillatum L.)

(Weber and Nooden, 1976) and giant duckweed (Spirodella polyrhiza) (Smart and

Trewavas, 1983). Unlike tubers, turions sprout from a single node at the tip of the

structure, although turions are comprised of several densely packed nodes (Yeo et

al., 1984). The outer portion of the turion is made up of leaf tissue, which swells and

envelopes the terminal apex, giving the structure a scale-like appearance. These

leaves accumulate carbohydrates, with an abscission layer forming at the base of the

lowermost leaves. The turion detaches from the mother plant and remains dormant

until conditions favorable for growth arise.

Turion formation by dioecious hydrilla is promoted by short-days (Haller et

L, 1976). This response has been shown to be phytochrome mediated (Klaine and

Ward, 1984). Phytochrome has been implicated in several plant processes, including

flowering, seed germination and tuber formation (Quail, 1991). However, recent

work provided evidence that phytochrome may regulate more fundamental plant

processes (Lissemore e aL, 1987), (Smith and Whitelam, 1990), (Kendrick and

Nagatani, 1991), (Seeley et aL, 1992) and (Edgerton and Jones, 1992). Phytochrome

appears to be involved in the activation of K+ channels (Lew et aL 1992) and has

been suggested to be a protein kinase (McMichael and Lagarias, 1990) (Doshi ti aL,

1992). Recent research also indicated that at least two types of phytochrome [Type

I (Phytochrome A) or Type II (Phytochrome B] can be present within plant tissues








16
(Abe It al., 1985) (Shimazaki and Pratt, 1985). Phytochrome A has been shown to

regulate stem elongation (Boylan and Quail, 1991), chloroplastic gene expression

(Sharrock et aL, 1988) and anthocyanin biosynthesis (Adamse et l., 1988).

Phytochrome B has been linked to photoperiodic responses (Vince-Prue and

Takimoto, 1987; Takimoto and Saji, 1984) with respect to floral induction.

Changes in hormone levels occur soon after photoperiodic induction of tuber

formation. In potato, changes in ABA, gibberellin, and cytokinin levels have been

reported. Gibberellins are high under long-day photoperiodic conditions and decline

under short days (Krauss, 1985), while the opposite effect is observed with abscisic

acid. Several researchers have reversed the effect of photoperiod with exogenous

applications of these hormones (i.e. preventing tuber formation under short-day

lengths with gibberellic acid while inducing tuberization with abscisic acid under long-

day lengths). However, a critical ratio or balance between these two hormones may

be needed for tuberization to occur (Menzel, 1980) (Vreugdenhill and Struik, 1989).

Cytokinins accumulate in response to tuberization but do not induce this process

(Krauss, 1985). In addition, increased nitrogen levels in the leaves have been shown

to delay or inhibit tuber formation in potato (Krauss, 1978), while increased

photosynthate concentration promotes this response (Wenzler et al., 1989).

Exogenously applied abscisic acid also induced turion formation in hydrilla

(Van et l, 1978), (Klaine and Ward, 1984). Klaine and Ward (1984) also reported

that turion formation in hydrilla was reduced by exogenous gibberellic acid, similar

to that observed for potato. However, cytokinin had no effect. Turion formation in







17
giant duckweed (Perry and Byrne, 1969) can also be induced by exogenous ABA

applications, suggesting that the role of abscisic acid in regulating vegetative

reproduction is similar for a variety of species.

The role of ABA and gibberellic acid in tuber formation is well documented

but specific changes related to initiation of this process is unclear. Hannapel et al.

(1985) showed exogenous gibberellin prevented the accumulation of patatin and two

other potato tuber proteins and correlated this with the inhibition of tuber formation.

Abscisic acid had an inhibitory effect on overall protein synthesis in Spirodela.

polyrhiza turion formation (Smart and Trewavas, 1984), and the authors suggested

that there were several novel proteins specific to induced tissue.

Quantification of abscisic acid has traditionally been accomplished through the

use of gas chromatography-mass spectrometry (Dumbroff egaiA, 1983; Reymond et

al., 1987). However, the use of enzyme-linked immunoassays (ELISA) for ABA

analysis has increased over the years. The advantage of ELISA is that limited

sample purification and increased number of samples can be analyzed within a given

time frame.

Several researchers have developed ABA antibodies (Weiler, 1980; Mertens

et al.. 1983; Ross geal 1987; Quarrie et 1988). Most monoclonal antibody

immunoassays were able to use crude aqueous extracts without interference,

providing accurate quantification comparable to GC-MS analysis (Leroux et.aL, 1985;

Quarrie et al., 1988; Soejima et al., 1990; Tahara et al., 1991). Some

radioimmunoassays can detect free and conjugated ABA; this is dependant on C1







18
or C4 ABA coupling to proteins for total or free ABA, respectively (Weiler, 1980).

However, ABA analysis with ELISA from plant tissues varies with plant

species and tissue type. Some extracts may require purification while other extracts

do not. Therefore, validation through internal standardization is recommended for

ABA analysis from a previously unvalidated tissue type.

Hydrilla is a perennial species, with regrowth from overwintering turions often

being the main source for re-establishment. Turions sprout in the spring, forming a

large, monotypic stand. The hydrilla then reproduces in the fall, replenishing the

turion supply within the hydrosoil. Therefore, turion depletion in the hydrosoil would

be the most effective means for long-term hydrilla control. However, herbicide

treatments during fall conditions (turion formation) would result in applications to

established hydrilla plants. However, fluridone has been shown to be very effective

for the control of hydrilla, and can reduce turion formation at concentrations greater

than 5 ppb. Fluridone will cause decreased ABA levels within plant tissues and this

may be the reason for the reduction in turion production.

The ability of hydrilla to reproduce by turions is the greatest restraint to the

control of this species. Therefore, a better understanding of the fundamental

processes involved in turion initiation and development would greatly assist in efforts

to effectively manage this species. To accomplish this goal the objectives of this

research were to 1) develop an ELISA based ABA analysis procedure for quantifying

ABA from hydrilla; 2) test the hypothesis that fluridone reduces turion formation

from a reduction in ABA content, not a lethal impact on plant growth; and 3)







19
compare the response (growth and turion development) of monoecious and dioecious

hydrilla biotypes cultured under controlled conditions to long and short-day

conditions, fluridone and exogenous plant growth regulators.












CHAPTER II
EVALUATION OF AN ENZYME LINKED IMMUNOASSAY PROCEDURE
FOR THE ANALYSIS OF ABSCISIC ACID IN HYDRILLA

Introduction

Abscisic acid (ABA) has been implicated in many plant developmental

processes, including control of gene expression, plasma membrane ion flux, and seed

dormancy (Neill et al, 1986; Barratt et al., 1989). In addition, ABA plays a vital role

in plant water relations, through the regulation of stomata and growth under water

stressed conditions (Saab et al., 1990; Saab et al., 1992). ABA has also been

implicated in turion formation and heterophylly of aquatic macrophytes (Smart and

Trewavas, 1984; Kane and Albert, 1987).

Abscisic acid is a 15-carbon compound with a molecular weight of 264.3. It

exists in two forms: 'cis' and 'trans', which isomerize in light. Cis-ABA is the

biologically active form, therefore quantification of ABA should take this fact into

account. The acidic nature of abscisic acid (pKa of 4.8) has been utilized in analysis

protocols (Jones and Davies, 1991).

Abscisic acid is derived from the terpenoid biosynthetic pathway, but the exact

synthesis route has only recently been elucidated. Earlier work suggested ABA was

formed through two separate pathways: a direct and indirect pathway (Walton,

1980). The direct pathway was suspected to involve a C15 precursor derived from

farnesyl-pyrophosphate, which may itself be xanthoxin (Milborrow, 1983). Xanthoxin







21
was discovered in the late 1960's (Taylor and Burden, 1973) as a precursor to ABA

and led to the indirect pathway hypothesis, as this compound is derived from the

breakdown of carotenoids. Therefore, ABA was also proposed to be derived from

carotenoid breakdown. However, elucidation of the exact biosynthetic pathway has

been extremely difficult due to the large number of potential ABA precursors versus

the minute amount of ABA (Cowan and Railton, 1986; Milborrow, 1983).

Subsequent research has provided the most definitive evidence that ABA is

produced via the indirect pathway of carotenoid breakdown. Biosynthetic inhibitors,

such as fluridone, have been shown to block carotenoid synthesis and also lower

ABA levels (Moore and Smith, 1984; Gamble and Mullet, 1986; Oishi and Bewley,

1990). In addition, carotenoid deficient mutants have lower levels of ABA (Wang

et al., 1984; Neill et al., 1986). Since these studies, most researchers have concluded

the synthesis of ABA involves epoxidation and isomerization of specific carotenoid

molecules, the formation of xanthoxin, and subsequent ABA formation (Zeevaart et

al, 1989; Li and Walton, 1990; Parry and Horgan, 1991).

Abscisic acid can be extracted from plant tissue through a variety of methods.

Goliber and Feldman (1989) used 80% methanol for extraction from Hippuris

vulgaris while Gamble and Mullet (1986) found adequate extraction with 100%

acetone. Loveys and vanDijk (1988) found boiling water sufficient for extracting

ABA from grape (Vitis spp.) leaves and Tahara et al. (1991) found extraction with

tris-buffered saline to provide excellent extraction of ABA from wheat (Triticum

aestivum L. em. Thell.) leaves.







22
Traditional methods of ABA quantification include high performance liquid

chromatography (HPLC) and/or gas-liquid chromatography (GC). These methods

provide excellent quantification but require a large amount of purification (Dumbroff

et al., 1983; Leroux et al., 1985; Soejima et al., 1990). Recently, enzyme-linked

immunoassays (ELISA) have been developed for ABA detection, greatly reducing the

rigorous purification required prior to analysis (Raikhel et al., 1987; Tahara et al.,

1991). In addition, the number of samples that can be processed at a single time is

much greater with ELISA.

Hydrilla [Hydrilla verticillata (L.f.) Royle] is a submersed aquatic plant that has

become a weed problem throughout much of southeastern United States and

California (Langeland, 1990). Hydrilla is a highly aggressive species, forming large,

monotypic stands that interfere with flood control, navigation and many recreational

water activities including boating and fishing (Haller, 1976; Langeland, 1990).

Hydrilla persists within a given area primarily through the production of specialized

vegetative reproductive structures called turions. Turion formation in hydrilla and

several other aquatic species is thought to be directly affected by ABA but the exact

role of ABA is not known (Van et al., 1978; Klaine and Ward, 1984). Therefore,

a better understanding of the affect of ABA on turion formation in hydrilla could

provide more effective management strategies for this noxious species.

Several researchers have found the enzyme-linked immunoassay (ELISA)

procedure specific enough to quantify ABA in crude extracts without purification

(Tahara et al,, 1991). However, each individual plant species must be tested and







23
validated if crude extraction is to be used (Zeevaart and Creelman, 1988). In

addition, plant tissue types may also vary in the content of interfering compounds,

particularly between chlorophyllous and achlorophyllous tissue. Therefore, the major

impediment to the universal applicability of ELISA is the unpredictable presence of

compounds that cross-react with the ABA-specific antibody. A procedure known as

internal standardization must be conducted on tissue prior to analysis for ABA using

ELISA. This technique allows a determination of whether purification of the sample

is necessary before ELISA quantification.

Although ABA has been implemented in hydrilla turion development, ABA

quantification from hydrilla tissue by ELISA has not been widely used. Therefore,

studies were conducted to develop a purification procedure for detecting ABA from

hydrilla and to determine if such a procedure was warranted for quantification by

ELISA.



Materials and Methods

Elution profiles and column/ABA characterization. Reverse-phase cartridges,

particularly C18, have been widely used for abscisic acid purification (Hubick and

Reid, 1980). Therefore, initial studies were performed to characterize the

relationship between eluant volume, methanol:water ratio and pH on the retention

of ABA to this type of column. Columns1 were prepared prior to use by washing


1 PrepSep; Fisher Scientific, Pittsburgh, PA 15219.







24
once with methanol and twice with deionized water. All samples were passed

through the column under vacuum (90 kPa) and the resulting eluant was collected

in 20 ml glass vials. During elution these vials were placed within a vacuum

manifold2.

In the first experiment, 100 yL of 1 mM ABA was added to the top of the
column and eluted with varying concentrations of methanol:water (20:80, 50:50,

70:30, 80:20, 100:0) in 10 mL aliquots up to 50 mL. The elution pH was

approximately 7, with the majority of the ABA molecules in the protonated form.

The ABA content was then determined spectrophotometrically at 250 nm. Recovery

was expressed as a percent of ABA added.

A similar experiment was conducted with varying concentrations of

methanol:water (0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 70:30, 80:20, and 100:0) in

10 ml aliquots up to 50 mL. In addition, the methanol:water elutions were acidified

with 1% formic acid to obtain a pH of less than 3.0. This resulted in most the ABA

in an unprotonated, neutral form. Recovery was expressed as a percent of ABA

added.

The preceding elution studies allowed the development of a purification and

concentrating procedure that was used in the following two experiments (see results

and discussion). An experiment was conducted to determine if the presence of

compounds) in hydrilla extract would interfere with accurate quantification of ABA


2 PrepTorr; Fisher Scientific, Pittsburgh, PA 15219.








25
using UV spectroscopy. Tissue extract contained 20 mg-fresh weight hydrilla shoot

tips which were homogenized in 10 to 15 mL 100% methanol and the extract filtered

under vacuum. This extract contained three levels of plant tissue (0, 12.5, and 25 mg-

fresh weight/sample). Abscisic acid was added to the plant extracts at concentrations

of 0, 8, 13, or 18 nM prior to column extraction. ABA was detected

spectrophotometrically at 250 nm.

A study was also conducted to determine if components in hydrilla extract

would influence the recovery of ABA purified using column chromatography. This

study involved fortifying hydrilla extract with different concentrations of ABA before

or after purification. Tissue extract was prepared using the aforementioned methods

and three concentrations (150, 250, or 350 nM) of ABA were used for fortification.

Abscisic acid recovered was detected spectrophotometrically at 250 nm.

ELISA internal standardization for ABA in hydrilla. An internal

standardization experiment was conducted to determine if purification of hydrilla

tissue was necessary prior to ABA quantification with ELISA. Antibody and

antibody tracer specific for ABA were obtained directly from the manufacturer .

Procedures for the preparation of ELISA plates, sample preparation and ABA

concentration determination are outlined in Appendices A-E.

Internal standardization was performed on crude extracts of hydrilla stem and

turion tissue utilizing ELISA. Due to the large range of values to be tested,


3 PhytodetekT, Idetek, Inc., 1245 Reamwood Ave., Sunnyvale, CA 94089.







26
standardization of each tissue type was performed in two experiments. In the first

experiment, 100 jL of 0, 0.02, 0.04, 0.06, 0.08, 0.2, 0.4, or 0.6 pmol ABA was added
to dilutions of hydrilla stem tissue (1:2 and 1:4) and turion tissue (1:4 and 1:8).

Dilutions were based on 20 mg-dry weight/sample as a 1:1 dilution and ABA was

added prior to quantification by ELISA. Samples were extracted according to the

procedures outlined in the previous section. The methanolic extract was then dried

under a stream of nitrogen gas and reconstituted in tris-buffered saline prior to

quantification by ELISA.

In the second experiment, 100 gL of 0.0, 0.2, 0.5, 1.0, 2.0, or 5.0 pmol ABA
was added to dilutions of hydrilla stem tissue (1:1 and 1:3) and turion tissue (1:1 and

1:4). Dilutions were based on 12 or 8 mg-dry weight/sample as the 1:1 dilution) for

stem or turion tissue, respectively. ABA was added prior to quantification by ELISA.

Tissue extracts for both experiments were prepared in a manner similar to plant

material used in the column extraction procedure.



Results and Discussion

Abscisic acid under neutral pH conditions showed little affinity for the C18

column when the mobile phase contained greater than 30% methanol. The majority

of ABA was recovered with only 10 mL of 30% methanol (Table 2.1). Much higher

concentrations of methanol were necessary to elute ABA from the C18 column when

the mobile phase was acidic (Table 2.2). Under low pH conditions ABA retention

increased at similar methanol concentrations to the previous experiment. However,

complete retention was observed at concentrations below 20% methanol.







27
Table 2.1. The effect of elution volume and methanol concentration on abscisic acid
C18 column retention under neutral pH conditions.


Cumulative Methanol Concentration (%)
Elution
Volume 0 10 20 30 40 50 70 80 100

mL ---------......------------------........ % ABA eluted ---------------------------------

10 831 504 772 872 941 943 89+5 763 80+4

20 333 272 182 2+2 0 0 -- 53 5+3

30 15+2 111 21 22 0 0 0 0 0

40 71 62 21 0 0 0 0 0 0

50 41 0 0 0 0 0 0 0 0


1 Means (four replications) followed by standard errors.







28
Table 2.2. The effect of elution volume and methanol concentration on abscisic acid
C18 column retention under low pH conditions.


Cumulative Methanol Concentration (%)
Elution
Volume 0 10 20 30 40 50 70 80 100

mL --------------------------- % ABA eluted ------ ------------

10 0 0 0 2+11 14+6 57+2 903 99+2 92+3

20 0 0 0 0 593 443 153 -- 11+4

30 0 0 0 84 134 0 0 0 0

40 0 0 0 284 0 0 0 0 0

50 0 0 0 22+3 0 0 0 0 0


1 Means (four replications) followed by standard errors.







29
Concentrations greater than 50% removed ABA from the column after 20 mL of

elution volume. Extraction of ABA using C18 solid-phase columns is highly

dependent on pH. Low pH conditions allows greater retention of the ABA to the

column while neutral conditions do not. These results agree with the work of Parry

and Horgan, 1991 and other researchers.

The purpose of these studies was to develop a two-stage procedure for the

purification and concentration of ABA for analysis by ELISA. In the first

purification step, it was imperative that the appropriate solvent strength (%

methanol) be achieved to allow the retention on the column of as many extract

components as possible while allowing the elution of ABA. Because the volume of

solvent increased in this step, it was then necessary to concentrate the purified ABA

extract. This was accomplished by determining the appropriate solvent strength of

the mobile phase that would allow the retention of ABA by the column. This

technique is known as solid phase extraction and has many advantages over liquid

to liquid extraction, which results in large quantities of waste solvent. In addition,

solid phase extraction is cheap, rapid and allows for the simultaneous preparation of

numerous samples. The results provided information necessary to devise a clean-up

procedure that could be utilized for ABA analysis. This procedure provided over

92% recovery for all concentrations when ABA was extracted in the absence of plant

tissue (data not shown). Details of this procedure can be found in Appendix F.

When this procedure was used with the addition of plant tissue, it resulted in

greater than 89% recovery for all concentrations of ABA added (Table 2.3). Visual









Table 2.3. The effect of plant tissue and column extraction on the recovery of
abscisic acid. Abscisic acid was added before or after column purification.


ABA Plant + ABA Plant + ABA
Added Before Column After Column Recovery

(nM) ------------------- absorbance ------------- ----%----
0 0.2760.401 0.2760.40 --

150 0.5800.20 0.632+0.34 92

200 0.703 0.09 0.752+0.45 93

250 0.786+0.18 0.8800.42 89


1 Means (three replications) followed by standard errors.







31
observations indicated that the column purification procedure provided good

separation of chlorophyll and carotenoids. However, anthocyanins were extracted

with the ABA, as indicated by a pinkish color during the second column extraction.

Furthermore, the presence of interfering compounds in hydrilla tissue extract

was not detected using this procedure (Figure 2.1). An internal standardization

procedure allows for the detection of an interfering compound. If the addition of

ABA to various extract dilutions does not cause a change in the rate of absorbance

then interfering components are not present. The absence of such a compound

would be indicated by parallel lines as the rate at which ABA detected as a function

of ABA added was the same regardless of the extract dilution. Although different

amounts of ABA were detected, dilutions as a function of ABA added was the same.

The column clean-up procedure developed reported herein provided good

recovery of ABA and separation of possible interfering compounds. Although

additional purification could be necessary with chromatographic methods of

quantification, this procedure provided an excellent initial purification step.

Furthermore, this procedure provided adequate purification of hydrilla tissue for

quantification by ELISA, if purification was needed.

Internal standardization of ELISA allowed for the detection of an interfering

compound (s) by dilution of the interfering compound (s) with the addition of higher

levels of ABA. The absence of interference would be indicated by parallel lines of

the two levels of plant tissue and/or the lack of a significant interaction (P > 0.05).

The results from hydrilla stem tissue indicated the absence of interfering compounds










0-OABA ALONE X
A-A DILUTION 1:2
0.8 X-X DILUTION 1:1 X
w 0

Z 0.6 -





0.2



0 5 10 15 0

ABA ADDED (nM)

Figure 2.1. Internal abscisic acid standardization for column cleanup
procedure with hydrilla tissue.
procedure with hydrilla tissue.







33
(Figures 2.2 and 2.3). Good separation of the two dilutions was observed in the first

experiment at 0.6 and 0.8 pM ABA. However, the ability to detect differences in

dilution was limited to a range of 0.2 to 0.5 pM in the second experiment. Hydrilla

turion tissue also appeared to be free of interfering compounds (Figures 2.4 and 2.5).

Although the lines do not appear to be absolutely parallel, statistically there was no

interaction (P >0.05) between ABA added and ABA detected for the different

extract dilutions. This implies that there was a consistent change in the amount ABA

detected regardless of the amount of ABA added (i.e. the lines representing

independent and dependent variables for the different dilutions were parallel

although differing in magnitude).

Furthermore, the presence of interfering compounds would result in response

convergence at a higher added ABA, because the interfering compound (s) would

react with the antibody, over-estimating ABA present. However, these data do not

fit this scenario. In addition, preliminary experiments were conducted by adding

ABA prior to purification and the same relationship between ABA added and ABA

detected was observed (data not shown). Based on these results analysis of ABA can

be accurately determined from hydrilla tissue by analysis of crude extraction.

However, prudence should be exercised during analysis to ensure proper dilution of

the respective sample; only those readings within the linear range of the standard

curve are valid. The internal standardization reported herein provided an accurate

determination within the range of 0.02 to 5.0 pmol per sample, allowing a broad

range of sample ABA concentrations to exist. This also provides a large degree of








1.5
STANDARDS y. 0.060 + 1.40x R2 0.89
A DILUTION 1:2 y 0.025 + 1.61x Rt 0.0
DILUTION 1:4 y = 0.037 + 1.49x R2 = 0.89


1 -A

o -'7


^ 0.5 ,-
< .,j



0
0 0.2 0.4 0.6 0.8

ABA ADDED

Figure 2.2 Internal standardization of abscisic acid quantification using
ELISA with hydrilla stem tissue. Range of values was 0.02 to 0.8 pmol.










5 A DILUTION 1:3 y 0.15 + 0.5x 0.g7


o 4

OL
co 3
0







0 1 2 3 4 5
ABA ADDED

Figure 2.3. Internal standardization of abscisic acid quantification using
ELISA with hydrilla stem tissue. Range of values was 0.2 to 5.0 pmol.









0.8


0 0.1


0.2


0.3


0.4 0.5 0.6


ABA ADDED


STANDARDS y 0.028 + 0.808x RF 0.85
A DILUTION 1:4 y 0.131 + 0.835x 2- 0.81
DILUTION 1:8 y 0.081 + 0.745x R2 0.85




7 -




A


....I. I.*. .. I. ..


0
LM

LM
w

Cr
CO
C()
0
<
<


0.6



0.4



0.2


Figure 2.4. Internal standardization of abscisic acid quantification using
ELISA with hydrilla turion tissue. Range of values was 0.02 to 0.6 pmol.









6
A 1:1 DILUTION y 0.29 + 1.04x R2 0.99
a 1:4 DILUTION y -0.18 + 0.95x R' -0.98
5

2 --




o1 -
0 .







0 1 2 3 4 5

ABA ADDED

Figure 2.5. Internal standardization of abscisic acid quantification using
ELISA with hydrilla turion tissue. Range of values was 0.2 to 5.0 pmol.







38
flexibility when undertaking ABA analysis from hydrilla tissue. Using this

methodology, 8.0 pmol per sample for crown tissue (20 mg dry weight) and 12 pmol

per sample for turion tissue (8 mg dry weight) were detected from these hydrilla

tissue types. From these data, the appropriate amount of tissue needed for ABA

extraction would be 10 to 20 mg dry weight.

However, this procedure should be used to correlate large differences in ABA

levels between tissue types, biotypes or seasonal hormone fluctuations. The minute

quantities of ABA found endogenously can be depleted by a multitude of factors

before actual analysis, therefore relative (large) differences only should be used when

determining the effect of ABA.












CHAPTER III
THE INTERACTIVE EFFECT OF PHOTOPERIOD AND FLURIDONE
ON THE GROWTH, REPRODUCTION, AND BIOCHEMISTRY OF
HYDRILLA [Hydrilla verticillata (L.f.) Royle]

Introduction

Hydrilla is an exotic, submersed vascular plant that has become one of the

most troublesome aquatic weeds in the state of Florida. Hydrilla was first observed

in a Miami canal and in Crystal River around 1960 and is thought to have been

introduced from Asia by the aquarium plant industry (Blackburn et al. 1969; Haller,

1976). Since introduction, hydrilla has spread throughout Florida, the southeastern

United States, and California (Langeland, 1990). Infestations have also been

reported in areas of Maryland, Virginia and Washington D.C., but this is thought to

be a separate introduction involving a different hydrilla biotype (Stewart et al., 1984).

Hydrilla is a major problem in many freshwater ecosystems, forming a dense,

entangled mat of shoots at the water surface (Haller and Sutton, 1975). This

extensive growth interferes with most recreational water activities, navigation, and

flood control. In addition, hydrilla effectively outcompetes most native vegetation,

changing the natural balance of the aquatic ecosystem. Changes in the native flora

and fauna occur that, ultimately impact the freshwater fishing industry of Florida.

Hydrilla regrows in the spring as the water temperature begins to rise (Haller,

1976). During the initial phases of growth, hydrilla shoots quickly reach the water







40
surface and begin to branch, forming a dense canopy (mat)(Van et al., 1977). This

mat effectively absorbs nearly 95% of the available sunlight (PPFD), limiting light

penetration to understory vegetation and reducing competition. In addition, hydrilla

can adapt to extremely low light levels. Bowes et al. (1977) reported the light

compensation point of hydrilla to be 12 to 20 1smol'm'2.sec-1. Further work by

Salvucci and Bowes (1983) showed that a change in photosynthetic metabolism, from

C3 to C4 like photosynthesis occurred under certain conditions. This ability,

coupled with the low light compensation point, allows hydrilla to outcompete other

species and maintain the monotypic mat.

Hydrilla is difficult to control because spread can occur through a variety of

mechanisms including stem fragmentation and turion development. In hydrilla,

turions are produced at the tips of positively geotropic rhizomes, which extend from

the root crown into the hydrosoil (Sculthorpe, 1967). These structures, termed

subterranean turions or tubers, become detached from the mother plant and can

remain dormant for 1 to 5 years (Van and Stewart, 1990). Turions may also be

formed in leaf axils and are termed axillary turions (Yeo et al., 1984). Axillary

turions are generally smaller than subterranean turions and are usually formed on

detached, floating plants (Haller, 1976; Miller et al.. 1993). Due to the smaller size,

axillary turions are thought to function in dispersal, whereas subterranean turions act

primarily in the persistence of hydrilla in a given area (Spencer et al., 1987; Thullen,

1990).







41
Hydrilla forms turions during the late summer and early fall in North Florida

(Haller, 1976; Miller et al, 1993). Van metal. (1978) reported that hydrilla responded

to photoperiod, with turions produced only under short-day conditions. Studies by

Klaine and Ward (1984) elucidated the role of phytochrome in controlling this

response. The level of ABA is thought to rise in response to short-day conditions,

preceding turion formation. ABA applied exogenously to hydrilla under long-day

conditions promotes turion production, but the differentiation between subterranean

and axillary turion formation has not been studied (Van et al., 1978; Klaine and

Ward, 1984).

Management of hydrilla is difficult and results are variable (Schmidtz et al.,

1987), with control efforts focusing primarily on herbicide use for logistical reasons.

Endothall, (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid), diquat, (6,7-

dihydrodipyridol[1,2-a:2',l'-c]pyrazinediium ion), and copper sulfate alone or in

combination (s) provide good initial control but hydrilla quickly regrows. A slow

acting herbicide, fluridone, (1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(1H)-

pyridinone), was introduced for aquatic weed control during the 1980's and has

shown great promise for the control of hydrilla (McCowan et al., 1979). However,

proper exposure periods and concentrations are necessary for effective control

(Netherland et al., 1993).

Fluridone inhibits carotenoid biosynthesis, causing photooxidation of

unprotected chlorophyll molecules (Bartels and Watson, 1978; Devlin et al 1978;

Maas and Dunlap, 1989). Fluridone has also been shown to inhibit the production







42
of ABA, which is thought to form from the carotenoid biosynthetic pathway (Li and

Walton, 1990; Parry and Horgan, 1991). Because of this, fluridone is used in

physiological experiments to study the effect of ABA on plant growth and

development (Hole et al., 1989; Oishi and Bewley, 1990; Saab et al., 1990).

Short-term control of hydrilla is often realized within a given area but long-

term eradication is rarely achieved. Fluridone provides excellent control of hydrilla

and is generally applied in the spring as the hydrilla begins to regrow. Good initial

control is observed but regrowth from dormant turions occurs during the mid to late

summer months. This regrowth provides substantial biomass for reproduction in the

fall and turion levels in the hydrosoil are replenished. Thus the cycle of spring

treatment but regrowth and subsequent turion formation in the fall never results in

effective, long-term management.

One management strategy would be to treat hydrilla during the fall before or

during turion formation. However, hydrilla may not be sensitive to herbicides at this

time. Herbicides are most active on young, rapidly growing plants. Hydrilla growing

in the fall would be near maturity and should require a higher dose to achieve

control. Fluridone has been shown to reduce turion formation at concentrations >

5 ppb (MacDonald et al., 1993). However, the actual mechanism (i.e., reduction in

ABA levels and subsequent turion inhibition or turion inhibition as a result of plant

death), is not known. MacDonald et al. (1993) postulated that lower ABA levels

result from sublethal fluridone concentrations and indicated a potential growth

regulator phenomenon.







43
Clearly, the key in long-term hydrilla management is turion depletion in the

hydrosoil. Two strategies. have been suggested concerning this issue; inhibition of

turion production or elimination of turion dormancy allowing precocious germination.

The objective of this study focused on the former strategy. Hydrilla was grown under

long (vegetative growth) and short-day (reproductive growth) conditions and

evaluated for changes in growth, turion formation and biochemical parameters in

response to varying rates of fluridone.



Materials and Methods

Hydrilla was initially planted on July 1, 1993 from apical stem segments and

grown under natural conditions in 900 L concrete vaults at the Center for Aquatic

Plants in Gainesville, FL. Stock hydrilla tissue was obtained from the Waccissa River

in north Florida; a pre-determined "fluridone-free" water system. Treatment plants

were grown in 10 x 10 cm plastic pots filled with organic potting soil4 and covered

with a 2 cm deep sand "cap" to prevent floating. Two 10 cm long apical hydrilla

sprigs were planted per pot and 6 pots were placed in 24 x 30 cm plastic dishpans.

On August 16, 1993 the plants were transferred to 540 L fiberglass vaults under short

(natural) or long (artificially extended) day greenhouse conditions. Environmental

conditions of the long-day greenhouse were 16 hr light/8 hr dark photoperiod, day

temperature of 30 5 C, night temperature of 25 5 C, with a mean quantum


4 Ace Hardware Corp., Oak Brook, IL 60521.







44
irradiance at noon of 1200 ltmol'm-2.sec1 (PPFD). Photoperiod was extended for

4 hours after sunset with incandescent bulbs [100 zmol'm2'2sec-1 (PPFD)]. The

short-day greenhouse was maintained at similar conditions under natural (short-day)

photoperiodic conditions.

A second population of young hydrilla plants was established on August 18,

1993 in a manner similar to the older plants and placed under the same conditions.

The 8 week old population represented summer hydrilla growth while the younger

plants represented hydrilla regrowing from overwintering turions. Throughout the

remainder of this chapter, mature and young will be used to denote the 8 week old

and newly established hydrilla plants, respectively.

Each greenhouse (photoperiodic condition) contained 8 vaults, 4 comprising

mature hydrilla plants, 4 containing younger hydrilla. Each vault contained a total

of 10 dishpans. Fluridone concentrations of 0, 1, 5, and 10 ppb were established

within each population and photoperiodic regime beginning Sept. 2, 1993. Periodic

flushing and insecticide treatment were necessary to maintain optimum growth

conditions. After flushing fluridone was re-applied to each vault to achieve the

desired concentration.

Hydrilla (one dishpan) was harvested from each vault on Sept. 2, 1993 and

harvests continued on a weekly basis for 8 weeks. Each dishpan contained 6

replications (1 replication = 1 pot). Parameters measured from both plant age

groups included fresh weight and subterranean turion number. Mature plants were

subsequently divided into apical stem, crown, and subterranean turion sections.







45
Subsamples were a composite from all replications. Immediately following

measurements and/or subsampling, young plants (entire plant) and mature plant

subsamples were frozen in liquid nitrogen and stored at -20 C. Samples were

lyophilized at -50C and dry weights recorded. The tissue ground was then to 0.5 mm

fineness and stored at 20 C prior to biochemical analyses.

Chlorophyll/carotenoid analyses. Approximately 10 mg dry apical stem tissue

was placed in a 20 ml glass scintillation vial and filled with 10 ml of

chloroform:methanol (2:1 v/v). Vials were placed on a rotary shaker (150 rpm) for

12 hours at 4 C. The homogenate was filtered and the crude extract dried under a

stream of nitrogen gas at room temperature under dim light (< 5 Amol'm2'"sec'1).

The residue was resuspended in 80% acetone and absorbances at 470, 646, and 663

nm were measured spectrophotometrically. Total chlorophyll concentration was

calculated using the formula: 17.32 x A646 7.18 x A663. Total carotenoid content

was determined using the formula: 1000 x A470 3.27 x Chla 104 x Chlb)/229

(Lichtenthaler and Wellburn, 1983). Chlorophyll a (Chla) and chlorophyll b (Chlb)

concentrations (pg/mL) were calculated from the following formulas: 12.21 x A663 -

2.81 x A646 and 20.13 x A646 5.03 x A663, for Chl a and b, respectively. Three

replications of the composite apical stem section subsample from mature plants only

(described above) were analyzed for chlorophyll and carotenoid content.

Anthocyanin analysis. Approximately 10 mg dry apical stem tissue was placed

into a 20 ml scintillation vial filled with 5 ml of 1% acidic methanol (v/v). Tissue

was extracted for 12 hours on a rotary shaker (150 rpm) at 4 C. The resulting







46
mixture was allowed to settle and absorbance of the supernate was measured

spectrophotometrically at 530 and 657 nm. Chlorophyll has some absorbance at 530

nm in acidic methanol, therefore corrections were made using the formula: A530 -

0.25 x A657, which assumes an extinction coefficient of 34,000 M^cm-1 (Jonsson et

al., 1984; Mancinelli, 1990). Anthocyanin content was determined from apical stem

section subsamples from mature plants only (described above) with 3 replications per

subsample.

Abscisic acid analysis. Tissue (10 to 20 mg dry weight) was extracted with

100% methanol for 12 hours on a rotary shaker (150 rpm) at 4 C. Methanol was

evaporated at -50 C and the residue resuspended in 0.5 to 1.0 ml of tris-buffered

saline and re-extracted as described previously. Following extraction, the

homogenate was centrifuged at 9000 rpm for 4 minutes. Analysis for ABA was

performed on the resulting supernate using an enzyme-linked immunoassay specific

for ABA. Details of the immunoassay procedure are presented in Appendices A-E.

A single sample from composite apical stem segments and crown subsamples of

mature were analyzed for ABA content. A single sample from 3 replicates of

younger plants 0, 2, 4, 6, and 7 weeks after treatment (WAT) were analyzed for ABA

content.

Statistical analysis. Data were initially analyzed by analysis of variance to test

for treatment effects (WAT, photoperiod, and fluridone concentration) and

interactions. The effect of photoperiod was separated with Fisher's protected LSD

test at the 0.05 level of significance. The effect of fluridone concentration was







47
compared to the untreated control using Dunnett's 'T' test at the 0.05 level of

significance. Means are presented with standard errors.



Results and Discussion

There was a significant (P < 0.05) three-way interaction between weeks after

treatment, photoperiod and fluridone concentration on the dry weight of mature and

young hydrilla plants. The dry weight of untreated plants increased over time in both

photoperiodic regimes for both mature and young hydrilla populations (Tables 3.1

and 3.2). Young plants showed a greater increase in biomass than the mature

hydrilla over the duration of the study. Seven WAT, the biomass of young plants

increased by 83 and 90% under long and short-day conditions, respectively.

However, mature plants only showed an increase of 39 and 25% for long and short

day photoperiods, respectively. The dry weight of mature plants was reduced by

fluridone concentrations of 5 and 10 ppb by more than 63%, regardless of

photoperiod at 7 WAT. Young hydrilla plant biomass was reduced by fluridone

concentrations of 5 and 10 ppb greater than 95% under short-day conditions and

greater than 68% under long days. Fluridone at 1 ppb under long-day conditions

caused 50 and 42% reduction in dry weight of mature plants compared to the

untreated control after 6 and 7 weeks of treatment, respectively. Short-day grown

mature hydrilla, however, did not show significant biomass reduction at 1 ppb

fluridone, while younger plants were significantly reduced at 5 and 7 WAT under

short day conditions.








48
Table 3.1. The effect of photoperiod and fluridone on the dry weight of mature
hydrilla plants.



Weeks After Treatment1
Photo- Fluridone
period2 (ppb) 1 2 3 4 5 6 7

------------------------g/plant----------- ----------
Long Day
0 4.6 3.9 11.9 9.6 3.4 7.4 7.5

1 4.4 4.6 8.0 5.3 3.6 3.7*3 3.2*

5 4.3 4.3 4.0* 3.5* 1.7 2.2* 1.1*

10 4.2 3.9 5.6* 1.8* 4.1 2.8* 2.8*

Short Day
0 5.3 4.7 5.5 4.8 4.6 6.9 7.1

1 4.3 4.1 5.1 3.5 4.1 6.0 4.3

5 3.9 3.5 3.8 2.3* 1.7* 1.9* 1.4*

10 4.9 3.3 4.5 1.6* 1.9* 2.3* 1.7*


Average hydrilla plant dry weight at the initiation of the study was 4.25 g.
2 LSDO.05 = 2.3 to separate the effect of photoperiod within week and fluridone
concentration.

Values within a week and photoperiod followed by are significantly different from
the control (Dunnett's 't' test 0.o5)-







49
Table 3.2. The effect of photoperiod and fluridone on the dry weight of young
hydrilla plants.



Weeks After Treatment1
Photo- Fluridone
period2 (ppb) 1 2 3 4 5 6 7

--------..------------. g/plant --------------------------
Long Day
0 0.17 0.17 0.40 0.36 0.58 0.75 0.66

1 0.20 0.26 0.37 0.44 '0.57 0.51*30.71

5 0.20 0.25 0.21* 0.19* 0.10* 0.07* 0.03*

10 0.30* 0.44* 0.20* 0.17* 0.23* 0.12* 0.21*

Short Day
0 0.28 0.43 0.55 0.60 0.90 1.38 2.97

1 0.25 0.34 0.41 0.57 0.49* 0.86 0.81*

5 0.33 0.43 0.42 0.42* 0.29* 0.27* 0.31*

10 0.28 0.23* 0.18* 0.14* 0.07* 0.07* 0.05*


1 Average hydrilla plant dry weight at the initiation of the study was 0.18 g.

LSDo.05 = 0.17 to separate the effect of photoperiod within week and fluridone
concentration.
2Values within a week and photoperiod followed by are significantly different from
the control (Dunnett's 't' test 0.05)-







50
There was a significant (P < 0.05) three-way interaction between weeks after

treatment, photoperiod, and fluridone concentration on the subterranean turion

production of mature and younger hydrilla plants. Turions were not produced during

the first 4 weeks of treatment for either mature or young plants (data not shown).

Mature hydrilla grown under short-day conditions produced a greater number of

turions at 0 and 1 ppb fluridone 5 WAT or longer than under long-day conditions

(Table 3.3). Subterranean turion production was reduced by more than 80% in

mature hydrilla by fluridone at 5 and 10 ppb at 4 to 7 WAT under both

photoperiods. Fluridone at 1 ppb also reduced turion production of mature plants

at 4 to 7 WAT under long and short day conditions, with the exception of 5 and 7

WAT under short days.

Subterranean turion production by younger hydrilla plants was reduced under

long day conditions and 5 and 10 ppb fluridone by nearly 100% (Table 3.4).

Fluridone at 1 ppb reduced turion production in the younger plant 6 and 7 WAT

under short and long photoperiods, respectively.

Young and mature hydrilla plants responded similarly to photoperiod and

fluridone in terms of growth and reproduction. Fluridone is very effective for

hydrilla control and rates of 5 and 10 ppb reduced dry weight and nearly eliminated

turion production when hydrilla was exposed for greater than 5 weeks, similar to

previous research (MacDonald et al., 1993). At 1 ppb fluridone, hydrilla growth was

arrested in the mature plant population, while young plants at this treatment

continued to accumulate biomass. Both mature and young plants gained biomass







51

Table 3.3. The effect of photoperiod and fluridone on the subterranean turion
production of mature hydrilla plants.



Weeks After Treatment
Photo- Fluridone
period' (ppb) 4 5 6 7

------------------------turions/plant -------------------------------
Long Day
0 3.5 2.2 2.0 4.0

1 0.3*2 0.3* 0* 0*

5 0.2* 0* 0* 0.2*

10 0.2* 0* 0.2* 0.5*

Short Day
0 4.3 6.3 8.0 11.7

1 3.0 5.8 3.0* 9.0

5 0.2* 0.2* 0.3* 2.3*

10 0.2* 0* 0* 0.8*


1 LSDo.o05 = 1.7 to separate the effect of photoperiod within week and fluridone
concentration.
2 Values within a week and photoperiod followed by are significantly different from
the control (Dunnett's 't' test 0.o5).








Table 3.4. The effect of photoperiod and fluridone on the subterranean turion
production of young hydrilla.



Weeks After Treatment
Photo- Fluridone
period1 (ppb) 4 5 6 7

------------------------ turions/plant -------------------------------
Long Day
0 0 0 1.7 0.7

1 0 0.7 0* 0.7

5 0 0 0* 0

10 0 0 0* 0

Short Day
0 0.8 4.0 6.2 12.0

1 3.3 4.2 5.0 4.0*

5 0.2 0* 0* 0*

10 0* 0* 0* 0*


1 LSDo.05 = 1.0 to separate the effect of photoperiod within week and fluridone
concentration.
2 Values within a week and photoperiod followed by are significantly different from
the control (Dunnett's 't' test o.05)-








53
in untreated conditions but the younger, more rapidly growing plants should have

been more susceptible than mature plants to fluridone treatment.

A possible explanation for this apparent contradiction may be related to light

intensity. Mature hydrilla plants have the majority of photosynthetic tissue near the

water surface, exposed to a greater amount of light and potential photooxidative

stress versus younger plant tissue emerging through the water column. In addition,

the removal of apical dominance would cause branching at the water surface for the

mature plant (Van et al., 1977). The new tissue produced by the mature hydrilla

plants would then be more susceptible to the affect of fluridone due to the higher

light intensity at the surface. This would result in a reduction of growth. However,

the loss of apical dominance in young hydrilla resulting from fluridone treatment

would cause branching near the hydrosoil, where light intensity and potential

photooxidative stress is much lower. Hydrilla can survive under very low light

intensities (Bowes et al., 1977) and this could explain the ability of the younger plants

to increase in dry matter over the study period. Even though carotenoid levels would

be lower as a result of fluridone treatment, so would potential photooxidative stress.

Another explanation could be the rapid growth of the younger plants as compared

to the mature hydrilla plants. The rapid growth could overwhelm the lethal affects

of fluridone, with continued growth before fluridone began to take affect.

Subterranean turion production appeared to be directly related to growth in

terms of dry matter for both young and mature plants, where reduction in growth

also resulted in a reduction of turion number. There was turion formation under







54
long-day conditions by the mature hydrilla plants. However, the number of turions

produced did not increase over time as in the short-day grown plants, which indicated

a breach in long-day extension, or pre-induction before transfer to greenhouse

conditions.

There was a significant (P < 0.05) three-way interaction between weeks after

treatment, photoperiod and fluridone concentration for total chlorophyll, carotenoid

and anthocyanin content. Chlorophyll and carotenoid content of the mature hydrilla

plants was decreased at 5 and 10 ppb fluridone after 2 weeks of treatment in both

long and short-day photoperiodic conditions (Tables 3.5 and 3.6). Chlorophyll and

carotenoid content under long-day conditions was also decreased by 1 ppb fluridone,

but the effect under short days was variable. There was also a general decline in

these pigment levels as a function of weeks after treatment, with greater decreases

observed at higher levels of fluridone. Conversely, anthocyanin content increased

over time and short-day conditions (Table 3.7). The effect of fluridone on

anthocyanin content was highly variable and anthocyanin content appeared to

decrease anthocyanin content at higher fluridone rates.

Fluridone decreased carotenoid levels at concentrations of 5 and 10 ppb after

1 week of exposure as expected, as the mechanism of action of this compound has

been demonstrated to be an inhibition of carotenoid biosynthesis (Bartels and

Watson, 1978). Chlorophyll content also decreased in a similar fashion. Earlier

work by Doong et al., 1993 demonstrated a 'lag' in the chlorophyll content of hydrilla

at 5 ppb fluridone following carotenoid decrease but this was not evident from the







55
Table 3.5. The effect of photoperiod and fluridone on the chlorophyll content of
mature hydrilla plants.



Weeks After Treatment1
Photo- Fluridone
period2 (ppb) 1 2 3 4 5 6 7

----------------- /g/g-fresh weight -----------
Long Day
0 673 847 679 695 543 450 489

1 981*3 529* 322* 398* 170* 364* ---

5 498* 310* 91* 94* 75* 198* 219*

10 299* 167* 134* 25* --- 62* 207*

Short Day
0 616 613 527 465 438 269 486

1 552 603 320* 489 278* 254 292*

5 534* 357* 207* 225* 46* 52* 128*

10 200* 85* 63* 278* 32* 29* 28*


1 Average hydrilla
fresh wt.


chlorophyll content at the initiation of the study was 904 ptg/g


2 LSDo.o5 = 58 to separate the effect of photoperiod within week and fluridone
concentration.

3 Values within a week and photoperiod followed by are significantly different from
the control (Dunnett's 't' test 0.05).







56
Table 3.6. The effect of photoperiod and fluridone on the carotenoid content of
mature hydrilla plants.



Weeks After Treatment1
Photo- Fluridone
period2 (ppb) 1 2 3 4 5 6 7

------------------ tg/g-fresh weight ---------
Long Day
0 68 74 86 92 87 65 92

1 91*3 56* 44* 54* 24* 72 70

5 49* 36* 15* 15* 12* 61 39*

10 26* 21* 23* 3* --- 11* 42*

Short Day
0 67 67 74 84 77 48 106

1 52* 63 44* 76 55* 46 47*

5 54* 50* 25* 44* 9* 12* 26*

10 19* 10* 10* --- 7* 9* 6*


1 Average hydrilla carotenoid content at the initiation of the study was 89 p.g/g fresh
wt.
2 LSDo.05 = 10 to separate the effect of photoperiod within week and fluridone
concentration.

Values within a week and photoperiod followed by are significantly different from
the control (Dunnett's 't' test 0.05).







57
Table 3.7. The effect of photoperiod and fluridone on the anthocyanin content of
mature hydrilla plants.



Weeks After Treatment1
Photo- Fluridone
period2 (ppb) 1 2 3 4 5 6 7

-------------.----- g/g-fresh weight ----------------
Long Day
0 58 76 69 76 123 76 113

1 70*3 69 75 128* 68 66 156*

5 52 41* 62 101* 94* 80 --

10 54 53* 86* 141* --- 30* --

Short Day 0 63 75 73 143 117 329 419

1 56 55* 85 131 206* 346 300

5 70 61* 143* 110* 95 81* 216

10 51 50* 83 109* 94 99* --


1 Average hydrilla
fresh wt.


anthocyanin content at the initiation of the study was 63 /g/g


2 LSDo.o5 = 39 to separate the effect of photoperiod within week and fluridone
concentration.

3 Values within a week and photoperiod followed by are significantly different from
the control (Dunnett's 't' test 0.05)-








58
present study. A possible reason for this effect could be that high light intensity

caused a greater degree of photooxidative stress and thus, more rapid chlorophyll

breakdown. Chlorophyll content of untreated plants declined throughout the

duration of the study in both photoperiodic regimes. Berg (1977) and Kar and

Choudhuri (1987) reported a loss of chlorophyll content in hydrilla during late

summer and hypothesized that a senescence had occurred. However, a decline in

chlorophyll was also observed under long-day conditions, when senescence should not

have been a factor.

The effect of fluridone on anthocyanin content was highly variable and did not

cause an increase in the level of this pigment, as demonstrated by earlier studies

(Doong et al., 1993). However, the level at which Doong et a. reported elevated

anthocyanin content was 50 ppb fluridone, far above the levels tested in this study.

Interestingly, anthocyanin levels increased dramatically under short-day conditions,

with higher fluridone concentrations suppressing the level of this pigment. Berg

(1977) reported an increase in anthocyanin content as hydrilla began to decline

during late summer and this was probably the case for the short-day grown hydrilla

plants. The decline in anthocyanin content from fluridone treatment probably

resulted from tissue deterioration.

There was no significant differences in the ABA content of crown tissue from

mature plants (P > 0.05 see Appendix G). The average ABA content of these

structures was 25 and 204 pmol/g fresh weight for crown and turion tissue types,

respectively. There was a significant interaction between weeks after treatment and







59
fluridone concentration for apical stem tissue but not a significant (P >0.05) effect

of photoperiod (Appendix H). Abscisic acid content increased in control and 1 ppb

fluridone treated plants 1 and 2 weeks after study initiation, respectively, while

fluridone at 5 and 10 ppb decreased ABA content (Table 3.8).

ABA content in younger plant tissue was only affected by photoperiod (P <

0.05), not by fluridone concentration and there was not an interaction (Appendix I).

Abscisic acid content in young plant tissue was highest under short-day conditions

when averaged over time and fluridone: 0.530.07 and 0.71+0.05 pmol/g-fresh
weight, long and short days, respectively.

The results of the ABA analyses performed in this study are highly variable

but follow similar findings of other researchers (Van et al., 1978; Klaine and Ward,

1984; Anderson et al., 1990 [unpublished]). ABA content was shown to rise soon

after treatment, but the increase was transient. In addition, fluridone reduced ABA

content and turion production in mature plants at 5 and 10 ppb, but this could also

be the indirect consequence of plant death. ABA was higher under short-day length

conditions, which favor turion formation. However, direct evidence linking

turionformation and increased ABA content was not established. Although ABA is

probably involved in some aspect of turion formation and/or development, the exact

mechanism is still unknown.

The results from this study provided further evidence that hydrilla can be

effectively managed by late summer to early fall applications of fluridone, which

prevent turion formation. Mature hydrilla tissue was shown to be as susceptible to







60
Table 3.8. The effect of fluridone concentration on the abscisic acid content of
mature hydrilla apical stem segments, averaged across photoperiodic regime.



Weeks After Fluridone (ppb)
Treatment1 0 1 5 10


-------------------- pmol/g-fresh weight2 -------

1 18 36 13 13

2 39 25 13 15

3 -- 18 11 5

4 19 13 11 11

5 -- 11 7 -

6 26 19 12 3

7 15 16 10 15


1 Average abscisic acid content at the initiation of the study was 15 pmol/g fresh wt.
2 LSDo.05 = 13 to separate the effect of fluridone concentration within week.







61
fluridone as younger tissue and fluridone concentrations of 5 ppb or higher will

dramatically reduce subterranean turion production. This contradicts the earlier

hypothesis of MacDonald etal. (1993) who proposed that sublethal concentrations

of fluridone could block turion formation. However, the present studies support the

argument that lowering ABA concentration sufficiently to block reproduction would

require a lethal reduction in carotenoids. Therefore, the reduction in turion

formation is probably a coincidental result of plant death. Prudence should be

exercised when applying these results to real-world situations, as factors such as

exposure time and critical concentrations have yet to be evaluated.












CHAPTER IV
THE INFLUENCE OF FLURIDONE, PHOTOPERIOD AND
PLANT GROWTH REGULATORS ON THE GROWTH AND
REPRODUCTION OF MONOECIOUS AND DIOECIOUS
HYDRILLA [Hydrilla verticillata (L.f.) Royle]

Introduction

Hydrilla is a submersed aquatic vascular plant that has become a major

aquatic weed problem in the southeastern U.S. (Haller, 1976; Langeland, 1990). This

species is particularly important in Florida, where it was introduced in the late 1950's

by the aquarium plant industry (Blackburn et al., 1969). Since introduction hydrilla

has spread throughout the southeastern United States and California.

There are both monoecious and dioecious biotypes of hydrilla which both can

be found in the United States. Until recently, each biotype was believed to grow in

distinct regions, but monoecious and dioecious have been reported in a common lake

in North Carolina (Coley et al., 1993). Dioecious hydrilla exists primarily in those

areas mentioned above, and consists of only the female plant. Monoecious hydrilla

was introduced during the late 1970's and can now be found in areas of Virginia,

Maryland, Washington D.C., and North Carolina (Stewart et al., 1984; Coley et al.,

1993).

Hydrilla causes problems in many freshwater ecosystems, displacing most

native vegetation. This highly aggressive species exists in large, monotypic stands,

which form a dense mat at the water surface (Haller and Sutton, 1975; Bowes et al.,







63
1977). Hydrilla's extensive growth interferes with navigation and flood control, and

severely limits most water recreation activities. Furthermore, hydrilla growth will

alter the flora and fauna of a given area, impacting freshwater fish populations.

Hydrilla possesses several physiological and morphological adaptations

including a low light compensation point, C4-like photosynthetic metabolism, and

specialized vegetative reproduction [turions] (Bowes et al., 1977; Van et al., 1976;

Salvucci and Bowes, 1983). Turions are specialized vegetative structures that

function in persistence and dispersal of many aquatic macrophytes (Sculthorpe, 1967).

In hydrilla, turions may be formed at the tips of rhizomes that penetrate into the

hydrosoil (subterranean turions), or in leaf axils (axillary turions)[Yeo et al., 1984].

Subterranean turions are slightly larger and function to insure persistence of hydrilla

within a given area, while the smaller axillary turions are thought to function in

dispersal (Haller, 1976; Spencer et al., 1987; Thullen, 1990).

Hydrilla turion production varies between biotypes, from both a physiological

and morphological standpoint. Dioecious hydrilla forms turions only under short-day

(< 12 hours light) conditions, while monoecious hydrilla appears to be day-neutral

(Klaine and Ward, 1984; Van et al., 1978; Anderson and Gee, 1986). Monoecious

hydrilla will produce a greater number of smaller turions than dioecious hydrilla

(Spencer and Anderson, 1987; Van, 1989). This size characteristic is true for both

axillary and subterranean turions. Turion formation in the dioecious biotype is

believed to be regulated by phytochrome (Klaine and Ward, 1984; Van etal, 1978).

This response has been associated with increased levels of abscisic acid (ABA) and







64
exogenous applications of this hormone have resulted in turion formation under non-

photoinductive conditions (Klaine and Ward, 1984; Van et al., 1978). However,

delineation between axillary and subterranean turion formation was not addressed.

Furthermore, the effect of ABA on monoecious hydrilla was not studied.

ABA has been associated with a number of plant processes including water

relations, growth and development, and seed dormancy (Neil et al., 1986; Zeevaart,

J.A.D. et al.. 1988; Saab et al., 1990). ABA has also been shown to cause

heterophylly in some aquatic plants (Kane and Albert, 1987; Goliber and Feldman,

1989).

Turion formation is similar to tuber formation in terrestrial species, such as

potato (Solanum tuberosum) and jerusalem artichoke (Helianthus tuberosum).

Tuber formation in potato involves changes in abscisic and gibberellic acid content,

with an increase in ABA during tuberization and a subsequent decline in gibberellic

acid levels (Krauss, 1985). Similar to turion formation in hydrilla, ABA will promote

tuber formation under non-photoinductive conditions while gibberellic acid will

inhibit tuberization under inductive conditions. However, most researchers feel there

exists a critical ratio between these two hormones, not simply an 'on-off

phenomenon (Menzel, 1980; Vreugdenhill and Struik, 1989). This interactive effect

has not been evaluated on either biotype of hydrilla.

Plant growth regulator experiments are usually performed under laboratory

conditions to limit variability. Axenic culture is sometimes employed to study various

plant processes under highly controlled conditions. Axenic culture has proven to be







65
a valuable technique for studying the physiology of aquatic plants (Kane and Albert,

1987). In addition, axenic culture permits plant growth under heterotrophic

conditions. This allows for the potential use of a compound that influences

photosynthesis and other autotrophic processes. Fluridone is often used to regulate

ABA biosynthesis, but is lethal under autotrophic conditions.

Monoecious and dioecious hydrilla biotypes differ in the process of turion

formation but little is known regarding the processes involved. Therefore, the

objectives of these studies were to evaluate the growth and reproductive development

of monoecious and dioecious hydrilla in axenic culture to 1) long and short-day

photoperiod, 2) fluridone under long-day conditions, and 3) exogenous abscisic acid

and gibberellic acid applications.



Materials and Methods

Axenic plant stock material was obtained from Dr. Michael E. Kane,

Environmental Horticulture Dept., University of Florida. From this material, stock

cultures of both biotypes were established from 3 to 5 cm long apical stem segments.

Stock cultures were maintained in aluminum foil capped 0.94 L glass vessels filled

with 150 cm3 quartz silica sand (as a rooting medium) + 500 ml of liquid growth

medium. Basal medium consisted of half-strength Murashige and Skoog mineral

salts1 and 0.75% (w/v) sucrose, adjusted to pH 6.5 with 1.0 N KOH prior to



1 Murashige and Skoog Basal Salt Mixture (1962); Sigma Chemical Co.,
St. Louis, MO 63178.







66
autoclaving. Growth medium was autoclaved at 22 psi and 250 F for 20 minutes.

Deionized water was added to the vessels containing sand prior to autoclaving to

prevent breakage. After autoclaving, medium was added to the vessels and plant

material transferred. All transfers were performed under sterile conditions. Stock

cultures were maintained on a monthly basis and sterility was checked by indexing,

according to the procedures of Kane (unpublished) and Knauss (1976). Stock

cultures were maintained in an environmentally controlled growth chamber set at the

following conditions: 11 + 1 hr light/6 + 6 hr dark photoperiod (1hr light

interruption at the middle of the dark cycle to maintain long-day conditions); 150

Mmol'm-2"sec"1 (PPFD); 30 5 C. The light source consisted of fluorescent and

incandescent bulbs.

Experimental plant material was grown in 38 x 300 mm glass tubes2 filled with

50 cm3 quartz-silica sand and 175 ml growth medium. Plant material was obtained

from stock plants. Establishment of hydrilla was accomplished by transferring a 3 to

5 cm long (0.005 g dry wt) apical stem section into each tube and forcing the basal

end into the sand 1 to 2 cm deep to enhance rooting. Each tube was considered a

replication. Experimental cultures were maintained in an environmentally controlled

growth chamber set at the following conditions: 9 + 1 h light/7 + 7 h dark

photoperiod (1 h light interruption at the middle of the dark cycle to maintain long-

day conditions); 600 jmol-m'2"sec"1 (PPFD); 30 5 C mean light temperature and


2 Bellco Glass, Inc., Vineland, N.J. 08360.







67
25 + 5 C mean dark temperature. Short-day grown plants were cultured in a

chamber maintained at: 10 h light/14 h dark photoperiod -- 200 ymol-m-2.sec-1

(PPFD) and 25 + 5 C mean light temperature and 20 5 C mean dark

temperature. Lighting in both chambers consisted of a mixture of incandescent and

fluorescent bulbs.

Photoperiodic response. Monoecious and dioecious hydrilla biotypes were

grown under long or short-day conditions for 8 weeks with a minimum of 6

replications. The experiment was repeated once. After 8 weeks, the plants were

harvested and subterranean and axillary turion number was determined.

Subterranean turion development was determined by the formation a rhizome

penetrating the sand substrate. Axillary turion formation was distinguished by a

swelling of shoot tissue, compact leaf development and the lack of prominent

internodes. The plants were then dried at 60 C for 48 h and dry weights recorded.

Effect of fluridone. A preliminary experiment was conducted to determine

if hydrilla could be grown heterotrophically using axenic techniques with

supplemental sucrose. Monoecious and dioecious hydrilla biotypes were grown under

long-day conditions for 12 weeks. The experiment was repeated once with 4

replications. Fluridone was prepared as a concentrated stock in water from technical

grade material and filter-sterilized (0.22 Am)4 prior to addition to the tubes.

Fluridone was added at concentrations of 0, 0.5, 1.0, 5.0, or 10 ppb at the time of



3 DowElanco, Inc., Indianapolis, IN 46268.

4 MSI, Fisher Scientific, Pittsburgh, PA. 15219.








68
establishment. After 12 weeks, the plants were harvested and the number of axillary

turions produced was determined. The plants were then dried at 60 C for 48 h and

dry weights recorded.

Effect of exogenous abscisic acid and gibberellic acid. Monoecious and

dioecious hydrilla biotypes were grown under long-day conditions for a total of 8

weeks. Biotypes were run as separate experiments and each experiment conducted

twice with a minimum of three replications. After 4 weeks of growth the growth

medium was exchanged and hormone treatments were applied. Abscisic acid (99%

mixed isomers)5 and gibberellic acid (90% GA3)5 were prepared as concentrated

stocks in water and filtered-sterilized prior to treatment. Small amounts of ethanol

was used to facilitate dilution and the appropriate ethanol concentration was added

to the control plants. Abscisic acid was applied at 0, 0.1, 1.0, or 10 pM and
gibberellic acid at 0, 5, 50, or 500 4M to establish a matrix of 16 total treatments.
Four weeks after treatment, the plants were harvested and the number of axillary

turions, subterranean turions, and flowers produced were counted. Axillary turions

were subsampled and the plants dried at 60 C for 48 h and dry weights recorded. No

attempt was made to distinguish between male and female flowers of the monoecious

biotype.

Statistical analysis. Data were initially analyzed by analysis of variance to test

for treatment effects biotypee, photoperiod, fluridone concentration, ABA or GA

content) and interactions. Treatment means were separated with Fisher's


5 Sigma Chemical Co., St. Louis, MO 63178.








69
protected LSD test or Dunnett's 'T test at the 0.05 level of significance. Means are

presented with standard errors.

Results and Discussion

Although plant material was obtained from axenic stocks, a certain degree of

contamination, bacteria and/or fungal, occurred in each of the experiments. Because

of the relatively short duration of the experiments, this contamination did not cause

plant death. There was no observed trend for any treatment to be more

contaminated than the others. Therefore, it is not the author's intent to claim axenic

culture was used or maintained, and conclusions drawn from these studies are not

intended to reflect that premise.

There was a significant (P < 0.05) interaction between biotype and

photoperiod for subterranean and axillary turion production and dry weight. Both

biotypes produced greater biomass under long-day conditions (Table 4.1). Dioecious

hydrilla grown under short days, produced less than 70% of the biomass produced

by long-day grown plants. There was a 20% difference for monoecious hydrilla.

Axillary turion production was not observed for dioecious hydrilla, while monoecious

hydrilla produced these structures under both photoperiods (Table 4.2). Dioecious

hydrilla produced subterranean turions only under short-day conditions, while the

monoecious hydrilla produced subterranean turions under both photoperiods (Table

4.3).

In this study, photoperiod had a tremendous effect on plant growth and

subterranean turion formation for dioecious hydrilla. Dioecious hydrilla has been








70
Table 4.1. The. effect of photoperiod on the growth of 8 week old dioecious and
monoecious hydrilla.


Photoperiod


Long-Day

Short-Day


Dioecious Monoecious

------------------- g dry/plant -- ----------

0.920.09' 1.21 0.05

0.28 +0.04 0.95 +0.05


Means followed by standard errors.








71

Table 4.2. The effect of photoperiod on the axillary turion production of 8 week old
dioecious and monoecious hydrilla.


Photoperiod Dioecious Monoecious

S--------- turions/plant------

Long-Day 0 0.880.401

Short-Day 0 2.71+1.41


1 Means followed by standard errors.









Table 4.3. The effect of photoperiod on the subterranean turion production of 8
week old dioecious and monoecious hydrilla.



Photoperiod Dioecious Monoecious

--------------- turions/plant ---------

Long-Day 0 0.750.491

Short-Day 1.29+0.42 1.000.53


1 Means followed by standard errors.








73
shown to undergo 'annual decline' (Berg, 1977), which is characterized by a loss of

biomass prior to the onset of winter. In these experiments short days may have

caused this phenomenon in dioecious hydrilla, thus limiting biomass production.

Several species of plants cease vegetative growth during the onset of reproduction

(Gardener et al., 1985). However, this does not seem to be the case with

monoecious hydrilla. Another possible explanation could be the lower light intensity

in the short-day chamber. Monoecious hydrilla also displayed significantly less

biomass in this environment, but to a much lesser extent than dioecious hydrilla.

Dioecious hydrilla has been reported to be a short-day plant in terms of turion

formation, with a critical photoperiod of 11 to 12 hours (Van et al., 1978; Miller et

al., 1993). This characteristic response was also observed in these studies. However,

axillary turion development was not observed under either day length. Haller (1976)

and Miller et al. (1993) reported axillary turion formation occurred on floating

hydrilla plants. Plants used in these experiments were intentionally rooted which

possibly precluded axillary turion formation. Monoecious hydrilla produced axillary

and subterranean turions under both photoperiods. This contrasts with earlier

research, which showed no turion formation under 14 to 16 hour daylengths or photo-

interruption of long nights (Anderson and Spencer, 1986; Spencer and Anderson,

1987).

Monoecious and dioecious hydrilla biotypes were adversely affected by

increasing concentrations of fluridone (Table 4.4). Ten ppb fluridone caused 91 and

82 % reduction in shoot biomass of dioecious and monoecious hydrilla, respectively.








74
Table 4.4. The effect of fluridone on the growth of dioecious and monoecious
hydrilla. Plants were grown under long-day conditions for 12 weeks.


Dioecious Monoecious

--------------------- g dry/plant -------------------

0.56+0.181 0.63+0.26

0.450.16 0.71+0.08

1.03+0.18 0.600.08

0.26+0.10 0.32+0.10

0.050.01*2 0.110.04*

0.383


1 Means followed by standard errors.
2 Values within a given biotype followed by are significantly different from the
control (Dunnett's 't' testo.05).

3 To separate the effect of photoperiod within a given level of fluridone.


Fluridone

(ppb)

0

0.5

1.0

5.0

10

LSDo.05








75
One ppb fluridone caused a 46 % increase in dioecious shoot biomass. Fluridone

decreased turion production at higher concentrations (5 and 10 ppb), while

stimulating turion production at 0.5 ppb in monoecious hydrilla (Table 4.5). This

observation is similar to results reported from earlier field experiments with

dioecious hydrilla (MacDonald et al., 1993). This indicates that these biotypes

behave similarly to fluridone in response to turion formation.

Fluridone is a very effective herbicide, inhibiting carotenoid biosynthesis and

causing subsequent photooxidation of unprotected chlorophyll molecules (Bartels and

Watson, 1978; Devlin et al., 1978). In this study, plant tissue became whitish in color,

indicating pigment loss. Monoecious hydrilla appeared to 'recover' as indicated by

green coloration, but still showed a decrease in biomass over the course of the study.

The lethal effect of fluridone has been demonstrated to be an inhibition of

photosynthesis and plant death due to starvation. However, plants grown with

sucrose amendments should have been able to survive fluridone treatment. If

hydrilla could have been grown heterotrophically then fluridone could have been

used to regulate ABA formation without the lethal effects from reduced

photosynthesis. This could have allowed a determination of whether fluridone acts

as a growth regulator at low concentrations or a herbicide at higher concentrations.

Hydrilla could not be grown heterotrophically and therefore separation of the growth

regulator and herbicidal effects of fluridone could not be ascertained.

In this experiment, fluridone decreased the growth of both monoecious and

dioecious hydrilla biotypes, with monoecious being affected to a lesser degree than









Table 4.5. The *effect of fluridone on the axillary turion production of monoecious
hydrilla. Plants were grown under long-day conditions for 12 weeks.


Fluridone


Turions/Plant


(ppb)


2.00.71

5.0+1.1*2


2.71.7

0.30.3


1 Means followed by standard errors.
2 Values followed by are significantly different from the control (Dunnett's 't'
testo.05)-







77
dioecious hydrilla. Although the goal of fluridone treatment in this experiment was

to lower ABA levels thus precluding turion formation, it is possible that lowered

ABA may be causing the reduction in growth. Studies with ABA-deficient mutants

have demonstrated lower growth rates, epinasty and wiltiness associated with these

types of mutations (Koornneef et al., 1982).

Fluridone at 5 and 10 ppb caused a deep purple coloration in dioecious

hydrilla. Fluridone at high rates has been shown to increase the anthocyanin content

of hydrilla but only in mature tissue (Doong et al, 1993). This phenomenon was

thought to be in response to oxidative stress due to the loss of carotenoids.

Anthocyanin production is also associated with high levels of carbohydrate (Creasy,

1968), present in mature tissue, and which was also present in this study.

There was not a significant (P > 0.05) effect of experiment for exogenous

applications of abscisic and gibberellic acid, therefore experiments for each biotype

were pooled. In dioecious hydrilla, exogenous applications of ABA alone induced

axillary turion formation at all concentrations (Table 4.6). The greatest number of

turions were observed following treatments of 0.1 and 1.0 IM ABA. The effect from
ABA was reversed by applications of GA, but not completely. ABA also stimulated

axillary turion production in monoecious hydrilla, but only at concentrations of 1.0

and 10 pM (Table 4.7). Similarly, GA at 50 and 500 uM completely reversed this
effect at 1.0 iM ABA.

Total subterranean turion weight per plant reflected the same trend observed

from turion number per plant (Tables 4.8 and 4.9). The size of dioecious turions

were always larger than monoecious hydrilla regardless of treatment (0.005 and 0.002









Table 4.6. Axillary turion production by 8 week old, long-day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.



Abscisic Gibberellic Acid (0iM)1
Acid (PM)' 0 5 50 500

------------------ turions/plant-- -----------

0 0 0 0 0

0.1 15.65.12 6.43+2.8 5.02.6 0

1.0 27.06.2 0.90.9 17.79.4 4.63.2

10 11.4+4.0 7.64.3 5.42.6 4.41.3



1LSDo.05 = 10.6 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.


2 Means followed by standard errors.









Table 4.7. Axillary turion production by 8 week old, long-day grown monoecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.



Abscisic Gibberellic Acid (;M)1
Acid (sM)1 0 5 50 500

------------------ turions/plant----------------------


42.614.72

52.3 + 17.1


9.36.1


35.610.6


22.4 10.2


4.1+3.1


1 LSDo.05 = 4.8 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.


2 Means followed by standard errors.









Table 4.8. Axillary turion weight per plant by 8 week old, long-day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.



Abscisic Gibberellic Acid (;M)1
Acid (gM)1 0 5 50 500

--------------------- mg dry/plant -- -----------

0 0 0 0 0

0.1 74312 3315 2012 0

1.0 128+45 3+3 65+40 5+3

10 5222 52+3 31+15 15+8



1 LSDo.05 = 0.06 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.


2 Means followed by standard errors.









Table 4.9. Axillary turion weight per plant by 8 week old, long-day grown
monoecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.



Abscisic Gibberellic Acid (IAM)1
Acid (SM)' 0 5 50 500

--------------------- g dry/plant -- ------------

0 0 0 0 0

0.1 0 0 0 0

1.0 0.231+0.0762 0.0200.013 0 0

10 0.1460.052 0.0570.025 0.0420.03 0.0060.005


1 LSDo.05 = 0.07
gibberellic acid or
abscisic acid.


2 Means followed by standard errors.


to separate the effect of abscisic acid within a given level of
to separate the effect of gibberellic acid within a given level of







82
g- dry wt turion for dioecious and monoecious hydrilla, respectively). The

percentage of total plant weight represented by turions also reflected the same

response as described for turion number (Tables 4.10 and 4.11). interestingly ,

monoecious hydrilla appears to partition a greater percentage of biomass for

reproduction than dioecious hydrilla, producing a greater number of smaller turions.

The effect of ABA and GA on the shoot dry weight of both hydrilla biotypes

was different than the effects on turion formation (Tables 4.12 and 4.13). Both ABA

and gibberellic acid inhibited shoot growth of dioecious hydrilla with GA being the

more inhibitory compound. At the highest concentration of both compounds, ABA

ameliorated the activity of GA. Monoecious hydrilla was inhibited equally by ABA

and GA in terms of shoot growth. However, these two compounds appeared to act

additively to inhibit shoot growth. Although a reduction in shoot growth was

observed on a dry weight basis, the plants treated with GA elongated to the top of

the tube. It is probable that the reduction in dry weight was caused by decreased

leaf production by the plants.

Dioecious hydrilla did not produce subterranean turions or flowers (data not

shown). Monoecious hydrilla produced an average of 5 subterranean turions per

tube only in the absence of hormone applications (untreated control). In addition,

monoecious hydrilla produced an average of 44 flowers per tube the addition of

gibberellic acid at 50 1M. Floral production has been reported to be influenced by
nitrogen and phosphorus (Pietrse et al., 1984). Flowering has been associated with

short day conditions in dioecious hydrilla, suggesting turion formation and flower









Table 4.10. Percent weight of axillary turions produced by 8 week old, long-day
grown dioecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.



Abscisic Gibberellic Acid (1M)1
Acid (AM)1 0 5 50 500

---------------- % of total dry weight ---------

0 0 0 0 0

0.1 12.06.22 0.80.09 0.72+0.08 0

1.0 22.5+8.1 0.410.07 0.580.14 0.42+0.12

10 0.650.10 0.62+0.13 0.44+0.10 0.500.12



1 LSDo.o05 = 8.95 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.


2 Means followed by standard errors.









Table 4.11. Percent weight of axillary turions produced by 8 week old, long-day
grown monoecious hydrilla as influenced by exogenous abscisic and gibberellic acid
applications.



Abscisic Gibberellic Acid (iM)1
Acid (tiM)1 0 5 50 500

--------------- % of total dry weight -- -----------


31.2+9.32


2.5+1.7


29.09.6 13.33.9 8.74.8


0

1.20.8


1 LSDo.05 = 9.8 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.


2 Means followed by standard errors.









Table 4.12. Shoot biomass production by 8 week old, long-day grown dioecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.



Abscisic Gibberellic Acid (/AM)1
Acid (jAM)1 0 5 50 500

-----------------------------g dry/plant----- ----------

0 0.800.042 0.730.15 0.660.10 0.350.14

0.1 0.580.11 0.80+0.09 0.72+0.08 0.500.04

1.0 0.690.13 0.410.07 0.580.14 0.420.12

10 0.65+0.10 0.62+0.13 0.44+0.11 0.500.12



1 LSDo.05 = 0.08 to separate the effect of abscisic acid within a given level of
gibberellic acid or to separate the effect of gibberellic acid within a given level of
abscisic acid.


2 Means followed by standard errors.









Table 4.13. Shoot biomass production by 8 week old, long-day grown monoecious
hydrilla as influenced by exogenous abscisic and gibberellic acid applications.



Abscisic Gibberellic Acid (jM)1
Acid (/pM)1 0 5 50 500

-----------------------------g dry/plant-------- -------

0 0.680.092 0.530.12 0.720.07 0.490.07

0.1 0.42+0.03 0.580.07 0.54+0.10 0.34+0.06

1.0 0.58+0.11 0.47+0.12 0.42+0.11 0.31+0.05

10 0.460.05 0.37+0.05 0.33+0.06 0.32+0.06


1 LSDo.05 = 0.23
gibberellic acid or
abscisic acid.


2 Means followed by standard errors.


to separate the effect of abscisic acid within a given level of
to separate the effect of gibberellic acid within a given level of







87

production are linked to a common physiological process. However, floral

stimulation by exogenous gibberellic acid, implied a different, separate mechanism

was involved in this process.

Several researchers have demonstrated the effect of exogenous ABA

applications on turion formation in dioecious hydrilla but little work has been done

with the monoecious biotype (Klaine and Ward, 1984; Van t al. 1978). Dioecious

hydrilla appeared to be more sensitive to ABA applications than monoecious hydrilla,

as evidenced by turion formation at 0.1 piM and reduced sensitivity to GA
applications. ABA has been shown to induce tuber formation in potato and this

effect can be reversed by application of GA (Vreugdenhill and Struik, 1989). The

increased sensitivity of the dioecious biotype to exogenously applied ABA could be

related to photoperiodic responses, whereby applications of ABA could induce a

cascade of reactions that lead to turion formation. However, this should have

resulted in subterranean turion formation, not in the production of axillary turions.

Interestingly, monoecious hydrilla produced subterranean turions without

treatment, similar to those plants in the photoperiod experiment. If ABA was

directly responsible for the formation of these structures, then exogenous applications

should have induced this process. This suggested an indirect role of ABA in turion

development, with the formation of the rhizome being the initial step and not related

to changes in ABA. ABA could be produced after rhizome differentiation to

maintain dormancy and induce swelling of the rhizome tip. In a normally developing

hydrilla plant, the amount of ABA produced during subterranean turion development







88
is sufficiently high that ABA could begin to accumulate in other tissues. These

tissues could be an active meristem, such as the tips of stems or buds in the leaf axils.

As ABA begins to accumulate, the tissue begins importing carbohydrate, forming a

turion. ABA has been shown to regulate assimilate flow in plants (Porter, 1981) and

could be acting to insure proper carbohydrate levels of the turion.

However, there appears to be critical ratio between ABA and GA, as

evidenced by turion size. Turions produced under any influence of GA, were

extremely small and probably would not have been able to regenerate a new plant.

Therefore, ABA must accumulate in a limited number of sites, ensuring proper

development of the propagule. This could be regulated by GA. Several researchers

feel there exists a critical ratio of ABA to GA at all times within the plant, similar

to the roles of auxin and ethylene (Menzel, 1980; Vreugdenhill and Struik, 1989).

Therefore, the effect of exogenous ABA is probably not direct, but rather alters the

ratio of ABA:GA. The relatively lower GA level induces the desired phenomenon

(personal communication, Dr. Anwar Khan, Cornell University).

Collectively, these studies provided evidence that hydrilla cultured under

controlled conditions responds similarly to hydrilla grown under field conditions. The

exogenous applications of hormones indicated that ABA alone does not control

hydrilla turion formation but rather a critical ABA:GA balance may be the key to

the regulation of this phenological process. These hormones also appear to regulate

floral production in monoecious hydrilla, and subterranean turion formation.

However, the role of ABA and GA in these processes warrants further research.